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PMC554763	Background ========== The yellowfin tuna, Thunnus albacares, has a global distribution in tropical and sub-tropical oceans. Annual catches of yellowfin tuna have averaged 1.2 million metric tons since 1998 with sizes ranging from 5 to 20 kg \[[@B1]\]. If the average size of a harvested fish is 10 kg, then the harvest of 1.3 million metric tons in 2002 represents approximately 130 million individual fish. In an earlier study, Scoles and Graves \[[@B2]\] were unable to find evidence of genetic differentiation between small samples of yellowfin tuna (Thunnus albacares) from the Atlantic (n = 20) and Pacific Oceans (n = 100) using an RFLP analysis of whole mitochondrial DNA (mtDNA). Subsequently, Ward et al. \[[@B3]\] found significant differentiation at the GPI-A\* allozyme locus but only weak evidence for genetic differentiation with an RFLP analysis of whole mtDNA. In the whole mtDNA study, much larger sample sizes were employed to increase the sensitivity of the mtDNA assay, but only two restriction enzymes were used and consequently, only a few restriction sites were analyzed. The GPI-A\* data were consistent with earlier studies that demonstrated genetic differentiation between Eastern and Western Pacific samples \[[@B4],[@B5]\]. Skipjack tuna (Katsuwonus pelamis) are found in tropical and warm temperate waters of the world\'s oceans. They are present in the three major oceans in large numbers and comprise approximately 40% of the annual catch of the world\'s tunas. Annual catches are on the order of 2 million metric tons or approximately 670 million individuals per year \[[@B1]\]. Despite their huge numbers, the skipjack tuna are not as well studied as most members of the genus Thunnus. Tagging studies have demonstrated limited seasonal movements, but not much transoceanic movement. Thus, they probably do not spawn at discrete locations \[[@B6]\]. In the Pacific, genetic studies using isozymes have demonstrated an East-West cline in a serum esterase allele \[[@B7]-[@B9]\]. Fujino et al. \[[@B10]\] also demonstrated differences in esterase allele frequencies in samples from the Atlantic, Indian, and Pacific Oceans. However, small samples of Atlantic (n = 7) and Pacific (n = 9) skipjack appeared identical when mtDNA was examined \[[@B11]\]. After the formation of the Isthmus of Panama, the potential contact between Atlantic and Indo-Pacific populations was limited to the waters around southern Africa. It appears that this separation of the Atlantic and Pacific Oceans resulted in significant genetic drift for many large pelagic fishes since these subpopulations are now genetically differentiated. Examples include bigeye tuna \[[@B12],[@B13]\], albacore \[[@B14],[@B15]\], swordfish \[[@B16],[@B17]\], blue marlin \[[@B18]\] and sailfish \[[@B19]\]. In addition, even more pronounced differentiation produced species pairs of Atlantic and Pacific bluefin tunas and the Atlantic white marlin and the Pacific striped marlin \[[@B19]-[@B22]\]. To measure the degree of genetic differentiation between the Atlantic and Pacific sub-populations of either yellowfin or skipjack tuna, we examined the hypervariable control region I (CR-I) and a segment of a coding region gene of the respective mitochondrial DNAs. Genetic differences were observed between the Atlantic and Pacific yellowfin tuna samples with PCR-RFLP data of the ATCO gene region, but not with CR-I sequence data. In contrast, no differences between the Atlantic and Pacific skipjack tuna samples were detected with either type of data. Information contained in the CR-I reveals very different demographic histories for yellowfin tuna and skipjack tuna. However, very large long-term female effective population sizes (N~e~) were estimated for both species, which may explain the observed levels of inter-oceanic genetic partitioning. Results ======= Control region nucleotide sequence analysis ------------------------------------------- A total of 333 bp of the nucleotide sequence of the mitochondrial DNA (mtDNA) control region was determined for 148 yellowfin tuna (Table [1](#T1){ref-type="table"} and GenBank accession numbers AY899520 -- AY899681). For the pooled sample of yellowfin tuna, 110 variable sites defined 130 haplotypes (h= 0.997) and a nucleotide diversity (π) of 3.5%. Diversity indices for each locality sampled were also high, although values of π and hwere slightly lower for the NW Atlantic yellowfin sample where five haplotypes were repeated twice. For skipjack tuna, a total of 394 bp was determined for 115 individuals (Table [2](#T2){ref-type="table"} and GenBank accession numbers AY899405 -- AY899519). In the pooled skipjack tuna sample, there were 157 variable sites defining 111 haplotypes, resulting in a very high value of haplotypic diversity (h= 0.999). Nucleotide diversity (π = 8.4%) was more than twice as high in skipjack tuna as in yellowfin tuna. All of the sampling localities of skipjack tuna had very high diversity values (π \>7.7%; h\> 0.998). For both species, the high haplotypic diversity values are consistent with the observed large census population sizes (N~c~). Phylogenetic analyses resulted in very different trees for each species. The CR-I gene-tree topology of skipjack tuna is much larger and better structured than the yellowfin tuna phylogeny, and contains multiple branches with high bootstrap proportion support (Figure [1](#F1){ref-type="fig"}). However, there is no obvious phylogeographic association in either species, with CR-I lineages from different basins scattered throughout the phylogenetic trees. As a consequence, in both yellowfin tuna (Table [3](#T3){ref-type="table"}) and skipjack tuna (Table [4](#T4){ref-type="table"}), the majority of the genetic variation corresponds to differences between individuals within populations, and only a minor fraction of the variation corresponds to differences among-groups. Thus, in both species, the control region sequences provide no evidence of genetic differentiation between Atlantic and Indo-Pacific sub-populations. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Genetic diversity indices and demographic parameters of yellowfin tuna CR-I. Nnumber of individuals; M, number of haplotypes; π, nucleotide diversity; h, haplotypic diversity. Tajima\'s D neutrality test and associated probability in parentheses. Mismatch distribution parameters τ, Θ~O,~Θ~1.~ ::: Population N M π h Tajima\'s D τ Θ~O~ Θ~1~ --------------- ----- ----- --------------- --------------- ---------------- ------- ------- ------ All 148 130 0.035 ± 0.018 0.997 ± 0.001 -1.588 (0.022) 8.516 0.047 566 NW Atlantic 31 26 0.027 ± 0.014 0.987 ± 0.012 -0.908 (0.185) 9.746 0.000 87 Ivory Coast 32 29 0.029 ± 0.016 0.994 ± 0.009 -1.254 (0.085) 8.531 0.008 1097 Pacific Ocean 41 40 0.033 ± 0.017 0.999 ± 0.006 -1.155 (0.111) 9.250 0.000 6655 Indian Ocean 44 41 0.032 ± 0.017 0.997 ± 0.006 -1.346 (0.059) 8.391 0.708 352 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Genetic diversity indices and demographic parameters of skipjack tuna CR-I. Abbreviations and notations as in Table 1. ::: Population N M π h Tajima\'s D τ Θ~O~ Θ~1~ ------------------ ----- ----- --------------- --------------- ---------------- ------- ------- ------ All 115 111 0.084 ± 0.041 0.999 ± 0.001 -0.419 (0.424) 15.82 12.34 3795 NW Atlantic 31 30 0.082 ± 0.041 0.998 ± 0.009 -0.343 (0.408) 15.32 13.50 6655 Brazil 17 17 0.084 ± 0.043 1.000 ± 0.020 -0.354 (0.431) 28.38 4.03 98 E. Pacific Ocean 32 31 0.077 ± 0.038 0.998 ± 0.008 -0.084 (0.537) 14.00 16.10 6655 Solomon Islands 35 34 0.083 ± 0.041 0.998 ± 0.007 -0.266 (0.445) 14.18 16.75 4683 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### AMOVA of the patterns of sequence variation contained in the CR-I of yellowfin tuna. Localities were assigned into three regional groupings: Atlantic, Pacific and Indian. The Atlantic region included two samples, NW Atlantic and Ivory Coast, whereas the Pacific and Indian regions included only one sample each. ::: Source of Variation Variance components Percentage variation Fixation Indices Probabilities --------------------------------- --------------------- ---------------------- ------------------ --------------- Among groups 0.00155 Va 0.31 Φ~CT~: 0.003 0.17 ± 0.01 Among populations within groups -0.00041 Vb -0.08 Φ~SC~: -0.001 0.51 ± 0.01 Within Populations 0.49818 Vc 99.77 Φ~ST~: 0.002 0.038 ± 0.006 ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### AMOVA of the patterns of sequence variation contained in the CR-I of skipjack tuna. Localities were assigned into three regional groupings: Atlantic, Pacific and Indian. The Atlantic region included two samples, NW Atlantic and Brazil, whereas the eastern Pacific and Indian Ocean (Solomon Islands) regions included only one sample each. ::: Source of Variation Variance components Percentage variation Fixation Indices Probabilities --------------------------------- --------------------- ---------------------- ------------------ --------------- Among groups 0.296 Va 1.89 Φ~CT~: 0.019 0.16 ± 0.00 Among populations within groups 0.349 Vb -2.23 Φ~SC~: -0.023 0.72 ± 0.01 Within Populations 15.712 Vc 100.34 Φ~ST~: -0.003 0.72 ± 0.01 ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Unrooted neighbor-joining (NJ) trees showing the relationship of 111 yellowfin tuna and 130 skipjack tuna haplotypes estimated from a matrix of Tamura Nei (α = 0.5) distances. Values of bootstrap proportion support above 60% are included and the geographical origin of haplotypes is identified with symbols (see inset). The bar indicates the size of a line representing differences of 2% with both trees drawn to the same scale. ::: ![](1471-2148-5-19-1) ::: RFLP analysis of the mitochondrial ATCO gene region --------------------------------------------------- We hypothesized that the high mutation rate of the control region might have resulted in homoplasy and high levels of haplotypic diversity that masked the actual genetic divergence of Atlantic and Pacific mtDNAs in these species. Analysis of a gene that has a lower mutation rate than that of the control region has been used to reveal genetic divergence in bigeye tuna \[[@B13]\]. Therefore, we performed a nucleotide sequence analysis on the yellowfin tuna mitochondrial ATCO gene region since variation had been shown previously for this gene \[[@B23],[@B24]\]. We found mutations at two positions that resulted in the loss of restriction sites for the enzymes DdeI and HpyCH4III, respectively. When amplified Atlantic and Indo-Pacific yellowfin ATCO DNAs were analyzed with these two enzymes, most DNAs were cut with both enzymes (Table [5](#T5){ref-type="table"}). However, DNAs that had lost either one of the two restriction sites were more common in the Atlantic sample than in the Indo-Pacific sample. When the haplotype distributions were compared, the differences were found to be significant with 7% of the variation occurring between samples (Table [6](#T6){ref-type="table"}). Thus, low levels of genetic differentiation have occurred between the two yellowfin sub-populations. When the haplotypes were mapped to a neighbour-joining tree of the control region sequences, the mutations affecting the restriction sites were clustered, suggesting that each mutation had occurred once prior to separation of the two sub-populations (data not shown). This result is consistent with a slower rate of mutation in the ATCO gene region compared to that of the control region. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Allele frequencies at the yellowfin tuna mtDNA ATPase6 locus ::: Sample N ATCO Haplotypes ---------- ----- ----------------- ------ ------ D H DH Atlantic 138 0.13 0.22 0.65 Pacific 96 0.02 0.12 0.86 Haplotype designations: D, cut with DdeI; H, cut with HpyCH4III; DH, cut with both enzymes. N is the sample size. ::: ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Analysis of molecular variance between Atlantic and Pacific yellowfin tuna samples ::: Source of Variation d\. f. Sum of Squares Variance Components Percentage of Variation --------------------- -------- ---------------- --------------------- ------------------------- Between Populations 1 1.19 0.015 Va 7.0 Within Populations 232 46.69 0.201 Vb 93.0 Totals 233 48.60 0.216 Fixation Index F~ST~: 0.070 Va and F~ST~: P(random value \> = observed value) = 0.00098+/-0.00098 ::: Similar experiments were performed with the skipjack tuna samples. Two variable sites had been observed in the mitochondrial cytochrome b (cytB) gene by Terol et al. \[[@B25]\]. Since one of these sites could not be assayed with restriction enzymes, we determined the nucleotide sequence of this portion of the cytBgene for each DNA in the Atlantic and Pacific skipjack tuna samples. Little additional variation was observed, and allele frequencies were similar in the two samples (Table [7](#T7){ref-type="table"}). When the haplotype distributions were compared, all of the variation occurred within samples. Thus, there is no evidence for genetic differentiation between the Atlantic and Pacific sub-populations of skipjack tuna. ::: {#T7 .table-wrap} Table 7 ::: {.caption} ###### Allele frequencies at the skipjack mtDNA cytBlocus ::: Allele\* ---------- ---- ---------- ------ ------ Region n AT AC GC Pacific 80 0.31 0.24 0.45 Atlantic 49 0.31 0.18 0.51 \Variable base at the PshA1 site and a position 19 bases upstream as determined by nucleotide sequence analysis. ::: Demographic history and effective population size ------------------------------------------------- The unimodal mismatch distribution for the pooled sample of yellowfin tuna (Figure [2](#F2){ref-type="fig"}) and a significant Tajima Dtest (Table [1](#T1){ref-type="table"}) both suggest the historical expansion of this population. In sharp contrast, the skipjack mismatch distribution is bimodal (Figure [2](#F2){ref-type="fig"}) and the neutrality test is not significant (Table [2](#T2){ref-type="table"}), suggesting that the effective size of the skipjack population has been large and stable for a long period. In fact, the value of θ~0~suggests that the number of females in the skipjack population was originally large (N~e0~= 320,000) whereas the effective number of yellowfin tuna females prior to expansion was small (N~e0~= 823). However, the values of θ~1~suggest large long-term numbers of effective female breeders (N~e1~) of about 98 million for skipjack tuna and about 10 million for yellowfin tuna. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Mismatch distributions for the entire sample of a) yellowfin tuna and b) skipjack tuna. The solid bars in the histograms represent the observed pairwise differences between haplotypes and the curves the expected distribution under the sudden expansion model. ::: ![](1471-2148-5-19-2) ::: Discussion ========== Compared to other scombroid species with cosmopolitan distributions, we found substantially less genetic differentiation between Atlantic and Pacific sub-populations of yellowfin tuna and no inter-oceanic genetic differentiation of skipjack tuna. For instance, in bigeye tuna, there are two highly divergent mtDNA clades, one is cosmopolitan and the other is endemic to the Atlantic \[[@B12],[@B13]\]. This asymmetric distribution of Atlantic and Pacific clades has been observed in other large pelagic fishes as well, including swordfish \[[@B16],[@B17]\], blue marlin \[[@B18]\], and sailfish \[[@B19],[@B26]\]. Similarly, the striped marlin (Pacific) and white marlin (Atlantic) and the Atlantic bluefin tuna and the Pacific bluefin tuna are considered to be pairs of sister species \[[@B19]-[@B21],[@B26]\]. Thus, significant differentiation has occurred between the Atlantic and Pacific populations of many large pelagic species. Why are skipjack and yellowfin tuna populations different? One possibility is that in contrast to other large pelagic species, sufficient gene flow occurs to prevent inter-oceanic genetic differentiation. However, this explanation is contrary to the distribution patterns of tunas and billfish \[[@B27],[@B28]\]. For instance, bigeye tuna has a distribution of catches that would suggest a population continuum from the Indian Ocean to South Atlantic waters along the east and west coasts of Africa. However, bigeye samples show a marked inter-oceanic differentiation \[[@B12],[@B13]\]. In the Indian Ocean, the presence of skipjack and yellowfin tuna south of 20° S, is confined to the warm waters associated with the Agulhas currents. Seasonal movements do occur around the Cape of Good Hope via the Agulhas current, but it appears that the migrant tuna and other pelagic species return to the Indian Ocean as the seasons change \[[@B13],[@B29]\]. In fact, the distribution patterns of these two species in the South Atlantic along the African coast of are remarkably similar to both blue marlin and sailfish, two species that show a pronounced differentiation between Indo-Pacific and Atlantic populations. It should be noted, however, that the presence of \"Pacific\" mitochondrial DNA clades in Atlantic subpopulations of many tuna species indicates that inter-oceanic migration has occurred in the past. However, contemporary levels of inter-oceanic genetic differentiation for these species indicate that current levels of gene flow are absent or severely reduced. Thus, current distribution patterns of skipjack tuna and yellowfin tuna are not consistent with substantial levels of inter-oceanic gene flow. Certainly, there is no evidence to suggest that gene flow is occurring or has occurred at levels higher in these two species than in any of the other pelagic species where interoceanic differentiation has been demonstrated. An alternative explanation to account for the lack of inter-oceanic differentiation in skipjack tuna and yellowfin tuna, is that the time since population expansion began has not been sufficient to allow for the populations to become differentiated. Assuming the very conservative mutation rate for CR-I of 4.9% per million years, a generation time 3.5 years, and the tau value 8.52, expansion of yellowfin tuna occurred about 522 Ky ago. By comparison, the estimated time for genetic differentiation of the Atlantic and Pacific populations of swordfish and of the Atlantic and Pacific bluefin tunas, using the same mutation rate, is very similar (450--470 Ky), and an even shorter time (170 Ky) is sufficient to explain the substantial genetic differentiation between the Atlantic and Mediterranean swordfish populations \[[@B30]\]. Thus, time since expansion cannot explain the absence of inter-oceanic differentiation in these species. Therefore, we propose that much larger effective population sizes are the primary factor responsible for the similarity of the Atlantic and Pacific sub-populations of skipjack and yellowfin tuna. Among large pelagic species, the female effective population size can be estimated from the demographic estimates obtained from the CR-I sequences of Atlantic bluefin tuna and swordfish. Assuming a mutation rate of 4.9% per million years and a generation time of 6 years, the female N~e~estimates are 900,000 for bluefin tuna and 800,000 for swordfish \[[@B30]\]. Effective population sizes have not been estimated for other species of tunas, but annual harvest data are generally used as a proxy for abundance since the commercial harvest of large fish has become quite efficient throughout the world\'s oceans and harvest restrictions have only incremental impact on the total harvest of any species. Furthermore, haplotypic diversities of tuna mitochondrial DNAs are approximately 99% in all tuna species \[[@B12],[@B31]\], suggesting that female reproductive variance is small. Accordingly, census population size can be expected to be proportional to effective population size in tuna species. The estimated number of skipjack and yellowfin caught in the year 2002 is 670 million and 130 million, respectively (Table [8](#T8){ref-type="table"}). The catch of all other tunas was 24 million individuals or less. Thus, the abundance of skipjack tuna is approximately 5 times that of yellowfin, and more than 300 times that of Atlantic bluefin tuna and swordfish. These estimates of abundance from fisheries data appear to correspond well with the estimated female N~ef~values. The observed correspondence does not take into consideration dramatic changes in abundance of some of these species over the last 20 years, nor sex ratio differences, or the age distribution of the catch (e.g., number of mature females). However, the comparison supports the hypothesis that skipjack tuna and yellowfin tuna, which are the two most abundant species, also have the largest effective population sizes, and the lowest amounts of genetic partitioning compared to other scombroid fishes. ::: {#T8 .table-wrap} Table 8 ::: {.caption} ###### Worldwide Tuna Catch Data for the Year 2002 \[1\] ::: Species Catch (metric tons) Ave. Wt. (kg) Est. Number Fish Harvested\ ------------------ --------------------- --------------- ------------------------------ skipjack 2.0 × 10^6^ 3 670 × 10^6^ yellowfin 1.3 × 10^6^ 5--20 130 × 10^6^ bigeye 0.43 × 10^6^ 15--20 24 × 10^6^ albacore 0.24 × 10^6^ 9--20 16 × 10^6^ longtail 0.125 × 10^6^ 15--20 6.9 × 10^6^ Pacific bluefin 0.024 × 10^6^ 7 3.4 × 10^6^ Atlantic bluefin 0.036 × 10^6^ 17 2.1 × 10^6^ \Number of Atlantic bluefin tuna harvested obtained from ICCAT \[51\]. ::: Bigeye tuna, and bluefin tuna have much greater levels of genetic differentiation between Atlantic and Pacific subpopulations when mitochondrial DNA control region sequences are compared \[[@B12],[@B31]\]. However, their population sizes are 6 and 15 times lower than that of yellowfin and 30 and 75 times lower than that of skipjack, respectively. Furthermore, since bluefin tuna are a more temperate species, their effective population sizes may have been significantly lower than those observed in recent times whenever the northern hemisphere experienced glacial maxima. Thus, the relative differentiation of the Atlantic and Pacific subpopulations is consistent with the demographics of these species of tuna. One exception to the patterns described above is albacore tuna. The distribution of albacore tuna mitochondrial DNAs does not appear to fit the patterns described above for the other temperate tuna species. Albacore abundance is similar to that of bigeye tuna and both species have two mtDNA clades. However, in albacore the two clades are not as well differentiated nor do they display the phylogeographic association observed for the bigeye tuna clades \[[@B12]\]. Instead, the bimodal mismatch distribution of pairwise differences in albacore mtDNA is concordant with very large long-term effective population sizes in contrast to the contemporary population size. Conclusion ========== Much lower levels of genetic differentiation were found among sub-populations of yellowfin tuna compared to those observed for other large tunas, probably due to the large population size of yellowfin tuna. Since skipjack tuna appear to have even larger population sizes than yellowfin tuna, it is not surprising that no genetic differentiation was observed between Atlantic and Pacific samples of these fish. Methods ======= Samples of yellowfin tuna were obtained from the eastern Pacific Ocean (near the equator at 110° W; n = 41), the Indian Ocean (n = 63), the Gulf of Mexico and the East Coast of Florida (n = 38) and the Gulf of Guinea (n = 100). Samples of skipjack tuna were obtained from the Northwest Atlantic (n = 31), off the coast of Brazil (n = 19), the eastern Pacific Ocean (n = 43), and the south Pacific near the Solomon Islands (n = 37). DNA isolation, mitochondrial DNA D-loop region amplification, and nucleotide sequence analyses have been described previously \[[@B12],[@B32]\]. The number of segregating sites (S) was estimated with MEGA. Values of haplotypic diversity (h) \[[@B33]\], nucleotide diversity (π) \[[@B34]\] and the mean number of pairwise differences (K) were computed in ARLEQUIN ver. 2.0 \[[@B35]\]. Mitochondrial DNA haplotype phylogenies were estimated using neighbour-joining analyses \[[@B36]\] with Tamura-Nei distances (α = 0.5)) in MEGA \[[@B37]\]. The pair-deletion option was used when missing data, insertions, or deletions were present. Maximum-Parsimony (MP) \[[@B38],[@B39]\] was carried using heuristic searches with the default options in PAUP\ 4.0b10 \[[@B40]\]. Statistical support for the nodes was estimated with 1000 non-parametric bootstrap replicates \[[@B41]\]. All trees were rooted at midpoint. Analyses of molecular variance (AMOVA) \[[@B42]\] were performed to estimate the partitioning of genetic variation in regional hierarchical arrangements using ARLEQUIN. The demographic history contained in the mtDNA CR-I sequence data was inferred using two approaches. First, the null hypothesis of neutrality may be rejected when a population has experienced population expansion \[[@B43]\]. Accordingly, Tajima\'s D test of neutrality \[[@B43],[@B44]\] and its significance levels were estimated using DnaSP 4.00 \[[@B45]\] based on 1000 simulated re-samplings replicates. Alternatively, a population that has experienced a rapid expansion in the recent past shows smooth wave-like mismatch distribution \[[@B46],[@B47]\]. Thus, mismatch distribution analyses, under the assumption of selective neutrality, were also used to evaluate possible historical events of population growth and decline \[[@B47],[@B48]\]. Past demographic parameters, including τ \[[@B49]\], θ~0~and θ~1~and their probabilities \[[@B47]\] were estimated in ARLEQUIN taking into account the heterogeneity of mutation rates \[[@B35]\]. For the analysis of the mitochondrial cytochrome b gene, a 650 bp fragment of the skipjack tuna cytochrome b gene was amplified using primers CB3 (GGCAAATAGGAARTATCATTC) and GLUDG (TGACTTGAARAACCAYCGTTG) \[[@B50]\]. Alleles were identified by determining the nucleotide sequence of the amplified fragment. For yellowfin tuna, the ATCO gene region was amplified using primers H9342 (GCCATATCGTAGCCCTTTTTG) and L8562 (CTTCGACCAATTTATGAGCCC) \[[@B24]\]. The amplified fragments were digested with either DdeI or HpyCH4III and the digestion products were resolved by electrophoresis in a 1.2% agarose gel. Authors\' contributions ======================= BE, JV, and JAB conceived the study, supervised the genetic studies, analyzed the data and wrote the manuscript. JAB, AL, and ET performed the DNA sequence analyses. BE, DB, ET, LL, and KC performed the DNA amplifications and RFLP analyses. Acknowledgements ================ We thank Bernard Stequert, Peter Sharples, the National Marine Fisheries Service, and the Inter-American Tropical Tuna Commission (IATTC) for providing the samples used in this study. Kurt Schaefer of IATTC graciously provided estimates of age at maturity for skipjack and yellowfin tunas and valuable information on the regional heterogeneity of these estimates. This study was funded in part by grant RT/F-1 to the Cooperative Institute for Fisheries Molecular Biology (FISHTEC) from NOAA.	PubMed Central	2024-06-05T03:55:53.911485	2005-2-22	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554763/", "journal": "BMC Evol Biol. 2005 Feb 22; 5:19", "authors": [ { "first": "Bert", "last": "Ely" }, { "first": "Jordi", "last": "Viñas" }, { "first": "Jaime R ", "last": "Alvarado Bremer" }, { "first": "Donna", "last": "Black" }, { "first": "Luciano", "last": "Lucas" }, { "first": "Kelly", "last": "Covello" }, { "first": "Alexis V", "last": "Labrie" }, { "first": "Eric", "last": "Thelen" } ] }
PMC554764	Introduction ============ The health situation in Sudan continues to be affected by long-lasting conflict and related humanitarian emergencies such as food crises and epidemics. Malaria is one of the major causes of morbidity and mortality. In Sudan, an estimated 7.5 million patients suffer from malaria each year and 35,000 die from this disease, which accounts for up to 20% of hospital deaths \[[@B1]\]. The problem appears to have worsened in recent years due to increasing levels of Plasmodium falciparumresistance against the two most commonly used antimalarials: chloroquine (CQ) and sulfadoxine/pyrimethamine (SP). CQ resistance in the northern and central part of the Sudan is near 50% \[[@B2]\]. In the South, CQ resistance below 15% was found in some isolated locations \[[@B3],[@B4]\], but was over 80% elsewhere \[[@B5],[@B6]\]. Resistance to SP was shown by presence of SP-resistant genotypes \[[@B7]\] and in vivostudies documenting of 0--11% resistance in Khartoum and the eastern part of the country \[[@B2],[@B8]\] and varying levels of 0, 16% up to 70% in different locations in the southern part of the country \[[@B4]-[@B6]\]. Amodiaquine (AQ) provided an alternative for CQ in Africa in recent years; this drug is not officially registered in the Sudan, and due to its limited use, was expected to have preserved good efficacy. The nationally recommended treatment protocol for P. falciparummalaria in Sudan at the time of this study was still CQ as first-line and SP as second-line. A change in protocol was however in progress, and the Sudanese Health authorities had indicated that artemisinin-based combination therapy (ACT) was the preferred new first-line treatment \[[@B9]\]. The World Health Organization (WHO), MSF and various other NGOs supported this viewpoint \[[@B10],[@B11]\]. In line with recommendations for several other African countries, two ACTs were proposed for Sudan: Artesunate (AS) +SP or AS+AQ. The third option, used in other African countries, Artemether-Lumefantrine (Coartem) was not seen as a viable option at the time, because of its cost. To provide information on the efficacy of ACT treatment in Sudan, the in vivotherapeutic efficacy of the first two combinations were studied in Malakal town, Upper Nile State. Patients and Methods ==================== Malakal is located in southern Sudan (on the edge of the Khartoum-governed area), which is an area is of medium to high malaria endemicity (main transmission season: June-November). This open-label study was done in Malakal paediatric hospital supported by Médecins Sans Frontières, from September 2003 to January 2004. The methodology used was in accordance with standard WHO procedures for antimalarial drug efficacy assessment in high transmission settings \[[@B12],[@B13]\]. Children aged between 6 months and 5 years with fever (axillary temperature of 37.5°C or more) and P. falciparuminfection with a density of 2,000 to 200,000 parasites/μl but no signs of severe malaria (WHO criteria, \[[@B13]\]) or any other serious health condition were included in the study (provided written parental consent was given). One of the two therapies was allocated (by sequential alternation), and given under supervision: either (1) AS+SP, 3 days AS 4 mg/kg body weight on day 0, 1 and 2 plus SP, 25/1.25 mg/kg, single dose on day 0 or (2) AS+AQ, 3 days AS 4 mg/kg on day 0, 1 and 2 plus AQ 10 mg/kg on day 0, 1, 2. Doses were calculated for weight categories in quarter tablets. In case of vomiting within 30 minutes, the treatment was repeated. Repeated vomiting led to exclusion from study and referral to the hospital for quinine treatment. Medications were normally given in the mornings and appointments for next day roughly matched 24 hr intervals. Artesunate was obtained from Dafra (Belgium) and SP and AQ from IDA (the Netherlands). The sample size of at least 116 inclusions per arm was calculated to detect a 12% difference between the two arms, assuming a 97% efficacy at day 28 for the AS+SP arm (based on 100% efficacy of SP found in a recent study performed at a nearby location, \[[@B4]\]) and 85% for the AS+AQ arm (internationally recommended level for change), with 80% power and 5% risk type I error, anticipating a loss to follow-up of 10%. Patients returned daily during the first 3 days of illness and weekly thereafter for 6 weeks (42 days) after treatment. Side effects of drugs and self-medication were recorded based on patient reports and actively asked for during visits. Cases which remained negative during follow-up were considered Adequate Clinical and Parasitological Responses (ACPR). Failures were classified as (1) Early Treatment Failures (ETF) in case of significant parasitaemia at day 2 or 3 or parasites and fever at day 3, (2) Late Clinical Failures (LCF) for cases with parasites and fever during follow-up after day 3 and (3) Late Parasitological Failures (LPF) for parasite infections without fever at day 42, all in accordance with WHO guidelines \[[@B13]\]. Rescue treatment for failures was quinine 10 mg/kg/eight-hourly for 7 days. PCR analysis was used to distinguish new infections from recrudescences. Slides were examined by two microscopists independently and 20% of slides were cross-checked in an external laboratory (at KEMRI, Nairobi). The density of parasites was determined by simultaneous count of white blood cells and parasites, assuming a standard density of 8,000 WBCs per μl \[[@B13]\]. Haemoglobin levels were evaluated at day 0, 14, 28 and 42, to observe return to healthy Hb-levels after effective antimalarial treatment. PCR analysis of the msp1and msp2loci was used to distinguish re-infections from recrudescences \[[@B14],[@B15]\]. In case of two or more identical alleles in pre- and post-treatment genotypes, the case was classified as recrudescent, or as a new infection if one or none of the alleles matched. Cases were excluded from the final analysis in case of loss to follow-up, self-medication with antimalarials and concomitant disease and when PCR results showed re-infections or were undetermined due to missing samples, undetermined genotypes or non-amplifiable DNA. The research protocol was reviewed and approved by the Sudan Research Directorate, the Malaria, Leishmaniasis and Schistosomiasis Directorate, as well as by the Ethical Review Board of MSF (comprising of MSF-external experts only). During recruitment of the children in study, informed written consent was obtained from their parents/guardians. Results ======= A total of 269 children were recruited during the 5-month study period. Of these, 134 were treated with AS+AQ and 135 with AS+SP. Baseline characteristics were similar in both treatment groups (Table [1](#T1){ref-type="table"}). Of the children included, 40 were excluded from the final analysis due to associated serious other febrile illness (n = 15), self-medication with antimalarials (n = 7), loss to follow-up (n = 13) or erroneous inclusion/classification (n = 5, i.e. three cases of disagreement over microscopy quality control, two cases retreated with parasites but no fever prior to day 42 in AS+AQ group). There were three children with Hb level of 5.0 g/dl included (limit of in/exclusion), based on their condition assessed by the medical doctor. Altogether, 113 (84%) completed the study after treatment with AS+AQ and 116 (86%) with AS+SP (Figure [1](#F1){ref-type="fig"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Baseline characteristics of patients at study-enrolment, per treatment arm, Malakal, Upper Nile, Sudan 2003--4. ::: AS + AQ(n = 134) AS + SP(n = 135) --------------------------------------------- ------------------------- ------------------------ Sex ratio (F/M) 68/66 64/71 Mean age (months) (SD, ranges) 33 (13, 7--59) 33 (15, 6--59) Mean weight (kg) (SD, ranges) 11.1 (2.4, 5.7--17.0) 11.3 (2.6, 5.4--18.0) Mean axillary temperature (°C) (SD, ranges) 38.7 (0.9, 37.5--40.9) 38.9 (0.9, 37.5--40.9) Mean haemoglobin value (g/dl) (SD, ranges) 7.8 (1.7, 5.0--12.2) 7.9 (1.8, 5.0--12.7) Parasitaemia geometric mean (/μl) (ranges) 20 952 (2 100--199 500) 24835 (2 000--198 000) ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Patient flow during recruitment and follow-up ::: ![](1475-2875-4-14-1) ::: Both artemisinin-combitherapies were highly effective to treat P. falciparuminfections and prevent parasite re-emergence. Only 1 case of ETF was found, in the AS+SP group (Table [2](#T2){ref-type="table"}). At day 28 and day 42, the proportion of patients still parasite-free was higher in the group treated with AS+SP than in the group AS +AQ (χ^2^test, p = 0.014 for results day 28; p = 0.0049 for day 42). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Treatment endpoints on day 28, 42 after treatment and PCR adjusted day 42, Malakal, Upper Nile, Sudan, 2003--2004 ::: AS + AQ AS + SP ---------------------------- ------------- ------------- ------------ ------- ---------- -------------- Day 28 Results 117 116 ACPR 105 89.7 82.8--94.6 114 98.3 93.9 -- 99.8 ETF 0 0 1 0.9 0.0--4.7 LCF 10 8.5 4.2--15.2 1 0.9 0.0--4.7 LPF 2 1.7 0.2--6.0 0 0 Day 42 Results 113 116 ACPR 96 85.0 77.0--91.0 112 96.6 91.4--99.1 ETF 0 0 1 0.9 0.0--4.7 LCF 15 13.3 7.6--20.9 2 1.7 0.2--6.1 LPF 2 1.8 0.2--6.2 1 0.9 0.0--4.7 PCR corrected day 42^\#^ 97 113 No PCR result (12) (2) Re-infections (4) (1) ACPR 96 99.0 94.4--100 112 99.1 95.2--100 ETF 0 0 0.0--3.7 1 0.9 0.0--4.8 LCF 1 1.0 0.0--5.6 0 0 0.0--3.2 LPF 0 0 0.0--3.7 0 0 0.0--3.2 ^\#^Cases with missing or undetermined PCR results and re-infections were excluded from the analysis. ACPR = Adequate Clinical Parasitological Response, ETF = Early Treatment Failure, LCF = Late Clinical Failure, LPF = Late Parasitological Failure. ::: PCR analysis was unable to generate results for a large part of cases (only 6/20 available, Figure [1](#F1){ref-type="fig"}). In the AS+AQ treatment group, of 17 failures, only 1 was confirmed to be recrudescent, whereas of the other cases, 4 were re-infections and 12 samples were not analyzable. Of the 3 \'failures\' in the AS + SP group, one was an early treatment failure, PCR identified 1 re-infection and 2 were non-analysable. Hence, with the PCR correction and exclusions of non-analysable and re-infection cases, AS+AQ resulted in 1.0% confirmed failures (1/97) and AS+SP in 0.9% true failures (1/113) (Table [2](#T2){ref-type="table"}). Extrapolation of the cases with known PCR results to the ones that remained undetermined (assuming 2/3 of parasitemia due to reinfection), would give a 95.3% efficacy of AS+AQ (101/106) and 98.2% of AS+SP (112/114). Alternatively, a \'worst case\' scenario, assuming that all non-analysable PCRs were actually recrudescences, would lead to 88.1% efficacy in the AS+AQ group (96/109) and 97.4% for AS+SP (111/114). Parasites were rapidly cleared in both treatment groups. At day 2 only 17% and 22% of patients still had a (low) parasitaemia in the AS+AQ and AS+SP groups, respectively. Gametocytes were found in 20% (52/262) of children on inclusion. During follow-up, 78% of these carriers at enrolment showed gametocytes again on one or more follow-up days (32/41), whereas 22% of the children without gametocytes at inclusion were later found to be gametocytemic (42/192). There was no difference between the age, temperature, parasitemia, haemoglobin on admission of the two groups, neither in rates of ACPR at study-endpoint. Gametocyte carriage was similar in both treatment groups and all gametocytes disappeared gradually from the blood during the first weeks after follow-up, and cleared by day 21 in 95% of cases (Figure [2](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Parasitaemia and gametocytaemia during 6-week follow-up. Symbols: AS+SP ▲ parasites △ gametocytes, AS+AQ ● parasites ○ gametocytes. Error bars indicate 95%CI. ::: ![](1475-2875-4-14-2) ::: The body temperature of all patients went down quickly; fever clearance (T\<37.5°C) at day 1 and day 2 was 86% and 97% in the AS + AQ group and 83% and 93% in the AS+SP, and at day 3 all but one (on AS+SP) were free from fever. Average haemoglobin levels for the AS+AQ and AS+SP treatment groups increased from under 8.0 g/dl to near 10 g/dl 6 weeks after treatment. The proportion of children classified as moderately anaemic (haemoglobin from 5 to 8 g/dl) reduced from 51% to 11% during follow-up period, similar in both arms. No serious adverse events were observed or reported on routine clinical examination during follow-up and none of the failures developed complicated malaria. Discussion ========== This is one of the first studies into the clinical outcomes of ACT combination therapy in Sudan and provides useful information for decision-makers working to ensure effective antimalarial protocols in this part of the country. Both of the artemisinin-based combination therapies tested here were found to be highly efficacious in the treatment of uncomplicated P. falciparummalaria in this area of the Sudan. AS+SP appears to be the better treatment option on the basis of non-PCR corrected responses, showing a lower percentage of patients returning with parasitaemia. The PCR analysis indicates the true efficacy is comparable between both treatments (near 99%), but is, however, limited by a high proportion of indeterminate cases. More realistically, an efficacy between 97--98% can be expected for AS+SP and 88--95% for AS+AQ, acceptable levels after the long follow-up of 42 days after treatment. A rapid parasite clearance and fever reduction was found following treatment with both ACTs. The rise in haemoglobin values and the reduction of the proportion of (moderately) anaemic children after treatment confirms that the malaria parasites were effectively removed from the blood and red blood cell levels rose after treatment. The ACTs tested also had an effect on gametocytes. In general, the gametocidal action of AS appears to work through preventing the development of new gametocytes rather than clearance of existing ones \[[@B16]\]. In our study, the 20% of gametocyte-carrying infections at enrolment cleared gradually by day 21. Newly detected gametocytes developed in 22% of cases after treatment, which is lower than after monotherapy, at least for SP. A previous study in Upper Nile \[[@B4]\] showed that 68% of patients (of all ages) treated with SP and 28% of those treated with AQ developed gametocytaemia during the 14 days after treatment, while gametocyte prevalence at admission was only 2%. Gametocidal effect is very important since the sexual stages of parasites are essential for person-to-person transmission of malaria via mosquito vectors. The main limitations of the study were that the number of patient exclusions were higher than anticipated (15%) due to concomitant febrile illnesses and loss to follow-up, as well as the lack of results for PCR-analysis, caused by samples missing or an inability to provide sufficient DNA on amplification due to low densities in post-treatment samples. Increasing the number of inclusions compensated losses to follow-up. Repeated attempts at PCR-analysis for problematic samples were only partly successful and, therefore, lead to extrapolation about the findings for the missing samples. At time of writing, a change of national protocols towards ACT in northern Sudan is in preparation -- coordinated among health authorities, NGOs and other relevant actors. The first line treatment recommended country-wide is AS+SP, based on reported high efficacy of SP. Northern Sudan is the only country in Africa, which has chosen the option AS+SP \[[@B17]\]. On the basis of the results of this study, this is justifiable. One other study in Sudan on AS+SP and AS+AQ efficacy was recently completed in the Nuba Mountains and also shows high efficacy of 91.2% and 92.7%, respectively, for these ACTs at 28 days after treatment \[[@B18]\]. ACTs with SP or AQ as companion drugs have shown to be very effective in other areas of Africa, provided that the companion drug still maintained a good level of efficacy \[[@B19]-[@B22]\]. In the Upper Nile area both SP and AQ were still an effective treatment for P. falciparumand SP has shown high efficacy in various areas of northern Sudan \[[@B2],[@B4],[@B23]\]. It remains to be seen whether AS+SP will be equally efficacious in other, e.g. more northern areas of the Sudan. In Southern Sudan AS+AQ has recently been put forward as the therapy of choice \[[@B17]\]. The use of two different therapies would seem a sensible option in a vast country with areas of different patterns of resistance. An advantage of AS+SP over AS+AQ is that it is more convenient to administer, as SP is given as a single dose and can be administered under observation in a health facility, whereas AQ requires 3 days to complete a course. SP tablets are also easier to take (AQ has a bitter taste). Blister packs of AS+SP, which combine the two drugs and clearly indicate daily tablets to be taken, are currently available for different age categories in Sudan. AS+SP is cheaper than AS+AQ. Implementation of the new national protocol with AS+SP will hopefully take place as quickly as possible to prevent rising morbidity and mortality. Vulnerable displaced populations in epidemic-prone areas and areas of high perennial malaria transmission should be prioritized. Introduction of ACT will have to go hand in hand with laboratory confirmed diagnosis (microscopy or rapid diagnostic tests) to prevent unnecessary use of valuable drugs (thus minimizing drug pressure) and ensure that non-malarial cases are appropriately treated. The change in guidelines should also filter through all health service providers, including the private sector and drug vendors to decrease the potential risk of SP monotherapy and incomplete dosages. The change will initially require more funds to be made available for malaria treatment. The international community -- including many NGOs, the WHO, donors, and the Global Fund -- has shown willingness to support countries to change antimalarial protocols \[[@B24],[@B25]\]. At present, MSF has already started to pilot AS+SP treatment in its project areas in Northern Sudan, i.e. Darfur, Upper Nile and Gedaref, on behalf of the Ministry of Health. The implementation of treatment as well as the future efficacy of AS+SP should be monitored carefully in a number of dispersed sentinel sites, as there is a possibility that SP resistance may further rise before the combination has been made available countrywide. Authors\' contributions ======================= IB was responsible for overall supervision, data analysis and writing of the paper, RA for field-implementation, communication as well as data analysis and writing, MB for preparation and set-up of study, support and advocacy. PH was responsible for microscopy laboratory work and quality. EA participated as field supervisor of clinical work and data gathering. EBH and MAF were responsible for day-to-day study procedures and supervision of clinical teams. JM did the PCR analysis and FC was involved in design of study, data analysis and writing of manuscript. All authors read and approved the final manuscript. Acknowledgements ================ We thank all Sudanese children who participated in this study and their parents, as well as the local authorities and MoH representatives who facilitated the execution of this study. The vital efforts of the MSF field staff are gratefully acknowledged. MSF-teams in Khartoum, Amsterdam and London and Epicentre in Paris had an invaluable input in the study providing efficient organization, logistics and support during the whole period. We thank KEMRI Nairobi for slide quality control. This study received funding from MSF and its donors.	PubMed Central	2024-06-05T03:55:53.914593	2005-2-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554764/", "journal": "Malar J. 2005 Feb 24; 4:14", "authors": [ { "first": "Ingrid", "last": "van den Broek" }, { "first": "Ribka", "last": "Amsalu" }, { "first": "Manica", "last": "Balasegaram" }, { "first": "Pamela", "last": "Hepple" }, { "first": "Engudaye", "last": "Alemu" }, { "first": "El Badri", "last": "Hussein" }, { "first": "Muhammed", "last": "Al-Faith" }, { "first": "Jacqui", "last": "Montgomery" }, { "first": "Francesco", "last": "Checchi" } ] }
PMC554765	Background ========== Immunization protocols rely on the capacity of the vaccine design to elicit long term protective, peptide specific, MHC restricted-CD4^+^and CD8^+^T cell responses in cancer patients. In preclinical studies, few vaccination strategies were shown to counteract tumor induced-tolerance and to promote immunity to cancer leading to tumor eradication \[[@B1]\]. One of the most promising approach recently developped is based on the adoptive transfer of mature dendritic cells (DC) pulsed with tumor peptides and control antigen \[[@B2],[@B3]\]. Dhodapkar et al. performed a pionnering clinical trial demonstrating the rapid generation of T cell immunity against recall antigens and Keyhole Limpet Hemocyanin in normal volunteers \[[@B4]\] and Schüler-Thurner et al. successfully promoted active, melanoma peptide-specific, IFNγ producing, effector CD8^+^T cells \[[@B5],[@B6]\] and helper CD4^+^Th1 lymphocytes \[[@B6]\] in the majority of patients with metastatic melanoma. If a single injection of CD34+ progenitor derived-DC vaccine can lead to induction of T cell immunity \[[@B7]\], correlations between clinical responses and CTL Tc1/CD4^+^Th1 activation have been rarely reported \[[@B8]-[@B10]\]. Therefore, it is conceivable that antigen spreading initiated by the specific T cells and/or intervention of alternate effectors also elicited by mature DC \[[@B11],[@B12]\] might account for this apparent discrepancy. DC process exogenous antigens in endosomal compartments such as multivesicular endosomes \[[@B13]\] which can fuse with plasma membrane, thereby releasing antigen presenting vesicles called «exosomes» \[[@B14]-[@B16]\]. Exosomes are 50--90 nm diameter vesicles containing antigen presenting molecules (MHC class I, class II, CD1, hsp70--90) tetraspan molecules (CD9, CD63, CD81), adhesion molecules (CD11b, CD54) and CD86 costimulatory molecules \[[@B17]-[@B19]\] i.e the necessary machinery required for generating potent immune responses. Exosomal MHC class I and II /peptide complexes are functional but require to be transferred to naive DC \[[@B20]-[@B23]\] to promote T cell activation leading to tumor eradication \[[@B16],[@B21]\]. Exosomes pulsed with tumor peptides are more efficient than peptides alone and as efficient as mature DC for the priming of MART1 -specific CTL and for tumor growth inhibition in the HLA-A2 transgenic mouse model \[[@B21]\]. The molecular characterization of DC derived-exosomes \[[@B24],[@B25]\] and the definition of quality control parameters for exosome purification and storage \[[@B26]\] allowed to conduct a Phase I clinical trial aimed at evaluating the feasability of exosome harvesting from autologous monocyte derived-DC cultures and the safety of exosome inoculation in melanoma patients. Secondary endpoints were the immunomonitoring of peptide specific CD4^+^and CD8^+^T cell responses restricted by exosomal MHC class II and I molecules respectively. Up to 41 ± 6.7 (9--115) id/sc vaccinations/patient at the lowest dosage (i.e 0.13 × 10^14^exosomal MHC class II molecules /vaccine) could be generated for all patients from one leukapheresis. One partial response and some other tumor regressions at skin and lymph node sites even in tumors that did not respond to other vaccine formulations were observed in the absence of toxicity. A case report of MART1 antigen spreading and MHC class I loss variant suggested that exosomes mediated bioactivity in vivo, supporting to conduct Phase II clinical trials. Patients, Material and Methods ============================== Protocol design, Patients\' characteristics and eligibility criteria -------------------------------------------------------------------- We report here about the first phase I trial (Fig. [1](#F1){ref-type="fig"}) which administers 4 exosome vaccinations intradermally and subcutaneously at 1 week intervals (Fig. [2](#F2){ref-type="fig"}). The study was approved by the Ethics Committee, local IRBs and regulatory authorities, and informed written consent was given by all patients. The authors were in compliance with the Helsinki Declaration. Fifteen patients bearing melanoma fullfilling the inclusion criteria were enrolled in the study (see [Additional file 1](#S1){ref-type="supplementary-material"} and Table [1](#T1){ref-type="table"}). Inclusion criteria were: biopsy-proven American Joint Committee on Cancer stage IIIB and IV metastatic melanoma, HLA-A1^+^, or -B35^+^and HLA-DPO4^+^phenotype (as defined by serological and molecular typing), tumor expressing MAGE3 antigen (assessed by RT-PCR, as described elsewhere \[[@B27],[@B28]\]), inclusion at least 4 weeks after cytotoxic chemotherapy, surgery, or radiation therapy, intradermal skin test positivity to one or more recall antigens using Pasteur Mérieux multi DTH test, age \> 18 years, Karnofsky performans status \>80%, lymphocyte counts \>1000 /mm^3^. Exclusion criteria were: prior chemotherapy or biotherapies \<4 weeks before trial entry, brain metastasis, pregnancy, concurrent steroids or immunosuppressive therapy, history of asthma, congestive heart failure, autoimmune disease, active infections. Three patients presenting with skin or LN involved sites declined conventional therapies and received exosomes upfront (see [Additional file 1](#S1){ref-type="supplementary-material"} and Table [1](#T1){ref-type="table"}). Patients received a 4 week outpatient vaccination course with antigen loaded DC derived-exosomes given intradermally (1/10^th^) and subcutaneously (9/10^th^) every week for a total of 4 vaccinations (Fig. [2](#F2){ref-type="fig"}). The injection sites were rotating between both thighs and forearms. The study was scheduled in two steps i.e 1) MHC class I peptide loading was «indirectly» performed on the DC culture at 10 μg/ml, and 2) MHC class I peptide loading was «directly» performed onto purified and acid eluted exosomes (Fig. [1](#F1){ref-type="fig"}). Indeed, our preclinical studies aimed at comparing the exosome immunogenicity after indirect versus direct peptide loading showed a superiority of the latter process \[[@B20],[@B22]\]. Exosomes (quantified as concentrations of MHC class II molecules, see below) or peptides that were pulsed onto exosomes were administered in a dose escalation design. In the first step, 0.13 or 0.40 × 10^14^MHC class II molecules were inoculated in a cohort of three patients each. In the second step, the concentration of peptide loading onto exosomes varied from 10 μg/ml to 100 μg/ml in a cohort of 3 and 6 patients respectively (see [Additional file 1](#S1){ref-type="supplementary-material"} and Table [1](#T1){ref-type="table"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Study design. Different weeks (W) of the study are described. Screening, HLA typing and tumor evaluation were performed within 2 weeks before lymphapheresis. DTH (Multi Mérieux skin test) was performed at the time of lymphapheresis, and exosome vaccine started 3 weeks later. ::: ![](1479-5876-3-10-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Schema of exosome purification processes. Exosomes were purified from monocyte derived-DC (MD-DC) culture supernatants according to a good manufacturing process already described \[26\]. In the second part of the trial, indirect loading of MHC class II peptides (MAGE3~247--258~.DP04, KKLLTQHFVQENYLEY) was performed on DC cultures followed, after exosome purification by a direct loading of MHC class I (MAGE3~168--176~.A1/B35; EVDPIGHLY) peptides at 10 or 100 μg/ml at pH 4.2 \[22\]. Quality control parameters included dosing of exosomal MHC class II molecules, flow cytometry analyses and functional assays using superantigens as described in material and methods. ::: ![](1479-5876-3-10-2) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Formulation of Product ::: Dose Groups Peptides loaded / HLA class Peptide loading method and concentration DEX dose (expressed as numbers of MHC class II molecules) ------------- ------------------------------- ------------------------------------------ ----------------------------------------------------------- A MAGE A3 (168--176) / class I Indirect (10 μg/mL) 0.13 × 10^14^ MAGE A3 (247--258) / class II Indirect (10 μg/mL) tetanus toxoid / class II Indirect (10 μg/mL) B MAGE A3 (168--176) / class I Indirect (10 μg/mL) 0.4 × 10^14^ MAGE A3 (247--258) / class II Indirect (10 μg/mL) C MAGE A3 (168--176) / class I Direct (10 μg/mL) 0.13 × 10^14^ MAGE A3 (247--258) / class II Indirect (10 μg/mL) tetanus toxoid / class II Indirect (10 μg/mL) D MAGE A3 (168--176) / class I Direct (100 μg/mL) 0.13 × 10^14^ MAGE A3 (247--258) / class II Indirect (10 μg/mL) ::: Clinical grade exosome production --------------------------------- ### MD-DC propagation Based on size and density, we could rapidly purify exosomes from monocyte derived-DC (MD-DC) culture supernatants according to a good manufacturing process already described \[[@B26]\]. Briefly, the adherent fraction of peripheral blood mononuclear cells (PBMC) from a leukapheresis is replenished with fresh medium (AIMV media discarded from haptoglobin and albumin through a 500 kDa MWCO hollow fiber cartridge ultrafiltration (UFP-500-C-4A from A/G Technology, Needham, MA) followed by sterile filtration through a 0.22 um Sartopore 2 membrane) containing rhuGM-CSF (50 ng/ml, Immunex, Seattle, WA) and rhu IL-4 (250 IU/ml, Schering Plough, Kennilsworth, NJ) until day 7. ### Clinical grade exosome purification Exosomes secreted in the supernatant of MD-DC cultures (1--4 liters) were purified to 175 fold the starting volume according to a GMP method previously described \[[@B26]\]. ### MHC class I peptide loading of exosomes This method has been previously described \[[@B21],[@B22]\]. Briefly, the indirect loading of peptides was performed for both MHC class I (Mage3~168--176~.A1/B35; EVDPIGHLY) and MHC class II peptides (Mage3~247--258~.DP04, KKLLTQHFVQENYLEY) in pharmaceutical quality (Multiple Peptide Systems, San Diego, CA) at 10 μg/ml at day 6 of the MD-DC culture and the exosomes were purified from day 7 culture supernatant as described above. The direct loading of MHC class I peptides at pH 4.2 has been reported \[[@B22]\]. Quality control parameters for exosome inoculation -------------------------------------------------- Insurance quality criteria allowing exosome release in patients were qualitative (expression of CD81 tetraspanins), quantitative (at least 1 × 10^14^MHC class II molecules in immunocapture assays) and included a functional assay (bioactivity in the superantigen SEE test of potency). ### Quantitation of exosome MHC class II concentration by adsorption ELISA and immunophenotyping These methods have been previously described \[[@B26]\]. ### Test of potency The bioactivity of exosomal MHC class II molecules was tested using a superantigen bioassay. Exosomes were incubated with femtoM dosages of SEE and washed by density cushion. SEE harboring exosomes were pulsed onto Raji cells. Exosome pulsed Raji cells were subjected to Jurkat cell lines that produced IL-2 in response to SEE. IL-2 concentrations in the supernatants of the Raji/Jurkat cocultures were assessed using a commercial IL-2 ELISA kit. Clinical monitoring ------------------- Adverse events were graded according to the WHO Toxicity criteria. All patients underwent assessment of tumor status at baseline and 2 weeks after the fourth exosome vaccination. Tumor evaluation was performed using the RECIST criteria. Disease progression was defined as \>20% increase in target lesions and/or the appearance of new lesions, partial response as a \> 30% decrease in the sum of the longest diameters of target lesions. Immunomonitoring assays ----------------------- Pre-, per- and post-immunization PBMCs were collected before vaccination (week 0, 5% of total leukocytes collected from the starting leukapheresis), after 2 (week 4, 50 ml heparinized blood sample) and 4 vaccines (week 7, by leukapheresis), isolated by Ficoll density gradient and conditioned either for immediat testing or kept frozen for further analyses. In patient \#12, single cell suspensions from dissociated cervical lymph nodes resected at the end of the induction and retreatment phases were also collected. ### Immunophenotyping Determination of peripheral lymphocytes subsets were performed by four color immunostainings using combinations of FITC-, PE-, Cychrome-, APC-labeled mAbs directed against CD2, CD3, CD4, CD5, CD8, CD19, CD25, CD27, CD45RA, CD56, CD69, CD122, HLA-DR (all purchased from Becton Dickinson, Pont de Claix, FR). Controls included isotype-matched immunoglobulins. Thawed PBMCs were stained for 15 min at 4°C, washed twice and fixed in 1X PBS with 0.1% paraformaldhehyde. Ten thousand viable PBMCs were acquired on a FACSCalibur cytometer (Becton Dickinson, BDIS, San Jose, CA, USA) according to standard FSC/SSC criteria and analyzed with the Cellquest software. ### Peptides and recall antigens for functionnal assays Reactivity of PBMCs was assessed in response to MAGE3~168--176~.A1/B35, MAGE3~247--258~.DP04 (Multiple Peptide System), and control viral peptides (HIV/nef~113--128~.A1, HIV/nef~73--82~.B35, FLU/pb1~591--599~.A1, FLU/np~265--274~.A2, EBV/bzlf1~54--64~.B35, EBV/bmlf1~259--267~.A2, CMV/pp65~495--503~.A2 TT/p2~830--844~, Eurogentec, Seraing, BEL) used according to the HLA classe I phenotype of patients, as well as, against tetanus anatoxin (at 100, 10 and 1 μg/ml), tuberculin (at 50, 5 and 0.5 IU/ml), and phytohemaglutinin (at 2.5, 0.5, and 0.1 μg/ml, PHA HA16, Murex Biotech, Dartford, UK). ### Proliferation assays Fresh PBMCs (2 × 10^5^cells/well) were cultured in triplicates in presence of graded doses of peptides (at 50, 5, and 0.5 μg/ml) or control antigens or medium alone. Proliferative capacity was determined after overnight pulsing with \[^3^H\]thymidine (1μCi/well, NEN, Paris, France) at day 5 of the coculture (earlier at day 3 for PHA). Cells were harvested onto 96-well Unifilter microplates, dried overnight and radioactivity counted on a microplate scintillation counter (TopCount-NXT, Packard, CA, USA). All determinations were made in triplicate wells and data were calculated as means ± SEM of cpm. ### Enzyme-linked Immunospot assays ELISPOT assay for the detection of antigen-specific IFNγ-producing T cells was performed as described previously \[[@B29]\]. Briefly, fresh or frozen PBMCs (5 × 10^5^cells/well) were cultured in triplicate in nitrocellulose-bottomed 96-well plates (MAHA S4510, Millipore, Saint Quentin- en-Yvelines, FR) precoated with 2 μg/ml of a primary anti-IFNγ mAb (1-D1K, Mabtech, Hamburg, GER) in presence of peptides (at 5 μg/ml) or control antigens in RPMI1640 medium supplemented with 8% human AB serum. After incubation for 48 hrs, wells were washed five times and incubated with a secondary biotinylated anti-IFNγ mAb (7-B6-1, Mabtech) for 2 h, washed and stained using an extravidine-alkaline phosphatase conjugate substrate kit (Biorad, Hercules, CA, USA). Spots were evaluated and counted using a computer-assisted video imaging analyser (Bioreader 2000, Biosys, Karben, GER). ### Microcultures for semiquantitative detection of Mage3.A1/B35 CTL precursors The microculture method developed by Coulie et al. \[[@B30]\] was used to assessed CTL precursors specific for MAGE3.A1/B35 peptide. Briefly, groups of 2 × 10^5^peptide-pulsed PBMC (20 μg/ml) were cultured in Iscove\'s medium with 10% human AB serum supplemented with IL-2 (20 IU/ml), IL-4 (10 ng/ml) and IL-7 (10 ng/ml) and restimulated at day 7 with replacement of 50% fresh medium with addition of MAGE3.A1/B35 peptide (10 μg/ml). On day 15, microcultures were screened for specific CD8^+^T cells using the relevant MAGE3.A1 or B3501 tetramers coupled to PE together with a control APC labeled-HLA-A1 containing an influenza peptide (all tetramers kindly given by D. Colau, Ludwig Institut for Cancer Research, Brussels, BE). This microculture procedure was also applied on patient \#12\'s tumor infiltrating LN to highlight CD8+ T cells specific for HLA-A2/MART1~26--35~, and HLA-B3501/MAGE3~168--176~. Flow cytometry acquisition and analysis of tetramer positive cells were performed as recommanded by P. Coulie \[[@B30]\]. Immunohistochemistry on lymph node tissues ------------------------------------------ Immunostainings were performed on sections obtained from formalin-fixed and paraffin-embedded lymph node samples. Sections were deparaffinized, placed in 10 mmol/L Na-citrate buffer (pH.7), and heated in a microwave during 20 min. Endogenous peroxidase was blocked with 1% hydrogen peroxide in methanol for 30 min. Slides were stained using anti-CD3 and anti-CD57 mAb (Pharmingen, France) and secondary antibodies with appropriate controls. Results ======= Feasability of exosome production in advanced melanoma patients --------------------------------------------------------------- Leukaphereses of 1.5 blood mass performed in the 15 metastatic melanoma patients enrolled in the study allowed the recovery of 9.7 × 10^9^± 0.8 PBMC (range: 4.4--15) containing 20.7% ± 1.8 CD14^+^cells (range: 12.4--33.0). These monocytes differentiated into immature MD-DC in rhu GM-CSF and rhu IL-4 (means: 313 × 10^6^± 100, range: 50--1200) as assessed at day 7 in flow cytometry highlighting loss of CD14 molecules, acquisition of CD1a, poor cell surface expression of CD83. In 3 patients out of 15, the first leukapheresis did not allow CD14^+^cells to adhere and consequently, a second leukapheresis was required to harvest exosomes. The GMP process allowed to purify a mean of about 5.22 × 10^14^± 0.9 (range: 1.2--15.0) exosomal MHC class II molecules from supernatants of day 6 to day 7 MD-DC required for up to 41 ± 6.7 (9--115) id/sc vaccinations/patient at the lowest dosage (i.e 0.13 × 10^14^exosomal MHC class II molecules /vaccine). As for the patients who benefited from continuation treatment requiring a second leukapheresis (pt \#3, \#12 and \#14), the second exosome recovery was not significantly different from the first one. The quality control parameters required for GMP exosome batch release are reminded in M&M. GMP exosomes were successfully produced from DC cultures derived from all melanoma patients enrolled in this phase I study, allowing at least 4 exosome inoculations. Safety and clinical outcome --------------------------- No major (\>grade II) toxicity was observed. A grade 1 fever was recorded in 5 patients. We noted slight inflammatory reactions at vaccine sites without outward delayed type hypersensitivity reactions. Some patients (\#4, \#8, \#11, \#12 and \#15) reported a transient swelling and sensitivity of cutaneous and lymph nodes metastases 48 hrs after each exosome inoculation. Among the six patients vaccinated in the first part of the study, only one patient (\#3) presenting with a stage III disease exhibited a minor response (disappearance of one sc lesion out of 3). This patient benefited from a continuation therapy with exosomes every other 3 weeks for 21 months and remained stable for up to24 months. It is noteworthy that this patient was progressing despite vaccination with MAGE3 protein prior to enrollment in the Phase I exosome trial. In the second part of the study («direct loading»), responses were only observed in the second group of patients receiving the highest dosages of peptides (100 μg/ml) pulsed onto exosomes. One female patient (\#12) presenting with five supraclavicular invaded lymph nodes after conventional chemotherapy (DTIC, 2 cycles) exhibited a partial response (PR) after the induction therapy. Interestingly, disappearance of arterial neovasculature concomittant with tumor shrinkage and necrosis as assessed by doppler pulsed ultrasonography could be demonstrated (Fig. [3](#F3){ref-type="fig"}). Halo of depigmentation around neavi appeared 10 months after the initiation of vaccination (Fig. [3](#F3){ref-type="fig"}). A continuation therapy with exosomes was administered for 4 months every other 3 weeks allowing stabilisation without toxicity supporting the indication of surgery that confirmed the partial response (Jul. 2002, Fig. [3](#F3){ref-type="fig"}) followed by a second leukapheresis allowing to pursue vaccination for 10 months. This patient relapsed in contralateral nodes six months after exosomes discontinuation but exhibited a slow pace of tumor growth. Two additionnal patients (\#11, \#14) presenting with only skin and /or LN lesions exhibited transient stabilisation and started a continuation therapy. Interestingly, patient \# 14 who displayed a SD with exosomes had previously progressed despite biotherapy with IFNα and vaccination with poxviruses recombinant for the MAGE 3A1 cDNA. It is of note that patient \#9 in a M1b stage exhibited a regression on a subcutaneous nodule but did progress in the pulmonary sites (mixed response). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Clinical outcome of patient \#12 during exosome-based vaccination. This patient presented with progressive supraclavicular lymph nodes containing MAGE3 expressing tumor cells in July 2001 when enrolled in the exosomes Phase I trial starting in October 2001. She underwent a first leukapheresis for exosomes production and vaccination (weekly injections in Nov. 2001 during induction therapy and from January 2002 to April 2002 on a three week basis in continuation treatment). The initial size of the target LN are indicated on the left and were followed up by doppler pulsed ultrasonography from Nov 2001 to Dec 2001 (sizes indicated on the right side at the end of the induction therapy). Continuation therapy with exosomes was indicated and maintained clinical stability until the last available exosome dose in Jul. 2002, when she underwent surgery for lymphadenectomy. Results obtained by the pathologists are indicated (N+ if node is invaded by tumor cells, N- if not, MAGE3+ as expression of MAGE3 mRNA in RT-PCR). A second leukapheresis was performed on Aug. 2002 allowing a second therapy with exosomes on a 3 week basis that was continuated until Jul 2004. Six months after exosomes discontinuation (Oct 2004), the patient relapsed in contralateral LN and presented with one lung metastasis. ::: ![](1479-5876-3-10-3) ::: Exosome therapy promoted 2 stable diseases, 1 minor response, 1 partial response and 1 mixed response in skin or LN sites even in patients progressing with biotherapies or alternate vaccines. Patient \#3 and \#12 with respectively minor and partial responses are still alive. Neither of these patients exhibited a delayed type hypersensitivity response to the immunizing epitopes after the completion of the 4 vaccines. T cell immunomonitoring ----------------------- The phenotypic analyses of lymphocyte subsets did not reveal any significant changes in the percentages nor absolute counts following exosome therapy (not shown). Interestingly, the CD122 molecule i.e IL-2Rβ chain was upregulated in both CD4 and CD8 T cell subsets after exosome therapy (p \< 0.05 using Student t\'test, week 7 versus week 1 for CD4+ T cells). Serial ex vivo ELISPOT analyses of PBMC before (W1, Fig. [4A](#F4){ref-type="fig"}) and after (W7, Fig. [4B](#F4){ref-type="fig"}) exosome vaccination did not reveal any significant Th1 (using TT or DP04 MAGE 3 peptides) or Tc1 (using MAGE 3. A1/B35 peptides) type immune responses induced by exosomes. Recall responses (proliferative responses to TT protein using autologous DC or restimulation with autologous DC pulsed with MAGE 3.DP04) did not allow to conclude that exosomes boosted MHC class II specific responses at these time points (not shown). In accordance with these findings, the skin reactivities to iterative injections of id/sc exosomes did not correspond to clinically significant Delayed Type Hypersensitivity responses. Extensive immunomonitoring by screening of 15 day-microcultures of MAGE3 peptide pulsed PBMC performed in the presence of IL-2, IL-4, IL-7 \[[@B30]\] using the MAGE3.A1/B35 tetramers allowed to detect and clone specific CTL precursors only in 3 patients (\#3, 9 and 11) but estimated at a low frequency (10^-6^- 10^-7^of the CD8^+^T cells). These studies did not allow to conclude for significant anti-MAGE3A1/B35 CTL responses induced by exosome therapy nor to suggest the clonal expansion of discrete T cell specificities (not shown) in patients\' peripheral blood. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Evaluation of Tc1/Th1 immune responses to melanoma and viral/recall antigens. A. Before exosome vaccination (W1). B. After exosome inoculation (W7). PBMC obtained at baseline (W1) and after 4 exosome injections are cultured 48 h with the immunizing melanoma antigens i.e Mage 3.A1/B35 or Mage3.DP04 (5 μg/ml) or with viral/recall control antigens (FluMP.A1, HIV.A1, EBV.B35, tetanus anatoxin, tuberculin) or with PHA. The three first patients were also assayed with the universal MHC class II restricted TT peptide. The specific T cell response in each of the evaluated patients is expressed as the number of IFNγ spot forming unit/5 × 10^5^PBMC. ::: ![](1479-5876-3-10-4) ::: Comprehensive analyses of a partial response -------------------------------------------- Patient\#12 presenting with five supraclavicular lymph nodes exhibited a partial regression after 4 exosome vaccines in November 2001 and underwent a continuation therapy from Jan. 2002 to April 2002 (Fig. [3](#F3){ref-type="fig"}). The HLA class I haplotype of the patient was A2.A26.B35.B44 and the initial tumor sites expressed both MAGE1, MAGE3 and other melanoma antigens such as Melan-A/MART1 and NA-17 (as assessed using semiquantitative RT-PCR). The antitumor effects did not correlate with an enhanced frequency of MAGE3 specific CTL precursors as demonstrated using microculture detection assays after the first 4 vaccines or after the continuation treatment (not shown). However, tumor shrinkage after the continuation therapy prompted a partial surgical resection in July 2002 and comprehensive analyses of tumor infiltrating lymphocytes (TILN) and tumor cells in 4 avalaible sites (\#1, 3, 5, 6 in Fig. [3](#F3){ref-type="fig"}). Site 5 was scored tumor free by the pathologists. Site 1 and 3 maintained the transcription of Mage3 mRNA. In these TILN sites, detection of HLA-A2/MART1~26--35~, -/gp100~209M-217~-/Tyrosinase~239--251~and HLA-B3501/MAGE3~168--176~-specific CD8+ lymphocytes using soluble fluorescent tetramers were performed directly ex\_vivoand after microculture restimulation assays using MART1~26--35~or MAGE3 peptides.Only high frequencies of MART1 specific CD8+ T cells were detected in TILN from site 1 and 6 (1.22 and 0.85 % respectively) and in peripheral blood (0.53% in May 2002 versus 0.15% in Oct. 2001) that were confirmed by expansion in all microculture assays (Fig. [5A](#F5){ref-type="fig"} and [5B](#F5){ref-type="fig"}). In parallel, halo of depigmentation around naevi appeared (Fig. [3](#F3){ref-type="fig"}). Moreover, a cell line established from site 1 (CUR1) did not express the HLA-A2 allele in flow cytometry using the MA2.1mAb but maintained the expression of HLA-BC molecules (Fig. [5C](#F5){ref-type="fig"}). In November 2003 while disease was slowly progressive, another LN was removed allowing to re-establish another cell line (CUR2) that no longer express HLA-BC molecules (as assessed in flow cytometry using W6.32 or anti-HLA-BC mAb, Fig. [5C](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Antigen spreading and MHC class I loss variant in patient\#12. Flow cytometry analyses on serial blood specimen (A) or tumor invaded lymph nodes (B) gating on CD8+ T lymphocytes stained using A2/Mart1 or A2/gag specific fluorescent tetramers. Ex vivo microcultures stimulated with Mart1 peptides and examined according to similar settings. (C) Flow cytometry analyses of two CUR tumor cell lines (pt\#12) after the first exosome therapy (continuation treatment) in clinical response and after the second exosome course (second leukapheresis) at relapse for MHC class I (anti-HLA-A2 mAb, anti-HLA-BC mAb and W6.32 Ab) expression. A positive control was included which consisted of a allogeneic HLA-A\0201melanoma line FON. ::: ![](1479-5876-3-10-5) ::: Immunohistochemistry performed on the primary (April 2001) and secondary (July 2002) tumor specimen revealed quantitative modifications of tumor infiltrating CD3+CD57+ T cells. While activated T cells were mostly surrounding the tumor bed initially, a two fold expansion or recruitment of activated T cells invade tumor areas after exosome vaccines (Fig. [6](#F6){ref-type="fig"}). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Lymphocyte recruitment and activation in patient\#12\'s melanoma. T cells that were double-stained by anti-CD3 and anti- CD57 mAb were counted in 2 sections of 6 lymph nodes available from the lymph node dissection specimen obtained before (A. April 2001) and after (B. July 2002) treatment (cf Fig. 3). Counts were performed in 12 sections in total as follows: cells that were double-stained by CD3 and CD57 were counted on the whole section including B cell areas, and the total count was reported to 1 mm^2^. Results showed an increase of the CD3+CD57+ cells after treatement (mean = 122/mm^2^) as compared to the count before treatment (mean: 58/mm^2^). ::: ![](1479-5876-3-10-6) ::: Discussion ========== This work is reporting for the first time the feasability and safety of DC derived-exosome-based vaccination in melanoma patients. From a single leukapheresis, up to 10^14^-10^15^exosomal MHC class II molecules could be purified in GMP conditions for all patients, allowing 9--123 sc/id vaccinations with 0.13 × 10^14^exosomal MHC class II molecules. The optimal dosages of exosomal MHC class II molecules required to trigger an efficient peptide-specific CTL response leading to tumor rejection was about 10^10^-10^11^molecules in preclinical studies in the HLA-A2 transgenic mouse model \[[@B21]\]. The optimal method for pulsing exosomal MHC class I molecules with peptides was a direct loading after acid elution of the exosomal pellet \[[@B20],[@B22]\]. A dose response was observed in vitro, prompting the use of 100 ug/ml for peptide pulsing onto GMP exosomes. As to MHC class II associated-peptide loading, pulsing immature day 5 DC with 10 ug/ml was optimal \[[@B22]\]. However, in this Phase I study, since no untoward clinical effect nor pharmacodynamic parameter appear to be dose-dependent, neither the peptide pulsing method nor the dosage of exosomes or peptides could help defining the maximal tolerated doses of exosomes. Interestingly, in the second Phase I trial initiated at the Dukes\' University in unresectable lung carcinoma, there was a trend for a better efficacy of exosomes directly pulsed with MHC class I peptides in long term survival \[[@B31]\]. Only one objective response (PR) according to the RECIST criteria was recorded. However, one minor response and two stabilisations prompted the continuation of treatment as well. Tumor regression was exclusively observed in skin and lymph node lesions, as already reported for peptide-based vaccines \[[@B32]\]. It is noteworthy that three of these patients had primarily resisted to alternate immunotherapy strategies (i.e IFN type I, ALVAC-Mage 3, Mage 3 protein) but it is also likely that such immunomodulators might have facilitated exosome efficacy. However, we were not able to detect significant Mage 3 specific CTL precursors in peripheral blood (nor lymph nodes and lesions) after exosome vaccination. Notewithstanding, MHC class I tumor loss variant and naevi depigmentation was observed in one case (pt \#12), suggesting melanoma antigen spreading mediated by HLA-A2-restricted CTL cells. It is conceivable that the Mart1-specific CTL monitored in blood and in LN TIL could have mediated part of these antitumor effects. No DTH responses to iterative inoculations of exosomes or MHC class II restricted-direct or recall responses (to TT epitopes (3 pts tested), or Mage DP04 epitopes (12 patients tested)) were detected at 4 and 6 weeks of exosome vaccination. It is noteworthy that peptides and not whole proteins (such as TT or KLH) were used to pulse DC in vitro, in contrast to what has been reported in most DC trials. However, in the transgenic Marilyn mouse model \[[@B23]\], id inoculation of I-A^b^harboring exosomes pulsed with H-Y epitopes could promote expansion of peptide specific CD4^+^transgenic T cells. Nevertheless, MHC class I restricted-CTL responses elicited by exosomes pulsed with Mart1 peptides in HLA-A2 transgenic mice were dramatically boosted by adjuvants \[[@B21]\]. Therefore, it is conceivable that injection of exosomes in the area of an inflammatory draining lymph node harboring mature APC might promote elicitation of T cell immune responses leading to tumor regressions in patients. Alternatively, we recently showed that regulatory C4+CD25+ T cells (Treg) restrict T cell responses elicited by DC derived-exosomes (Chaput et al, in preparation). Therefore, high yields of Treg at start might severely impair the efficacy of exosomes to prime or boost T cell responses. Supporting this view, our unpublished data demonstrate synergistic T cell dependent-antitumor effects between peptide pulsed-exosomes and immunopotentiating dosing of cyclophosphamide. Since no specific CD4+ or CD8+ T cells generated by the exosome vaccines could be detected and could account for the tumor regression in patient\#12, what could have been the primary effectors accounting for antigen spreading? We were able to show enhanced NK cell effector functions following exosome administration in peripheral blood of 8/13 patients including patient \#12 for whom CD3-CD57+ cells expressing PEN5 (mostly expressed on CD56^dim^NK cells) were markedly infiltrating the tumor after therapy (N. Chaput, manuscript in preparation). Therefore, it is conceivable that DC derived-exosomes from melanoma patients are specifically endowed with NK cell stimulatory capacity in vivo. This hypothesis deserves further investigations and should be pursued in the next clinical studies. Moreover, since our preclinical data in tumor bearing mice highlighted a critical role of suppressor T cells in restricting exosome-mediated specific T cell responses, T regulatory cells should be monitored and combination of cyclophosphamide together with exosomes could be invisioned (N. Chaput, in preparation). Phase II/III trials will address the potential of exosomes to enhance time to progression in advanced non small cell lung carcinoma patients. Abbreviations ============= DC: Dendritic cells; MD-DC: monocyte-derived DC; Dex:Dendritic cell -derived exosomes; CTL:Cytotoxic T Lymphocytes; GMP:good manufacturing processes; MHC:Major Histocompatibility Complex; LN*: lymph nodes. Authors\' contributions ======================= Bernard Escudier^1•^was the principal investigator, Thierry Dorval^2•^, Sophie Piperno^2^, Caroline Robert^3,9^were the clinicians ensuring patients\' enrolment, follow up and clinical care. ^•^BE and TD equally contributed to this work Marie-Pierre Cabi^4^, Sophie Novault^3^, Christophe Leboulaire^3^, Mojgan Movassagh^3^and Christophe Leboulaire^3^were monitoring T cell responses corrdinated by Olivier Lantz^4^and Eric Angevin^1^ Nathalie Chaput^3^, Fabrice André^3^and Caroline Flament^3^monitored NK cell responses Catherine Boccaccio was in charge of the cell therapy unit at the Institut Gustave Roussy^5^ Sebastian Amigorena^4^, Christian Bonnerot^4^and Laurence Zitvogel^1^were the scientists at the source of preclinical data orienting the study design. Thomas Tursz^1^was heading the Host and Tumor Development Programm of the Institut Gustave Roussy, initiating the clinical study. Vincent Serra^6^, Nancy Valente^7^, Olivier Dhellin^6^were the Director of Anosys SA, the medical director of Anosys Inc. and the Pharmacist in charge of production, regulatories and quality insurance in Anosys SA respectively. Jean-Bernard Le Pecq^6,7^was the Chief and Scientific Officer of Anosys leading the research and development of the exosome programm. Alain Spatz^8^, Christophe Borg3 were in charge of the immunohistochemitry analyses on tumor specimen. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Dosing, demographic, base line data and clinical outcome ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We are indebted to Marie-Françoise Avril for patients\'follow up, to Eric Vivier for sending the PEN5 Ab and the critical review of data, to Di Hwei Hsu for preclinical studies, to patient \#12 for systematic consent in all investigations performed without personal benefit, to Prs T. Boon and P. Coulie from the Ludwig Institute for Cancer Research, Brussels Branch for careful examination of the immunomonitoring data. NC has been supported by the QLRT-2001-00093 and AP-HP as well as ARC. FA has been supported by the Young Investigator Award, ASCO 2002. The work has been funded by the EC Cell factory programm project QLRT-2001-00093, Ligue labellisée contre le cancer and ARC.	PubMed Central	2024-06-05T03:55:53.917257	2005-3-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554765/", "journal": "J Transl Med. 2005 Mar 2; 3:10", "authors": [ { "first": "Bernard", "last": "Escudier" }, { "first": "Thierry", "last": "Dorval" }, { "first": "Nathalie", "last": "Chaput" }, { "first": "Fabrice", "last": "André" }, { "first": "Marie-Pierre", "last": "Caby" }, { "first": "Sophie", "last": "Novault" }, { "first": "Caroline", "last": "Flament" }, { "first": "Christophe", "last": "Leboulaire" }, { "first": "Christophe", "last": "Borg" }, { "first": "Sebastian", "last": "Amigorena" }, { "first": "Catherine", "last": "Boccaccio" }, { "first": "Christian", "last": "Bonnerot" }, { "first": "Olivier", "last": "Dhellin" }, { "first": "Mojgan", "last": "Movassagh" }, { "first": "Sophie", "last": "Piperno" }, { "first": "Caroline", "last": "Robert" }, { "first": "Vincent", "last": "Serra" }, { "first": "Nancy", "last": "Valente" }, { "first": "Jean-Bernard", "last": "Le Pecq" }, { "first": "Alain", "last": "Spatz" }, { "first": "Olivier", "last": "Lantz" }, { "first": "Thomas", "last": "Tursz" }, { "first": "Eric", "last": "Angevin" }, { "first": "Laurence", "last": "Zitvogel" } ] }
PMC554766	Background ========== In 1988, an unusually high incidence of cancer in the Cape Cod region of Massachusetts prompted a series of epidemiological studies to investigate possible environmental risk factors associated with the region, including tetrachloroethylene-contaminated drinking water \[[@B1]-[@B7]\]. Tetrachloroethylene (or perchloroethylene, PCE) entered the drinking water when it leached from vinyl liners of water distribution pipes introduced in the late 1960s. When the contamination was discovered, the Massachusetts Department of Environmental Protection began flushing and bleeding the pipes in 1980. At that time, the suggested limit set by the Environmental Protection Agency (EPA) was 40 ppb \[[@B8]\], but has since been lowered to a mandatory Maximum Contaminant Level (MCL) of 5 ppb. A population-based case-control study was undertaken to investigate the association between tetrachloroethylene exposure from public drinking water and breast cancer \[[@B5]\]. The study defined exposure using a cumulative measure Webler and Brown termed the relative delivered dose (RDD) \[[@B9]\]. Calculations for the RDD use the rate at which PCE leached from the pipe liner, the surface area of the interior of the pipe, and the upstream load. The RDD is relative to the total delivered mass of PCE entering each residence over time, but the constants and variables assumed to be constant were dropped from the analysis. While this allowed for grouping of the population into exposure categories, the RDD value computed is not an actual water concentration. Refer to Webler and Brown for a detailed description of the RDD model \[[@B9]\]. Because PCE is a volatile organic chemical that readily escapes from water into air, the amount of PCE inhaled during showers and baths, as well as the amount ingested and dermally absorbed, was relevant. The RDD measure does not consider these exposure pathways, which could potentially result in bias from exposure misclassification. Using personal exposure factors such as tap water consumption and bathing habits, we constructed a dose model to quantify the relative amount of PCE taken in by each subject, which we refer to as the personal delivered dose (PDD). The dose values calculated by the PDD model were subsequently used to measure the strength of the association between PCE exposure and the risk of breast cancer. The objective was to see if additional information contained in individual survey data affected associations between breast cancer and PCE exposure. Methods ======= Study Population ---------------- The population-based case-control study was designed to evaluate the association between breast cancer and tetrachloroethylene (PCE) exposure from public drinking water \[[@B5]\]. During the period 1987--1993, the Massachusetts Cancer Registry recorded 672 incident cases of female breast cancer among permanent residents of the Massachusetts towns Barnstable, Bourne, Brewster, Chatham, Falmouth, Mashpee, Provincetown, and Sandwich, where pipes with PCE-containing vinyl liners had been installed. Female controls were chosen to represent the underlying population that gave rise to the cases. Selection criteria required controls to be permanent residents of the same towns during 1987--1993. Controls were frequency matched to cases on age and vital status. Because many of the cases were elderly or deceased, three different sources of controls were used: (1) random digit dialing identified living controls less than 65 years of age; (2) Centers for Medicare and Medicaid Services, formerly the Health Care Financing Administration, identified the living controls 65 years of age or older; and (3) death certificates identified controls who had died from 1987 onward. The resulting 616 controls provide an estimate of the exposure distribution in the underlying population. Subjects or their next-of-kin completed extensive interviews, which provided information on demographics (e.g., age, sex, marital status, education), a 40-year residential history, and potential confounders (e.g., age, family history of breast cancer, age of first live or still birth, oral contraceptive use). Next-of-kin served as proxies for cases and controls who were deceased or too ill to participate in the interview. \"Index years\" were randomly assigned to controls to achieve a distribution similar to that of cases\' diagnosis years and only exposures before the diagnosis year (for cases) and index year (for controls) were counted. The analysis considered a range of latent periods: 0, 5, 7, 9, 11, 13, 15, 17, and 19 years. For a detailed description of the methods, see Aschengrau et al. \[[@B5]\]. Dose Model ---------- If individual behavior in water use is an important element in a person\'s exposure, using the relative delivered dose (RDD) could bias the results. The RDD quantifies the amount of PCE in the drinking water, but does not consider exposure from inhalation, dermal absorption, and ingestion. PCE is a volatile organic compound and daily indoor inhalation exposure to contaminated water from showering can be up to six times greater than exposure from ingestion \[[@B10]\]. To further quantify dose and reduce exposure misclassification, a number of personal factors (e.g., bottled water consumption, duration and frequency of showers and baths) were considered. Non-proxy cases and controls were interviewed about many of these factors: the number of glasses of tap water consumed per day, including drinks made with tap water, such as coffee or lemonade; the use of bottled water; and the temperature, frequency, and duration of showers and baths. Information on a subject\'s physical characteristics, such as height and usual weight, was also obtained. Certain model parameters not provided by the questionnaire were obtained from the current scientific literature (e.g., inhalation rate, water flow rate, air exchange rate). We used this information to construct a personal delivered dose (PDD) model that considered three exposure routes: inhalation, dermal absorption, and ingestion. The RDD value was converted into an annual concentration and used as the initial water concentration for the PDD model (mg/L). The amount of PCE contributed by inhalation is a function of the temperature, frequency and duration of baths and showers, and the concentration of PCE in the bathtub/shower stall air. To determine the amount of PCE that volatilized from the water, the two-resistance theory was applied to temperature dependent physical and chemical properties of PCE \[[@B11]\]. The dermal absorption component of the model estimated each subject\'s surface area (from her height and weight) and determined the amount of PCE absorbed during baths and showers using Fick\'s first law \[[@B12]\]. The amount of PCE that a subject ingested was dependent on the volume of tap water consumed. By summing the total amount of PCE from the three exposure routes over all exposed residences, we arrived at a personal delivered dose (PDD) for each subject. A detailed description of the dose model is provided in [Additional file 1](#S1){ref-type="supplementary-material"}: Dose Model Appendix. Data Analysis ------------- Questions regarding tap water use and bathing habits were not asked in proxy interviews so the PDD analysis was restricted to non-proxy subjects (n = 885, Table [1](#T1){ref-type="table"}). To accurately compare results from the RDD and PDD analyses, we first recalculated associations using the original RDD exposure measure for only the non-proxy subjects. Women with cumulative RDD exposures were compared with never-exposed women. Never-exposed women did not live downstream of vinyl-lined pipes. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Number of subjects by proxy/non-proxy, PCE-exposed/unexposed, and case/control status. ::: Non-Proxy Subjects Proxy Subjects Total Subjects ----------------- ------------------------ -------------------- -------------------- PCE-exposed 189 102 291 Cases 101 54 155 Controls 88 48 136 Unexposed 696 301 997 Cases 360 157 517 Controls 336 144 480 Total 885 403 1288 Cases 461 211 672 Controls 424 192 616 ::: We defined a series of four exposure levels based on the exposure distribution of exposed controls. The lowest exposure level included all exposed subjects with RDD values less than or equal to the 50^th^percentile. The remaining exposure levels were nested and included all RDD values greater than the 50^th^percentile, greater than the 75^th^percentile, and greater than the 90^th^percentile. Therefore, a subject exposed at \> 90^th^percentile was also considered exposed at \> 75^th^and \>50^th^percentiles. We chose to nest exposure categories because there were too few subjects for mutually exclusive categories. There are no previous studies comparing nested exposure categories to mutually exclusive exposure categories. Exposure groups were further categorized for latent periods that ranged from 0 to 19 years. Each exposure level was treated as a binary variable in separate multiple logistic regression models. Odds ratios (ORs) were calculated for each exposure level relative to never-exposed cases (n = 360) and controls (n = 336). The adjusted analysis controlled for a group of core confounders: age at diagnosis or index year, family history of breast cancer, personal history of breast cancer (before current diagnosis or index year), age at first live birth or stillbirth, and occupational exposure to PCE. These factors were chosen as confounders a prioribased on the current scientific literature. Additional potential confounders were added to the logistic regression models along with the core confounders, including history of benign breast disease; past use of diethylstilbestrol, oral contraceptives, and menopausal hormones; cigarette smoking history; alcohol drinking history; history of ionizing radiation treatment; quetlet index (measure of obesity); race; marital status; religion; education level; and physical activity level. None of these additional variables changed the adjusted estimates by more than 10%, and so the final models included only the core confounders. Adjusted analyses were not performed if there were fewer than three exposed cases and three exposed controls in an exposure level \[[@B5]\]. We calculated 95% confidence intervals (CIs) for the adjusted ORs using maximum likelihood estimates of the standard errors \[[@B13]\]. We then repeated the crude and adjusted analyses using each subject\'s personal delivered dose (PDD) as an exposure measure. The PDD distributions of the exposed controls were used to define the same four exposure levels: less than or equal to the 50^th^percentile, greater than the 50^th^percentile, greater than the 75^th^percentile, and greater than the 90^th^percentile. The referent category remained never exposed cases and controls. We also conducted a goodness-of-fit analysis to compare the RDD and PDD exposure measures and to determine which model performed better \[[@B14]\]. We compared the deviance of the models at different exposure levels and latencies. Lastly, we performed a nonparametric rank test to determine if the ranks of the subjects\' PDD exposures differed significantly from the ranks of their RDD exposures. Results ======= RDD analysis ------------ We were interested in comparing the results of the Aschengrau et al. RDD analysis using all subjects to the restricted analysis performed on only non-proxy subjects. The distributions of core confounders were similar among non-proxy and all subjects, except non-proxy subjects were younger than all subjects (Table [2](#T2){ref-type="table"}). The number of exposed subjects was reduced by 35% when proxies were removed (from 291 to 189) when no latency was considered. The number of unexposed subjects used as a common reference group for all analyses was reduced by 30% (from 997 to 696). The median, 75^th^percentile, and 90^th^percentile RDD values for the non-proxy exposed controls were similar to the values for the exposed controls among all subjects (Table [2](#T2){ref-type="table"}). We compared analyses for ever vs. never PCE-exposed and found that the odds ratios were similar for the non-proxy subjects and all subjects (Table [3](#T3){ref-type="table"}) \[[@B5]\]. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Distribution of selected confounders of breast cancer subjects (%) and RDDs of PCE-exposed controls. ::: Characteristic Non-ProxyCases (n = 461) Non-ProxyControls (N = 424) AllCases (n = 672) AllControls (N = 616) ---------------------------------- ------------------------------ --------------------------------- ------------------------ --------------------------- Age at diagnosis or index years 1--49 years 19.7 (91) 20.6 (87) 16.5 (111) 16.7 (103) 50--59 years 13.7 (63) 17.0 (72) 12.2 (82) 13.6 (84) 60--69 years 33.0 (152) 31.1 (132) 31.5 (211) 29.9 (184) 70--79 years 28.8 (133) 25.2 (107) 28.4 (191) 26.0 (160) 80+ years 4.8 (22) 6.1 (26) 11.4 (77) 13.8 (85) Age at first birth or stillbirth \< 30 years 60.6 (279) 65.7 (278) 61.0 (410) 66.8 (411) 30+ years 13.8 (64) 12.4 (53) 14.5 (97) 12.8 (79) Nulliparous 25.6 (118) 21.9 (93) 24.5 (165) 20.4 (126) Prior breast cancer 5.6 (26) 3.3 (14) 5.4 (36) 4.8 (30) Family history of breast cancer 24.3 (112) 15.8 (67) 25.6 (172) 15.5 (95) Occupational exposure to PCE 16.3 (75) 16.0 (68) 15.5 (104) 14.8 (91) RDD Exposure (for no latency) Minimum \-\-- 0.001 \-\-- 0.001 Maximum \-\-- 206.9 \-\-- 243.8 Median \-\-- 2.9 \-\-- 2.5 75^th^percentile \-\-- 11.9 \-\-- 12.1 90^th^percentile \-\-- 31.0 \-\-- 29.2 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Tetrachloroethylene exposure history of breast cancer subjects, adjusted^a^odds ratios, and 95% confidence intervals. ::: Latency period, years Exposed Cases Exposed Controls Adjusted ORs (95 % CI) ---- ----------------------- --------------- ------------------ ------------------------ 0 Non-proxy Subjects 101 88 1.1 (0.8--1.5) All Subjects 155 136 1.1 (0.8--1.4) 5 Non-proxy Subjects 87 69 1.2 (0.9--1.8) All Subjects 129 107 1.2 (0.9--1.6) 7 Non-proxy Subjects 71 61 1.1 (0.8--1.6) All Subjects 111 96 1.1 (0.8--1.5) 9 Non-proxy Subjects 63 57 1.1 (0.7--1.6) All Subjects 97 85 1.1 (0.8--1.5) 11 Non-proxy Subjects 49 43 1.1 (0.6--1.7) All Subjects 79 65 1.2 (0.8--1.7) 13 Non-proxy Subjects 43 32 1.3 (0.7--2.1) All Subjects 61 45 1.3 (0.9--2.0) 15 Non-proxy Subjects 30 21 1.4 (0.7--2.6) All Subjects 44 31 1.4 (0.9--2.3) 17 Non-proxy Subjects 15 15 1.0 (0.4--2.2) All Subjects 21 21 1.0 (0.6--2.0) 19 Non-proxy Subjects 6 6 1.1 (0.3--3.5) All Subjects 9 9 1.1 (0.4--2.9) ^a^The OR was calculated relative to never-exposed cases (n = 360 for Non-Proxy, n = 517 for ALL) and controls (n = 336 for Non-Proxy, n = 480 for All). Controlled for age at diagnosis or index year, family history of breast cancer, personal history of breast cancer (before current diagnosis or index year), age at first live-birth or still birth, occupational exposure to PCE, and vital status at interview (for Allanalysis only). ::: PDD analysis ------------ The distribution of cumulative RDD and PDD values ranged by five orders of magnitude, equivalent to a range from micrograms to hundreds of milligrams. Of the 189 exposed subjects in the no latency analysis, the personal delivered dose model changed the exposure categories of 39 subjects. However, the result from a non-parametric signed rank test indicates that the subjects\' RDD ranks and PDDs rank are not significantly different (p = 0.81). In general, odds ratios from the PDD analysis were slightly higher than the RDD analysis for exposure levels above the 50^th^and 75^th^percentiles at shorter latency periods (see [Additional file 2](#S2){ref-type="supplementary-material"}: Table 4). At longer latencies, the ORs for the lowest and highest exposure groups in the PDD analysis were slightly higher than the RDD analysis, but small numbers of exposed subjects limited the adjusted analyses. The odds ratios for breast cancer increased with increased latency and higher exposure categories, although the odds ratios were not statistically significant. The confidence intervals were generally the same width for both the RDD and PDD analyses; they included the null value in both analyses, and grew wider as the exposure level and latency increased. Overall, the results from the PDD analysis did not differ greatly from the RDD analysis and any differences were well within the variation present in the RDD data, which formed the \"input\" to the PDD analysis. The best fitting model is often but not always the one that produces the higher odds ratio \[[@B14]\]. The deviance measure of goodness-of-fit was smaller for the PDD than the RDD model at shorter latencies and lower exposure levels and larger at longer latencies and higher exposure levels. However, the close agreement between the goodness-of-fit measures suggests that there is little difference between the two models (see [Additional file 3](#S3){ref-type="supplementary-material"}: Table 5). Further evidence of this is provided by the results of the nonparametric rank test, which indicated the two exposure rankings were not statistically different. Discussion ========== The dose model was constructed to reduce nondifferential exposure misclassification due to variations in personal behavior. In the RDD analysis, exposure was based solely on subjects\' RDD values and did not take into consideration factors such as bathing habits and bottled water consumption. Nondifferential exposure misclassification should bias results towards the null when the exposure is dichotomous. Based on this reasoning, we expected the moderate elevations in risk observed in the RDD analysis by Aschengrau et al. \[[@B5]\] to increase further in the current PDD analysis. The results show that, in general, this was not the case. Overall, the risks calculated from the PDD analysis differed only slightly from the RDD analysis, if at all. The fact that the PDD model did not increase the odds ratios may be due to a number of reasons. A possible explanation is that no association exists between exposure to PCE and breast cancer, but there is a fairly large body of literature now that supports a carcinogenic effect for PCE in humans. The biologic rationale for a breast cancer effect stems from a hypothesis described by Labreche and Goldberg that organic solvents such as PCE may act either directly as genotoxic agents or indirectly through their metabolites to increase the risk of breast cancer \[[@B15]\]. More likely, the impact of variations in personal habits was small in comparison to variations in characteristics of the drinking water distribution system, or the questionnaire information did not accurately account for individual variations. Errors in estimating the RDD values used in the dose model may explain why the model made little difference in determining risk. Improper assumptions or incorrect input variables in the Webler-Brown model led to errors in the RDD values \[[@B5]\]. The resulting exposure misclassification would not be corrected using the dose model. As a result, the dose model would still be biased. Furthermore, both RDD and PDD are measures of cumulative exposure, where exposure was summed over a subject\'s residences on Cape Cod. One subject may have been exposed at a high intensity for two or more short residency durations while another subject with the same exposure value may have been exposed at a low intensity for one long residency duration. The exposure pattern can influence cancer risk if, for example, a threshold intensity of PCE must be reached in order to cause breast cancer or if breast cancer induction requires prolonged continuous exposure \[[@B16]\]. Another limitation of the analysis was the restriction to subjects with non-proxy interviews, which reduced the sample size by 31%. When all subjects were included in the RDD analysis, small to moderate increases were observed among women whose exposure level was greater than the 90^th^percentile \[[@B5]\]. When only non-proxy subjects were included, we no longer observed moderate increases. This difference may be due to the fact that the maximum RDD value was higher for all subjects than for non-proxies. Therefore, the use of only non-proxy subjects may not accurately reflect population risk. Imputing values for proxy subjects is a possible option for future analyses. Faulty recall in the behavioral data is another possible reason why the PDD model did not strengthen the association between breast cancer and PCE. Subjects were asked to remember details about bathing habits and drinking water that occurred up to forty years before the interview. As a result, the exposure data obtained at interview may not be accurate. The inputs that most heavily influenced the PDD model were initial water concentration and duration of exposure. These variables were also included in the RDD model. In this study population, personal factors like bath and shower temperature, bathing frequencies and durations, and water consumption did not differ greatly among subjects. Therefore, including these characteristics in the PDD model did not significantly improve the exposure measure or change which subjects were considered exposed and to what level they were exposed. Conclusion ========== In an attempt to characterize PCE exposure more precisely, we constructed a dose model that considered exposure from inhalation, ingestion, and dermal absorption. The model incorporated personal information on tap water use and bathing habits obtained from study interviews. The dose values calculated by the model were subsequently used to measure the strength of the association between PCE exposure and the risk of breast cancer. Although our results from the PDD analysis did not differ greatly from the RDD analysis, it remains important to assess exposure as accurately as practical in an epidemiological investigation. Many factors such as tap water use and bathing habits could be considered when determining exposure to volatile chemicals in domestic water supplies, but our analysis suggests that the use of such ancillary data does not always result in an improvement in exposure accuracy if the ancillary data are inaccurate or if they have little effect on an individual\'s exposure level. Abbreviations ============= PCE, Tetrachloroethylene; RDD, Relative Delivered Dose; PDD, Personal Delivered Dose; EPA, Environmental Protection Agency; MCL, Maximum Contaminant Level; ORs, Odds Ratios; CIs, Confidence Intervals Competing Interests =================== The author(s) declare they have no competing interests. Authors\' Contributions ======================= VV created the dose model, conducted the statistical analyses, and drafted the manuscript. AA provided the data and assisted in epidemiologic analysis and editing. DO participated in the design of the study and the editing of the manuscript. All authors read and approved the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 This document describes the dose model in more detail. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 This document provides a table of adjusted odds ratios for breast cancer by tetrachloroethylene exposure levels in RDD and PDD analyses. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 This document provides a table of deviance measures for logistic regression models by tetrachloroethylene exposure levels in RDD and PDD analyses. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This work has been supported by grant 2P42 ES07381 from the National Institute of Environmental Health Sciences (NIEHS) with funds from the U.S. Environmental Protection Agency (EPA). Its contents are solely the author\'s responsibility and do not necessarily represent the official views of the NIEHS or the EPA.	PubMed Central	2024-06-05T03:55:53.920499	2005-2-25	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554766/", "journal": "Environ Health. 2005 Feb 25; 4:3", "authors": [ { "first": "Verónica", "last": "Vieira" }, { "first": "Ann", "last": "Aschengrau" }, { "first": "David", "last": "Ozonoff" } ] }
PMC554767	Background ========== The contribution of surveillance systems in providing valuable information for injury prevention and control is widely recognized; for example, surveillance data can be used to highlight the burden of injury, set priorities for prevention, and evaluate preventive strategies \[[@B1],[@B2]\]. Estimates of the population burden of injuries differ, though, depending on how information is obtained. Detailed trauma registries and special surveillance systems \[e.g., \[[@B3]\]\] contain rich contextual information on particular subsets of injuries, but since such databases are generally not population-based, they cannot be used to estimate the incidence of injury. Although population-based surveys can yield estimates of the total burden of non-fatal injuries across a broad spectrum of injury severity, they often include insufficient sample sizes for studying small population subgroups \[[@B4]\], and are subject to recall errors \[[@B5]\]. Administrative health care databases, due to their presumed near complete coverage of injuries requiring medical care and their lack of reliance on self-reports, may be particularly useful for injury surveillance. Such databases allow for local or regional estimates of the burden of injury, which has been identified as an important goal \[[@B2],[@B6],[@B7]\], and since they are pre-existing, they are cost-efficient. Administrative data also provide an opportunity to examine health care use for injury. Administrative databases only capture injuries that receive medical care, however, and since surveillance using administrative data is often based on hospitalization data alone, only relatively severe injuries are included. Decisions regarding whether to seek medical care and where to seek care for an injury may be influenced by outside factors (such as access to care, care-seeking, and practice patterns), which may lead to selection biases \[[@B8]-[@B10]\]. In Ontario, Canada, administrative health care databases that may provide information on the incidence of non-fatal injuries include hospital discharge and physician billing data. Although hospital discharge data have been widely used in Canada to study injury, the feasibility of using physician billing information for injury surveillance has rarely been investigated \[[@B11]\]. These data, if valid, may help to expand the coverage of administrative databases to include more minor injuries, capturing care delivered in physicians\' offices and emergency departments. Although minor injuries have less impact on individuals and are less costly to the health care system on a per-injury basis, minor injuries have a large impact in terms of total population morbidity due to their frequent occurrence \[[@B12],[@B13]\]. Expanding coverage by including physician billing data would thus serve to provide a more comprehensive picture of the total health care burden of injury, and may also reduce selection biases in the surveillance data. It is not clear how the injury information provided by administrative databases compares with that obtained from population-based surveys. A study of adolescent injuries we conducted using data from the Ontario Health Survey (OHS) \[[@B14]\] presented a unique opportunity to explore such a comparison; a subset of the 1996--1997 OHS data was linked by respondent to Ontario administrative health care databases, including both hospital discharge and physician billing data. The overall purpose of this study was to explore the feasibility and value of using physician billing data for Ontario, Canada, along with hospitalization data, for the surveillance of adolescent injuries. The first objective was to document the burden of adolescent injury based on administrative health care data, focusing on the relative contribution of physician billing information and comparing overall estimates with surveillance information from survey data. The second objective was to examine data quality issues, by directly comparing adolescent injuries identified using administrative health care databases (\"administratively-defined injuries\") with those identified using self-report survey data (\"self-reported injuries\"). Methods ======= Sample and data sources ----------------------- The study sample included adolescents aged 12 to 19 years who participated in the health component of the 1996--1997 OHS (N = 3331), which was part of the National Population Health Survey (NPHS) \[[@B14]\]. Survey responses were linked to administrative health care datasets through unique health card number, respondent name, address, sex, and birthdate. Although over 95 percent of OHS respondents agreed to allow their survey responses to be linked to administrative databases, sufficient information for linkage was available for only 66 percent, including 2067 (62%) of the adolescent participants, due to missing demographic information for respondents. This subgroup provided an opportunity to examine injury occurrence using multiple data sources within the same sample. A unique set of sampling weights was created for the linked subsample of the OHS, to improve representativeness. The linked sample of adolescents was similar to the full OHS sample in terms of gender, rural/urban status, and age. Self-reported information was collected through telephone interviews. Proxy respondents provided survey information for 35 of the 2067 participants. Inpatient hospitalizations for injuries were identified using the Discharge Abstract Database (DAD) of the Canadian Institute for Health Information (CIHI) \[[@B15]\]. All Ontario hospitals are included in the computerized DAD, which contains clinical, demographic, and administrative data for each hospital discharge. Physician care for injuries, and specifically injuries cared for in emergency departments and in physicians\' offices or other outpatient facilities, was identified using physician billing data. Approximately 94% of physicians in Ontario are paid on a fee-for-service basis, through billings to the Ontario Health Insurance Plan (OHIP) \[[@B16]\]. The computerized OHIP claims database captures basic information on these services (Ontario Ministry of Health and Long-Term Care). Injury measures --------------- ### 1. Self-reported injuries (survey data) OHS respondents were asked a series of questions related to acute injuries in the past 12 months that were, from the perspective of the respondent, serious enough to limit normal activities (examples given by the interviewer included \"\...a broken bone, a bad cut or burn, a sprain, or a poisoning\") \[[@B14]\]. Participants reporting that they had experienced one or more such injuries were considered to have a self-reported injury. ### 2. Administratively-defined injuries #### i) Hospital visits for injury (identified using hospital discharge data) Inpatient hospitalizations for injury were identified for the 365-day period prior to the OHS interview, for each adolescent. An adolescent was considered to have an injury-related hospitalization if, during the one-year period, he or she had at least one documented hospital discharge with an External Cause of Injury Code (E Code) in the range 800--999 (excluding codes 870--879 and 930--949, related to medical/surgical misadventures and adverse effects of the therapeutic use of medications), based on the International Classification of Disease (ICD), 9^th^revision \[[@B17]\]. #### ii) Physician care for injuries (identified using physician billing data) The OHIP physician claims database does not contain codes representing causes of injury. Rather, injuries were identified based on codes that reflect billable services (\"procedure codes\"), and the diagnoses associated with such services (\"diagnostic codes\"). To improve sensitivity, a combination of both diagnostic and procedure codes from the database was used to create an injury algorithm that would identify physician care for injury during the one-year study period, based on the methods of Tamblyn and colleagues \[[@B11]\]. The development of the algorithm was based on a pilot study involving 200 adolescents (further details regarding the algorithm and pilot study findings are available from the authors upon request). Two lists of codes were initially created from the full listing of diagnostic and procedure codes used in the database \[[@B18],[@B19]\]. The first list (\"definite injuries\") included diagnostic and procedure codes that were viewed as being definitely related to acute injury for adolescents. Since some diagnostic and procedure codes used in the claims data were non-specific, a second list (\"possible injuries\") was also developed. The initial code lists were reviewed by three physicians with experience in family medicine and/or emergency care. The lists were then expanded and further reviewed by three researchers, including a primary care physician and a researcher with physiotherapy experience. All physician claims with diagnostic or procedure codes on the definite injury list were considered to represent injuries. Based on the pilot test results, claims representing possible injuries were considered injury-related only if they represented care for an adolescent who also had a definite injury claim within a two-day period, and if the possible claim could be considered to represent care for the same injury as the definite claim. The physician billing database also provided information on the location of physician care (e.g., physician\'s office or emergency department) for each claim. #### Summary of administratively-defined injury measures Adolescents who had at least one documented injury in either the hospitalization or the physician billing database were considered to have an administratively-defined injury. Adolescents with documented injury-related physician care at a physician\'s office or outpatient facility were considered to have a physicians\' office visit for injury. Adolescents with documented injury-related physician care at an emergency department were considered to have an emergency visit for injury. Two alternative injury outcomes were created to examine the impact of decisions made in developing the physician billing algorithm. These outcomes were based on diagnoses that were relatively common and were captured only as possible injuries in the algorithm, including non-specific conditions of the musculoskeletal system and adverse effects of drugs and medications. Data analysis ------------- ### Burden of injury First, the injury algorithm was used with the full sample of 2067 adolescents, resulting in estimates of adolescent injury-related physician care by gender and location of care. Descriptive analysis was then carried out for adolescents with non-missing data on important OHS variables (N = 2047). In addition to examining differences in the overall observed burden of adolescent injury between administrative and survey data, we also explored whether there were differences within demographic subsets of the sample where the types of injuries experienced or the types of injury care received were likely to differ. Thus, we examined the proportion of adolescents with administratively-defined and self-reported injuries separately by gender, age group, and rural versus urban residence. Since numerous comparisons were possible within the results related to the burden of injury, we chose to report 95% confidence intervals around each proportion, rather than presenting statistical tests. Because the OHS used a complex sampling design to yield a provincially representative sample, weighted proportions were calculated. Variance estimates were adjusted using bootstrap replicate weights to account for clustering within the sample \[[@B14]\]. ### Data quality exploration Two analyses were conducted to explore data quality. First, as a sensitivity analysis for the physician billing database, we examined the impact of re-classifying two common \"possible injury\" diagnoses as actual injuries in the physician billing database. These diagnoses included i) non-specific musculoskeletal system diagnoses, and ii) adverse reactions to drugs and/or medications. These diagnoses were viewed as potentially problematic because they were not specific enough to injuries to warrant inclusion as \"definite\" injuries, but we believed that they might be commonly used by physicians providing injury-related care. Secondly, we directly compared self-reported and administratively-defined injuries at an individual level, to provide further insight into data quality. Not all self-reported injuries identified using the survey data would be expected to have led to medical treatment, and conversely, it is possible that some medically treated injuries may not have led to a restriction in normal activity. Some overlap between the survey data and administrative data was expected, though, in terms of injuries identified. Thus, we examined discrepancies between the injury variables at the level of the individual adolescent (i.e., the extent to which adolescents with administratively-defined injuries were likely to also have self-reported an injury during the same time period). Odds ratios, based on two-way data tables, were used as a measure of association for these direct comparisons of injury variables across data sources. We also explored possible reasons for the discrepancies observed, including potential recall error in the survey data, and potential error in both datasets resulting from overlap between acute injuries and repetitive strain injuries. These exploratory analyses involved, where appropriate, descriptive statistics (e.g., mean or median values) or odds ratios (as a measure of association for two dichotomous variables). All of the data quality analyses were used to examine within-sample methodologic issues. Therefore, as we did not wish to generalize the results from these analyses to a target population, unweighted analyses were conducted, and confidence intervals were not included. Results ======= The burden of adolescent injury ------------------------------- During the one-year period prior to the OHS interview, there were a total of 13501 physician visits for any cause among the 2067 adolescents in the initial sample (where a visit represents all of the care provided by a physician to a patient on the same date, at any location), based on the physician billing data. Of these, 1390 visits were identified as being related to injury, representing 10.3% of all physician care. The proportion of visits due to injury varied across locations of care. For example, 8.6% of physicians\' office visits (n = 604) and 45.6% of emergency department visits (n = 402) were identified as injury-related. A greater proportion of physician care was injury-related among males (14.2%), relative to females (7.3%). Within the final sample (N = 2047), while 18.8% of adolescents self-reported an activity-limiting injury in the past year, 25.0% had at least one administratively-defined injury, based on the hospitalization and physician billing databases (Table [1](#T1){ref-type="table"}, weighted proportions). While 17.1% had at least one physicians\' office visit for injury, 13.4% had one or more emergency department visits or inpatient stays for injury (these two outcomes were combined because only 18 adolescents had documented inpatient care for injury). Among adolescents with physician care for injury, the majority had one or two identified visits in the one-year period. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Sample characteristics and prevalence of injuries by data source (weighted) ::: Total (Unwtd N = 2047) Males (Unwtd N = 1081) Females (Unwtd N = 966) --------------------------- ------------------------ ------------------------ ------------------------- -------------- ------ -------------- Rural 17.2 (15.7, 18.7) 17.2 (15.1, 19.2) 17.2 (14.9, 19.6) Age group 12--14 years 34.0 (31.5, 36.6) 35.0 (31.6, 38.4) 33.0 (29.2, 36.8) 15--17 years 39.4 (36.7, 42.0) 38.9 (35.3, 42.5) 39.9 (36.0, 43.8) 18--19 years 26.6 (24.1, 29.1) 26.1 (22.8, 29.3) 27.2 (23.3, 31.1) Injury measures Self-reported^1^ 18.8 (16.9, 20.7) 22.1 (19.1, 25.0) 15.3 (12.7, 17.8) Administratively-def^2^ 25.0 (22.9, 27.1) 28.0 (24.9, 31.0) 21.7 (18.6, 24.7) Physician\'s office^3^ 17.1 (15.2, 18.9) 19.5 (16.8, 22.1) 14.4 (11.9, 17.0) ED/inpatient^4^ 13.4 (11.8, 15.0) 15.5 (13.1, 18.0) 11.0 (8.9, 13.1) 1--2 injury visits^5^ 16.5 (14.8, 18.3) 18.2 (15.7, 20.8) 14.6 (12.2, 17.0) \> = 3 injury visits^5^ 8.2 (6.7, 9.6) 9.7 (7.6, 11.9) 6.4 (4.5, 8.3) CI = Confidence Interval; def=defined; ED = emergency department; unwtd = unweighted ^1^Self-reported injury, identified using survey data ^2^Administratively-defined injury, identified in the hospitalization or physician billing databases ^3^At least 1 documented physician\'s office visit for injury within 1 year prior to the interview ^4^Any documented emergency department or inpatient visits for injury within 1 year prior to interview ^5^Number of physician visits (any location) for injury (based on physician billing data only) ::: Table [2](#T2){ref-type="table"} shows the proportion of adolescents with each injury outcome, separately for each age group and by rural/urban status. A higher proportion of males were injured relative to females, across injury outcomes and subgroups. An exception was emergency department or inpatient attended injuries among rural adolescents, although in this case, the estimated proportion for females had high sampling variability. A small decrease in the proportion injured was observed with increasing age. This decrease was less apparent for emergency department and inpatient injury visits; again, sampling variability was high. For self-reported injuries, a higher proportion of rural adolescents was injured relative to urban adolescents, particularly for females. Rural/urban differences were not apparent for administratively-defined injuries overall. By location of care, the proportion with a physician\'s office visit for injury was higher for urban adolescents, while the proportion with emergency department or inpatient injury care was higher for rural adolescents. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Distribution of injury outcomes (weighted) ::: Self-reported injury (survey data) ------------------------------------------------------------------------------ ------------------------ ------------------------ ------------------------- -------------- -------- -------------- Total (Unwtd N = 2047) Males (Unwtd N = 1081) Females (Unwtd N = 966) \%^1^ 95% CI \%^1^ 95% CI \%^1^ 95% CI Age 12--14 years 21.2 (17.6, 24.8) 23.4 (18.0, 28.8) 18.6 (13.7, 23.5) Age 15--17 years 18.9 (15.8, 22.1) 23.7 (19.0, 28.4) 13.9 (9.7, 18.0) Age 18--19 years 15.6 (12.0, 19.2) 17.8 (12.5, 23.1) \13.3 (8.2, 18.3) Rural 21.9 (17.5, 26.3) 23.1 (17.0, 29.2) 20.5 (14.0, 26.9) Urban 18.2 (16.0, 20.4) 21.8 (18.4, 25.2) 14.2 (11.3, 17.1) Administratively-defined injury (hospitalization and physician billing data) Total (Unwtd N = 2047) Males (Unwtd N = 1081) Females (Unwtd N = 966) \%^1^ 95% CI \%^1^ 95% CI \%^1^ 95% CI Any injury^2^ Age 12--14 years 27.2 (23.1, 31.2) 29.5 (24.2, 34.9) 24.4 (18.5, 30.4) Age 15--17 years 25.1 (21.6, 28.6) 27.4 (22.4, 32.5) 22.6 (17.9, 27.3) Age 18--19 years 22.0 (18.1, 25.8) 26.7 (20.6, 32.7) 17.0 (12.0, 21.9) Rural 25.2 (20.5, 29.9) 26.6 (20.2, 33.0) 23.7 (17.1, 30.3) Urban 24.9 (22.5, 27.3) 28.3 (24.7, 31.8) 21.2 (17.9, 24.6) Phys. office injury^3^ Age 12--14 years 19.8 (16.2, 23.3) 21.6 (16.7, 26.5) 17.6 (12.5, 22.8) Age 15--17 years 16.9 (13.9, 19.9) 19.8 (15.2, 24.3) 13.8 (10.0, 17.6) Age 18--19 years 13.9 (10.5, 17.3) 16.2 (11.1, 21.2) \11.5 (6.8, 16.3) Rural 14.0 (10.5, 17.5) \16.2 (10.8, 21.6) \11.6 (6.9, 16.3) Urban 17.7 (15.6, 19.8) 20.1 (17.1, 23.2) 15.0 (12.0, 18.1) ED/inpatient Injury^4^ Age 12--14 years 13.8 (10.8, 16.9) 15.9 (11.5, 20.4) \11.4 (7.4, 15.4) Age 15--17 years 13.6 (11.1, 16.2) 14.5 (10.9, 18.2) 12.7 (8.9, 16.4) Age 18--19 years 12.4 (9.4, 15.4) 16.4 (11.7, 21.1) \8.2 (4.7, 11.7) Rural 18.0 (13.9, 22.1) 17.6 (12.5, 22.8) \18.4 (12.1, 24.6) Urban 12.4 (10.6, 14.2) 15.1 (12.3, 17.9) 9.5 (7.3, 11.7) CI = Confidence Interval; ED = emergency department; phys. = physician\'s; unwtd = unweighted ^1^Row percentages (for example, of 12--14 year-olds, the percent who experienced the injury outcome) ^2^Any documented injury in the administrative databases (hospitalization and physician billing data) ^3^At least 1 documented physician\'s office visit for injury within 1 year prior to the interview ^4^Any documented emergency department or inpatient visits for injury within 1 year prior to the interview \ Proportion should be interpreted with caution due to high sampling variability ::: Sensitivity analysis and comparison of injuries across data sources ------------------------------------------------------------------- As a sensitivity analysis for the physician billing data algorithm, the impact of re-classifying two common \"possible injury\" diagnoses as actual injuries was examined. When the non-specific musculoskeletal system diagnoses were added to the injury dataset, the proportion of adolescents with administratively-defined injury increased from 25.0% to 29.3%. When adverse reactions to drugs and/or medications were added, the proportion increased only slightly, to 25.9% (weighted proportions). The results of the within-sample analysis used to compare injuries identified using different data sources are shown in Table [3](#T3){ref-type="table"} (unweighted). Section i) of the table shows the direct comparison of administratively-defined and self-reported injuries at the individual level, for the total sample, and then separately by gender, and by location of care for administratively-defined injuries. For example, of the 2047 adolescents in the final sample, 550 had a documented administratively-defined injury, while 1497 had no such injury. Of the 550 adolescents with administratively-defined injury, 213 (38.7%) self-reported an injury, compared with 193 (12.9%) among the 1497 adolescents with no administratively-defined injury. The odds ratio for the relationship between administratively-defined and self-reported injury was 4.3. There was a higher congruence between the two data sources in terms of identified injuries for females (odds ratio 5.7), relative to males (odds ratio 3.4). To examine the congruence with self-reported injury separately by location of care for the administratively-defined injuries, the third and fourth columns of the table (\"administratively-defined injury\") were restricted to those identified as having specifically received care at either a physician\'s office or an emergency department/inpatient facility. For example, 358 adolescents had a documented physician\'s office visit for injury, and 134 (37.4%) of these adolescents also self-reported an injury. When compared with the 12.9% of adolescents who self-reported an injury but had no administratively-defined injuries, the resulting odds ratio was 4.0. There was a higher congruence with self-reported injuries for emergency department or inpatient care for injury (odds ratio 5.8). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Exploring self-reported versus administratively-defined injury (unweighted) ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- i\) Direct comparison of injuries identified using different data sources: self-reported injury for those with and without administratively-defined injury ------------------------------------------------------------------------------------------------------------------------------------------------------------ --------- -------------------- ------------------ ----------------------- ------ -------------------- ------------------ Administratively-\ No administratively-\ defined injury defined injury Total N N Self-report N(%) N Self-report N(%) Odds ratio Total 2047 550 213 (38.7) 1497 193 (12.9) 4.3 Males 1081 318 126 (39.6) 763 123 (16.1) 3.4 Females 966 232 87 (37.5) 734 70 (9.5) 5.7 Phys. office^1^ 1855 358 134 (37.4) 1497 193 (12.9) 4.0 ED/ inpatient^2^ 1812 315 145 (46.0) 1497 193 (12.9) 5.8 ii\) Time from most recent administratively-defined injury to OHS interview (N = 550)^3^ N Mean (\# days) Median (\# days) Adolescents with self-reported injury 213 141 125 Adolesents without self-reported injury 337 173 171 Total 550 160 146 iii\) Self-reported repetitive strain injuries, by self-reported & administratively-defined acute injuries (N = 2045) N Repetitive Strain\ Odds Ratio Injury N (%) Self-reported acute injury 406 57 (14.0) 1.9 No self-reported acute injury 1639 129 (7.9) Administratively-defined acute injury 550 88 (16.0) 2.7 No administratively-defined acute injury 1495 98 (6.6) Administratively-def. & self-rep. acute injury 213 35 (16.4) 1.1 Administratively-def. & no self-rep. acute injury 337 53 (15.7) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- def = defined; ED = emergency department; OHS = Ontario Health Survey; phys = physician\'s; self-rep. = self-reported ^1^At least 1 documented physician\'s office visit for injury within 1 year prior to interview, based on the administrative data. Adolescents with emergency department or inpatient visits but no physicians\' office visits for injury are excluded from the denominator. ^2^Any documented emergency department or inpatient visits for injury within 1 year prior to interview, based on the administrative data. Adolescents with physicians\' office visits but no emergency department or inpatient visits for injury are excluded from the denominator. ^3^Analysis includes only those adolescents (N = 550) with administratively-defined injury ::: In order to investigate the possibility that recall error may have led to underreporting in the survey, we investigated the relationship to recall time (Table [3](#T3){ref-type="table"}, section ii). For adolescents with at least one administratively-defined injury, those who self-reported an injury had a shorter recall time from the most recent documented administratively-defined injury to the OHS interview (median 125 days) compared with adolescents who did not self-report an injury (median 171 days). Finally, to explore whether some acute injuries may have been misreported as repetitive strain injuries in the OHS (based on a series of questions on repetitive strain injuries that preceded those on acute injuries), and to explore whether the algorithm used with the physician billing database may have led to misclassification of some repetitive strain injuries as acute injuries, we examined the relationship to self-reported repetitive strain injuries. Both self-reported acute injury and administratively-defined acute injury appeared to be related to self-reported repetitive strain injury (odds ratios 1.9 and 2.7 respectively, Table [3](#T3){ref-type="table"}, section iii), although the relationship for administratively-defined injury was stronger. Among those with administratively-defined injury, there was no strong evidence of a relationship between self-reports of acute injury and repetitive strain injury; repetitive strain injury was reported by 16.4% of the 213 adolescents with self-reported acute injury, and a similar 15.7% of the 337 adolescents without self-reported acute injury (Table [3](#T3){ref-type="table"}, section iii, last two rows). Discussion ========== Contribution of physician billing data to injury surveillance using administrative databases -------------------------------------------------------------------------------------------- This exploratory study focused on the potential value of using physician billing data in combination with hospital discharge data to document the burden of injuries among adolescents. The results suggest that adding physician billing claims to hospitalization information is a feasible method of improving the comprehensiveness of healthcare administrative datasets. Approximately 10 percent of all physician care for adolescents in the study was identified as injury-related. Although a smaller proportion of physicians\' office visits was identified as injury-related, relative to emergency department physician visits, office care actually represented a larger number of visits. Thus, these relatively more minor injuries represent a large component of adolescent injury morbidity that would be missed if estimates relied on hospital data alone or even on a combination of hospital and emergency care information. The observed differences in the rural/urban distribution of adolescent injuries by location of care (Table [2](#T2){ref-type="table"}), reflecting potential difference in injury severity or access to care between rural and urban adolescents, also highlight the importance of capturing information across the full spectrum of care. Comparison of administrative databases and self-reports: value for injury surveillance -------------------------------------------------------------------------------------- A higher proportion of adolescents was identified as having administratively-defined injury relative to self-reported injury. One might expect the definition of self-reported injury used in the survey (injuries that limit normal activities) to capture a broader spectrum of injury severity compared with the administrative data (since some activity-limiting injuries may not receive medical care). The higher proportion of adolescents with administratively-defined injury, though, suggests that there may also be a subset of medically treated injuries that do not in fact limit normal activities; in other words, perhaps the definition of injuries used in the survey was actually more restrictive. Although neither data source can be viewed as a \"gold standard\", these results suggest that administrative health care data may actually provide a more sensitive means of ascertaining injuries, relative to self-reported survey data. Injuries identified as medically treated using administrative data may also be viewed as representing the health concerns of the person seeking care, and they have an impact on the health care system. These findings highlight the potential importance of administrative databases as a source of population-based injury information that can be used for affordable ongoing surveillance and for examining health care system issues such as patterns of service delivery. Despite these advantages, a limitation of many claims datasets, including the OHIP database, is a lack of detail on the circumstances surrounding the occurrence of injuries. The billing data contained no external cause information, such that description of injuries by mechanism and intent was not possible. Exploring data quality issues: injury outcomes in administrative databases and self-reports ------------------------------------------------------------------------------------------- Although the distribution of injuries, particularly for gender and age, was fairly similar for self-reported and administratively-defined injuries (Table [2](#T2){ref-type="table"}), congruence of injury outcomes was relatively low at the individual level (Table [3](#T3){ref-type="table"}, section i). This may in part reflect the different definitions of injury represented in the datasets (medically treated injuries in the administrative data, versus activity-limiting injuries in the survey data). Our exploration of data quality issues, however, revealed potential errors in both databases that may have contributed to the discrepancies. In the survey data, we found some evidence of recall errors (Table [3](#T3){ref-type="table"}, section ii). This finding is supported by previous research documenting recall errors in self-reports for a variety of health outcomes, including chronic conditions \[e.g., \[[@B20],[@B21]\]\] as well as injuries \[e.g., \[[@B5],[@B22],[@B23]\]\]. For example, in a study of parental recall of non-fatal injuries in children and adolescents, estimates of annual injury rates were found to decline as the recall period for injuries increased from two weeks to 12 months \[[@B5]\]; this suggests that the 12 month recall period used in the 1996--1997 OHS may have led to underreporting. In the study of parental recall referred to above, more severe injuries (resulting in surgery or hospitalization; or resulting in restriction to bed or school absence) appeared to be less subject to recall errors, relative to minor injuries \[[@B5]\]. This may partly explain the stronger association we found between self-reported injuries and administratively-defined injuries when administratively-defined injuries were restricted to those identified as having received emergency department or inpatient care. Studies that have directly compared self-reported health care use with health care use identified in medical records across a variety of health services have also tended to find that both males and females underreport physician visits to a greater extent than hospital or emergency care, particularly as the recall period increases \[e.g., \[[@B24]-[@B26]\]\]. In addition to recall error in the survey data, inaccuracies in the administrative databases may have played a role in contributing to the discrepancies between the administrative and survey data. Errors in the DAD have been identified \[[@B27]\], although this likely had little impact, due to the small number of injury hospitalizations. The method used to identify injuries in the physician billing database was exploratory. The sensitivity analysis and results related to repetitive strain injury (Table [3](#T3){ref-type="table"}, section iii) highlight the need to further validate the physician billing data algorithm, ideally using comparisons with medical charts. With respect to repetitive strain injury, its stronger observed relationship with administratively-defined injury (as compared with self-reported acute injury) suggests that the physician billing data algorithm may have led to the inclusion of some repetitive strain injuries. The lack of evidence for a relationship between self-reported acute injury and repetitive strain injury among those with administratively-defined injury (Table [3](#T3){ref-type="table"}, section iii) suggests that confusion with repetitive strain injury in the survey did not lead to underreporting of acute injuries. Strengths and limitations ------------------------- Strengths of our study included the detailed exploration of the methods used to identify injuries using physician billing data, and the unique comparison of injuries across datasets within the same sample of adolescents. In addition to the need to further validate the algorithm used with the physician billing data, study limitations included the small sample size, particularly for investigating the distribution of injuries resulting in emergency and inpatient care, and the incomplete linkage of the survey and administrative datasets. Although the incomplete data linkage may reduce the generalizability of the study findings, the linked sub-sample was similar to the full Ontario survey sample across demographic characteristics (gender, rural/urban status, and age), and the unique sampling weights created for this sub-sample may have improved representativeness. Finally, because this study capitalized on an opportunity presented by a larger study on youth injuries, we focused specifically on adolescents. Further research could examine the generalizability of both the approach and the findings to other age groups, where the types of injuries experienced and care-seeking patterns may differ. Studies in jurisdictions with similar medical claims databases would also help in assessing the generalizability of our research. Conclusion ========== Collectively, our findings allow us to draw two main conclusions. First, the results suggest that there is potential value in using physician billing data along with other administrative health care databases for the surveillance of injuries among adolescents. Although they are lacking in details about the circumstances surrounding injuries, comprehensive administrative injury datasets may be particularly useful for describing the overall occurrence of injury at local or regional levels, and for describing the economic implications of injury for the health care system. Secondly, we identified data quality concerns in both the survey and administrative databases that suggest a need for improvement and further study; for example, further research could help to identify appropriate recall periods and question wording for minimizing errors in survey data, and to determine the level of detail needed to accurately identify injuries in administrative databases. Because various sources of data are susceptible to different limitations, it remains important to consult multiple sources of information to fully document the burden of injury \[[@B2],[@B4],[@B7]\]. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= BKP participated in the design and coordination of the study and the development of the physician billing algorithm, carried out all statistical analyses, and drafted the manuscript. DM participated in the design, coordination, and supervision of the study and development of the physician billing algorithm, as well as revisions to the manuscript. KNS participated in the design, coordination, and supervision of the study, as well as revisions to the manuscript. IAG participated in the design of the study and development of the physician billing algorithm, as well as revisions to the manuscript. MKC participated in the design of the study and revisions to the manuscript. JJK participated in the design of the study and revisions to the manuscript, and provided statistical guidance. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6963/5/15/prepub> Acknowledgements ================ This study was supported by a doctoral research award from the Canadian Institutes of Health Research (BKP) and was completed as part of Dr. Potter\'s dissertation in the Department of Epidemiology & Biostatistics, University of Western Ontario. The authors gratefully acknowledge assistance from the Institute for Clinical Evaluative Sciences (ICES), Toronto, Ontario.	PubMed Central	2024-06-05T03:55:53.923204	2005-2-18	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554767/", "journal": "BMC Health Serv Res. 2005 Feb 18; 5:15", "authors": [ { "first": "Beth K", "last": "Potter" }, { "first": "Douglas", "last": "Manuel" }, { "first": "Kathy N", "last": "Speechley" }, { "first": "Iris A", "last": "Gutmanis" }, { "first": "M Karen", "last": "Campbell" }, { "first": "John J", "last": "Koval" } ] }
PMC554768	Background ========== The current World Health Organization classification for tumors of the nervous system includes medulloblastoma, medulloepithelioma, ependymoblastoma, supratentorial primitive neuroectodermal tumor (sPNET) and atypical teratoid/rhabdoid tumor (ATRT) in the category of embryonal brain neoplasms \[[@B1]\]. These tumors are united by their primitive cytological appearance and the ability to differentiate into multiple cell types. However, recent studies indicate that these lesions are genetically, and to some extent clinically, separable. ATRT are defined by the presence of rhabdoid cells, contain INI1mutations, and cause particularly grim clinical outcomes \[[@B2]\]. Medulloblastomas commonly contain isochromosome 17q, but this chromosomal alteration is rarely detected in sPNET or ATRT \[[@B3]\]. Global gene expression profiles also suggest that medulloblastoma, sPNET and ATRT are distinct entities \[[@B4]\]. Little is known about the differences in p53 expression and function among the various embryonal brain tumor subtypes. Initial reports on the p53 tumor suppressor gene suggested it was mutated in 10% or less of medulloblastomas \[[@B5]-[@B8]\]. However, Frank and colleagues have recently shown that the p53 pathway is inactivated by mutation of p53, methylation of p14ARF, or deletion of INK4/ARF in 21% of medulloblastomas \[[@B9]\]. In their study, 5 of the 6 medulloblastomas with alterations abrogating p53 function had significant anaplasia. Large cell/anaplastic changes in medulloblastoma are prognostic of significantly worse clinical outcomes \[[@B10]-[@B12]\]. Interestingly, p53 protein accumulation, which is often associated with loss of functionality, has been found by some \[[@B13]-[@B15]\], but not others \[[@B16],[@B17]\], to predict shorter survival in medulloblastoma patients as well. Several other lines of evidence also support a role for the p53 pathway in medulloblastomas. First, medulloblastomas sometimes arise in the context of Li Fraumeni syndrome, in which p53 germline mutations predispose patients to a wide range of neoplasms \[[@B18]\]. Second, inactivation of p53 accelerates the formation of medulloblastomas in transgenic mouse models \[[@B19]\]. Finally, inactivation of p53 and Rb simultaneously, either through genetic disruption or overexpression of viral T antigen, results in medulloblastomas in rodents \[[@B20]-[@B22]\]. It has been suggested that viral infection of human CNS tissues could promote formation of brain tumors by inhibiting p53 and Rb activity \[[@B23]\]. Some researchers have reported the presence of JC virus or other oncogenic polyomaviruses in human brain tumor specimens, including medulloblastomas \[[@B24],[@B25]\]. Large T antigen expressed by these viruses binds and inactivates p53 \[[@B26]\]. This process results in the accumulation and immunohistochemical detection of p53 protein. In human neural tissue this is best demonstrated in progressive multifocal leukoencephalopathy, in which JC virus infected oligodendroglial cells are strongly p53 immunopositive \[[@B27],[@B28]\]. It is therefore possible that the accumulation of p53 protein in some human medulloblastomas is caused by viral infection. In order to confirm the association between p53 immunopositivity, clinical outcome, and embryonal tumor subtype, we stained a tissue array containing representative cores from 80 embryonal brain tumors for p53. We also investigated JC virus infection as a possible mechanism for accumulation of p53 protein by searching for viral sequences using a highly sensitive quantitative real time polymerase chain reaction (PCR) assay. We found an association between p53 immunoreactivity, clinical outcome, and tumor subtype, but did not detect JC virus in medulloblastoma or supratentorial PNET. Methods ======= Clinical material ----------------- Medulloblastomas and other embryonal brain tumors diagnosed at the Johns Hopkins University Department of Pathology were identified through review of departmental records. Classic, desmoplastic/nodular and large cell/anaplastic medulloblastomas were classified using World Health Organization guidelines \[[@B1]\]. Nuclear size, cell morphology and the frequency of mitosis and apoptosis were used as previously described to grade anaplasia \[[@B11]\]. 80 Tumors from 78 patients were used to create a tissue array as previously described \[[@B29]\]. Patients ranged from 8 months to 55 years of age, with a median age of 9 years. Microscopic examination of the array confirmed that the appearance of tumor tissue cores corresponded to donor blocks. Frozen tumor tissue obtained from medulloblastomas resected at the Johns Hopkins Hospital was snap-frozen in liquid nitrogen and stored at minus 80°C prior to nucleic acid extraction. DNA was extracted using Trizol and further purified using a DNeasy column (Qiagen, Valencia, CA) according to the manufacturer\'s instructions. This study was approved by the Johns Hopkins University Institutional Review Board. Immunohistochemistry -------------------- The tissue array was sectioned at four microns, deparaffinized, and subjected to antigen retrieval by steaming (20 minutes at 80°C). Slides were then incubated at room temperature for 45 minutes with monoclonal antibody directed towards p53 (1:2000, clone DO-7, DAKO, Carpinteria, CA). Primary antibody was detected using the avidin-biotin complex (ABC) method with diaminobenzadine serving as the chromagen. We semiquantitatively graded staining intensity as negative, weak, or strong. Carcinomas with mutations leading to p53 stabilization were used as positive controls. No staining was seen in the absence of primary antibody (negative control). Detection of virus by real time PCR ----------------------------------- JC virus sequences were amplified from DNA using the forward primer PEP-1 (5\'-AGT CTT TAG GGT CTT CTA CC-3\') and reverse primer PEP-2 (5\'-GCC AAC CTA TGG AAC AG-3\') \[[@B30]\]. Additional specificity for detection of JC virus was achieved using the FAM/Black Hole Quencher-1 (FAM/BHQ-1) labeled TaqMan probe (5-/56-FAM/ CCA ACA CTC TAC CCC ACC T /3BHQ\_1/-3) \[[@B31]\]. This probe does not cross-react to the closely related human BK polyomavirus, or simian SV40 polyomavirus. Fifty microliter reaction volumes were used, comprised of 1X universal master mix (Applied Biosystems, Foster City, CA), 0.05 μM probe, 0.4 μM PEP-1, 0.4 μM PEP-2, and 16--288 ng tumor DNA. Amplifications were performed using a Biorad ICycler with the following thermal profile: 95°C for 10 minutes, then 50 cycles at 95°C for 15 seconds and 57°C for 1 minute. Quantitation of JC virus in tumors was determined using linear regression with an external standard curve included on each plate containing a 5-fold dilution series of known input JC virus plasmid diluted in a constant background of human placental DNA (70 ng/μl). To normalize for sampling variability, we quantitated the total cell equivalents in each sample by amplifying a human endogenous retrovirus gene (ERV-3) in an equal amount of tumor DNA \[[@B32]\]. Conditions for ERV-3 amplification were: 0.4 μM forward primer (PHP10-F: 5\'-CAT GGG AAG CAA GGG AAC TAA TG-3\'), 0.4 μM reverse primer (PHP10-R: 5\'-CCC AGC GAG CAA TAC AGA ATT T-3\'), and 0.25 μM TaqMan probe labeled with FAM and BHQ (PHP-P505/ERV-3 Probe: 5\'-/56-FAM/TCT TCC CTC GAA CCT GCA CCA TCA AGT CA/3BHQ\_1/-3\'). ERV-3 amplification was performed on an ABI 5700 using the following thermal profile: 95°C for 10 minutes, followed by 50 cycles of amplification at 95°C for 15 seconds and 60°C for 30 seconds. DNA extracted from the diploid cell line ATCC CCL 205 diluted in a constant background of 50 ng/μl salmon sperm DNA was used to construct the ERV-3 standard dilution curve. Statistical analysis -------------------- p53 statistical analyses were performed using GraphPad PRISM4 software (GraphPad Software, San Diego, CA). A two tailed Fishers Exact test was used to compare immunohistochemical staining profiles between groups. Significance of survival differences was assessed using log-rank analysis of Kaplan-Meier curves. The formula 1-(alpha)^1/N^was used to calculate the fiducial (exact) 95% upper bound of JC virus prevalence. Results ======= p53 protein levels are increased in anaplastic medulloblastoma, ATRT and sPNET ------------------------------------------------------------------------------ We used immunohistochemistry to examine p53 protein expression in representative cores from 80 embryonal brain tumors on a tissue array. Cores from 5 of the cases could not be evaluated due to crush artefact, cautery, or low cellularity. Of the remaining 75 tumors, 64 were medulloblastomas; 31 of these medulloblastoma were of the classic subtype, 13 were nodular/desmoplastic, and 20 were large cell/anaplastic. The tissue array used in this study also contained 11 other CNS embryonal tumors, including 8 sPNET and 3 ATRT. Among the sPNET were 3 with the long epithelial surfaces characteristic of medulloepithelioma. Immunoreactivity for p53 was present in a minority (35%) of the 75 embryonal tumors, and a relatively small subset of cells stained in most of the positive cases. In all positive cases the majority of the immunoreactivity was in tumor cell nuclei. An example of a nodular medulloblastoma with weak, scattered p53 immunoreactivity is shown in Figure [1A](#F1){ref-type="fig"}. A medulloblastomas and a sPNET with strong immunoreactivity are shown in Figure [1B](#F1){ref-type="fig"} and [1C](#F1){ref-type="fig"}, respectively. Only 16% (5/31) of classic medulloblastomas and 23% (3 /13) of nodular medulloblastomas were immunopositive for p53, and staining was weak in all of these cases. In contrast, 45% (9/20) of anaplastic medulloblastomas, 67% (2/3) of ATRT, and 88% (7/8) of sPNET were positive for p53. The intensity of staining in the anaplastic medulloblastomas and extracerebellar embryonal tumors was strong in many cases (Figure [2](#F2){ref-type="fig"}). The increase in p53 immunoreactivity in anaplastic medulloblastomas was statistically significant when compared to non-anaplastic ones, including classic and nodular lesions (P = 0.03, Fisher\'s Exact test). Other embryonal tumors (sPNET and ATRT) also had a significant increase in p53 expression compared to non-anaplastic medulloblastoma (P = 0.0001, Fisher\'s exact test). p53 expression was often widespread in the severely anaplastic medulloblastoma, ATRT, and sPNET groups, with 6 of 12 tumors showing immunoreactivity in over 25% of cells, while classic and nodular medulloblastomas always had fewer than 25% immunopositive cells (Table [1](#T1){ref-type="table"}). Staining for p53 was distributed evenly in most lesions, rather than being concentrated in focal groups of tumor cells. p53 Immunopositivity is significantly associated with worse clinical outcomes ----------------------------------------------------------------------------- We next examined whether p53 immunopositivity was associated with worse clinical outcomes in embryonal brain tumor patients. Of the 75 tumors from 73 patients scored on the array, survival data was available for 66 individuals. 61 percent of patients were alive at last contact, with follow up times ranging from 3 to 215 months (median 47 months). When survival of the entire embryonal brain tumor cohort was analyzed, 67% (31/46) of patients with p53 immunonegative tumors were alive, as compared to 45% (9/20) of patients with immunopositive tumors. Log rank analysis of Kaplan Meier survival curves confirmed the significance of this difference (P = 0.02). When only the 56 medulloblastoma patients with clinical follow up were analyzed, 71% of individuals with p53 negative tumors survived, compared to 58% of individuals with p53 positive tumors. However, these differences were not significant on log rank analysis (P = 0.25). The 6 patients with intensely immunoreactive tumors (3 anaplastic medulloblastoma, 2 sPNET, 1 ATRT) all died from their disease in less than two years. JC virus infection does not account for increased levels of p53 protein ----------------------------------------------------------------------- To address the possibility that p53 accumulation in medulloblastoma and other embryonal brain tumors is due to viral infection, we used quantitative RT PCR to search for viral sequences in tumor DNA. Of the cases used on the tissue array, 19 had material frozen suitable for high-quality DNA extraction. Four of these cases were p53 positive. Also available in our frozen tumor bank was tissue from 14 additional embryonal lesions (4 classic medulloblastoma, 5 anaplastic medulloblastoma, 1 nodular medulloblastoma and 4 sPNET). We did not detect JC virus sequences in any of these 33 tumors. We repeated the analysis on DNA extracted from a different tumor tissue fragment in 5 cases, but these spatially distinct regions also failed to contain viral DNA. ERV-3, an endogenous human retroviral element, served as a control and was easily amplified from tumor DNA samples (Figure [3](#F3){ref-type="fig"}). ERV-3 copy numbers ranged from \~2 × 10^3^- 2 × 10^5^per PCR assay (mean 3.4 × 10^4^). Because two copies of ERV-3 are present per human genome, DNA from at least 1000 cells was assayed in each PCR reaction. In contrast, all of the specimens tested were completely negative for JC virus. The quantitative Taqman assay was able to detect a minimum of \~10 copies of JC virus per PCR reaction as determined using a standard dilution curve. Thus the number of JC virus genomes should not exceed 10 per 1,000 tumor cells. Given our 0% detection rate, the 95% confidence limit for the largest value of the \'true\' underlying JC virus prevalence is no more than 7.6% (1-(0.05)^1/38^). Discussion ========== To investigate the prognostic potential and pathological role of p53 expression in embryonal brain tumors, we analyzed this protein in 75 medulloblastoma, ATRT and sPNET using immunohistochemistry. Overall, we found significant increases in p53 immunoreactivity in anaplastic medulloblastomas (45% positive) as compared to non-anaplastic ones (18% positive). The percentage of p53 immunopositive medulloblastomas in our study (27%) fell within the previously reported range of 3% to 53% \[[@B13],[@B16],[@B17],[@B33],[@B34]\]. This wide variation in published values is likely due to differences in antibodies used, their dilutions, and antigen retrieval protocols. Cuttoffs for a calling a tumor \"positive\" also varied among previous investigators, with some scoring only intensely positive lesions. Only 5% of our medulloblastomas fell into this strong staining category, and all of these were anaplastic. Mutation of the p53 gene often results in a stabilized protein of altered functionality that accumulates in the nucleus of tumor cells \[[@B35]\]. Our data are thus consistent with those recently reported by Frank and colleagues, who found that the TP53 pathway was frequently disrupted in large cell/anaplastic medulloblastomas \[[@B9]\]. Interestingly, supratentorial PNET and ATRT were also more commonly p53 immunopositive than non-anaplastic medulloblastoma in our study. While extracerebellar PNET were included in several earlier studies, p53 immunoreactivity was not reported separately for these lesions \[[@B14],[@B15]\]. Ho and colleagues documented p53 mutations in 6 of 14 sPNET but did not examine protein expression \[[@B36]\]. In another relatively large series, only 1 of 12 sPNET contained a mutation in the p53 gene \[[@B37]\]. Finally, Postovsky and colleagues described an unusual p53 mutation in a case report of a sPNET \[[@B38]\]. With regard to rhabdoid lesions, Berrak and colleagues documented faint p53 immunoreactivity in 6 of the 7 ATRT of the CNS they examined \[[@B39]\]. Malignant rhabdoid tumors arising outside the CNS are also commonly p53 immunopositive, and mutations predicted to inactivate p53 function have been documented in some \[[@B40]\]. Thus while the number of sPNET and ATRT we examined was relatively low, our data, combined with earlier reports, suggests that p53 function may be commonly altered in embryonal tumors arising outside the cerebellum. Clinical outcomes were significantly worse for embryonal brain tumor patients in our study whose lesions were p53 immunopositive. However, cases most commonly positive for p53 (anaplastic medulloblastomas, sPNET, and ATRT) are all more clinically aggressive than non-anaplastic medulloblastomas, making it difficult to infer causality resulting from p53 accumulation. p53 expression did not predict outcome within the group of medulloblastoma patients, although all 3 strongly p53 immunopositive tumors were severely anaplastic and associated with quite short survival. Interestingly, in a recently published report, Ray and colleagues found that p53 immunoreactivity was the only biological marker predictive of poor outcome on both univariate and multivariate analyses in a group of 112 medulloblastoma patients \[[@B41]\]. We also examined the potential role of viral infection as a mechanism for p53 protein stabilization in medulloblastoma. None of the four p53-positive cases from our tissue array with frozen material available contained viral sequences. We also tested nine additional samples of histological subtypes that were more commonly associated with p53 immunopositive staining (anaplastic medulloblastomas and sPNETs) where p53 staining was not performed. None of these tumors were JCV positive. While sample availability precluded complete testing of all confirmed p53 positive tumors for JCV, the available data do not support a causal role for JCV infection in p53 accumulation and development of embryonal brain tumors. The impact of viruses on medulloblastoma pathogenesis is controversial. The most commonly implicated agents are the polyomavirus family members JC virus, BK virus and SV40 virus. Of these, JC virus, which infects approximately 80% of the pediatric population and can cause CNS disease in immunosuppressed patients, has been most studied. It was shown decades ago that inoculation of JC virus into rodents resulted in formation of medulloblastoma-like cerebellar tumors \[[@B20],[@B22],[@B42],[@B43]\]. Transgenic mice containing JC virus early region sequences also develop medulloblastomas \[[@B44]\]. Del Valle and colleagues isolated JC virus large T antigen sequences from 11 of 23 human medulloblastomas they examined, and suggested inactivation of p53 and Rb by viral T antigen could be important in the pathogenesis of human embryonal brain tumors \[[@B24]\]. A second gene (Agno) from JC virus was later reported to be present in 11 of 16 medulloblastoma samples by the same group \[[@B25]\]. We failed to detect JC virus sequences in the 33 cases examined, using a sensitive and specific technique which should identify as few as 10 viral genomes per PCR reaction. This suggests the p53 immunopositivity we observe is not caused by JC virus infection in the majority of cases. Our data are also not consistent with the hypothesis that ongoing JC virus infection is common in medulloblastoma. Other recent studies have also reported a lack of JC virus DNA in medulloblastomas. Hayashi and colleagues failed to identify JC viral sequences in 13 medulloblastomas \[[@B45]\]. Kim and colleagues similarly failed to identify JC virus in 15 medulloblastomas, 5 sPNET and 2 medulloblastoma cell lines \[[@B46]\]. Rollison and colleagues examined 225 brain tumors, including 20 medulloblastomas, for JC, BK and SV40 viruses at two different laboratories \[[@B47]\]. No tumor tested positive in both laboratories. Finally, Weggen and colleagues failed to detect JC virus sequences in any of 116 medulloblastomas analyzed, although 2 of these cases were SV40 positive \[[@B48]\]. These reports do not rule out the possibility of a \"hit and run\" process in which virus participates initially in the formation of a lesion and is then lost. They do strongly suggest that ongoing JC virus infection is not common in human medulloblastomas, as was initially suggested. Interestingly, it has recently been shown that the putative SV40 infection of human mesotheliomas can be accounted for by contamination of samples with small amounts of common laboratory plasmids containing regions of the T-antigen gene \[[@B49]\], calling into question the true association of SV40 with human cancers, including brain tumors. Conclusion ========== In summary, we find significantly increased p53 protein levels in anaplastic medulloblastomas, sPNET, and ATRT as compared to classic and nodular medulloblastoma. JC virus was not detected in the 33 tumors examined, suggesting that T-antigen binding does not appear to be an ongoing factor in the pathobiology or p53 protein accumulation of embryonal brain tumors. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= Drs. Eberhart, Shah and Gravitt planned the study and wrote the initial manuscript draft. Dr. Eberhart, Ms. Chaudhry and Ms. Khaki collected samples, isolated DNA, and performed immunohistochemical staining. Dr. Gravitt supervised and R Daniel performed the JC virus copy number analysis in tumors. All authors reviewed and commented on the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/5/19/prepub> Acknowledgements ================ We would like to acknowledge the Lee-Min Duh and Eleni Goshu for technical assistance. This work was supported by NINDS K08NS43279 and a Burroughs Wellcome Career Award to CGE. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### p53 Immunostaining in CNS embryonal tumors.Most p53 immunopositivity was relatively faint and present only in a small fraction of the tumor. This nodular medulloblastoma had weak staining in less than 25% of cells (A). Some anaplastic medulloblastomas had strong p53 immunostaining in a larger fraction of cells (B). Most AT/RT and supratentorial PNET contained p53 immunopositive cells, like this lesion metastatic within the CNS in which almost all cells are strongly positive (C). Note the lack pf staining in non-neoplastic stroma (Asterisk). ::: ![](1471-2407-5-19-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### A statistically significant increase in p53 immunopositivity in AT/RT and sPNETMost classic and nodular medulloblastoma (MB) contained no p53 immunopositive cells. In contrast, p53 positive cells were present in many anaplastic MB, supratentorial primitive neuroectodermal tumors (sPNET) and atypical teratoid rhabdoid tumors (ATRT). Triangles represent individual tumors with negative (0), weak (1) or strong (2) p53 immunostaining. Bars represent the mean and standard error of the mean. The increase in p53 immunopositivity in the sPNET/ATRT category as compared to classical MB was statistically significant (Mann Whitney test). ::: ![](1471-2407-5-19-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### JC virus sequences are not detected in embryonal brain tumors.JC virus plasmid DNA is detectable over a wide range of dilutions when added to genomic DNA (A), but was not identified in DNA extracted from a range of embryonal brain tumors (B). In contrast, the ERV-3 endogenous retrovirus is easily detected both using standard dilution curves (C) and in tumor DNA (D). ::: ![](1471-2407-5-19-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Intensity and extent of p53 immunopositivity in CNS embryonal tumors ::: Case Tumor Subtype p53 Intensity p53 Positive Cells ------ -------------------------- --------------- -------------------- 1 Classic MB 1 5 to 25% 2 Classic MB 1 5 to 25% 3 Classic MB 1 5 to 25% 4 Classic MB 1 5 to 25% 5 Classic MB 1 5 to 25% 6 Nodular MB 1 5 to 25% 7 Nodular MB 1 5 to 25% 8 Nodular MB 1 5 to 25% 9 Moderately Anaplastic MB 1 5 to 25% 10 Moderately Anaplastic MB 1 5 to 25% 11 Moderately Anaplastic MB 1 5 to 25% 12 Moderately Anaplastic MB 1 5 to 25% 13 Moderately Anaplastic MB 1 5 to 25% 14 Moderately Anaplastic MB 1 5 to 25% 15 Severely Anaplastic MB 2 51 to 75% 16 Severely Anaplastic MB 2 51 to 75% 17 Severely Anaplastic MB 2 26 to 50% 18 sPNET 1 5 to 25% 19 sPNET 1 5 to 25% 20 sPNET 1 5 to 25% 21 sPNET 2 5 to 25% 22 sPNET/Medulloepithelioma 1 5 to 25% 23A sPNET/Medulloepithelioma 2 51 to 75% 23B sPNET/Medulloepithelioma 2 76 to 100% 24 AT/RT 1 5 to 25% 25 AT/RT 2 26 to 50% MB -- medulloblastoma; sPNET -- supratentorial PNET; AT/RT -- atypical teratoid/rhabdoid tumor; p53 Intensity (0 -- negative, 1 -- weak, 2 -- strong). :::	PubMed Central	2024-06-05T03:55:53.928676	2005-2-17	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554768/", "journal": "BMC Cancer. 2005 Feb 17; 5:19", "authors": [ { "first": "Charles G", "last": "Eberhart" }, { "first": "Aneeka", "last": "Chaudhry" }, { "first": "Richard W", "last": "Daniel" }, { "first": "Leila", "last": "Khaki" }, { "first": "Keerti V", "last": "Shah" }, { "first": "Patti E", "last": "Gravitt" } ] }
PMC554769	Background ========== The increased use of microarray expression profiling to study both the molecular biology of cancer and the cellular physiology of difficult-to-isolate cell types has led to a growing need for methods that allow the use of limiting quantities of RNA. Small surgical biopsies, fine needle aspirates, cyto-lavages, punch biopsies and blood samples often yield only 1--4 μg quantities of RNA as starting material for expression profiling. This limitation has prompted the development of amplification methods that produce the quantities of RNA required for microarray analysis. Changing requirements for the type and quantity of amplified RNA, driven by evolving microarray technologies, have led to the development of novel amplification strategies. While current methods are capable of delivering high-yield RNA amplification, this is often only achieved after complex priming strategies (for example, involving 4 or more primers) are coupled with multiple rounds of PCR and/or in vitrotranscription, resulting in time consuming and costly protocols. Here, we present an overview of RNA amplification strategies, identify key limitations to existing techniques, and describe a simple, robust, and cost-effective strategy for single round amplification of RNA in the sense orientation. RNA amplification methods ------------------------- Early attempts to amplify RNA employed a strategy based upon the Polymerase Chain Reaction (PCR) \[[@B1]-[@B4]\]. These methods relied on the terminal transferase activity of reverse transcriptase to allow addition of primer sites to the 3\' end of reverse-transcribed, first-strand cDNA. Multiple rounds of PCR primed from this site and from the poly-(A)^+^sequence on the second-strand cDNA could then be used to facilitate amplification. These methods were confounded by differential amplification of cDNA and by introduction of errors by Taqpolymerase. This problem prompted the development of a linear, T7-based in vitrotranscription (IVT) method by Van Gelder and Eberwine \[[@B5]-[@B7]\]. In what has now become known as the \"Eberwine Method,\" RNA templates are primed with an oligo(dT) primer that has been 5\' modified to contain a promoter for the T7 RNA polymerase and are subsequently reverse transcribed into first-strand cDNA. The RNA-cDNA hybrid is then treated with E. coliRNAse H, and priming for second-strand cDNA synthesis occurs by either RNA nicking and priming or cDNA hairpinning^6^. Second-strand cDNA synthesis is carried out with E. coliDNA polymerase and E. coliDNA ligase followed by blunt-ending with T4 DNA polymerase. Transcription and amplification are then accomplished using the T7 RNA polymerase, which binds to the T7 promoter introduced during first-strand cDNA synthesis, producing antisense RNA (aRNA). Technical revisions of the Eberwine method have included changes in first-strand primer concentration to minimize the appearance of non-sequence dependent RNA in the amplified product \[[@B8]\], supplementation of second-strand priming with random primers to improve its efficiency, and modifications that allow multiple rounds of IVT to augment yield \[[@B6],[@B9],[@B10]\]. Concerns regarding the fidelity of amplification with these methods stem from the 3\' bias introduced by the use of the promoter-modified oligo(dT) primer during first-strand cDNA synthesis, and questions remain over the degree to which this amplified RNA reflects the true transcriptome of the unamplified sample. To correct for this potential bias, three alternatives have been developed to the Eberwine protocol. One such alternative \[[@B11]\] is based upon the Eberwine approach, but second and subsequent rounds of amplification are primed with random nonamer primers modified by the addition of an upstream T3 promoter sequence (T3N9 primer). IVT from this T3 promoter prevents serially compounding the 3\' bias introduced by the oligo(dT) primer across multiple rounds of amplification. The T3N9 primer has also been used to prime the initial round of reverse transcription, a modification that is useful for amplifying partially-degraded samples of RNA \[[@B12]\]. In this case, the method sacrifices the ability to selectively amplify mRNA for the versatility generated by the random priming and subsequent amplification of any RNA sequence present in the sample. A second alternative to the Eberwine method is the \"template-switching\" (TS) strategy \[[@B13]\]. This technique centers on the observation that the Moloney murine leukemia virus reverse transcriptase (MMLV-RT) adds an oligo(dC) region to the 3\' end of first-strand cDNA after reaching the terminal end of an RNA template \[[@B14]\]. When an oligo(G) primer is added to the second strand RT reaction, it will hybridize with this oligo(dC) sequence, and the MMLV-RT will switch strands (the \"template-switch\") and continue the reverse transcription reaction. This strategy can be used to append a T7 promoter to the 5\' end of the oligo(G) primer\[[@B13],[@B15]\], facilitating RNA amplification by IVT. Yield can be further improved by combining this technique with PCR amplification after cDNA synthesis \[[@B16]\] for bulk production of amplified RNA \[[@B17]\]. Several variations on this theme involving changes in the primers and in the details of the PCR amplification have been described, all of which rely on a combination of TS primers and PCR-based amplification to produce large amounts of amplified RNA \[[@B18],[@B19]\]. A third recently introduced alternative is Ginsberg\'s Terminal Continuation (TC) technique \[[@B20]\]. In this approach, the initial reverse transcription reaction is primed with a mixture of an oligo(dT) primer and a modified TC primer. The former primes the reverse transcription of mRNA, while the latter is essentially a T7 promoter-containing GC-rich sequence that primes second strand cDNA synthesis. According to Ginsberg, the \"likely mechanism (for this incorporation) is that the TC primer binds preferentially to GC-rich CpG islands flanking 5\' regions of DNA that contain promoter sequences.\" \[[@B21]\] Initial reports of the results of TC amplification show promise for linear amplification of high-quality RNA, but extensive validations of this method have yet to be conducted. Validity of amplified RNA in microarray applications ---------------------------------------------------- The degree to which the pool of amplified RNA generated by these methods reflects the unamplified sample from which it is derived is an obvious concern for microarray applications and other downstream analyses, and the history of attempts to validate RNA amplification methods is summarized by Nygaard et al. \[[@B22]\]. Briefly, they note that a combination of Northern blotting, dot blot differential screening of cDNA probes synthesized from aRNA, internal RNA standards, hierarchical clustering, qRT-PCR, subgroup analysis, and ratio-intensity (RI) plot analysis has lead to the conclusions that relative expression levels are well-preserved after 1--3 rounds of amplification, that important over- or under-expressed genes are detectable after amplification, and that amplification may actually improve detection of RNA present in low copy number. However, they noted that few studies quantitatively compare total RNA and amplified RNA, that the true sources of amplification biases have not been thoroughly investigated, and that the degree to which \"noise\" affects the differences in these profiles has not been adequately assessed. Accordingly, they conducted multiple hypothesis testing based upon t-tests and ANOVA analysis of several technical parameters to study the nature and the magnitude of biases and variability associated with the use of amplified RNA in microarray expression profiling. They observed that approximately 10% of genes showed statistically significant differences in relative expression level between amplified and their unamplified counterparts and noted that neither technical replication of amplifications nor molecular characteristics of the sample were the likely cause of these observed differences. Despite these differences, they stated that the increased quantity and purity of mRNA hybridized in studies using amplified RNA increases overall fluorescence intensity and improves detection of low-abundance messages. Because they noted that more that 50% of the amplified RNA showed log~2~(ratio) differences within ± 0.5 of the unamplified RNA, they concluded that RNA amplification is useful in expression profiling and is likely to assist in the measurement of low-abundance RNA. Despite the methodological validation provided by these studies, only recently have statistically-based analyses been used to compare RNA amplification methods in a critical, head-to-head fashion. A study by Zhao \[[@B23]\] examined differences between T7-based amplification protocols, including the Eberwine method and the template switch (TS) technique, where these methods were used to amplify RNA extracted from tumor samples rather than from the idealized situation of cell lines. They observed that the use of TS strategies does not improve the fidelity of RNA amplification versus the Eberwine method, that there is good correlation between samples after amplification, that the overall bias introduced by T7-based amplification strategies is uniformly low, and that the results are reproducible. Their overall conclusion was that T7-based amplification protocols generate high-fidelity, amplified antisense RNA that is suitable for use in cDNA microarray analysis. Limitations of current amplification protocols ---------------------------------------------- It is currently accepted that RNA amplification strategies based on the Eberwine method maintain relative mRNA levels between samples when amplifying from either total RNA or poly(A)^+^RNA \[[@B24],[@B25]\], are useful with low microgram quantities of starting RNA \[[@B26]\], and are capable of preserving differential expression profiles when used in conjunction with microarray analysis \[[@B13]\]. Accordingly, efforts have become increasingly focused upon developing an optimized protocol that minimizes amplification bias, provides versatility, and reduces technical complexity. While current commercial kits and published protocols are making progress to these ends, we feel that several important dimensions have been overlooked, limiting their utility. First, most commercial protocols produce RNA only in the antisense orientation. With the emergence of spotted oligonucleotide arrays and mixed cDNA/oligo arrays \[[@B27]\], appropriate orientation of amplified RNA is an important experimental consideration. While TS and TC protocols can be modified to produce sense RNA, this generally comes at the cost of increased technical and biological complexity but adds no demonstrable benefit over the Eberwine strategy. Second, many current protocols rely on multiple rounds of IVT and/or PCR to produce amplified RNA, and even a few rounds of this amplification has the potential to introduce sequence error \[[@B6]\] or systematic bias \[[@B28]\]. While some applications, such as laser capture microdissection, may produce only picogram quantities of RNA and thus require extensive amplification, it should be noted that even small biopsy samples frequently yield low microgram quantities of RNA and thus may require only a single round of amplification. Finally, the increasing complexity of many amplification protocols creates logistical problems when implementing them for large-scale projects. Protocols that require multiple rounds of amplification can accumulate material and labor costs that quickly exceed those associated with the actual microarray analysis, and amplification can become a \"rate-limiting\" (or \"cost-limiting\") factor in study design. Additionally, the number of enzymes, reagents, and custom primers continues to increase as protocols become more complex, a situation which is equally undesirable. We believe that a protocol that addresses these limitations and provides a versatile and robust method for RNA amplification is needed. Goals for a revised RNA amplification protocol ---------------------------------------------- Our goal was to develop a strategy based upon the Eberwine method but with the ability to produce sense RNA from small quantities of total or poly-(A)^+^RNA extracted from both ideal samples (e.g. cell line RNA) and \"real-world\" samples (e.g. tumors or tissues). This protocol should avoid the need for PCR steps and should require a minimum number of primers (two). Additionally, the protocol should be cost-effective, efficient, and technically simple to conduct. Finally, the method should give results consistent with similar amplification techniques when used with subsequent microarray analysis. We believe these criteria have been met with our protocol, which consequently will be useful in a variety of laboratory applications. Results ======= Priming efficiency and cDNA yield --------------------------------- Quantitation of first strand cDNA after hydrolysis of the cDNA/RNA hybrid shows that priming with the T7-Oligo(dT) promoter/primer followed by reverse transcription produces cDNA that totals 5--10% by mass of the original mass of total RNA (data not shown). This is consistent with the average percentage of mRNA in the total RNA sample and therefore suggests that priming proceeds efficiently and that first strand cDNA synthesis successfully copies the population of mRNA in the original total RNA sample. Quantitation of second strand cDNA after priming with the T3N9 promoter/primer indicates a 120% increase in mass versus the mass of the first strand cDNA (data not shown). This suggests that second strand priming proceeds efficiently and that the first strand cDNA is successfully converted to double stranded cDNA by the second strand synthesis reaction. The extra mass (in excess of 100%) is attributable to the use of random nonamer primers which initiates synthesis at multiple positions along each first-strand cDNA template. Median transcript length ------------------------ While inherent constraints of reverse transcription limit the amount of truly \"full-length transcript\" produced during any RNA amplification scheme, it is nonetheless desirable for an amplification method to favor the production of long transcript. RNA amplified using our method has a median length of 794 nucleotides (range: 70--9000 nt) suggesting that this method is capable of preserving long transcript during amplification (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Yield and transcript length of amplified RNA produced by three amplification methods. Values in parentheses represent p-values from t-test comparing the value against that of the study protocol. ::: Study Protocol *RiboAmp Protocol (p-value vs. Study Protocol)* *BDSMart Protocol (p-value vs. Study Protocol)* --------------------------------------- -------------------- ----------------------------------------------------- ----------------------------------------------------- Mean yield from 4 μg total RNA (, μg) 26.29 71.56 (\<0.04) 4.87 (\<\<0.0001) Median amplified RNA length (nt) 794 507 (\<0.003) 764 (0.88) ::: Quantitative yield ------------------ Our method produced an average (![](1471-2164-6-27-i1.gif)) of 26.3 μg of amplified sense mRNA from 4 μg of total RNA (Table [2](#T2){ref-type="table"}). Assuming that 5% of the original sample of total RNA was mRNA, this reflects a 130-fold amplification. The protocol has been tested with as little as 1 μg of starting total RNA and produces sufficient mRNA for microarray analysis (\~2 μg) under these conditions (data not shown). Amplification bias ------------------ Any amplification will introduce some degree of bias into the population of amplified RNA. While recent reports suggest that the magnitude of this bias may be relatively unimportant as long as the bias is highly reproducible \[[@B29]\], we feel that aggressively limiting any source of experimental bias improves the quality of subsequent microarray data. We systematically evaluated the performance of RNA amplified using this protocol against identical hybridizations from unamplified RNA. Log~2~(ratio) plots of fluorescence intensity in amplified versus unamplified samples were constructed and analyzed for correlation as described above. Our results show that the amplification bias introduced by this method is small, as reflected in the high average correlation coefficient (![](1471-2164-6-27-i2.gif)) between expression profiles from amplified and unamplified RNA samples (![](1471-2164-6-27-i2.gif) = 0.8009, Table [3](#T3){ref-type="table"}, Figure [2](#F2){ref-type="fig"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Statistical analysis of microarray data generated by comparing hybridizations of amplified RNA samples to identical hybridizations of unamplified (total RNA) samples. Values in parentheses represent P-values from t-test comparing the value to that of the study protocol. ::: Study Protocol *RiboAmp Protocol (p-value vs. Study Protocol)* *BDSmart Protocol (p-value vs. Study Protocol)* ------------------------------------------------------------------ -------------------- ----------------------------------------------------- ----------------------------------------------------- Mean Correlation Coefficient () 0.8009 0.7202 (\<0.01) 0.7679 (0.23) Mean % of elements with absolute difference of ± 2 log~2~units 0.34 1.34 (\<0.02) 0.60 (0.56) Mean % of elements with absolute difference of ± 1.5 log~2~units 1.31 4.05 (\<0.05) 1.63 (0.67) Mean % of elements with absolute difference of ± 2 log~2~units 5.44 12.54 (\<0.02) 6.52 (0.61) Mean % of amplified array elements within ± 1 log~2~unit 94.56 87.46 (\<0.02) 93.48 (0.61) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Correlation between expression profiles of RNA samples amplified using the study protocol and corresponding unamplified RNA samples demonstrates a low degree of amplification bias. ::: ![](1471-2164-6-27-2) ::: Reproducibility --------------- We evaluated the reproducibility of this method and the consistency of the propagation of the amplification bias by comparing the expression profiles of hybridizations of three independently amplified RNA samples. Log~2~(ratio) plots of fluorescence intensity were constructed and analyzed as described above. Our results demonstrate a high degree of correlation between independent replicates (![](1471-2164-6-27-i2.gif) =.9446, Figure [3](#F3){ref-type="fig"}) and a narrow dispersion (σ^2^= 0.00023, CV = 1.60%), suggesting that the amplification is highly reproducible and that the amplification bias is introduced consistently when the protocol is repeated. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Correlation between independent replicates of expression profiles of RNA samples amplified using the study protocol demonstrates the high degree of reproducibility of the method. ::: ![](1471-2164-6-27-3) ::: Comparison with commercial amplification kits --------------------------------------------- We compared these results to results from the RiboAmp mRNA amplification kit (Arcturus), which produces antisense RNA using a version of the Eberwine method, and from the BDSmart mRNA amplification kit (BD Biosciences), which produces sense RNA using a template-switching strategy. Both protocols were carried out according to the manufacturer\'s specifications using 4 μg of the pooled cell line RNA and the glioblastoma RNA as the starting material for amplification. The final yield of each method was measured using the NanoDrop spectrophotometer, the median size of the amplified mRNA from each kit was assayed using the Bioanalyzer 2100 (Agilent), and the amplification bias of each method was assessed using microarray analysis (as described above). Three independent replicates were conducted for each method, and the reproducibility of the amplification was assessed as described above. The mean correlation coefficient (![](1471-2164-6-27-i2.gif)) and the dispersion of each group (σ^2^, CV) were calculated, demonstrating a high degree of reproducibility for both the RiboAmp (![](1471-2164-6-27-i2.gif) = 0.950, σ^2^= 0.0004, CV = 2.09%) and the BDSmart (![](1471-2164-6-27-i2.gif) = 0.895, σ^2^= 0.0010, CV = 3.47%) protocols. These results suggest that subsequent comparisons of our amplification method to these commercial techniques are not confounded by individual variability among replicates. Additionally, the strong correlation between independent replicates of our method (see above) and of these commercial techniques suggests that performing additional independent replicates would not add significant statistical power to subsequent comparisons. The average yield from our method (![](1471-2164-6-27-i1.gif) = 23.6 μg) was less than that of the Arcturus RiboAmp method (![](1471-2164-6-27-i1.gif) = 71.6 μg, p \< 0.04) but greater than that of the BDSmart method (![](1471-2164-6-27-i1.gif) = 4.87 μg, p = 1.7 × 10^-14^). The decreased yield versus the Arcturus RiboAmp method is expected, because IVT with the T7 promoter (production of antisense RNA, Arcturus method) proceeds more efficiently than IVT with the T3 promoter (production of sense RNA, our method). The improved yield over the BDSmart method is notable considering that both of these methods involve production of amplified sense RNA (Table [2](#T2){ref-type="table"}). The median length of the amplified RNA from our method (794 nt) was greater than that of the Arcturus RiboAmp method (507 nt, p \< 0.003) and comparable to that of the BDSmart amplification method (764 nt, p = 0.88) (Table [2](#T2){ref-type="table"}). The correlation between expression profiles of amplified and unamplified RNA was 10.6% better following amplification with our method (![](1471-2164-6-27-i2.gif) = 0.8009) versus amplification with the Arcturus RiboAmp method (![](1471-2164-6-27-i2.gif) = 0.7202, p \< 0.01) and was comparable to the correlation after amplification with the BDSmart method (![](1471-2164-6-27-i2.gif) = 0.7679, p = 0.15) (Table [3](#T3){ref-type="table"}). Discussion ========== The emergence of methods to study the transcriptome initially necessitated the production of relatively large quantities of RNA from experimental systems, and Eberwine and Van Gelder\'s T7-based system of In vitroTranscription^5--6^became the first standard protocol for RNA amplification designed to fulfill these requirements. The evolution of microarray technology and other RNA techniques have imposed requirements on the type, length, and/or orientation of the starting RNA. Additionally, the growing interest in medical and microbial genomics now requires that data be collected from samples that are becoming progressively smaller and more difficult to acquire. This changing role for RNA amplification has catalyzed the development of multiple institutional and commercial amplification protocols, all of which claim to be high-yield, low-bias techniques capable of amplifying RNA with the specific characteristics required for downstream applications. Template switching, terminal continuation, and other novel techniques for RNA amplification that have been developed over the past five years have become increasingly technically complex, and we feel that this is a significant disadvantage. In particular, they stray from the Eberwine approach, which has been extensively validated, and often rely on the use of proprietary (i.e. unspecified) enzymes, primers, and reaction components, which limit the utility of the methods (as well as protocol modification and fine-tuning). While proponents of these methods argue that the limitations are offset by improved yield, production of long transcript, reproducibility, and reduced amplification bias, we have found in practice that no single protocol is able to perform consistently in all four areas. The technique that we have described affords the advantages of all of the aforementioned protocols while eliminating the major limitations and controlling the technical complexity. Several aspects of this protocol make it a robust tool that we believe will be useful in conjunction with a variety of experimental systems and downstream applications. First, the method we present is based upon the Eberwine technique. The methods are both straightforward and validated and do not require the use of custom enzymes, multiple (more than 2) proprietary primers, or PCR steps to complete the amplification. Second, this new method produces amplified mRNA in the sense orientation and can be used for production of aRNA with minor modification. This corrects for the downstream limitations imposed by antisense amplification in both the original Eberwine method and the Arcturus RiboAmp kit and allows amplified mRNA produced by our method to be used in conjunction with both cDNA (sense and antisense orientation) and long oligonucleotide (sense orientation) arrays. Third, our method requires only one round of amplification to produce RNA in the sense orientation. We believe that this represents a significant advantage over previously described, T7-based amplification strategies (most notably the method of Xiang \[[@B11]\]), where the modifications necessary for producing sense RNA require a minimum of two rounds of amplification. Fourth, our method produces sufficient amplified sense RNA for multiple microarray analyses after a single round of amplification from as little as 1 μg of total RNA. This yield is significantly higher than the BDSmart amplification kit and we have demonstrated that our method produces amplified mRNA that performs at least as well as the BDSmart amplification kit and superior to the Arcturus RiboAmp protocol in microarray applications. Another advantage of our protocol is that it is readily adaptable to a variety of experimental systems. The approach that we have described synthesizes amplified RNA from eukaryotic total RNA in the sense orientation. However, because this method adds unique promoters to both the first and second cDNA strand (T7 and T3, respectively), either orientation of RNA can be produced from the same population of cDNA simply by selecting the appropriate RNA polymerase for IVT. Moreover, changing the two primers allows the protocol to be adapted to produce sense or antisense RNA from either prokaryotic or eukaryotic total RNA without any additional adjustments. The protocol can also be modified for use with either T3 or T7 IVT systems by changing the promoter sequence of the promoter-modified primer, it can be used to salvage partially-degraded RNA (as described by Xiang \[[@B11]\]), and it can be used for indirect RNA labeling protocols by substituting modified ribonucleotide bases into the IVT mix. Table [4](#T4){ref-type="table"} summarizes the primer pairs used for these alternate strategies, and Table [1](#T1){ref-type="table"} gives the necessary primer sequences. We have successfully tested a variety of these strategies and have achieved similar degrees of bias, reproducibility, and yield (data not shown). We believe that the ease with which this protocol can be adapted to a variety of experimental systems is a major advantage. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Primer sequences for study protocol and its variations. ::: Primer Name Sequence Concentration (ng/μL) ------------------ ------------------------------------------------------------------------- --------------------------- T3N9 5\'-GCGCGAAATTAACCCTCACTAAAGGGAGANNNNNNNNN-3\' 100 T7N9 5\'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGNNNNNNNNN-3\' 100 Oligo(dT)~24~ 5\'-TTTTTTTTTTTTTTTTTTTTTTTT-3\' 100 T3-Oligo(dT)~24~ 5\'-GCGCGAAATTAACCCTCACTAAAGGGAGATTTTTTTTTTTTTTTTTTTTTTTT-3\' 100 T7-Oligo(dT)~24~ 5\'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3\' 100 Random Hexamer 5\'-NNNNNN-3\' 3000 ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Primer strategies for alternate versions of the study protocol. ::: Type of Total RNA First Primer Second Primer IVT System^†^ Type of Product Orientation of Product --------------------------------------------- --------------------------------- ------------------- ------------------- --------------------- ---------------------------- Eukaryotic Oligo(dT)~24~, T7-Oligo(dT)~24~ T3N9 T3 mRNA Sense T3-Oligo(dT)~24~ Random Hexamer T3 mRNA Antisense\* *Prokaryotic (or Partially Degraded\\)* Random Hexamer T3N9 T3 Total RNA Sense T3N9 Random Hexamer T3 Total RNA Antisense ^†^T7 IVT systems may be used by changing the promoter sequence of the modified primer to a T7 binding sequence \* Modified Eberwine Amplification \\ As described by Xiang^11^ ::: In clinical and research settings using laser capture microdissection or other strategies where very small amounts of starting RNA (10--100 ng) are available, multiple rounds of amplification may still be necessary. A final advantage of our protocol, as discussed previously, is that a second round of amplification can be easily incorporated and can be used to produce additional RNA in either the sense or antisense orientation. Future testing of this protocol will focus on the quantity and fidelity of the RNA population after multiple rounds of amplification and will include appropriate comparisons to commercial, multi-round techniques. However, the purpose of the present technique is to minimize bias while maintaining yield after a single round of amplification. In this setting, our results show that our method outperforms two commercial, single-step protocols when using identical quantities of initial RNA. We believe the performance and versatility of this method make it particularly beneficial to investigators attempting to conduct microarray analysis from 1--4 μg of initial RNA. Neither this nor any other current strategy for RNA amplification is optimally suited to every experimental setting, and each technique has its own advantages and limitations. The challenge of selecting an amplification strategy is choosing a method that provides sufficient amplification and appropriate RNA orientation for downstream requirements yet minimizes the labor, cost, and bias introduced by the process. The method that we have described has been designed and validated for use in experiments where microarray analysis is to be performed with approximately 1 μg of starting total RNA. In this setting, our method produces a population of amplified RNA having minimal and consistent amplification bias, orientation compatible with both spotted cDNA and oligonucleotide arrays, and quantity sufficient for several downstream assays from a single amplification. Additionally, the protocol we present here can be easily adapted for use in a variety of experimental systems. This includes production of either sense or antisense RNA in prokaryotic or eukaryotic systems (simply changing one or both primers). None of these variations require multiple rounds of amplification to produce RNA of a specific orientation, but the protocol can easily be modified to enable multiple rounds when necessary. Finally, we would like to note that there are some limitations to the method we have outlined here. Readers may note that several amplification strategies have been reported to produce greater than 1,000-fold increases in RNA yield, one aspect of our method that may be viewed as a limitation is its \~130-fold amplification. However, most higher-yield amplification strategies use multiple rounds of amplification and are subject to additional amplification bias. While we recognize that researchers using submicrogram quantities of starting material may consider strategies incorporating multiple rounds of amplification, in our experience even small tissue biopsies yield more than 1 μg of total RNA. The ability to amplify this quantity of RNA efficiently and effectively was one of our major design considerations. In this setting, 130-fold amplification produces more than enough RNA for multiple downstream microarray analyses and confers the benefit of reduced bias inherent in a single-round amplification protocol. Conclusion ========== We have developed a robust method for amplification of mRNA in the sense orientation. This protocol is based upon the Eberwine method and incorporates the advantages of more recent amplification techniques while eliminating many of the limitations of these strategies. Our method allows the production of sense mRNA with one round of IVT, yields 130-fold amplification, preserves long transcript, and produces mRNA that is well suited for downstream microarray applications. Microarray assays performed with RNA amplified using our protocol demonstrate that the method results in low amplification bias and is highly reproducible. Additionally, our method is readily adaptable for the production of sense or antisense, labeled or unlabeled RNA from intact or partially-degraded samples of prokaryotic or eukaryotic total RNA. The method outperforms several commercial RNA amplification kits and can be used in conjunction with a variety of downstream microarray platforms (cDNA arrays, oligonucleotide arrays, Affymetrix GeneChip™ assays). We feel that these advantages make our method a robust tool with the potential for application in a variety of research settings. Methods ======= Overview of the method ---------------------- The protocol that we have designed is based on the Eberwine method but uses novel priming strategies to produce amplified RNA in the sense orientation. First-strand cDNA synthesis from total RNA is primed with an Oligo(dT)~24~primer followed by heat denaturing and then cooling to facilitate primer annealing. Reverse transcription produces cDNA-mRNA hybrids which are then subjected to alkali hydrolysis to remove template mRNA. First-strand cDNA (fs-cDNA) is separated from residual enzymes, nucleotides, and mRNA fragments using a spin column technique. Second-strand cDNA synthesis is primed with random nonamers containing an upstream T3 promoter sequence (T3N9). The sample is heated to denature the fs-cDNA and to eliminate secondary structure. The temperature is rapidly dropped to the upper limit of the annealing range and then ramped more slowly to a final temperature of 4°C. We believe that this strategy minimizes \"self-priming\" of the fs-cDNA and, in the absence of competitive priming from RNA fragments, facilitates optimal annealing of the second-strand primer. Second-strand cDNA (ss-cDNA) synthesis is carried out using E. coliDNA polymerase and ligase followed by blunt-ending with T4 DNA Polymerase. The double-stranded cDNA is purified using a spin column technique, and in vitrotranscription (IVT) from the T3 promoter sequences incorporated into the ss-cDNA produces amplified RNA in the sense orientation (Figure [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Overview of study method for amplification of RNA in the sense orientation ::: ![](1471-2164-6-27-1) ::: Cell line RNA ------------- Total RNA used in this study was derived from three cell lines: PA-1 ovarian teratocarcinoma, CaOV-3 ovarian adenocarcinoma, and U118MG glioblastoma. All cell lines were grown in a monolayer under Dulbecco\'s minimum essential medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cell lines were held at 37°C in an atmosphere of 4% CO~2~and maintained accordingly. When culture plates achieved \~90% confluence, RNA was extracted and purified following TIGR standard operating procedures based on the Trizol method (Invitrogen) \[[@B30]\]. RNA extracted from all three cell lines was pooled to create a RNA mixture with a final relative composition of 42.56% CaOV-3 RNA, 29.89% PA-1 RNA, and 27.55% U118MG RNA. Purity was verified by measurement of OD~260/280~and OD~230/260~in TE (pH 8.0) using a NanoDrop spectrophotometer, and RNA integrity was assessed by measurement of the 28S/18S ratio with the Agilent Bioanalyzer 2100 using the Agilent Total RNA Nano chip assay. The final values for the RNA pool were: OD~260/280~= 2.04, OD~230/260~= 2.20, 28S/18S = 2.13, indicating high purity, non-degraded RNA. Superas-In RNAse inhibitor (Ambion) was added to a final concentration of 1U/μL according to manufacturer\'s recommendations. RNA was stored at -80°C until it was used in the RNA amplification process. RNA amplification protocol -------------------------- All temperature controlled steps in this protocol were conducted using a Peltier thermal cycler (PTC225 DNA Engine Tetrad, MJ research) in thin-walled, nuclease-free PCR tubes (BioRad). All enzymes, buffers, and other reaction components were purchased from Invitrogen unless otherwise noted. Four (4) μg of pooled total cellular RNA (described above) was as starting material for the amplifications. Priming for first strand cDNA synthesis is carried out by combining the total RNA with 1 μL of T7-Oligo(dT) primers (100 ng/μL, Ambion) and 1 μL of Superas-In RNAse inhibitor (20U/μL, Ambion). The mixture is diluted to 17.8 μL in DEPC treated water, heated at 70°C for 10 minutes, and cooled to 4°C for five minutes. First strand cDNA synthesis is accomplished by adding 6 μL of 5× First Strand Buffer, 3 μL 0.1M DTT, 1.2 μL of dNTP mix (50 mM), and 2 μL of SuperScript II reverse transcriptase followed by incubation at 42°C for 2.5 hours. After incubation, the reaction is cooled to 4°C for 2 minutes. Template RNA is hydrolyzed by adding 10 μL of 1N NaOH (Sigma) and 10 μL of 0.5M EDTA (Sigma) followed by incubation at 65°C for 15 minutes. The pH is subsequently neutralized by adding 10 μL of 1N HCl (Sigma). Hydrolyzed RNA and residual dNTPs are removed using the Minelute reaction cleanup kit (Qiagen) according to the manufacturer\'s protocol. Two rounds of elution from the column, each in 10 μL of Buffer EB (Qiagen) are performed to improve recovery. Priming for second strand cDNA synthesis is accomplished by combining the eluted first-strand cDNA with 2 μL of random nonamer primers modified by the addition of a T3 promoter sequence at the 5\' end (T3N9 primer, 100 ng/μL, Table [1](#T1){ref-type="table"})^11^(Operon) followed by incubation at 95°C for 3 minutes. Immediately following this incubation, the samples temperature is decreased as quickly as possible to 50°C followed by a temperature ramp from 50°C to 4°C at the rate of 0.4°C/s (\~120s ramp time) and then a hold at 4°C for 2 minutes. Second strand cDNA synthesis is accomplished by adding 7μL of 5× Second Strand Buffer, 2 μL of dNTP mix (50 mM), 4 μL of E. coliDNA polymerase I (10U/μL), and 1 μL of E. coliDNA ligase (10U/μL) followed by incubation at 16°C for 2 hours. After incubation, blunt-ending is carried out by adding 2 μL of T4 DNA polymerase (5U/μL) and incubating at 16°C for 5 minutes. The reaction is terminated by adding 3.5 μL of 0.5 M EDTA (Sigma). Double stranded cDNA is purified using the Minelute reaction cleanup kit (Qiagen) following the manufacturer\'s protocol. Two rounds of elution with Buffer EB (Qiagen) are carried out to improve yield. The first elution is in 10 μL of Buffer EB, and the second is in 7 μL of Buffer EB. This provides the eluted cDNA in a final volume of 16 μL (corrected for 1 μL of retention on the column), which is the appropriate volume for use in the subsequent IVT reaction. In vitrotranscription is achieved using the T3 MegaScript kit (Ambion). A double reaction is used to improve yield, so 16 μL of NTP mix, 4 μL of 10× Reaction Buffer, and 4 μL of T3 MegaScript Enzyme Mix are added to the sample. The reaction is incubated at 37°C for 14 hours (overnight). The amplified RNA is purified using either the RNEasy Mini or the RNEasy Minelute kit (Qiagen) according to the manufacturer\'s protocol. Superas-In RNAse inhibitor (Ambion) is added to the amplified RNA to a final concentration of 1U/μL according to the manufacturer\'s recommendations. Assessment of products and intermediates ---------------------------------------- cDNA and RNA quantity and purity were assessed by measurement of OD~260/280~and OD~230/260~in the corresponding elution buffers (pH 8.0) using a NanoDrop spectrophotometer. RNA length distribution and integrity were assessed with the Agilent Bioanalyzer 2100 using the Agilent Total RNA Nano chip assay. Agilent\'s Bioanalyzer Expert 2100 software was used to quantify transcript length, and the software\'s automatic integration tool was used to determine the area under the curve. The median transcript length for each method was taken as the point at which half of the integrated area was achieved. Microarray analyses ------------------- Microarray analysis was conducted to determine the magnitude of amplification bias. Pooled human cell line RNA (as described above) as well as RNA extracted from one human glioblastoma were amplified as described. The amplified sense RNA (2 μg) was used as the starting material for synthesis of cDNA target, and a spiking control consisting of RNA transcripts of 10 genes from the Arabidopsis thaliana genome was added to each sample prior to cDNA synthesis in order to provide a consistent positive reference in subsequent hybridizations^28^. cDNA target synthesized from the cell line RNA was indirectly labeled with Cy-5 while target derived from the glioblastoma was labeled with Cy-3. The labeled cDNA from the cell line pool and from the glioblastoma were cohybridized to human 32,000 element spotted cDNA arrays. The array production, cDNA synthesis, target labeling, and hybridization were conducted following TIGR standard operating procedures \[[@B31]\]. Independent replicates were conducted for all amplifications and hybridizations. In order to compare our method to commercially-available RNA amplification kits, microarray analysis was conducted using identical pooled cell line and glioblastoma RNA that was amplified with two commercial products (RiboAmp kit, Arcturus; and BDSmart mRNA Amplification kit, BD Biosciences). Amplification of 4 μg of total RNA was carried out according to the manufacturer\'s protocol for both kits, and 2μg of amplified RNA from each sample was used for microarray analysis as described above. Independent replicates were conducted for all amplifications and hybridizations. Unamplified total RNA from both the pooled cell line and the glioblastoma was used as the starting material for the control arrays. Ten (10) μg of total RNA from each sample was used for cDNA synthesis, and the remainder of the protocol was carried out as described for the amplified samples. Data analysis ------------- Microarray data was analyzed using the TIGR TM4 software package for microarray analysis \[[@B32]\]. TIGR Spotfinder was used to isolate spots on the arrays, correct for background, and assess the reliability of spots for downstream analysis. TIGR MIDAS was then used to adjust the data by applying LOWESS normalization \[[@B33],[@B34]\] followed by standard deviation regularization \[[@B33],[@B35]\]. The Cy5 channel (pooled cell line sample) was taken as the reference for all transformations. Normalized values of fluorescence intensity (FI) were log~2~transformed, and ![](1471-2164-6-27-i3.gif) was calculated for each array element. Assessments of bias were conducted by comparing the log~2~(ratio) values for each element in the amplified sample arrays to their counterparts in the unamplified control arrays. Measurements of reproducibility were conducted by comparing the log~2~(ratio) values of corresponding elements in independent replicates. Statistical methods ------------------- The degree of bias introduced by an amplification method was assessed by plotting ![](1471-2164-6-27-i4.gif) against the ![](1471-2164-6-27-i5.gif) and calculating the correlation coefficient (r) for each independent replicate. The mean correlation coefficient (![](1471-2164-6-27-i2.gif)) was calculated for each method, and differences between ![](1471-2164-6-27-i2.gif) for each amplification method were tested for statistical significance. Welch\'s T-Test \[[@B36]\] was used for all comparisons to control for the possible heteroscedastic nature of the original array data. The degree of reproducibility of our amplification method was assessed by plotting the log~2~(ratio) values for paired hybridizations derived from independent amplifications of the same starting RNA, calculating the correlation coefficient (r) for a series of independent replicates, and computing the mean correlation coefficient (![](1471-2164-6-27-i2.gif)). Reproducibility was further investigated by calculating the variance (σ^2^) and the coefficient of variation ![](1471-2164-6-27-i6.gif) for ![](1471-2164-6-27-i2.gif) (CV), which describe the dispersion of the values contributing to ![](1471-2164-6-27-i2.gif). Authors\' contributions ======================= This method is based on an idea by Norman Lee. John Quackenbush and Nicholas Marko further developed the approach. Marko reduced this to practice in the laboratory, greatly expanding on the method and creating a robust protocol. Bryan Frank contributed significantly to the development, implementation, and optimization of the protocol. Marko conducted the data analysis and was largely responsible for the present manuscript. Acknowledgements ================ The authors wish to thank Nirmal Bhagabati for thoughtful discussion of the manuscript, Tove Andersson and Jennie Larkin for their assistance with the testing of variations of this protocol, and Julie Barber for her technical assistance in the laboratory. NFM is a Howard Hughes Medical Institute Medical Student Fellow and is funded in part by a grant from the Howard Hughes Medical Institute. Additional support for this work was provided by grants to JQ from the US National Cancer Institute and the National Heart, Lung, and Blood Institute.	PubMed Central	2024-06-05T03:55:53.930606	2005-3-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554769/", "journal": "BMC Genomics. 2005 Mar 1; 6:27", "authors": [ { "first": "Nicholas F", "last": "Marko" }, { "first": "Bryan", "last": "Frank" }, { "first": "John", "last": "Quackenbush" }, { "first": "Norman H", "last": "Lee" } ] }
PMC554770	Background ========== Although genetic variation for protein content has been found in forage plants, this variability is narrower than that observed for other traits such as digestibility \[[@B1]\]. Since the major protein components in monocot forage and silage crops are involved in metabolic activity, and hence are not \"true\" storage proteins, it has been argued that it is not feasible to make major changes in protein quality or protein composition by conventional breeding \[[@B1]\]. However, genetic engineering may allow improvement in protein quality and content through expression of a storage protein not found in grass vegetative tissue. Genes encoding seed storage proteins of various plant species have been transgenically expressed to test for improvement of nutritional quality. Most experiments were conducted with tobacco and legume species including alfalfa, soybean, canola, clover and lupins. For nuclear-targeted genes, accumulation of these seed storage proteins in vegetative tissue of transgenic plants was either undetectable or very low. These included pea vicilin \[[@B2],[@B3]\], soybean conglycinin \[[@B4]\], sunflower seed agglutinin \[[@B5],[@B6]\], and phaseolin \[[@B7]\]. The instability of seed proteins in non-seed tissues of transgenic plants was frequently attributed to protein targeting to protease-rich vacuoles in the vegetative cells, and subsequent degradation \[[@B5],[@B7],[@B8]\]. The greatest accumulation of a seed storage protein from a nuclear-targeted gene was achieved using zeins, a maize seed storage protein that is targeted to \"protein bodies\" directly from the endoplasmic reticulum (ER), thus avoiding the secretory route to the cellular vacuole. Transgenic expression from the CaMV 35S promoter in tobacco resulted in the formation of these protein bodies containing the zein within vegetative tissues \[[@B8],[@B9]\]. Alternatively, \"short-circuiting\" the protein-targeting route by addition of an ER retention signal to the storage-protein coding region also increased protein accumulation up to 100× \[[@B5],[@B6],[@B10],[@B11]\]. In many legumes, accumulation of specific vegetative storage proteins (VSPs) in leaves and stems is the main source of increased nitrogen content \[[@B12]-[@B14]\]. Use of VSPs instead of seed storage proteins to increase vegetative protein content in monocots may provide an advantage since they have evolved to function in vegetative cell types. Legume vegetative cells that accumulate VSP proteins contain multiple vacuole types and storage proteins are targeted to specific vacuoles where they are not rapidly degraded \[[@B15]\], and do not interfere with cellular metabolic processes. It is uncertain if monocots can produce similar vacuoles or successfully target a VSP to them. The most studied VSPs are the soybean VSPα and VSPβ proteins, which are lysine-rich glycosylated vacuolar proteins that accumulate abundantly in leaves, stems and pods, but not in seeds \[[@B12]-[@B14]\]. Recently, soybean vspAand/or vspBgenes fused to the 35S promoter were expressed in transgenic tobacco to study their accumulation in a heterologous dicot plant. Nuclear targeted genes produced VSP ranging between 2 and 6% of the soluble protein in leaves of the transgenic plants \[[@B16]\]; whereas, targeting to both the chloroplast and the vacuole within the same plant resulted in VSP comprising greater than 10% soluble protein \[[@B17],[@B18]\]. Soybean VSP is therefore an excellent candidate for use in transgenic improvement of plant protein status, particularly grasses that contain limited levels of lysine \[[@B16]\]. However, it remains to be determined if VSPs can be expressed and accumulated in monocot plants since storage protein stability is dependent on post-translational events that may differ between monocots and eudicots. This manuscript presents the transgenic expression of soybean VSPβ in the leaves of transgenic maize and discusses this expression in relation to the developmental stage of the plant. Results and Discussion ====================== Development of primary (R~0~) transgenic maize expressing vspB ---------------------------------------------------------------- Out of 101 plants, twenty, belonging to five independent lines (71-1, 45-1, 45-3, 44-1, and 4-1) were shown by Southern blot analysis to contain a 1.5 kb hybridizing band corresponding to the intact bargene (Fig. [1](#F1){ref-type="fig"}). All 20 plants also contained the expected 1.9 kb band that hybridized to the vspBgene. The same probe detected two bands in EcoRI restricted Soybean genomic DNA, \~5.7 and 8.6 Kb, corresponding to the highly homologous genes vspAand vspB\[[@B13]\]. Western blot analysis was used to detect the VSPβ polypeptide in leaf extracts from fourteen primary (R~0~) transgenic maize plants at vegetative stage (7 weeks old). A distinct VSPβ band was not visible in silver-stained SDS-PAGE separated maize extract proteins due to complexity of the total protein pattern and the relatively low level of VSPβ expression (Fig. [2a](#F2){ref-type="fig"}). However, some of these plants expressed VSPβ at a level high enough to be detected by Western blot analysis (Fig. [2b](#F2){ref-type="fig"}). Two plants (71-1-53 and 45-3-1) had the highest level of VSPβ, while four plants (71-1-23, 71-1-20, 45-1-4, and 45-1-7) accumulated a lower level of VSPβ. Although a faint lower MW peptide band was visible in negative control maize extracts, VSP was clearly only present in transgenic lines. Faint detection of bands in untransformed maize (none of identical size to soybean VSPβ) is consistent with previous reports of cross-hybridizing of soybean VSP antibodies with proteins from monocots \[[@B14]\]. In soybean extracts used as a positive control, the antibody for VSPβ recognized both VSPα and VSPβ polypeptides (Fig. [2b,c](#F2){ref-type="fig"}) Computer analysis of digital images of the Western blots was used to detect differences in relative band intensity of the immunologically detected VSPβ peptide. Because the native soybean VSPs (VSPα plus VSPβ) were easily visible on total protein stained gels (Fig. [2a](#F2){ref-type="fig"}), relative quantification of total stained proteins in the soybean samples indicated that the VSPs represented about 10% of the total soluble protein in young soybean leaves. This is close to the previously reported value of 15 % \[[@B12]\]. The intensity of the transgenic maize 45-3-1 VSPβ band on Western blots was 44% of the soybean VSP\'s band (digital image pixel quantification of Fig. [2b](#F2){ref-type="fig"}). Accounting for the differences in total protein applied to the gel (less soybean total protein was loaded), the VSPβ protein was estimated to have accumulated to 0.5 % of the total soluble protein. This is similar to the highest level of seed storage protein accumulation observed with the ectopic expression of zein \[[@B8],[@B9]\], but remains less than the 1% minimal expression level predicted by Wandelt et al. \[[@B11]\] to be needed to directly alter the nutritional quality of the leaves. Although the 0.5% of total soluble protein was too low to alter nutritional value, detection of VSPβ in 45-3-1 allowed monitoring of VSPβ level in leaves and stems during plant development. Presence of vspBin R~1~plants ------------------------------- R~1~plants were produced by back-crossing the R~0~plants with Hi II control non-transformed pollen. Back-crossing was performed because R~0~plants directly regenerated from tissue culture did not have synchronized production of pollen and receptive female flowers. The R~1~families segregating for barexpression were analyzed for the presence of the vspBgene by Southern blot analysis. From 57 R~1~plants analyzed, Southern blot analysis showed that 35 (61%) contained the vspBgene integrated into the genome. Figure [3](#F3){ref-type="fig"} shows examples for several R~1~from five R~0~parental lines. The \~1.9 kb vspBband can be seen in nine of the 16 R~1~lines. There are also fainter bands one slightly larger than the 1.9 kb band and one or two migrating between 4 and 5 kb. These are often observed as incomplete restriction of all Eco RIsites internal to plasmid DNA that is integrated into the maize genome. Similar bands are observed even with the plasmid control. Total RNA was isolated from leaves of young R~1~plants at the vegetative stage (7 weeks after planting). Eighteen transgenic R~1~plants, including the ones originating from R~0~plants representing different levels of VSPβ accumulation (high VSPβ accumulators: 45-3-1, 71-1-53; mid-level VSPβ accumulators: and 71-1-23, 71-1-20, 45-1-4, 45-1-7 and low VSPβ accumulators: 44-1-1- 4-1-2), were analyzed for vspBtranscripts. Because of high sequence similarity (85%) between vspAand vspBcDNAs, the same probe hybridized in soybean with both mRNAs as demonstrated by Staswick \[[@B13]\]. The transgenic plants 71-1-53A, 71-1-20A, 71-1-20E, and 71-1-20I produced a hybridizing band of approximately the expected size of 1.1 kb indicating transgene expression (Fig. [4a](#F4){ref-type="fig"}); however, most did not. Use of the Ubi-1 promoter for both the vspBand bargene probably led to a high number of transgenics with the vspBsilenced, and a higher level of VSPβ accumulation will likely result in future work using a combination of different promoters. Immunodetection of VSPβ protein in transgenic young plant leaves showed variation in accumulation in comparisons between the parental (R~0~) and their R~1~progeny, with the greatest variation observed with the highest VSPβ expressing R~0~plants (Fig [4b](#F4){ref-type="fig"}). The parental line 71-1-53 had a relatively high level of expression of VSPβ, but the only transgenic offspring from this line, 71-1-53A, had almost undetectable VSPβ; in contrast, a low level accumulator, 71-1-20, produced R~1~lines expressing different levels of VSPβ, although none of them expressed at higher levels than the parent. Quantification of the relative level of VSPβ expressed in the R~1~leaves showed that the highest level measured was only 0.03% of total soluble protein. Despite the overall low level of VSPβ expression, for the purposes of this work, VSPβ accumulation in several of the plants was high enough to study the relationship of plant developmental stage and vspB/VSPβ accumulation. Both the transcript abundance and VSPβ protein accumulation were determined in the R~1~lines using real-time quantitative RT-PCR and Western blot immunodetection, respectively. Real-time RT-PCR is more sensitive that Northern blot analysis and was able to detect transcripts that were not seen with standard total RNA blotting methods. The vspBtranscript was quantified in leaves from immature plants (prior to tassel formation) and silage stage plants (plants with developing seeds at the 18 DAP-days after pollination stage), as well as, stems from the silage stage plants, (Figure [5](#F5){ref-type="fig"}). The vspBtranscript was detected in all five transgenics (four of which had RNA not detectable using standard Northern blot methods). The relative level of RNA among the different plant samples was not consistent across the different lines with some having more transcripts in the young leaves while others had more in the older leaves and stems. When vspBRNA abundance was compared to VSPβ protein accumulation there was no correlation between the two (Fig. [5a, b, c](#F5){ref-type="fig"}). Antiserum to VSPβ detected VSPβ only in leaves from young maize plants that had not yet flowered. No VSPβ was detected in leaves and stems of silage stage corn that had developing seed. The soybean VSPβ peptide was the primary band reacting with the antiVSPβ antiserum in young leaves of transgenic maize, however, in silage stage stems and to a lesser extent the silage stage leaves, there were multiple bands detected at a different size than the VSPβ. These were also detected in the non-transgenic control plant samples and therefore, were not due to different modifications of the soybean VSPβ. Although the VSPβ protein dropped below detectable levels in the silage stage 71-1-20A, 45-3-1F and 45-3-1-G plants, the vspβtranscript was still detectable. In fact, in the 71-1-20A plants the transcript level was highest in the silage stage leaves that had no detectable VSPβ. Therefore, post-transcriptional events (i.e., changes in either the translational efficiency of the vspBtranscript or the protein stability of VSPβ) were altered in the silage stage leaves and stems as compared to the leaves of immature plants. Conclusion ========== The vspBgene was successfully introduced into R~0~regenerated maize and transferred to the R~1~progeny, of which vspBtranscript and VSPβ protein were detected and studied. This is the first report on introduction and expression of a legume vegetative storage protein in a monocot plant. The inability to detect VSPβ from the maize vegetative tissue at the time of seed development, even when the vspBtranscript was still expressed, must have arisen from either reduced translational efficiency of the vspBtranscript or a decrease in the stability of the VSPβ protein. The reduction of seed storage protein level in leaves of transgenic eudicots was also observed with expression of vicillin in alfalfa \[[@B11]\] and tobacco \[[@B2]\]; however, it was not observed with the expression of VSPα in tobacco \[[@B16]\] or ovalbumin in alfalfa \[[@B30]\]. These data suggest that factors controlling developmental change in vegetative tissue protein accumulation are a combination of host plant traits and innate characteristics of the ectopically expressed protein. It is interesting to speculate that if, in maize (a monocot), soybean VSPβ was degraded in a manner that provided amino acids that were translocated to the seed to support seed development, then development of high level VSP expressing monocots may be a way to improve nitrogen content of the seed/grain produced by the plant. Methods ======= Plant material and tissue culture methods ----------------------------------------- Plants of hybrid \"Hi-II\" maize were established in a greenhouse and immature tassels were used for embryogenic type II callus production, as described by Armstrong \[[@B19]\]. Transgenic plant development ---------------------------- Microprojectile bombardment of callus was performed using the procedure of Somers et al. \[[@B20]\]. Calli were cobombarded with equal amounts of pRSVP-1 (Shatters Jr., unpublished) and pAHC25 \[[@B21]\]. Plasmid pRSVP-1 was constructed by restricting the soybean vspBcDNA clone (998 bp) from pKSH3 \[[@B22]\] with EcoRI, blunt ends of this fragment were produced using S1 nuclease and the fragment was cloned into similarly blunt ended BamHI restricted pAHC17 \[[@B21]\]. As a result, the vspBcoding region was inserted downstream of the Ubi-1promoter and a 5\' untranslated region (exon) and intron; and upstream of the nosterminator sequence. The plasmid pAHC25 carried the bargene and the uidAreporter gene, both under the control of the Ubi-1promoter. Bombardments were performed with a Biolistic^®^PDS-1000/He Particle Delivery System (Bio-Rad Laboratories, Hercules, CA) and an osmotic treatment was applied to reduce the cell damage caused by the gene transfer method \[[@B23]\]. Putative transgenic maize were regenerated from glufosinate resistant callus as described by Armstrong \[[@B19]\], and grown in five-gallon pots containing sterile sand and Metromix-350 (1:1). Plants were fertilized weekly with Peter\'s 20-20-20 with micronutrients (Division of United Industry Corp., St. Louis, MO). Southern blot analysis ---------------------- One gram of frozen young leaf tissue was ground in liquid nitrogen and genomic DNA was extracted using the Dellaporta procedure \[[@B24]\]. Twenty micrograms of genomic DNA were digested with EcoRI, which released a 1.9 Kb fragment containing the vspBgene, the nosterminator and part of the Ubi-1promoter. DNA was separated on a 0.8% agarose gel, blotted onto Hybond N^+^membrane (Amersham Pharmacia Biotech, Inc. Piscataway, NJ) by capillary blotting \[[@B25]\], and UV cross-linked. The non-radioactive digoxigenin system (Roche Molecular Biochemicals, Indianapolis, IN) was used for labeling and detection of the transgene. Blotted DNA was probed with either a 611 or a 843 bp of vspBgene segment amplified from pRSVP-1 and gel purified. The forward and reverse primers 5\'-GTTCTTCGGAG GTAAAAT-3\' and 5\'-TTCGCCTCTGTGGT-3\' were used, respectively, to amplify a 611 bp segment, and the primer pair 5\'-GCAGGCTACCAAAGGT-3\' and 5\'-TAGGTGACTTACCCACAT-3\' was used to amplified the product of 843 bp. For identification of bartransgenic plants, the DNA was digested with EcoRI, which released a fragment of \~1.5 Kb that contained part of the Ubi-1promoter, the bargene, and the nosterminator, and was identified with a 419 bp digoxigenin labeled probe produced by PCR amplification of pAHC25 using the forward and reverse primers: 5\'-GGCGGTCTGCACCATCGT-3\' and 5\'-GCCAAGTTCCCGTGCTTGA-3\', respectively. Northern blot analysis ---------------------- Total RNA was isolated from 2 g of tissue using acid guanidine isothiocyanate-phenol-chlorophorm extraction \[[@B26]\], resuspended in T~10~E~1~and treated with 2 μl RNasin^®^(4U/μl) RNAse inhibitor (Promega, Madison, WI) and stored at -70°C until use. Thirty micrograms of total RNA were separated on 1.2% agarose formaldehyde gels and transferred to Hybond N^+^membrane by capillary blotting overnight or by pressure blotting for one hour using a PosiBlot^®^30-30 pressure blotter (Stratagene, La Jolla, CA) according to the manufacture\'s instructions. Membranes were UV-crosslinked, and probed with the same probes as described for Southern blot analysis. Chemilluminescence was captured using a Kodak Digital Science Image Station 440 CF (Eastman Kodak Company, Rochester, NY). Real-Time RT-PCR ---------------- Total RNA extractions for real-time RT-PCR were performed using 500 mg of tissue ground to fine powder using mortar and pestle in the presence of liquid nitrogen, then processed with the RNeasy midiprep Kit (Qiagen, Germany), following the manufacturer\'s protocol. Trace DNA contamination was removed from total RNA by a combination of acid phenol: chloroform 5:1 pH= 4.7 extraction and Dnase I treatment (Ambion, Texas). Real-time RT-PCR was performed on a Rotor-Gene RG-3000 (Corbett Research, Australia) using the Quantitect SYBR Green real-time RT-PCR kit (Qiagen, Germany), and the manufacturers protocols with 300 ng of Dnase I treated total RNA. Primers were designed to amplify a 108 bp fragment of the soybean vspBusing the following primers: 5\'-TGGTTCAACGCACTCTTC-3\' and 5\'-GGCTATGGTGAGCGTTCTTC-3\'. Reverse transcription was performed for 30 min at 50°C followed by a 15 min denaturing at 95°C, and 40 cycles of 40 s at 95°C, 40 s at 58°C and 40 s at 72°C. Quantification was based on relative abundance to maize 18S RNA by amplifying a 174 bp fragment with primers: 5\'-CCTGCGGCTTAATTGACTC-3\' and 5\'-GTTAGCAGGCTGAGGTCTCG-3\', and using the comparative quantification function of the Rotor-Gene RG-3000 software. All real-time RT-PCR experiments were conducted in triplicate and on triplicate RNA preparations for each sample. Melting curve analysis and agarose gel electrophoresis were performed to verify single product formation. Western blot analysis --------------------- Protein was extracted from 100 mg of leaves and stems with 0.5 ml of phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na~2~HPO~4~, 2 mM KH~2~PO~4~) supplemented with 1 tablet/10 ml buffer of the Complete-Mini protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN) by homogenizing in the presence of zirconia/silica 0.1 mm dia. beads. Centrifuged extract supernatants were removed and used for protein concentration determination \[[@B27]\]. Because of the low protein yields in stem extracts, they were precipitated with 10% trichloroacetic acid (TCA), washed with ice-cold acetone and resuspended prior to SDS-PAGE analysis. Thirty micrograms of protein were separated on 12% SDS-polyacrylamide gels. Proteins in the gel were either stained with silver nitrate \[[@B28]\] or transferred to Hybond-P (PVDF) membranes (Amersham Pharmacia Biotech, Inc. Piscataway, NJ) using Trans-Blot SD Semi-Dry Transfer Cell blotter and recommended protocols (Bio-Rad Laboratories). Soybean VSP was immunologically detected on the PDF membranes with anti VSPβ serum (provided by P. Staswick, University of Nebraska, produced as previously described \[[@B29]\] used at a 1:5,000 dilution. Detection was performed using a luminol substrate and the NEN Life Science Products, Inc. (Boston, MA) Reinascence kit. Chemiluminescent signal was captured by a Kodak Digital Science Image Station 440 Cf, and analyzed with Kodak 1D Scientific Image Software. Quantification of band intensity was always compared relative to samples from the same gel. List of abbreviations ===================== RT-PCR, reverse transcriptase-polymerase chain reaction; VSP, vegetative storage protein Authors\' contributions ======================= MFG participated in experimental design, carried out the transgenic plant development, plant crosses, and molecular blotting/detection methods, and participated in manuscript draft preparation. RLS participated in experimental design, and provided guidance and training in development of transgenic maize. CM performed RT-PCR experiments. BTS participated in experimental design and provided expertise and training in plant crosses. RGS conceived of the study, participated in experimental design, coordinated the experimental plan, and wrote the draft manuscript. All authors read and approved the final manuscript. Acknowledgements ================ This work was supported, in part, by grants from the Florida Dairy Research Council. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Southern blot analysis of primary (R~0~) maize transformed with pAHC25 and pRSVP-1. Twenty μgs of EcoRI restricted genomics DNA were electrophoresed through 0.8% agarose and blotted to Hybond membrane. The membranes were probed with either digoxigenin labeled bar(a) or vspB(b). Numbers and lines on sides of blots indicate location of molecular size markers (the number represents size in kilobases). The \"a\" and \"b\" sections are aligned so that the same genomic DNA samples are vertically aligned and represented by the same lane label. Plasmid lane is the plasmid containing the either the baror the vspβclones used as a positive control. Untransformed controls are lanes containing genomic DNA from untransformed Hi-II maize. Soybean indicates lanes containing restricted soybean genomic DNA. The two panels within each section represent separate blots hybridized with the same probe. ::: ![](1471-2229-5-3-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Western blot detection of VSPβ in primary (R~0~) transgenic maize. Thirty micrograms of protein from each sample were separated by SDS-PAGE. (a) Silver-stained 12% SDS-PAGE polypeptide profile for six of the 14 analyzed R~0~plants. (b) Immunodetection of VSP protein in SDS-PAGE separated transgenic R~0~maize extracts transferred to Hybond-P membranes and immunodetected using VSPβ antiserum and the Reinascence kit chemiluminescent detection method (NEN Life Sciences Products, Inc). (c) Underexposed western blot showing two distinct bands corresponding to VSPα and VSPβ polypeptides in soybean leaves. ::: ![](1471-2229-5-3-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Southern blot detection of vspBin R~1~transgenic maize. Twenty micrograms of EcoRI restricted genomics DNA was electrophoresed through 0.8% agarose and blotted to Hybond membrane. The membrane was probed with digoxigenin labeled vspB. The alphabetical labels of each R~1~family represent individual R~1~plants. Control sample represent genomic DNA from untransformed maize Hi-II. Two positive control samples are also included: plasmid, pRSVP-1 restriction digested to liberate the vspBcDNA clone (only partial digestion occurred as evidenced by the two bands at higher molecular weight than the vspBfragment), and genomic DNA from the 45-1-2 R~0~plant, previously shown to contain the vspBsequence. The Eco RIrestricted soybean genomic DNA is also included as a positive control for hybridization of the soybean vspBprobe. ::: ![](1471-2229-5-3-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### $a) Northern blot detection of vspBtranscripts in R~1~transgenic maize. Thirty micrograms of total RNA were separated on 1.2% agarose formaldehyde gels and blotted to Hybond N^+^membranes. The vspBtranscripts were detected by hybridized with a digoxigenin labeled vspBprobe. (b) Western blot detection of VSP in R~0~and their progeny (R~1~) transgenic maize. Thirty micrograms of protein in extracts from leaves of 7 weeks old plants were separated on 12% SDS-PAGE, blotted onto Hybond-P membrane, and VSP was immunodetected using VSPβ antiserum and the Renascence kit chemiluminescent detection method (NEN Life Sciences Products, Inc). ::: ![](1471-2229-5-3-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### a$ Real-time RT-PCR quantification of vspBtranscripts in R~1~transgenic maize tissue. Two hundred micrograms of total RNA from the indicated tissue was used as the template source for real-time RT-PCR detection of vspBtranscripts. Reactions were performed in 15 uL volume using the Qiagen Quantitect SYBR Green kit. Expression values are calculated by normalizing all threshold cycles (C~t~) for vspBto the 18S rRNA C~t~and converting this value to fold-increase over the value for the lowest expressing tissue, \*44-1-1-stem, which was arbitrarily set at 1.0. b,c) Developmental changes in VSPβ level in transgenic maize vegetative tissues. Soybean VSPβ was immunodetected in Western blots of 30 μgs of total protein from crude extracts separated by 12% SDS-PAGE and blotted to Hybond-P membranes. Crude Extracts were prepared from: YL, leaves from preflowering plants; SL, leaves from silage stage plants; and SS, stems from silage stage plants. (b) Coomassie blue stained SDS-PAGE separated proteins from crude extracts; (c) VSP was immunodetected using VSPβ antiserum and the Renascence kit chemiluminescent detection method (NEN Life Sciences Products, Inc). The arrows indicate the VSP-β protein band. ::: ![](1471-2229-5-3-5) :::	PubMed Central	2024-06-05T03:55:53.934181	2005-3-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554770/", "journal": "BMC Plant Biol. 2005 Mar 2; 5:3", "authors": [ { "first": "Magali F", "last": "Grando" }, { "first": "Rex L", "last": "Smith" }, { "first": "Cristina", "last": "Moreira" }, { "first": "Brian T", "last": "Scully" }, { "first": "Robert G", "last": "Shatters" } ] }
PMC554771	Background ========== Acute infection of the amniotic fluid and chorioamnion is a common cause of preterm delivery before 34 weeks gestation. Consistent with previous reports \[[@B1]\], an Australian study found that 31% of preterm births (PTB) before 34 weeks had histologic chorioamnionitis \[[@B2]\]. An inverse relationship between delivery gestation and histologic chorioamnionitis was shown with histologic chorioamnionitis identified in 66% of births before 25 weeks. Chorioamnionitis usually results from ascent of pathogenic organisms such as Group B streptococcus and Escherichia coli\[[@B3]\], from the lower genital tract. A T helper cell type1 (Th1) cytokine host response occurs with the production of pro-inflammatory cytokines such as (IL-1β and TNF) produced mainly by activated monocytes/macrophages in amnion, chorionic and decidual tissue. The increased levels of these cytokines trigger prostaglandin F2 and E2 biosynthesis by the decidua and amnion, inducing uterine contractions and labour \[[@B4]\]. In the setting of infection, IL-1 and TNF have a synergistic effect on the acute phase cytokine IL-6 with increased production in trophoblasts and chorion and to a lesser extent decidua \[[@B5]\]. The intensity and duration of the inflammatory response is modulated by T helper cell type 2 cytokines and anti-inflammatory mediators such as IL-4, IL-10, IL-1 receptor antagonist (IL-1ra) and transforming growth factor beta-1 (TGFβ-1). The innate host defense system is also an important factor in pregnancy and the body\'s response to infection. Mannose-binding lectin (MBL), a protein of the complement system, opsonises pathogens independently of antibody for phagocytosis and influences inflammatory pathways \[[@B6]\]. There are several common polymorphisms in the promoter and coding region of the MBL2gene that profoundly influence circulating levels of functional, multimeric MBL. These genetic variants and low MBL levels are associated with risk and severity of infection in a variety of clinical contexts \[[@B6]\]. Fas, a cell surface receptor of the TNF/nerve growth factor superfamily, mediates apoptosis after binding Fas ligand \[[@B7]\]. Activated leucocyte populations, including T cells, express high amounts of Fas and evidence suggests that trophoblast Fas-mediated apoptosis is increased in women with chorioamnionitis \[[@B8]\]. The immune system operates as a complex regulatory network of cytokines and other mediators with a degree of redundancy that changes as pregnancy progresses \[[@B9]\]. This makes it difficult to assess the importance of single cytokines in inflammation and response to infectious agents when examined in isolation. The genes encoding these mediators contain coding and non-coding polymorphisms that influence level or function of the encoded mediators \[[@B10]\]. These polymorphisms may influence susceptibility to chorioamnionitis and adverse pregnancy outcome. Single nucleotide polymorphisms (SNPs) for IL1+3953 and TNF-308 are associated with human chorioamnionitis in in vitrostudies \[[@B11]\] and carriage of the TNF-308 A allele has been reported as a risk factor for clinical chorioamnionitis in pregnant women \[[@B12]\]. However these reports were restricted to small numbers of cytokine genes and did not include the analysis of polymorphisms in molecules important in apoptosis and host defence. Examination of a range of immunoregulators and SNPs concurrently is more likely to identify which genes and alleles are of primary importance in response to intra-uterine infection. We hypothesized that polymorphisms in immunoregulatory genes may influence susceptibility to chorioamnionitis and subsequent preterm labour. To examine this hypothesis, we investigated multiple polymorphisms in immunoregulatory genes in a population of Australian Caucasoid women experiencing spontaneous PTB before 35 weeks and histologic chorioamnionitis. Methods ======= This case-control study was approved by the Women\'s and Children\'s Hospital Ethics and Research Committee. A total of 368 unrelated women of child-bearing age who had obstetric management at the Women\'s and Children\'s Hospital (WCH) and history of PTB before 35 weeks gestation were identified from the hospital data base. All women were approached in the first instant by letter of introduction and invited to participate in the study. For women living in the Adelaide metropolitan area, the study nurse made a home visit to those who volunteered for eligibility assessment. Geographical distance excluded 50, 66 did not respond, 40 were not eligible and 31 declined. Of the women enrolled some had more than one PTB. The PTB fitting the study criteria and closest to the enrolment date became the index birth. Written consent was obtained and a 9 ml peripheral blood sample was collected for isolation of leucocytes and DNA extraction. 181 women with a history of spontaneous preterm labour and subsequent PTB between 20 and 35 weeks in the index pregnancy had histological examination of the placenta performed. Histopathology staff members were unaware of the genetic results. Clinical data recorded included gestational age at delivery (calculated by ultrasonography at approximately 18 weeks). Obstetric factors known to place a woman at risk of preterm labour or preterm prelabour rupture of membranes (PPROM) (defined as prelabour rupture of membranes 3 or more hours before preterm delivery) were also recorded. Placental examination and swabbing was performed by staff histopathologists \[[@B13]\] and included examination of the placental tissue in blocks, extra-placental membranes in a \"roll\" (amnion, chorion and decidua) and the umbilical cord in cross section. Chorioamnionitis was defined as a dense polymorphonuclear leukocyte infiltration of the chorionamniotic component of the placenta and membranes (amnion, chorion, amniochorial membranes and/or chorionic plate (top part of the fetal side of the placenta). Inflammatory reaction confined to either the decidua or subchorial intervillous space of the placental disc, without infiltration of the chorion or amnion was recorded as negative chorioamnionitis \[[@B3]\]. Funisitis was defined as inflammation in one or more of the umbilical cord vessels (vasculitis) with or without inflammation in the Wharton\'s jelly (the supporting soft tissue around the vessel in the cord). The definition of acute chorioamnionitis included funisitis and/or amnionitis. In both groups a number of women had PPROM, occasional bleeding during pregnancy, recorded clinical chorioamnionitis or a history of prior term births. Tocolytic therapy and intravenous antibiotics were used according to obstetric protocols. All participants were Caucasoid, unrelated and at least 18 years of age at enrolment. Women with diagnosed autoimmune disease, pre-eclampsia, in vitro fertilisation treatment, confirmed uterine malformations, no spontaneous labour, ultrasound-confirmed fetal abnormality, insulin dependent diabetes, or multiple pregnancy were excluded. Demographic characteristics, a comprehensive medical and obstetric history, age, any smoking, alcohol consumption, substance use and clinical chorioamnionitis (defined as any three of the following -- white blood cell \>1500 × 10\^9/L, three consecutive C reactive protein readings ≥ 15mg/L, pyrexia ≥ 38°C, uterine pain or tenderness) were verified from case records. Clinical chorioamnionitis was not used as a subgroup for polymorphic analysis because this did not include women with sub-clinical infection. Since the risk of chorioamnionitis is not as great in PTB after 35 weeks, women with PTB between 35 and 37 weeks were not enrolled \[[@B2]\]. Genes were chosen if reported to be associated with inflammatory disease\[[@B10]\] and if existing functional data suggested a role in chorioamnionitis and preterm delivery\[[@B5],[@B11]\]. SNPs were selected where the minor allele has a frequency \>10%. Several genes were known to contain polymorphisms at multiple sites that are of functional importance (e.g. the 5\'and coding variants of MBL2) \[[@B14]\], thus multiple variants were genotyped and the haplotype recorded. SNPs in the IL1 gene cluster, TNF, IL4, IL6, IL10, TGFB1, TNFRSF6, and MBL2genes and their relationship with histologic chorioamnionitis were therefore examined in a cohort of women with spontaneous PTB. Peripheral blood samples were blinded and tested at the Australian Red Cross Blood Service, Adelaide (ARCBS). Genomic DNA was extracted from ethylenediaminetetraacetic acid anticoagulated venous blood using standard methods as previously described \[[@B15]\]. SNPs for TNF(+488,-238,-308) \[[@B16]\], IL1A(-889), IL1B(+3962, -511), IL4(-590), IL10(-1082, -819, -592), TGFB1(-800, -509) \[[@B17]\], MBL2\[-550, -221, codon 52 (Arg → Cys), codon 54 (Gly → Asp), codon 57 (Gly → Glu)\] \[[@B18]\], IL6(-174), IL-1 receptor antagonist IL1RN(+11100), IL-1 type 1 receptor IL1R1(+970) and TNFRSF6(-1377, -670) \[[@B19]\] were genotyped using the polymerase chain reaction and sequence specific primers (PCR-SSP). Multiple polymorphisms were examined in the IL10, TNFRSF6, TNF, TGFB1and MBL2genes by PCR haplotyping. The term \"haplotype\" denotes ordered combination of alleles on a single chromosome, and is used where multiple polymorphisms are genotyped by PCR-haplotyping for a single gene. For PCR haplotyping combinations of forward and reverse allele-specific-primers were used to directly amplify alleles on the same chromosome \[[@B20]\]. This technique also provides a degree of redundancy, in that multiple reactions genotype a polymorphism, thus confirming results. All PCR-SSP reactions have been extensively used in the same laboratory. Internal control primers for conserved regions of the DRB1(major histocompatibility complex, class 11 DR beta 1) and APC(adenomatous polyposis coli) genes were included in every PCR mix to verify successful amplification. All genes except the IL1Ahad a repeat genotyping rate of less than 1%. The IL1Ahad a repeat rate of 10%. The sample size was calculated by using the statistical software package EpiInfo 6 (Version 6.04d, Centre for Disease Control and Prevention, Atlanta, GA). We estimated the study had sufficient power (1-β = 80%, P = 0.05) to detect a minimum of 17% difference in genotype frequency between chorioamnionitis and no chorioamnionitis for frequencies of approximately 10% (ie. 10% versus 27%). For a genotype frequency of 3% the minimum detectable difference was 10% (3% versus 13%). Allele and haplotype frequencies for polymorphisms were determined by direct counting from each chromosome. Frequencies of genetic variants were recorded as allele (proportion of positive chromosomes), genotype (proportion possessing homozygous or heterozygous combinations of alleles), and \'carriage\' of an allele or haplotype (either in homozygous or heterozygous state). Alleles were said to be in Hardy-Weinberg equilibrium if the observed genotype frequencies did not differ significantly (P \> 0.05) from those expected when analysed by Chi square. Gene variant frequencies were compared using 2 × 2 contingency table analysis and the Chi square test for independence using EpiInfo 6. Mean birth weights were compared by t-test. Multivariable analysis was performed by stepwise backwards multiple logistic regression using SYSTAT 9.0 software (Systat version 1999 SPSS Inc). The basic model included genetic variables with P values \< 0.15 obtained in univariate analyses. In each multivariable model, only one genotype or haplotype of each gene was selected for analysis. Selection of genotype/haplotype for the model was based on strength of association, the previously reported importance of a gene variant and homozygosity of alleles. At each backward step in the regression analysis P values ≥ 0.07 were rejected. Kaplan-Meier survival analysis (SYSTAT 9.0) was used to identify cumulative probability in maintaining pregnancy by gestation stratified by chorioamnionitis and gene variables. Breslow-Gehan Chi square was used to weight each gestational period by the total number at risk at that time so that earlier gestations receive greater weight than later gestations. Results ======= Sixty-nine (38%) women had histologic evidence of acute chorioamnionitis, and 112 (62%) had no such evidence. There were no significant differences between those with and without histologic chorioamnionitis for maternal age, race, history of smoking, alcohol consumption, substance use, new partner, cervical cerclage, placenta praevia after 22 weeks with bleeding, history of two or more PTBs before 35 weeks, miscarriage between 12 and 20 weeks or PPROM (Table [1](#T1){ref-type="table"}). Histologic chorioamnionitis was positively associated with clinical chorioamnionitis \[Univariate Odds Ratio (OR) 6.1, 95% Confidence Intervals (CI) 2.4--17, uncorrected P ≤ 0.0001\], PTB before 29 weeks (OR 7.6, CI 3.4--18, P ≤ 0.0001) and positive placental culture (OR 3.0, CI 0.9--11, P = 0.04). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Demographic and clinical characteristics of women with preterm birth before 35 weeks. ::: Variable Histologic Chorio-amnionitis n = 69 No Histologic Chorio-amnionitis n = 112 Significant OR (95% CI), and Uncorrected P values ----------------------------------------------- ----------------------------------------- --------------------------------------------- ------------------------------------------------------- Maternal age (years) 29 (16--41) 30 (17--44) Delivery gestation (weeks) 28 (20--34) 30 (23--34) \* Mean birth weight 1399 (± 676) 1897 (± 504) \< 0.0001 Maternal age ≥ 35 years 11 (16%) 18 (16%) ns Maternal age ≤ 17 years 2 (3%) 2 (2%) ns Any smoking 22 (31%) 29 (26%) ns Any alcohol 44 (64%) 73 (65%) ns Any substance use 7 (10%) 10 (9%) ns Partner change for 14 (20%) 20 (18%) ns pregnancy Cervical cerclage 3 (4%) 2 (2%) ns Previous mid-trimester 4 (6%) 12 (11%) ns miscarriage (12--20 weeks) 2 or more preterm births 11 (16%) 20 (18%) ns Placenta praevia after 22 weeks with bleeding 1 (1%) 2 (2%) ns Documented clinical chorioamnionitis 22 (32%) 8 (7%) 6.1 (2.4--17), \< 0.001 PPROM ≥ 3 hours 39 (57%) 74 (66%) ns Gestation \< 29 weeks 33 (48%) 12 (11%) 7.6 (3.4--18), \< 0.001 Documented positive placental culture 10 (14%) 6 (5%) 3.0 (0.9--11), 0.04 Neonatal sepsis 10 (14%) 0 19 (3 -- 823), \< 0.001 Neonatal death 6 (9%) 3 (3%) ns Post partum endometritis 5 (7%) 3 (3%) ns Descriptive statistical results are mean (± SD) or number (%) as appropriate, and maternal age and gestation as median (range). Characteristics are from index birth. PPROM = Preterm premature rupture of membranes 3 hours or more before delivery \< 35 weeks. Univariate analyses were tested using Chi square analysis of 2 × 2 table, and uncorrected P values, Odds Ratio (OR), and 95% Confidence Intervals (CI), P values ≤ 0.05 are presented. Mean birth weights were tested by two-sample t-test, assuming unequal variances. \* See Figure 1, ns, not significant. ::: All alleles were in Hardy-Weinberg equilibrium. All gene variants were tested for associations between the two groups. In preterm women without histologic chorioamnionitis frequencies for the IL10-1082A/-819T/-592A (ATA) haplotype \[[@B17]\], MBL2codon 54Asp (the MBL \'B\' allele) \[[@B21]\], TNFRSF6-1377A/-670G (AG) haplotype \[[@B22]\] and TGFB1-800G/-509T (GT) homozygosity \[[@B17]\] were similar to reported Caucasian controls. In a comparison of women with chorioamnionitis and those without chorioamnionitis a comprehensive listing of gene carriage, alleles, genotype and haplotype frequencies is provided ([Additional file 1](#S1){ref-type="supplementary-material"}). Univariate analysis revealed that carriage of the IL10-1082A/-819T/-592A (ATA) haplotype (present in 49% of women with chorioamnionitis vs 33% without chorioamnionitis, OR 2.0, P = 0.03) and the IL10-819T and -592A alleles (50% versus 33%, OR 2.0, P = 0.03) were positively associated with histologic chorioamnionitis, as was carriage of the MBL254Asp (B allele) (39% versus 25%, OR 1.9, P = 0.04) (Table [2](#T2){ref-type="table"}). Although the frequency of the common TNFRSF6-1377 G/G genotype was higher in women with histologic chorioamnionitis (0.87 versus 0.73, OR 2.4, P = 0.03), the variant TNFRSF6-1377A/-670G (AG) haplotype (OR 0.4, P = 0.03) and homozygosity for the TGFB1haplotype -800G/-509T (GT) (OR 0.2, P = 0.03) were negatively associated with chorioamnionitis (Table [2](#T2){ref-type="table"}). No other significant associations with gene variant frequencies were seen in univariate analysis. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Genes, alleles, haplotypes and genotypes in women with preterm birth before 35 weeks and chorioamnionitis. ::: Gene Histologic Chorio-amnionitis n = 69 No Histologic Chorio-amnionitis n = 112 Univariate analysis OR (95% CI) Uncorrected P value Multivariable analysis OR (95% CI), P value --------------------- ----------------------------------------- --------------------------------------------- --------------------------------------------------------- ------------------------------------------------- *IL10* Allele-819 T 34 (0.50) 37 (0.33) 2.0 (1.0--3.8), 0.03 -592 A 34 (0.50) 37 (0.33) 2.0 (1.0--3.8), 0.03 Haplotype -1082A/-819T/-592A 34 (0.49) 37 (0.33) 2.0 (1.0--3.8), 0.03 1.8 (1.0--4.0), 0.05 Genotype -819 C/T 28 (0.41) 28 (0.25) 2.1 (1.0--4-1), 0.03 -592 A/C 28 (0.41) 28 (0.25) 2.1 (1.0--4-1), 0.03 *MBL2* Codon 54Asp (\'B\') 27 (0.39) 28 (0.25) 1.9 (1.0--3.9), 0.04 2.0 (1.0--4.0), 0.04 *TNFRSF6* Allele-1377A 9 (0.13) 30 (0.27) 0.4 (0.2--1.0), 0.03 Haplotype -1377A/-670G 9 (0.13) 30 (0.27) 0.4 (0.2--1.0), 0.03 0.3 (0.2--0.9), 0.03 Genotype -1377 G/G 60 (0.87) 82 (0.73) 2.4 (1.0--6.3), 0.03 *TGFB1* Haplotype -800G/-509T 2 (0.03) 14 (0.13) 0.2 (0.02--1.0), 0 .03 0.2 (0.04--0.9), 0.04 Homozygosity Data are presented as number and frequencies of alleles, genotypes and haplotypes. Univariate analyses were tested using Chi square analysis of 2 × 2 table, and uncorrected P values, Odds Ratio (OR), and 95% Confidence Intervals (CI), P values ≤ 0.07 are presented. Multivariable analyses included genetic variables with P values ≤ 0.15 using backward step multiple logistic regression. Multivariable model included carriage of IL10 ATA, MBL2codon 54Asp, TNFRS6-1377A/-670G (AG) haplotype, and homozygous TGFB1-800G/-509T, TNF+488 G/A, and IL1R1+970 T/T genotypes. MBL2codon 54Asp (allele \'B\') is a single nucleotide polymorphism in exon 1 codon 54 (Gly/Asp). TNFRSF6is the gene symbol for Fas; ns, not significant. ::: For confirmation of independence of association, a multivariable model was used including univariate variables with P values ≤ 0.15. This model included the above genetic variables (Table [2](#T2){ref-type="table"}) plus TNF+488 G/A genotype (25% versus 15%, OR 1.8, P = 0.11) and IL1R1+970 T/T (6% versus 14%, OR 0.4, P = 0.08) from [Additional file 1](#S1){ref-type="supplementary-material"}. Multivariable analysis confirmed that carriage of the variant IL-10 ATAhaplotype \[Multivariable Odds Ratio (MOR) 1.9, P = 0.05)\] and MBL2codon 54Asp (MOR 2.0, P = 0.04) were independently associated with histologic chorioamnionitis, while homozygosity for the variant TGFB1-800G/-509T (GT) haplotype (MOR 0.2, P = 0.04), and carriage of TNFRSF6-1377A/-670G (AG) haplotype (MOR 0.4, P = 0.03) remained negatively associated with histologic chorioamnionitis. The remainder of SNPs tested showed no significant differences. To examine the cumulative probability of maintaining a pregnancy and effect of chorioamnionitis on gestation at delivery in the overall cohort, Kaplan Meier survival analysis was performed. Figure [1](#F1){ref-type="fig"} shows that chorioamnionitis was more common in PTB at earlier gestations (Breslow-Gehan, Chi square 36 with 1 degree of freedom, P ≤ 0.001). In addition, the cumulative probability of maintaining a pregnancy was stratified on the carriage of IL10 ATAhaplotype (Figure [2](#F2){ref-type="fig"}). Women with this IL10 ATAhaplotype were more likely to deliver at earlier gestations (Breslow-Gehan, Chi square 3.5 with 1 degree of freedom, P = 0.06) (Figure [2](#F2){ref-type="fig"}). There was no evidence of the effect of MBL2, TGFB1, or TNFRSF6SNPs on gestation of PTB. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Relationship of chorioamnionitis with gestation at delivery.Kaplan-Meier survival estimates in 181 women where all gestations proceeded to delivery \< 35 weeks. Effect of chorioamnionitis (C) versus no chorioamnionitis (NC), P \< 0.001. Significance was tested using Breslow-Gehan Chi square analysis with 1 degree of freedom. ::: ![](1471-2393-5-4-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### *Relationship of IL10 ATAhaplotype with gestation at delivery.Kaplan-Meier estimates in 181 women where all gestations proceeded to delivery before 35 weeks. Effect of IL10 ATAhaplotype versus No ATA, P = 0.06. Significance was tested using Breslow-Gehan Chi square analysis with 1 degree of freedom. ::: ![](1471-2393-5-4-2) ::: Discussion ========== These results extend prior reports investigating gene polymorphisms and the pathogenesis of PTB and chorioamnionitis \[[@B11]\]. In this study we found significant positive associations between the carriage of the IL10 ATAhaplotype, MBL2codon 54Asp and histologic chorioamnionitis, and negative associations with TNFRSF6and TGFB1. This is the first study to investigate TNFRSF6and TGFB1in combination with such a wide range of cytokine SNPs in histologic acute chorioamnionitis. Also, this is one of the few studies that have used multivariable and survival analyses for investigating adverse pregnancy outcomes such as chorioamnionitis and PTB. The differences in genotype distribution for IL10and MBL2may be of biologic importance in the pathogenesis of histologic chorioamnionitis. The function of IL-10 in pregnancy is of particular interest because of its role in the homeostatic control of an inflammatory immune response \[[@B23]\]. Although there have been conflicting reports the majority suggest that the IL10 ATApromoter haplotype and alleles comprising the haplotype are associated with low protein levels in Caucasians \[[@B24]\]. Low levels of IL-10 have been associated with PTB in rat models \[[@B25]\]. In a pregnant rhesus monkey model, combined intra-amniotic and maternal systemic IL-10 administration inhibited IL-1β-induced preterm uterine contraction \[[@B26]\]. Survival analysis showed that the IL10 ATAhaplotype may influence the timing of PTB. IL-10 antagonises the synthesis and actions of pro-inflammatory cytokines such as IL-1 \[[@B27]\]. In chorioamnionitis, diminished IL-10 levels may provide an ineffective counter regulatory response to elevated maternal prostaglandin stimulated by the pro-inflammatory cytokines IL-1, IL-6 and TNF. Elevated levels of these pro-inflammatory cytokines in utero may serve as a maternal \[[@B28]\] and fetal \[[@B29]\] signal for the onset of premature parturition. The higher proportion of early PTB in women with the IL10 ATAhaplotype may also be a reflection of failure of tocolysis in the presence of infection \[[@B26]\]. Our finding that the carriage of the IL10 ATAhaplotype is more common in women with histologic chorioamnionitis suggests that this haplotype may be important in the pathogenesis of histologic chorioamnionitis and subsequent PTB. MBL is an important innate defense molecule active against a broad range of bacterial, viral, fungal and protozoan pathogens \[[@B30]\]. The MBL2variant alleles in exon 1 correlate with decreased circulating MBL. The single nucleotide substitution of an adenine for a guanine in codon 54 results in replacement of aspartic acid for a glycine in the MBL \[[@B31]\]. Functional MBL is multimeric, consisting of tetramers, pentamers and hexamers of triplets of the basic MBL peptide. This higher order structure is necessary for high avidity interactions between MBL carbohydrate recognition domains and repeated oligosaccharide moieties on pathogens. The MBL codon 54Asp \'B\' allele disrupts assembly of MBL2peptide trimers resulting in lower stability and serum levels \[[@B32]\]. Low serum levels of MBL and MBL2genetic variants are associated with infection in a range of contexts including recurrent infection in children and adults \[[@B6],[@B14]\], recurrent vulvovaginal candidiasis \[[@B33]\], recurrent miscarriage \[[@B34]\] and autoimmunity in common variable immunodeficiency disease \[[@B18]\]. The association between MBL2codon 54Asp and histologic chorioamnionitis suggests that this allele may result in low MBL levels, impaired opsonisation and clearance of pathogens, thus facilitating the development of chorioamnionitis. Fas protein is expressed on the surface of many cell types, such as lymphocytes, epithelial, fibroblasts and certain endothelial cells, and cytokines \[[@B7]\] and hormones \[[@B35]\] found in the placental microenvironment may modulate the immune response by regulating the expression of Fas and Fas ligand. In this study two polymorphisms in the Fas (TNFRSF6) gene promoter were studied at positions -1377 and -670, and the -1377A/-670G (AG) haplotype was negatively associated with histologic chorioamnionitis. The -1377G/A is situated at the transcription factor SP-1 binding site \[[@B22]\], and the -670A/G substitution is located in the enhancer region and abolishes the binding site of the nuclear transcription element GAS (Gamma Interferon Activation Sequence). The functional significance of these polymorphisms has not been fully elucidated but they may modulate TNFRSF6transcription, Fas expression and thus apoptotic and inflammatory responses. Recent evidence suggests that cells are more sensitive to Fas-mediated apoptosis when levels of Fas expression increase \[[@B36]\]. Apoptotic cell death can modulate host containment of pathogens \[[@B19]\] and thus genetically determined variation in Fas-mediated cell death may influence histologic chorioamnionitis. Reported studies on gene polymorphisms and adverse pregnancy outcome have mostly examined SNPs in the genes encoding the pro-inflammatory cytokines TNF and IL-1. The TNFpromoter -308A allele was associated with clinical chorioamnionitis in a small number of women \[[@B12]\]. The in vitrofunctional data of the effects of the TNFSNPs are conflicting and may be context dependent and influenced by strong linkage disequilibrium with other intra-and-extra-genic polymorphisms (e.g. with lymphotoxin alpha and classical HLA genes \[[@B37]\], not examined in this study). In contrast, in this study the TNF-308A could not be identified as a susceptibility or severity factor for chorioamnionitis. SNPs of the IL1 cluster have been examined from different ethnic groups, in particular IL1Band IL1RNin women with a history of recurrent pregnancy loss \[[@B38]\], PTB \[[@B39]\], and in in vitrostudies\[[@B11]\]. We did not find associations between genes of the IL1 cluster or IL1R1and histologic chorioamnionitis and this may partly be explained by differences in linkage disequilibrium patterns across different ethnic populations that can lead to different association results \[[@B40]\]. Our data support the role of IL10, MBL2and TNFRSF6variants in determining the risk of histologic chorioamnionitis. However there are limitations to the study, such as small sample size. The range and number of gene polymorphisms examined and analysed by univariate and multiple logistic regression in this study is notable, but some correction for multiple analyses is required \[[@B41]\]. The appropriate method still remains a contentious issue. The Bonferroni method was not conducted, as this is a conservative method and leads to overcorrection \[[@B42]\]. Also our study is a hypothesis-generating exploratory study. SNP frequencies in women with and without chorioamnionitis are available to be included in meta-analysis to confirm or reject these findings ([Additional file 1](#S1){ref-type="supplementary-material"}). Conclusion ========== In conclusion, the frequency differences in this study highlight the significance and biological relevance of genetic factors, IL-10, MBL and Fas regulation among women with acute chorioamnionitis and adverse pregnancy outcome. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= MAcontributed to study design, recruited study subjects, abstracted medical records, entered data, performed preliminary statistical analyses and drafted the manuscript. PHprovided design regarding immunoregulatory genes, interpretation of results and preparation of manuscript. CMconceived methodology and supervision of PCR testing, interpretation of test results and preparation of manuscript. SHsupervised preparation of DNA and performed all PCR tests. JRprovided clinical authorisation and support for the study. Provided input regarding clinical database design, and provided critical analysis for obstetric correctness. HM*conceived and coordinated the study, conceived medical record review, reviewed statistical analyses and preparation of manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2393/5/4/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Gene alleles, haplotypes and genotypes in Caucasoid Australian women with preterm birth before 35 weeks gestation and histologic chorioamnionitis. Frequency distribution of immunoregulatory gene alleles, haplotypes and genotypes in Caucasoid Australian women with preterm birth before 35 weeks gestation stratified by histologic chorioamnionitis. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We thank the WCH Clinical Information Services staff, midwife F Gatley, Dr P Bardy from the ARCBS and women participating in this study. The study was supported by grants from Channel 7 Children\'s Research Foundation of SA, Women\'s & Children\'s Hospital Perinatal Pathology Fund, Flinders Institute of Health Research, and the National Health & Medical Research Council (NHMRC) (Australia). M Annells is a NHMRC (Australia) Dora Lush Postgraduate Research Scholar.	PubMed Central	2024-06-05T03:55:53.936827	2005-2-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554771/", "journal": "BMC Pregnancy Childbirth. 2005 Feb 21; 5:4", "authors": [ { "first": "Margaret F", "last": "Annells" }, { "first": "Prue H", "last": "Hart" }, { "first": "Charles G", "last": "Mullighan" }, { "first": "Susan L", "last": "Heatley" }, { "first": "Jeffrey S", "last": "Robinson" }, { "first": "Helen M", "last": "McDonald" } ] }
PMC554772	Background ========== The definition of bacterial species, and a concept of species applicable to all bacteria, are problems that have long exercised systematists and microbiologists \[[@B1]-[@B4]\]. While species names have been assigned to groups of organisms sharing many common phenotypic traits, and a certain minimum level of genomic similarity, attempts to define species using DNA sequences have been relatively unsuccessful. The existence of very different levels of sequence diversity among named species, and the variable extent of gene flow within and between bacterial taxa \[[@B5]\], complicates species concepts and definitions. Indeed, for many, bacterial species are constructs of the human mind, arising from our desire to impose order on the bacterial kingdom \[[@B6],[@B7]\], rather than natural subdivisions imposed by underlying genetic processes, and a central question is not so much how species should best be assigned, but whether such entities exist and can be delineated. Molecular approaches to assigning bacteria to species began with the introduction of DNA-DNA hybridization, which allowed an objective assessment of the extent of sequence similarity among a set of genomes, and remains the systematicist\'s gold standard, defining bacterial species as those isolates whose genomes show at least 70% hybridization under standardized conditions \[[@B3]\]. However, few laboratories now use this method and, in practice, novel isolates (and particularly those that presently are unculturable) are usually compared to each other, and to known taxa, by assessing the sequence similarities in their 16S rRNA genes. 16S rRNA sequences are highly conserved and do not provide sufficient resolution to explore the relatedness among closely related bacterial populations and less conserved genes need to be used to delineate similar species. Individual isolates of a named species differ in gene content \[[@B8]\] and the distribution of these genes is key to understanding the variable properties of isolates of a species, particularly among bacterial pathogens. These auxiliary loci exist alongside the set of genes that are present in all isolates of the named species (the core genome) and which include those that encode enzymes with house-keeping functions \[[@B9]\]. Besides being present in all isolates of a species, the genetic variation in core house-keeping genes is considered to be largely neutral, and thus provides a more reliable indication of genetic relatedness than variation in genes that are subject to strong selection \[[@B10]\]. We would expect any reasonable definition of a species to delineate a cluster of isolates that have very closely-related house-keeping loci that are present in all isolates of a species (as has also been proposed for eukaryotes \[[@B11]\]). However, single house-keeping loci are unlikely to have sufficient variation to allow confident resolution of the different lineages. For recombinogenic bacteria, and arguably all bacteria, multi-locus approaches are required, as these provide increased resolution, and also reduce the impact of \'inter-species\' recombination. Thus, a localized interspecies recombinational event at one locus, which distorts the true relatedness between species, is buffered by the more reliable indications of relatedness provided by the other loci. Furthermore, attempts to observe whether or not species exist, and how sharply they can be defined, requires the analysis of large populations of each candidate species and not just one or a few reference isolates. A multilocus approach has recently been applied to small numbers of isolates of several relatively distantly related named species of enterobacteria \[[@B10]\], and other bacteria \[[@B11]\], and to larger numbers of isolates of related bacteria that are believed to have relatively low rates of recombination \[[@B12]-[@B14]\]. However, it is unclear whether species can be resolved using a multilocus approach in the more challenging case of highly recombinogenic bacteria colonising the same body site. Ideally, we would like to know if, in a large collection of such isolates that are believed to include examples of a number of closely related named species, we can resolve well delineated clusters, and the extent to which any clusters relate to the species names assigned by standard microbiological procedures. Can such populations diverge into distinct populations, and stay distinct, in the face of frequent and promiscuous recombination? In this study we have evaluated the ability of seven individual house-keeping gene sequences, and of the concatenated sequences of these genes, to resolve a large sample of human pathogenic and commensal Neisseriainto genotypic clusters. We chose this example because Neisseriaare naturally transformable, are among the most recombinogenic bacteria, and there is good evidence for relatively frequent localised recombination between the named Neisseriaspecies \[[@B15],[@B16]\] through transformation. We demonstrate that individual genes are incapable of identifying consistent clusters among the Neisseriaisolates, but the tree based on the concatenated sequences effectively resolves the three major named species within the sample, although the boundaries are fuzzy due to the presence of a small number of intermediate genotypes. Results ======= The widespread use of multilocus sequence typing (MLST) \[[@B17]\] for epidemiological purposes provides the sequences of seven house-keeping gene fragments from thousands of isolates of several bacterial pathogens. However, few of the available MLST databases include any substantial numbers of isolates of multiple closely related named species. An exception is the public NeisseriaMLST database, which includes several thousand sequence types (STs) of N. meningitidisand smaller numbers assigned to several other named human Neisseriaspecies \[[@B18]\] on the basis of standard phenotypic tests. The first 500 STs of N. meningitidiswere compared with all STs assigned to the other human Neisseriaspecies. The sequences of the seven gene fragments were concatenated in-frame and a tree was constructed (using third codon position sites) using Mr Bayes \[[@B19]\]. Figure [1](#F1){ref-type="fig"} is the majority rule consensus of 10 000 trees generated from the posterior probability at stationarity. All 67 STs of N. gonorrhoeae, and all but two of the 171 STs of N. lactamica, descend from single well-supported nodes (the remaining two N. lactamicaclustered very anomalously and have probably been incorrectly identified). The great majority of N. meningitidisalso formed a single well-resolved cluster, but a few arise from the branch leading to the N. lactamicaisolates. Very similar clustering of these three species was observed using other sets of 500 N. meningitidisSTs from the database, and in a neighbour-joining tree constructed using all STs in the NeisseriaMLST database (data not shown). The high levels of recombination in the Neisseriamake the fine structure of the tree meaningless (Figure [1](#F1){ref-type="fig"}), and here we use the tree-building software first and foremost as a clustering tool. Analysis of the individual gene trees shows that these fail to resolve the named species and highlights many examples where interspecies recombination has resulted in anomalous clustering (Figure [2](#F2){ref-type="fig"}). The clear inability of single locus trees to resolve the named species, which are well resolved using the concatenated sequences, establishes that multiple loci are required to buffer against the distorting effect of inter-species recombination at the individual loci. Although the concatenated sequences resolve three named species, N. gonorrhoeae, N. meningitidisand N. lactamica, their boundaries are not perfectly defined and a number of isolates are placed on the branch between N. lactamicaand N. meningitidis, representing intermediate genotypes. The small numbers of STs assigned to other human Neisseriaspecies do not cluster clearly. A significant separation is observed between two subtrees (A and B in Figure [1](#F1){ref-type="fig"}), although these both contain isolates assigned as N. sicca, N. mucosaand N. subflava. Multiple minimum-evolution trees constructed using all STs of these other Neisseriaspecies and randomly selected samples of ten STs from each of N. meningitidis, N. lactamicaand N. gonorrhoeae, showed the same deep split between these subtrees, which was also observed in trees constructed from all NeisseriaSTs (all species) in the MLST database, using Neighbour-Joining, minimum evolution and UPGMA tree-building approaches (data not shown). Discussion ========== Current molecular definitions of species use rules or cut-off values (e.g. ≥ 70% DNA-DNA hybridization) and rarely take account of the genotypic diversity within and between populations \[[@B3]\]. A more natural and pragmatic approach is to analyse large populations of related isolates, that are believed to cover multiple species, and to observe whether suitable molecular methods can resolve distinct clusters in sequence space that can be given appropriate names \[[@B11]\]. This approach has not yet been rigorously applied to bacteria. Consequently we have no idea whether large populations of related bacteria can invariably be divided into discrete clusters using suitable molecular methods or, alternatively, whether many groups of related bacteria fall into a genetic continuum where clear divisions do not exist. Sequence-based approaches should help us answer this question. However, most studies have focused on single loci and small numbers of isolates, whereas multilocus approaches with large populations are essential as the history of individual genes (including rRNA operons \[[@B20]\]) may be obscured by interspecies recombination, and clusters observed using a small number of isolates may merge when larger numbers of isolates are considered. Comparison of the tree based on the concatenated sequences with the individual gene trees clearly illustrates the inadequacy of single loci for resolving N. meningitidisand N. lactamica(Figure [2](#F2){ref-type="fig"}). The concatenation of the seven housekeeping loci shows that multiple loci can buffer against the distorting effects of inter-species recombination and that the boundaries between the three dominant species in the NeisseriaMLST database can be resolved. Network based methods (e.g. Neighbor-Net \[[@B21]\], Splitstree \[[@B22]\]) applied to both the concatenates and individual loci produce output with numerous reticulations, indicating the conflicting signals in the data, such that the implied relationships between STs within clusters have no phylogenetic meaning. Nevertheless, the use of multiple loci enables us to observe the species clusters even in the presence of conflicting signals. The three main clusters coincide well with the species names derived by standard microbiological procedures and the present definitions of N. meningitidis, N. lactamicaand N. gonorrhoeaeare reasonably secure; the two N. lactamicathat clustered highly anomalously probably represent species mis-identification. The most critical test of the multilocus approach is the ability to resolve N. lactamicafrom N. meningitidissince these colonise the same body site, the nasopharynx. Resolution of these named species was remarkably good, although the boundaries between N. lactamicaand N. meningitidisare somewhat fuzzy, due to the existence of intermediate forms. This is to be expected as recombinogenic bacteria have mosaic genomes, resulting from the occasional replacement of chromosomal segments with those from related populations. Thus, in any large dataset, there may be isolates in which one or more of the loci used in a multilocus approach to species definition will have been recently introduced from a related population. Single unusually divergent replacements, or replacements at more than one of the multiple loci, may place isolates away from the majority of isolates of the species. However, only seven STs in Figure [1](#F1){ref-type="fig"} fell into this category (of 667 STs from isolates identified as either N. meningitidisor N. lactamica), and there was no overlap between these two named species (i.e. a region containing isolates identified as both species interspersed with one another). Sorting the human commensal Neisseriainto species has been difficult, with frequent revisions of species names \[[@B23]\]. We gain some insight into the extent and source of this difficulty in Figure [1](#F1){ref-type="fig"}, where isolates assigned as N. mucosa, N. siccaand N. subflavaeach fall in very different parts of the tree, and the subtree shown in Figure [1A](#F1){ref-type="fig"} contains several closely related isolates that have been assigned to these three different named species. Additional studies of the human commensal, Neisseria(and of other groups plagued with similar problems, such as viridans streptococci) using the multilocus approach with large datasets, should clarify whether they fall into distinct clusters, or whether the difficulties in defining species by phenotypic methods reflect an underlying genetic reality in which resolved clusters are not evident. If necessary, further resolution between apparent clusters may be attempted by increasing the numbers of loci sequenced. Provided that the alleles at these loci show a degree of specificity to a given species cluster, then the resolution of that cluster will be enhanced. If this cannot be demonstrated, then it is likely that the isolates under test do not genuinely form separate populations, and should not be considered to be distinct species. This approach lends itself to \"electronic taxonomy\", in which systematic classification may be evermore finely elucidated through the accumulation of online sequence databases. The work described here obviously begs the question of what forces or mechanisms could generate such separation among recombining bacteria. We offer a simple model for recombining organisms as follows: consider two populations freely recombining within themselves and with each other. New mutations arising in one population will readily spread to the other, and to an observer they appear to form one cluster of related strains. If a barrier to recombination should be erected between them, such that isolates are much more likely to undergo recombination with their own population, then the rate of generation of new genotypes within each population may increase beyond the rate at which such genetic innovation is shared and the two populations begin to diverge. As the populations diverge, decreasing sequence identity will further impede recombination, thus reinforcing the effect of the original genetic barrier and creating a permanent separation \[[@B24],[@B25]\]. It is not difficult to suggest candidate mechanisms. Niche separation is one example, and almost certainly underlies the tight well-defined cluster of N. gonorrhoeae. Unlike the other human Neisseria, which colonise the nasopharynx, the primary niche of the gonococcus is the genital tract, and it has been proposed that gonococci arose relatively recently due to the successful invasion of the genital tract by a nasopharyngeal Neisserialineage \[[@B26]\]. Similarly, what appears to be single body site (e.g. the human nasopharynx) may contain multiple niches that can be exploited, leading to opportunities for speciation. Restriction-modification systems \[[@B27]\], limitation of transformability by differences in pheromone-type \[[@B28]\] and similar processes are feasible alternatives. The point at which such a group is described as a species is a matter more of human interest and attention than any intrinsic evolutionary process. The properties of the species clusters we observe will be determined by the diversification of those strains sharing the speciation loci (i.e those that determine gene flow). Because speciation is gradual, we should be able using estimates of recombination within and between groups derived from multilocus data, to define nascent species which if they continue to diversify in isolation, are expected to form distinct sequence clusters, ie species, in the future. Conclusion ========== The bacterial domain of life is not uniform. Instead we see clumps of similar strains that share many characteristics, and with an innate human urge to classify, we have defined these as species. This work shows that by applying a simple approach using sequence data from multiple core housekeeping loci, we can resolve those clusters, provided such clusters exist. However, these species clusters are not ideal entities with sharp and unambiguous boundaries; instead they come in multiple forms and their fringes, especially in recombinogenic bacteria, may be fuzzy and indistinct. A multilocus approach using large numbers of isolates will provide data that help us to develop theoretical models of how species emerge, and relate these to the observed population genetic structure of bacteria. This should be enormously helpful to taxonomists, whose foremost duty will remain to provide us with pragmatic species designations which attempt to reflect the underlying genetic reality. Methods ======= Strains ------- The contents of the publicly accessible NeisseriaMLST database \[[@B17],[@B18]\] were used to explore the validity of the approach described here for other species. Alleles at the seven MLST loci of all isolates defined as Neisserial species other than N. meningitidis(67 isolates of N. gonorrhoeae, 171 of N. lactamica, 5 of N. sicca, 3 of N. mucosa, 5 of N. cinerea, 7 of N. polysacchareae, 3 of N. flava, 4 of N. perflava, 4 of N. subflavaand 1 isolate of N. flavescens) were concatenated as described below, and analysed together with the concatenated sequences of N. meningitidisstrains with ST numbers from 1 to 500. Species definitions were as recorded at \[[@B17],[@B18]\], and were according to standard clinical microbiological schema. The sequences of the individual alleles at the seven loci in the above Neisseriawere also used to construct individual gene trees. Phylogenetics and population genetics ------------------------------------- MLST loci were concatenated in-frame to form a 3267 bp sequence, of which only third position sites were used in subsequent analyses. To illustrate clustering in this dataset, a tree was constructed using Mr Bayes 3.0b4 \[[@B19]\]. A starting tree was determined in PAUP (version 4 beta 10) \[[@B29]\] using the Neighbour-Joining method with distances corrected using the HKY85 model. The starting tree was input into Mr Bayes, and four Markov Chain Monte Carlo chains were run with default heating parameters until convergence and 10 000 trees were sampled from the posterior probability distribution. These were then used to produce a 50% majority rule consensus tree. Minimum evolution trees for individual loci were constructed in MEGA 2.1 \[[@B30]\]. Third position sites were used with the Kimura 2-parameter distance correction. List of abbreviations ===================== rRNA ribosomal RNA MLST Multi Locus Sequence Typing ST Sequence Type UPGMA Unweighted Pair Group Method with Arithmetic Mean Authors\' contributions ======================= BGS conceived of the study and drafted the manuscript, CF participated in study design and analysis of results, WPH designed the study, carried out the analyses and interpreted the results, and drafted the manuscript. Acknowledgements ================ This publication made use of the Neisseria Multi Locus Sequence Typing website \[[@B18]\] developed by Keith Jolley and Man-Suen Chan. The development of this site has been funded by the Wellcome Trust and European Union. We acknowledge the support of the Wellcome Trust (W.P.H. and B.G.S.) and the Royal Society (C.F.). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### *A tree constructed from concatenated MLST data resolves species clusters in Neisseria.The 50% majority rule consensus tree shown was produced in Mr Bayes using third position sites within the concatenated sequences of Neisserialspecies as described in methods. Assigned species names are indicated by the symbols. Two subtrees, A and B, are shown. These both contain strains identified as N. mucosa, N. siccaand N. subflava, showing that these named species do not form single clusters by this method. In these subtrees, the clades containing exclusively N. lactamicaor N. gonorrhoeaehave been collapsed. ::: ![](1741-7007-3-6-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Single locus trees fail to resolve species clusters.Individual minimum evolution trees constructed in Mega 2.1 for a) abcZ, b) adk, c) aroE, d) fumC, e) gdh, f) pdhCand g) pgm*. The assigned species names of the strains in which these alleles occur are shown. ::: ![](1741-7007-3-6-2) :::	PubMed Central	2024-06-05T03:55:53.939674	2005-3-7	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554772/", "journal": "BMC Biol. 2005 Mar 7; 3:6", "authors": [ { "first": "William P", "last": "Hanage" }, { "first": "Christophe", "last": "Fraser" }, { "first": "Brian G", "last": "Spratt" } ] }
PMC554773	Background ========== In eukaryotes, many cellular processes are regulated at the level of transcription. Initiation of transcription by RNA polymerase II requires the assembly of the basal transcription apparatus at the core promoter, a region of about 70 bp flanking the transcription start site (TSS) \[[@B1]\]. Interactions mediated by components of the basal machinery and transcription factors that recognize specific cis-regulatory elements, frequently located upstream of the core promoter, ensure efficient and regulated transcription by RNA polymerase II at Class II promoters \[[@B2]\]. Class II core promoters often contain conserved DNA elements recognized by components of the basal transcription machinery, the general transcription factors. The best-described core promoter DNA element is the TATA box, which is recognized by TATA-binding protein (TBP). The TATA box is a T/A-rich sequence usually located 25--35 base pairs upstream of the TSS \[[@B3]\]. Recruitment of TBP and TBP-associated factors, all part of the TFIID complex, directs assembly of the pre-initiation complex (PIC), a highly regulated process that ensures precise initiation of transcription. The directionality of the PIC is likely to be provided by the presence of another conserved element, present in a large fraction of Class II promoters, the BRE (IIB recognition element) \[[@B4],[@B5]\]. In addition, Initiator (Inr) elements are often present at the site of initiation of transcription in a number of eukaryotic core promoters. The Inr is a loosely conserved element containing an adenosine at the TSS and a C as the nucleotide preceding it (position -1), surrounded by a few pyrimidines \[[@B2]\]. The function of the Inr and the components of the basal transcription machinery that recognize this element remain poorly defined. In spite of the availability of a large number of computational programs that predict the presence of plant genes and their architecture (reviewed in \[[@B6]\]), accurately identifying core promoters solely based on genome sequence analysis remains a daunting task. Although no known DNA-sequence motif is present in all plant core promoters, TATA and Inr motifs represent two elements that are often present \[[@B7]\]. A main limitation in the analysis of plant core promoters is the insufficient amount of information available regarding TSSs, and hence the location of core promoters in genomic sequences. Over the past few years, several efforts have initiated the high-throughput production and analysis of full-length (FL) ArabidopsiscDNAs \[[@B8],[@B9]\]. These FL-cDNAs have dramatically improved the annotation of the Arabidopsisgenome \[[@B10]\], providing a powerful tool for the identification and analysis of core promoter elements. Here, we describe the analysis of core promoters of ca. 12,750 Arabidopsisgenes, using publicly available FL-cDNA sequences. Our objectives for this study were to i) identify motifs characteristic of Arabidopsiscore promoters; ii) determine how often Arabidopsiscore promoters contain a TATA box, and iii) compare the architecture of Arabidopsiscore promoters with those of Drosophila, the only higher eukaryote for which such a genome-wide analysis has been performed. We examined the presence, distribution and consensus sequence of conserved motifs proximal to the TSS. In addition to TATA elements, we identified several other motifs, primarily representing microsatellite elements, some of them overrepresented in particular regions of core promoters. Using Nucleotide Frequency Matrices (NFM), we carried out a genome-wide analysis for the presence and position of TATA-box elements. Our studies show that only about 29% of all Arabidopsisgenes contain a recognizable TATA element. The position of the TATA motif with respect to the TSS and correlations between the presence of a TATA with EST abundance and 5\' UTR lengths are discussed. Results and discussion ====================== Obtaining core promoter and 5\' UTR sequences for 12,749 Arabidopsisgenes --------------------------------------------------------------------------- As a first step towards identifying core Arabidopsispromoters, we queried TAIR\'s Gene Search with the condition of a FL-cDNA entry. We retrieved a total of 13,964 non-redundant hits, derived from over 28,000 total FL-cDNAs deposited at TAIR. The locus Ids for these 13,964 FL-cDNAs was used to retrieve the 5\' UTR corresponding to 12,749 genes. The remaining 1,215 genes for which a 5\' UTR was not retrieved corresponded to FL-cDNAs that differed between the annotations at TAIR and TIGR, sequences for which no 5\'UTR was annotated or sequences with 5\' UTR regions corresponding to alternative gene models. The \[-500, -1\] and \[-50, -1\] regions of all 12,749 genes was directly retrieved from the TAIR 500 bp upstream dataset. To obtain the \[+1, +50\] regions, we first checked the length of the 5\' UTRs, which was shorter than 50 bp for 2,649 genes and which was interrupted by introns in 2,179 genes. To include into our analyses these cases, three different strategies were followed. If the 5\' UTR was longer than 50 bp, and no introns were present in the corresponding \[-50, -1\] region (10,100 genes), a direct retrieval of the \[+1, +50\] region was performed from the TAIR 5\' UTR dataset. If the 5\' UTR was shorter than 50 bp and no intron interrupted this region (2,617 genes), we extended the 5\' UTR to 50 nt with a fragment of the immediately adjacent downstream coding sequence using the TIGR cDNA dataset. Finally, if the 5\' UTR was shorter than 50 nt and an intron interrupted this region (32 genes), we manually retrieved the \[+1, +50\] region from the genomic sequence using TAIR\'s SeqViewer. After these analyzes, we were able to generate datasets corresponding to the \[-500, -1\], \[-50, -1\] and \[+1, +50\] regions from a total of 12,749 genes. These datasets were used for all the subsequent analyzes in this study. Identification of conserved motifs in core promoters ---------------------------------------------------- To identify sequence motifs overrepresented in Arabidopsiscore promoters, we first searched for DNA elements conserved in the \[-50, -1\] and \[+1, +50\] regions of the 12,749 Arabidopsisgenes. The search was carried using both MEME and AlignACE (see Methods). Motifs correspond to short sequences (6--10 bp), often recognized by a DNA-binding protein, and which can be represented by a consensus sequence. While the total number of motifs retrieved per region with these algorithms was 16 and 32 respectively, only motifs detected in at least 50 sequences with either MEME or AlignACE are shown (Figure [1](#F1){ref-type="fig"}). A comprehensive list and sequence of the remaining motifs is provided as [Additional File 1](#S1){ref-type="supplementary-material"}. From 20 motifs present in 50 or more sequences in the \[-50, -1\] or \[+1, +50\] regions, seven were present in both regions (Motifs 1, 2, 4, 5, 8, 9 and 10; Figure [1](#F1){ref-type="fig"}), and thus were given the same numbers. Motifs 5 and 12 are reverse-complements of each other, and they are shown separately because they are over-represented in different regions of the core-promoters (Figure [1](#F1){ref-type="fig"}). Overall, the expectation maximization method MEME appears to be a more robust motif search algorithm than the Gibbs sampling method, AlignACE, since MEME resulted in a significant higher rate of identification for most of the motifs (Figure [1](#F1){ref-type="fig"}). Two motifs identified by MEME (Motifs 10 and 12, Figure [1](#F1){ref-type="fig"}) were not identified by AlignACE in any significant number of sequences. The distribution of the different motifs within the \[-50, -1\] or \[+1, +50\] regions was also investigated (Figure [1](#F1){ref-type="fig"}). In a few cases, there was a clear enrichment of motifs at particular positions. For example, Motif 3, only present in the \[-50, -1\] region, was clustered in the -30 to -45 region, Motif 9, present in both regions, clustered closer to the TSS and Motif 7 showed an enrichment in the vicinity of the -50 position (Figure [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Analysis of motifs present in the \[-50, -1\] and \[+1, +50\] regions of 12,749 Arabidopsisgenes. Motifs are numbered from 1 to 13 and ordered by the number of occurrences, indicated by the numbers under the motif name. The first numeral corresponds to the number of hits using MEME, the second to the number of hits using AlignACE. For example, 2417/1852 indicates a motif found 2,417 times using MEME and 1,852 times with AlignACE. The second column for each motif shows the nucleotide frequency distribution graphed using WebLogo, where the sizes of the characters represent the frequencies of occurrence. The third column provides a graphic representation of the frequency distribution (y-axis) of each motif in the \[-50, -1\] or \[+1, +50\] regions (x-axis). ::: ![](1471-2164-6-25-1) ::: Overrepresentation of motifs in the \[-50, -1\] or \[+1, +50\] regions ---------------------------------------------------------------------- To investigate whether the number of sequences containing each one of these motifs was accurately predicted by MEME or AlignACE and to establish which of these 13 motifs was significantly overrepresented in the \[-50, -1\] or \[+1, +50\] regions, we retrieved nucleotide frequency matrices (NFMs) for each one of these motifs from the results of the MEME search (see Methods). The NFMs for each of these motifs, provided as [Additional File 2](#S2){ref-type="supplementary-material"}, were used to determine their presence in the \[-50, -1\] or \[+1, +50\] regions. To establish whether the motifs were overrepresented in these regions, we used two background models. The first background model corresponded to an identical number of random sequences (columns 4 and 6 in Table [1](#T1){ref-type="table"} labeled Random) with the same nucleotide composition as the \[-50, -1\] or \[+1, +50\] regions. Because biological sequences are not random and intragenic sequences are richer in homopolymeric A/T than predicted by a random model with identical nucleotide composition, we used as the second background model the 12,749 non-core promoter \[-500, -450\] regions. The results are shown in Table [1](#T1){ref-type="table"} (column 2 in Table [1](#T1){ref-type="table"} labeled Real). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Motif frequency in the \[-50, -1\] and \[+1, +50\] regions of 12,749 Arabidopsisgenes compared to background models ::: \[-500, -450\] \[-50, -1\] \[+1, +50\] -------------- -------------------- ----------------- ----------------- ------------- ------------ Real Real Random Real\* Random Motif 1 1388 3817 778 4379 (3769) 806 Motif 2 1558 2195 235 3018 (2312) 323 Motif 3 543 1899 289 314 (289) 243 Motif 4 241 1288 109 1665 (1427) 114 Motif 5 88 382 49 421 (361) 54 Motif 6 275 477 56 894 (690) 81 Motif 7 59 153 34 28 (27) 51 Motif 8 157 282 83 421 (279) 106 Motif 9 208 385 111 519 (416) 163 Motif 10 168 270 21 519 (340) 23 Motif 11 548 460 297 1352 (213) 362 Motif 12 137 253 57 346 (308) 61 Motif 13 175 183 113 343 (241) 111 \Number in parentheses indicate the frequency of the motif in the 10,100 \[+1, +50\] 5\' UTR sequences without introns or coding regions. ::: Motifs 3 and Motifs 7 showed a clear Overrepresentation in the \[-50, -1\] interval. Motif 3 has all the characteristics of a TATA box (Figure [1](#F1){ref-type="fig"}), and was detected in 1,899 genes using the NFM, representing approximately 15% of all the genes investigated. A more detailed characterization of this motif is described below. Motif 7 was detected in a much smaller number of genes (153), and the corresponding motif with the A A/G GCCCA T/A consensus was shown before to be overrepresented in upstream regions versus coding regions of Arabidopsisgenes \[[@B11]\]. Consistent with our findings that show an increased accumulation of this motif towards the left border of the \[-50, -1\] interval (Figure [1](#F1){ref-type="fig"}), this motif was previously shown to have a strong positional preference for the \[-250, -50\] interval \[[@B11]\]. Interestingly, in Arabidopsisthis motif is associated with dark-induced genes and is over-represented in genes under circadian regulation \[[@B12]\]. Three motifs were also found to be overrepresented in the \[+1, +50\] region. Motif 10 resembles the (GAA)~n~microsatellite represented at least two fold higher in the \[+1, +50\] region, compared to the \[-50, -1\] or the \[-500, -450\] regions (Table [1](#T1){ref-type="table"}). This overrepresentation cannot be explained by the modest difference in nucleotide composition between these regions, consistent with the comparable distribution in the randomly simulated datasets (Table [1](#T1){ref-type="table"}). As described above, 2,649 of the \[+1, +50\] regions contain coding regions in addition to short 5\' UTRs. To investigate whether the coding sequences contributed to the overrepresentation of this motif, we analyzed the presence of this motif in the 10,100 \[+1, +50\] \"clean\" 5\'UTR regions, which do not contain any coding or intron sequences (shown between brackets in Table [1](#T1){ref-type="table"} under \[+1, +50\] Real). In these 10,100 sequences, Motif 10 was found in 340 \[+1, +50\] sequences, the same frequency as in the original dataset (519/12,749). Thus, this (GAA)~n~microsatellite is overrepresented in the \[+50, +1\] region, irrespective of whether it is coding or 5\' UTR. (GAA)~n~microsatellites have been extensively researched in humans \[[@B13]\], but not yet associated with any functional role in Arabidopsis. Motif 13, with the consensus T/A CCGGCGA (Figure [1](#F1){ref-type="fig"}), was detected by both MEME and AlignACE only in the \[+1, +50\] region (Table [1](#T1){ref-type="table"}). This motif, however, was not identified as the binding site for any known transcription factor, as deduced from searching the PLACE \[[@B14]\], TRANSFAC \[[@B15]\] and AGRIS \[[@B16]\] databases (not shown). Finally, Motif 11, present in a significant number of sequences (Figure [1](#F1){ref-type="fig"}), fits the Kozak consensus (ACCATGG) for a translation start ATG codon \[[@B17]\]. Consistently, 1,139 out of the 1,352 sequences in which we found Motif 11 have a short 5\' UTR, reflected in that this motif is present in just 213 5\' UTR \[+1, +50\] sequences (Table [1](#T1){ref-type="table"}). While this motif is irrelevant to our analysis, it provides a good internal control regarding the sensitivity and comprehensiveness of our search for motifs in the \[-50, -1\] and \[+1, +50\] regions. Motifs 1, 2, 4, 6, and 9 correspond to microsatellites commonly found in Arabidopsis\[[@B18]\], displaying similar frequency distributions in the \[-50, -1\] and \[+1, +50\] regions. From these 5 motifs, only Motif 2 does not seem to be significantly overrepresented in these two regions, when compared to the \[-500, -450\] sequences (Table [1](#T1){ref-type="table"}). The potential participation of microsatellites in the control of gene expression is unclear, but according to recent studies in rice and Arabidopsis, their distribution may follow a gradient in the direction of transcription \[[@B18]\]. Motif 8 conforms to a (CG)~n~microsatellite, frequent in monocots such as rice, but not often found in Arabidopsis\[[@B18]\], which is consistent with a low but comparable frequency in all three regions studied here (Table [1](#T1){ref-type="table"}). The apparent higher frequency of Motif 8 in the \[+1, +50\] region, compared to the \[-50, -1\] (421 versus 282, respectively), is likely to correspond to an increased G/C content of the 5\' UTR (see Methods), as reflected by the increased distribution of this motif in a random simulation of sequences with the same nucleotide composition of the corresponding \[+1, +50\] region (Table [1](#T1){ref-type="table"}). Motif 9, corresponding to a (CA)~n~microsatellite (with n = 5), was found to be only slightly overrepresented in the \[-50, -1\] region, compared to the \[-500, -450\] background model (Table [1](#T1){ref-type="table"}). Interestingly, however, this motif is significantly clustered in the \[-35, -10\] region (Figure [1](#F1){ref-type="fig"}). A similar clustering was not observed in the \[+1, +50\] region, where this motif is significantly overrepresented, compared to the background models (Table [1](#T1){ref-type="table"}). Motif 5, with the consensus sequence AAACCCTA (Fig. [1](#F1){ref-type="fig"}), and similarly overrepresented in the \[-50, -1\] and \[+1, +50\] regions, compared to the random or \[-500, -450\] background models (Table [1](#T1){ref-type="table"}), does not conform to a typical microsatellite sequence. Interestingly, however, the sequence of Motif 5 is precisely the reverse complement of Motif 12, which with the TAGGGTTT DNA-consensus fits the sequence of the Arabidopsistelomeric sequence \[[@B19]\], and of the telobox, the binding site for a MYB-related telomeric DNA-binding protein previously described in proteins from yeast, plants and animals \[[@B20]\]. This element, present in the 5\' UTR or promoter region of many genes encoding products associated with the translational apparatus \[[@B21]\], was also shown to participate in the expression of Arabidopsisroot meristem genes \[[@B22]\]. Our analysis suggests that the number of sequences containing the telobox motif in either the forward or reverse-complement configuration is much larger than previously reported \[[@B23]\]. Consistent with previous studies \[[@B23]\], only a few genes (8) contain Motif 5 or 12 in both the \[-50, -1\] and \[+1, +50\] regions. We also investigated for the presence of motifs previously shown to be overrepresented in the \[-60, +40\] regions of Drosophilacore promoters \[[@B24]\]. Using the corresponding NFMs, we searched our databases for DRE (DNA-replication related element) and DPE (downstream promoter element), usually found \~30 nt downstream of the TSS \[[@B25],[@B26]\]. Although the \[-60, +40\] region is shifted 10 bp towards the 5\' end from our selection, the positional clustering of the DRE and DPE motifs \[[@B24]\] still falls under the \[-50, +50\] region investigated here. In our analyses, neither one of the two motifs was represented at a level significantly higher than in the random models (not shown). A CCAAT box NFM \[[@B7]\] did not result in any significant distribution change between real and randomly generated datasets for both regions (not shown). This was expected because CCAAT boxes usually cluster around the -75 position \[[@B27]\], which is outside of the \[-50, +50\] interval investigated here, corresponding to what is generally recognized as the core promoter region. Similarly, none of the motifs identified here appeared to correspond to Inr elements. We conclude that, with the exception of the TATA box, the elements involved in the architecture of core promoter in Arabidopsisand Drosophilaare overall different. Distribution of TATA motifs in core Arabidopsispromoters ---------------------------------------------------------- According to our analysis for conserved core promoter elements, Motif 3 (Figure [1](#F1){ref-type="fig"}) is likely to represent the TATA box characteristic of many Class II promoters. Consistent with this idea, Motif 3 is significantly overrepresented in the \[-50, -1\] region (Table [1](#T1){ref-type="table"}) with a clear clustering in the -30 to -45 region (Figure [1](#F1){ref-type="fig"}). Surprisingly, however, Motif 3 was only detected in 15% of the 12,749 core promoters investigated, lower than found in previous studies, which suggested that 57% of plant genes had a TATA box \[[@B7]\]. To investigate this striking difference between previous estimates for the frequency of a TATA box in Arabidopsispromoters and our own analyses, we utilized the previously described TATA NFM \[[@B7]\]. With this NFM, MotifScanner identified 3,679 TATA motifs in the \[-50, -1\] region, significantly higher than the number of hits in the \[+1, +50\] region, or in the corresponding background models (Table [1](#T1){ref-type="table"}). Thus, according to this analysis, 28.8% of all Arabidopsisgenes contain a TATA, comparable to the number of Drosophilacore promoters suggested to contain a TATA box (28--34%) \[[@B24]\], but still significantly lower than previously reported for the analysis of 305 plant promoters \[[@B7]\]. Interestingly, however, if these prior studies are restricted to just the 63 sequences from Arabidopsis, only 23 showed the presence of a TATA, representing a frequency of 36.5%, comparable to our own results. Previous studies also suggested that plant TATA-less promoter were the exception \[[@B28]\], and that TATA-less promoters were mainly restricted to photosynthetic \[[@B28]\] and plastid ribosomal genes \[[@B29]\]. Our results, however, indicate that TATA-less promoters are found more frequently than TATA-containing promoters. We cannot rule out that Arabidopsisis the exception among the plants, a possibility to be considered given the much lower percentage of TATA-containing promoters in Arabidopsiscompared to other plants \[[@B7]\]. More likely, however, the lack of a good knowledge of the position of the TSS may have resulted in previous studies in a very significant over-estimate of the presence of TATA elements. As an example, if the search for TATA elements is carried out on the 12,749 \[-500, -1\] regions, 6,316 sequences (using the MEME NFM) or 8,776 (using the expanded PlantProm NFM) are retrieved as containing a significant hit to a TATA element (Figure [2A](#F2){ref-type="fig"}), corresponding to 49.5% and 70% respectively, much closer to previous, yet likely incorrect, estimates \[[@B28]\]. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Position of TATA motifs in Arabidopsispromoters. A, The analysis of the 12,749 \[-500, -1\] regions with the MEME-derived NFM (Table 3) resulted in 6,316 sequences containing a significant hit (indicated by the red curve), 1,768 of them clustered in the \[-50, -1\] region. A similar analysis with the expanded and improved PlantProm-derived NFM (Table 4) resulted in 8,776 hits (blue curve), 2,507 of them clustered in the \[-50, -1\] region. B, Expansion of the \[-50, -1\] region indicating with a vertical green line that the average distance of the TATA motifs present in the \[-50, -1\] region is 31.7 nt from the TSS (using the first conserved T as the reference position). ::: ![](1471-2164-6-25-2) ::: The sequences from all these putative TATA-containing promoters were retrieved and the NFMs were retrained with this new information. The new matrix obtained from 1,899 sequences gathered using our MEME NFM (Figure [1](#F1){ref-type="fig"}) is shown in Table [3](#T3){ref-type="table"}. Similarly, the PlantProm TATA NFM was retrained with the 3,679 sequences, resulting in an improved and expanded NFM (Table [4](#T4){ref-type="table"}). These NFMs provide robust tools for the identification of additional plant TATA motifs. The two NFMs are significantly better than previously available plant TATA NFMs, with regards to the addition of flanking sequences that permit to expand the TATA consensus, and because of the much larger number of sequences used to build them. They have very similar nucleotide distributions, probably the biggest difference being at position 8, were the matrix derived from our MEME analysis has a much stronger requirement for an A (compare Tables [3](#T3){ref-type="table"} and [4](#T4){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### TATA NFM derived from 1,899 motifs. ::: Derived from MEME* ----------------------- -------- -------- -------- -------- ------- ------- ------- ------- --------- ------- --------- ------- ------- -------- -------- -------- -4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10 11 12 A 0.227 0.259 0.244 0.245 0.003 0.997 0.001 0.994 0.408 0.994 0.358 0.906 0.241 0.439 0.302 0.393 C 0.244 0.262 0.230 0.398 0.001 0.001 0.002 0.003 0.001 0.001 0.001 0.003 0.294 0.228 0.269 0.204 G 0.125 0.180 0.113 0.153 0.002 0.001 0.000 0.001 0.001 0.002 0.001 0.090 0.193 0.153 0.160 0.161 T 0.403 0.300 0.413 0.203 0.994 0.001 0.997 0.002 0.590 0.003 0.641 0.002 0.272 0.180 0.270 0.242 t n t c T A T A T/A A T/A A n a n a ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### TATA NFM derived from 3,679 motifs. ::: Derived from PlantProm ---------------------------- -------- -------- -------- -------- ------- ------- ------- ------- --------- ------- --------- ------- ------- -------- -------- -------- -4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10 11 12 A 0.246 0.262 0.248 0.243 0.058 0.917 0.000 0.998 0.493 0.943 0.417 0.655 0.197 0.399 0.340 0.383 C 0.246 0.247 0.242 0.434 0.030 0.000 0.049 0.001 0.000 0.001 0.020 0.093 0.349 0.286 0.244 0.212 G 0.118 0.184 0.126 0.111 0.000 0.001 0.000 0.000 0.000 0.038 0.000 0.100 0.221 0.159 0.141 0.141 T 0.391 0.308 0.384 0.213 0.911 0.083 0.951 0.001 0.507 0.018 0.563 0.153 0.232 0.156 0.275 0.264 t n t c T A T A T/A A T/A A c a a a ::: The new NFMs were used to scan the \[-500, -1\] region and establish where each of them localized a TATA with the highest probability. As shown in Figure [2A](#F2){ref-type="fig"}, both NFMs showed a significant peak in the \[-50, -25\] region, consistent with the position expected for TATA elements. To establish the average distance of TATA elements to the TSS, the MEME and PlantProm TATA NFMs were used to scan the 12,749 \[-50, -1\] regions and the positions of the corresponding TATA boxes were recorded and graphed (Figure [2B](#F2){ref-type="fig"}). The average distance of a TATA (position 1 in Tables [3](#T3){ref-type="table"} and [4](#T4){ref-type="table"}) to the TSS is 31.7 nt (indicated with a green line in Figure [2B](#F2){ref-type="fig"}). Thus, the position of the TATA box in Arabidopsisis more similar to what is typically the case in animal promoters, usually 25--30 nt from the TSS \[[@B2]\] than what is found in yeast, where the TATA box has a variable position in the \[-100, -40\] region \[[@B30]\]. We investigated whether the presence of TATA motifs correlated with other properties of the corresponding genes. Based on our analysis of the 12,749 FL-cDNAs, we determined that the average size of the 5\' UTR of Arabidopsisgenes is 129 nt (Figure [3](#F3){ref-type="fig"}). Interestingly, when we compared the average length of the 5\' UTRs of TATA-containing versus TATA-less genes, we found that TATA-containing genes had an average of 108 nt in their 5\' UTRs, compared to 138 nt in TATA-less genes. This difference in the length of the 5\' UTRs between these three populations of genes is evident in the sway towards shorter 5\' UTRs in the TATA-containing population (Figure [3](#F3){ref-type="fig"}). The reason for this difference in 5\' UTR length between TATA-containing and TATA-less promoter is not clear, although it is possible that the longer 5\' UTR provide additional features that contribute to PIC assembly. We also investigated whether the presence of a TATA element made a difference in the times that each gene was represented in ESTs, an approximate indication of the relative level of expression of the corresponding gene. While each Arabidopsisgene is represented in average by 9.48 ESTs (see Methods), the 12,749 sequences utilized here are represented in average by 13.02 ESTs, suggesting that the available FL-cDNAs are likely to correspond to genes expressed at a higher level than the average Arabidopsisgene. Interestingly, however, TATA-containing genes were represented in average by 17.6 ESTs (17.68 using the MEME NFM and 17.52 using the PlantProm NFM, Tables [3](#T3){ref-type="table"} and [4](#T4){ref-type="table"}), whereas TATA-less genes were represented by just 11.23 ESTs. These results suggest that the presence of a TATA is generally associated with genes expressed at a higher level. Gene Ontology analyses (see Methods) did not provide any insights on possible cellular functions associated with these gene clusters (not shown). An analysis of the sequences flanking the TSS, and likely containing the Inr element, did not reveal any significant difference in nucleotide composition between TATA-containing and TATA-less promoters (data not shown). Thus, the assembly of the PIC is likely to occur in ArabidopsisTATA-less promoters solely through the Inr, or regulatory elements outside of the \[-50, +50\] region investigated here also participate in the recognition of the core promoter by components of the basal transcriptional machinery. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Length distribution of 5\' UTRs in TATA-containing and TATA-less genes. The length of the 5\' UTR of all 12,749 genes (orange bars) shows an average of 129 nt. Promoters lacking a TATA box (TATA-less, red bars) have in average 5\' UTRs 138 nt long. The 5\' UTR of TATA-containing genes (blue bars) are in average 108 nt long. ::: ![](1471-2164-6-25-3) ::: Conclusion ========== Understanding the architecture of core promoters is central to establishing the mechanisms by which the basal transcriptional machinery assembles and facilitates formation of the pre-initiation complex. We provide here the first genome-wide analysis of Arabidopsiscore promoters. We have identified several motifs overrepresented in core promoters, with respect to background models consisting of random sequences of identical nucleotide composition or intergenic regions. With the exception of microsatellites similarly distributed in the \[-50, +1\] and \[+1, +50\] regions and the TATA element, for which an in-depth analysis was carried out, most other overrepresented motifs were present in only a small subset of the sequences analyzed. Our studies provide robust NFMs corresponding to TATA elements and other conserved motifs, and show that only 29% of all Arabidopsispromoters contain a TATA element located in average approximately 32 nt upstream of the TSS. The absence of a TATA correlates with a lower representation of the corresponding gene in public EST collections as well as with longer 5\' UTR sequences. However, the absence of a TATA is not compensated for by the overrepresentation of any one of several motifs present in Drosophilacore promoters, suggesting significant differences in the organization of core promoters from animals and plants. Methods ======= Retrieval of core promoter and 5\' UTR sequences ------------------------------------------------ To obtain the sequences of the region of promoters spanning the first 500 nt upstream of the TSS \[-500, -1\] and the corresponding 5\' UTRs, we used the TAIR Gene Search web tool \[[@B31]\]. The TAIR database was queried for all genes having a full-length cDNA (FL-cDNA) entry. The corresponding 5\' UTR and the \[-500, -1\] regions datasets were downloaded from TAIR \[[@B32]\], last updated on February 28, 2004. The FL-cDNA sequences were obtained from the June 10, 2004 realese of the TIGR\'s cDNA dataset \[[@B33]\]. The locus Ids of the gene queries were checked against the 5\' UTR, \[-500, -1\] and FL-cDNA files to reject erroneous annotations. We divided the 100 bp region flanking the TSS in upstream \[-50, -1\] and downstream \[+1, +50\] sub-regions of 50 bp each. The \[-50, -1\] and \[+1, +50\] intervals of the confirmed genes were directly retrieved from the downloaded TAIR files, when possible. In those cases when the 5\' UTR region was shorter than 50 bp, the TIGR file was used to extend the region to the necessary length by appending a fragment of the immediately adjacent coding sequence. When an intron interrupted the 5\' UTR, we manually extracted the 50 bp region from the Arabidopsisgenomic sequences using the SeqViewer tool at TAIR. Motif discovery and motif search -------------------------------- To characterize core promoters, we first investigated features represented by conserved regions or motifs. From several algorithms available \[[@B34]\], we chose the expectation maximization method MEME (version 3.0.8) \[[@B35]\] and the Gibbs Sampling method AlignACE \[[@B36]\]. MEME and AlignACE were run for the \[-50, -1\] and \[+1, +50\] regions separately for the entire set of genes. For MEME, a fixed minimum motif length of 5 and a maximum of 10 was set and 20 motifs were requested using the zero or one occurrence per sequence model. For AlignACE, only the background fractional GC content of the input sequences was supplied, and all the other parameters were left at default values. MEME and AlignACE were run in the Itanium 2 Cluster at the Ohio Supercomputer Center. The results obtained with MEME were compared with those obtained with AlignACE. Motifs consisting of single nucleotide repeats (i.e. A~n~) were manually parsed out independent of the number of occurrences or positional preferences. The obtained motifs were plotted according to their positions within the regions and their consensus sequences were graphed using WebLogo version 2.7 \[[@B37]\]. To find pre-defined motifs in the \[-50, -1\] and \[+1, +50\] regions, we used the higher order probabilistic model MotifScanner from MotifSampler version 3.0 \[[@B38]\]. The searches were fed with the nucleotide frequency matrices (NFMs) of the selected motifs obtained from the MEME search, and a background model of order 1 accounting for single- and di-nucleotide distributions for each set. The prior probability of finding one instance of the motif was left to the default value of 0.2. We also ran the motif search with elements conserved in core promoters of other organisms. The first two corresponded to the TATA and CCAAT elements obtained as NFMs from PlantProm \[[@B7]\]. The other two corresponded to the Downstream Promoter Element (DPE) and the DNA-replication Related Element (DRE) described for Drosophilacore promoters \[[@B24]\]. For these new four elements, we performed the same analysis as described before, using the \[-50, -1\] and \[+1, +50\] region datasets and the corresponding randomly generated dataset. Generation of random sequence models ------------------------------------ After establishing that the distribution of nucleotides in the Arabidopsis\[-50, -1\] and \[+1, +50\] regions are \~65% A/T to \~35% C/G and \~61% A/T to \~39% C/G, respectively, a pseudo-random set of 50 bp sequences was generated for each region to be tested with the matrices as a way of determining the chances of finding the motifs candidates in a stochastic environment. This information was then used together with the search results obtained from the real data to support the confidence of the findings. Analysis of TATA elements ------------------------- For the analysis of the TATA motif, the TATA NFM previously described \[[@B7]\] was used against the NFM reported by our own motif search. Using MotifScanner, the distribution of TATA elements in the upstream vicinity of the TSS was investigated. After determining the location of the putative TATA motifs in the \[-50, -1\] region, the NFMs were retrained with the new retrieved TATA motifs. Analysis of gene ontology and expression level based on EST abundance --------------------------------------------------------------------- To determine whether the occurrences of the discovered motifs were associated with specific gene functions or products we retrieved the Arabidopsis Gene Ontology Database \[[@B39]\] (last update July 20, 2004) and correlated the annotated molecular function, biological process or cellular component of Arabidopsisgenes with the ones found in the motif clusters. Under the assumption that the contribution of a gene to transcription activity is related to the number of its detected ESTs, we downloaded a dataset from TAIR that accounts for the number of ESTs submissions per locus \[[@B40]\] (last update July 23, 2004). With this, we then established the relative expression levels based on the ratio of the genes containing a particular motif and the overall EST frequency per gene. List of abbreviations ===================== bp, base pair; EST, expressed sequence tag; FL-cDNA, full-length cDNA; Inr, Initiator element; NFM, nucleotide frequency matrix; nt, nucleotide; PIC, pre-initiation complex; TBP, TATA-binding protein; TSS, translations start site 5\' UTR, 5\' untranslated region Authors\' contributions ======================= C.M. carried out all the analyses and interpreted the results. E.G. was involved in the design and supervision of the project. C.M. and E.G. jointly wrote the manuscript. Both authors read and approved the final manuscript. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Frequency of TATA frequency in the \[-50, -1\] and \[+1, +50\] regions of 12,749 Arabidopsisgenes compared to background models ::: \[-500, -450\] \[-50, -1\] \[+1, +50\] --------------- -------------------- ----------------- ----------------- ---------- ------------ Real Real Random Real Random MEME 543 1899 289 314 243 PlantProm 1526 3678 1431 1084 1209 ::: Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Complete list of the motifs present in the \[-50, -1\] and \[+1, +50\] regions of 12,749 Arabidopsisgenes. The analysis was carried out as described for the results shown in Figure [1](#F1){ref-type="fig"}. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Nucleotide Frequency Matrices for all the motifs shown in Figure [1](#F1){ref-type="fig"}. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ The analysis of plant promoters is supported in part by grants MCB-0210413 and MCB-0418891 (to E.G.) from the National Science Foundation and by a seed grant from the Ohio Supercomputer Center. We appreciate helpful comments on this manuscript by Nicole Matthews, Ralf Bundschuh, George Heine, Marcela Hernandez and Rebecca Lamb. We thank Uwe Ohler for sharing with us the position probability matrices for motifs overrepresented in Drosophilacore promoters.	PubMed Central	2024-06-05T03:55:53.941431	2005-2-25	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554773/", "journal": "BMC Genomics. 2005 Feb 25; 6:25", "authors": [ { "first": "Carlos", "last": "Molina" }, { "first": "Erich", "last": "Grotewold" } ] }
PMC554774	Background ========== The randomised controlled trial (RCT) has a number of important features that make it the \'gold-standard\' evaluation method. One of the most important aspects of random allocation is that it eliminates selection bias. Randomisation ensures that the two or more groups formed are similar, except for chance differences, in all aspects. Nevertheless unless trials are undertaken in a rigorous manner biases can be introduced that negate the effect of random allocation. Indeed, a poorly conducted RCT can be worse than a good observational study as the latter is interpreted in the light of possible confounding whereas the results of an RCT might be uncritically accepted. Random allocation can take place either at the level of the individual level or at a higher group or cluster level. In a cluster randomised trial groups of people are allocated to receive an intervention or not. In some areas of evaluation (e.g., education) the cluster is the natural method of allocation. For example, a trial among school children may well randomise by class or by school rather than by individual child. Allocation by cluster may be preferable for a number of reasons. There may be practical reasons: for instance, teaching a novel curriculum will be easier to use existing classes than form new ones through randomisation. There may be contamination issues. Individuals allocated to a control treatment may inadvertently receive some aspects of the intervention if they are in proximity to the treated group. Allocation by cluster has some important statistical issues that have been addressed 65 years ago in the educational trial literature \[[@B1]\] and subsequently widely in medical statistics \[[@B2]\]. In brief, analysis of cluster trials needs to take into account the clustered nature of the data otherwise the risk of a Type I error (i.e., erroneously concluding there was a statistically significant difference) increases. However, more seriously in our view is the potential of cluster trials producing a biased estimate of treatment effect. Randomisation should eliminate selection bias. Selection bias can be reintroduced within any trial if there is high loss to follow-up or failure to use intention to treat analysis. In cluster trials selection bias can also be introduced through participant recruitment. Because cluster trials often recruit their participants after the clusters have been randomly allocated this can lead to selection effects\[[@B3],[@B4]\]. There are a number of potential reasons for this. Foreknowledge of allocation --------------------------- If the person recruiting participants has both knowledge of the clinical characteristics of the participants and of the allocation schedule biased recruitment can occur. Subversion, within individually randomised trials, can occur by recruiting participants with poor prognostic characteristics so that they are more likely to enter the \'unfavoured\' group \[[@B5],[@B6]\]. Evidence for the biasing effects of allocation foreknowledge has been shown on treatment effect sizes \[[@B7],[@B8]\]. Consequently a rigorously designed individually randomised trial ought to conceal the allocation schedule from the people who are recruiting participants. Cluster randomised trials often do not, or cannot, conceal treatment allocation. For example, a trial was undertaken to reduce violence among children randomised by school \[[@B9]\]. After allocation the children were recruited into the study and the intervention was delivered. The allocation could not be concealed from the teachers researchers or children. This has two potentially unfortunate consequences. Awareness of the allocation can lead to biased recruitment in cluster trials \[[@B10]\]. Alternatively, or in addition, participants can differentially refuse consent to participate in the trial and this could be another source of selection bias. For example, in a cluster trial evaluating the use of advanced end of life directives among residents of nursing homes there was a differential in participant rates of 83% among people in the intervention homes compared with 92% in the control arm \[[@B11]\]. Such differential participation rate can lead to selection bias. Treatment effects on recruitment -------------------------------- Recruitment of participants with different clinical characteristics is not necessarily a sign of subversion it could be simply a consequence of the cluster level intervention. For example, in an evaluation of an educational package for the treatment of back pain primary care physicians were trained in \'evidence based\' management of back pain \[[@B12]\]. This training was associated with an increased recruitment rate among practices allocated to training compared with no training. Because training involved recognition and diagnosis of back pain with hindsight we should not be unsurprised that differential recruitment would occur in this instance. As well as having more potential, than individually randomised trials, for the introduction of selection bias. Cluster randomised trials also may be at more risk of dilution bias. Because consent for treatment is often not obtained until after randomisation more participants, than in an individually randomised study, may refuse treatment and this will consequently dilute any treatment effects. For example, Kendrick and colleagues in a cluster randomised trial to prevent accidental injuries among young children found that only 75% of the group allocated to the experimental group actually received the intervention \[[@B13]\]. Whatever the underlying reasons for differences in recruitment the consequences are potentially the same: selection bias has been introduced. Figure [1](#F1){ref-type="fig"} shows the potential sources of bias that can occur after cluster randomisation. With the introduction of selection bias trial results are unreliable. In this paper we examine some evidence for this phenomenon and make recommendations on how to design this problem out of future cluster trials. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Sources of bias in cluster trials ::: ![](1471-2288-5-10-1) ::: ### Evidence for recruitment bias among individual trials A recent review identified a sample of 36 cluster randomised trials from three major general medical journals, between 1997 and 2002 \[[@B4]\]. This review identified all cluster randomised trials published in three major medical journals over a period of five years. In this review 15 of the trials could have experienced bias in their recruitment of participants. Of these 15 trials seven showed some evidence in the published papers of consenting differential numbers of participants or excluding participants in a selective fashion. One of the remaining 8 trials, whilst having no evidence of bias in the original published paper was later subsequently found to have experienced recruitment bias \[[@B10]\]. Therefore, 25% of cluster trials published in major clinical journals suffered potential selection bias. On the other hand a review of 152 cluster trials undertaken in primary care found that only 8 (5%) were found were the authors reported differential recruitment \[[@B14]\]. However, unlike Puffer and colleagues each trial was not carefully scrutinised to ascertain whether or not there was a problem of biased recruitment (Eldridge, personal communication). Although Puffer and colleagues noted that some trials had significant differences in recruitment and retention rates between groups they did not investigate whether or not this had an impact on important treatment covariates. To assess whether observed differences in recruitment could have had an effect on important predictors of outcome we examined the age differences between treatment groups. We chose age for two reasons: first, it is a commonly reported baseline characteristic and, second, is the most likely common confounder across different disease groups. Nevertheless, we acknowledge that biased recruitment may not manifest itself in terms of age differences \[[@B12]\]. From the 36 trials we identified 14 that reported, either directly or indirectly, the mean age and standard deviation of the treatment groups. Of the 14 trials that were included nine stated they had taken clustering into account in their sample size calculation, two had not and the remaining three it was not clear whether they had adjusted their sample size. We then grouped the 14 trials according to whether Puffer et alconsidered there was evidence for differential recruitment. Eight out of the 14 trials had been regarded as potentially biased. In Figure [2](#F2){ref-type="fig"} we plot the standardised mean age differences between treatment groups (ie., age difference divided by the pooled within group standard deviation). Negative age differences were all converted to positive differences as we were uninterested in the direction of the bias. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Standardised mean differences of patient age ::: ![](1471-2288-5-10-2) ::: As can be seen in Figure [2](#F2){ref-type="fig"} the age difference between treatment groups tend to be larger in the potentiallybiased group. The mean age difference was greater than 10% of their standard deviation in 3 out of the 8 potentially biased trials. The pooled standardised mean difference in the biased group was also twice as large as that in the non-biased group. A test for the difference using a meta-regression resulted in a non-significant p-value of 0.15; therefore, the difference observed in this instance was not conclusive. Age imbalances for any single trial could be due to chance as it is more difficult to achieve balance in cluster randomised trials compared with individually randomised trials due to the smaller number of allocated units. On the other hand, cluster trials, like individually randomised trials should be balanced across allcluster trials if there was no bias present. In Figure [3](#F3){ref-type="fig"} we plot the standardised mean age differences by whether or not the trial showed a statistically significant effect. The significance was determined as p-value \< 0.05. In all 14 trials the analysis took the clustering effect into account through various methods The figure suggests that significant trial results were associated more often with potentially biased recruitment with larger baseline differences in age, even though a formal test for interaction did not show a statistical significance (p-value 0.3) as this was not adequately powered. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Standardised mean difference by bias group and treatment significance ::: ![](1471-2288-5-10-3) ::: ### Evidence for bias from a systematic review Cluster randomised trials often answer different questions to individually randomised trials or cannot use individual allocation. Therefore, it is difficult to make a direct comparison between individual and cluster randomised trials in terms of the likely differences in effect sizes within the same subject area. However, within the area of hip protection for fracture prevention there are trials using both individual and cluster allocation. The most recent Cochrane review of hip protectors has identified 13 RCTs of hip protectors with hip fracture outcomes \[[@B15]\]. In addition, there is a large individually randomised trial that has not yet been included in the review (i.e., 14 in total) \[[@B16]\]. In figure [4](#F4){ref-type="fig"} the effect sizes from these trials are plotted against their sample size. The sample sizes were adjusted for the design effect for the cluster trials and therefore the sample sizes for these trials are the effective sample sizes (sample size divided by the design effect). One out of the five cluster randomised trials reported their design effect, from which we estimated an intra-cluster correlation coefficient (ICC) and applied this to the other four studies, as they are all similar trials, to calculate a correction factor. As the figure shows the resulting funnel plot indicates little evidence of effect from individually randomised trials. In contrast, all of the cluster trials show a substantial benefit when using the cluster design. This suggestion of bias could be as a result of publication bias. On the other hand, there are a number of alternative explanations. First, the cluster trials might have been undertaken in a different setting than the individually randomised trials and this might account for the observed differences in effect. Second, the intervention (hip protection) may work better using a clustered design. Third, there might be treatment contamination in the individually randomised trials: biasing the treatment effect towards the null. Fourth, the observed differences may be due to poor implementation of cluster trial methodology, which biases the results of those trials towards the positive. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Funnel plot of individually and cluster randomised trials ::: ![](1471-2288-5-10-4) ::: There is a tendency for the cluster trials to be largely undertaken among residents of nursing homes compared with individually randomised trials -- although one of the largest individually randomised trial was in a nursing home setting. It is possible that compliance might have been better in a nursing home setting and this could account for the difference in effect. However, the compliance rates were not that different from those trials using individually randomisation. The second reason that hip protection might work in a clustered design might be that the intervention is delivered as a \'package\' of care and their use alerts the clinical staff responsible (e.g., nursing home staff) to the dangers of falls and this encourages other anti-fracture interventions. It is possible that in the individually randomised trials the control group could have been \'contaminated\' by accessing hip protection by, for example, buying the product themselves. In a large individually randomised trial we undertook of hip protectors \[[@B16]\] some participants in the control arm did purchase hip protectors; however, the prevalence of this was very low. An alternative explanation of the difference was poor implementation of the cluster trial methodology: including selective recruitment, differential loss to follow-up and failure to use intention to treat analysis. For example, the largest cluster randomised trial had a 30% difference in the population that were included in the trial after random allocation \[[@B17]\]. ### Preventing biased recruitment In order for cluster randomised trials to provide unbiased evidence for treatments we must design out any sources of recruitment bias. In this section we will consider design suggestions that should minimise this threat. ### Use individual allocation Often cluster randomisation is used to overcome the perceivedthreat of contamination between the treatment groups. Although in many instances this threat is real in some cases there may be little contamination. Indeed, even if there are quite high contamination rates (e.g., 20%) it may still be more efficient in sample size terms to randomise more patients in an individual trial and accept a diluted effect size \[[@B3]\]. Therefore, one solution to avoiding biased recruitment is to avoid using cluster trial methods if at all possible. ### Prior identification of participants In some instances it may be possible to identify participants before cluster allocation. For example, if we consider a school based evaluation of a health promotion curriculum. Children within schools or intact classes can be identified before the cluster allocation. Children and their parents can be asked to participate in the study and are presented with the alternatives under consideration. Once consent has been obtained to take part in the study then the schools or classes are randomised to the different curricula. ### Independent recruitment Evaluation of an intervention for incidence disease cases means prior identification is not possible. For example, consider a trial of educating primary care physicians for the treatment of acute shoulder pain. Because the condition has an incident nature it is necessary to recruit participants in a prospective manner. Should the primary care physician undertake this then selection bias is likely to ensue. Therefore, to reduce this possibility an \'independent\' person needs to recruit participants. Consider a recent example of such an approach. In a trial of educating GPs for the identification and treatment of depression in primary care trial participants were recruited by practice receptionists. Because the receptionists from both intervention and control practices had been exposed to the same amount of trial training then the potential for selection bias is reduced, although never eliminated \[[@B18]\]. Discussion ========== The use of cluster randomised trials has significantly increased in medical research in recent years \[[@B19]\]. Despite Lindquist outlining an appropriate approach to the analysis of cluster trials in 1940 \[[@B1]\] -- many fail to undertake the analysis taking the clustering effect into account. Consequently the attention of many medical statisticians has been directed at the appropriate analysis and sample size issues with less attention to more serious problems with the design of cluster trials. Whilst inappropriate analysis will give misleading precision (i.e., smaller confidence intervals and lower p values) it will rarely change the point estimate of a treatment effect. In contrast, bias can give a misleading effect size estimate. Cluster randomised trials are potentially more susceptible to some forms of bias than individually randomised trials. Biased recruitment can be a problem in some cluster randomised trials. One symptom of biased recruitment is differential recruitment rates. However, one trial noted significant selection bias even when there were similar recruitment rates \[[@B10]\]. Therefore, even when recruitment rates appear similar between treatment arms selection bias can be introduced. We have examined the issue of bias in cluster trials by comparing a sample of trials against similar individually randomised studies from a review of hip protectors. This suggested a difference in effect size that was dependent upon the type of study design. However, the sample size was small and there are alternative explanations to the apparent differences in effect sizes: not least the explanation of chance. We have also looked at baseline differences in ages of people in cluster trials that appeared to be free of bias with those that seem to have had bias introduced due to poor methodological application of design. There was a difference in age imbalance, which was suggestive of an interaction with statistical significance of trial results although a formal statistical test failed to show a significance of the difference. Our sample size was relatively small and we could have missed a statistically significant difference through lack of statistical power. Nevertheless, this paper does raise concerns about the design of cluster trials and signals that such trials should be used with caution. If there are important confounding variables, stratification, matching or regression models for clustered data are required. Studies with evidence of biased recruitment might try methods of analysis that allow for observed confounding. For example, if there were imbalances in patient or cluster level covariates between the randomised groups multi-level or hierarchical models explicitly model the treatment effect adjusting for the confounding, provided that there is a fairly large number of clusters. However, even the most sophisticated statistical analysis cannot adjust for the unmeasured or unknown confounder, which is one of the main reasons we undertake random allocation. Therefore, it is crucial that we avoid the introduction of bias into our cluster designs. Future cluster randomised trials should endeavour to either identify participants before randomisation or use an independent person, preferably blind to allocation, to recruit participants. Furthermore, cluster randomised trials ought to be undertaken such that loss to follow-up is similar between groups and intention to treat is always used. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= SH undertook the statistical analysis and wrote sections about statistical methods and implications. SP wrote the first draft and contributed to the original data collection. DT had the original idea of the paper and undertook revisions to the original draft. SW contributed to the original review and collected additional data for the paper. All authors contributed to commenting on drafts of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2288/5/10/prepub>	PubMed Central	2024-06-05T03:55:53.945477	2005-3-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554774/", "journal": "BMC Med Res Methodol. 2005 Mar 2; 5:10", "authors": [ { "first": "Seokyung", "last": "Hahn" }, { "first": "Suezann", "last": "Puffer" }, { "first": "David J", "last": "Torgerson" }, { "first": "Judith", "last": "Watson" } ] }
PMC554775	Background ========== Immunocytochemistry (ICH) is an important method for identification of proteins in cells and in tissues. Since the biological activity of many proteins is dependent on their phosphorylation status, a challenge for immunocytochemistry is to characterize the protein form and not just the total amount \[[@B1]\]. p27^Kip1^(p27) is a key inhibitor of cell division that protects tissues from excessive cell proliferation \[[@B2]\]. As a consequence of an altered balance between synthesis and degradation, the amounts of this protein are abnormally low in advanced and poorly differentiated neoplasms \[[@B3]\]. Since p27 expression is readily assessed by ICH, this protein is a prognostic marker quite popular in histopathology \[[@B4]\]. However, little is known on its in vivoregulation. p27 cellular levels, copious in quiescent cells undergoing terminal differentiation, are scanty in cycling cells \[[@B2]\]; in these cells p27 is phosphorylated on Thr 187 by cyclin-dependent kinase (cdk) 2 in late G1 \[[@B5]\]. This event leads to enhanced ubiquitination and p27 proteolysis by the proteasome, which marks the restriction point and promotes cell proliferation \[[@B5]\]. Therefore un-phosphorylated (\"plain\") p27 is representative of the total protein amounts only in quiescent cells, whereas in cycling cells a fraction of p27 is transiently present in the pThr187 form before degradation \[[@B5]\]. Recently, Montagnoli et alraised an antibody (Ab), specific for pThr187-p27 that was reactive in immunoprecipitates from proliferating cells and negative in quiescent cells \[[@B6]\]. Even more recently this Ab was shown to be reactive also on paraffin sections \[[@B7],[@B8]\]. The present study was undertaken to assess pThr187-p27 Ab staining pattern in a wide range of normal, dysplastic and neoplastic tissues; its expression was correlated to those of MIB-1, a standard marker of proliferation, and of \"plain\" p27. The relationship between the two forms of p27 was also studied at a sub cellular level by double immunofluorescence (IF) and laser scanning confocal microscopy (LSCM). Here we show that p27 expression is not restricted to quiescent cells but that it also occurs in proliferating cellular compartments, where it is detectable by regular ICH only in its pThr187 form. Therefore, to fully assess p27 tissue expression both antibodies should be used. Methods ======= Antibodies ---------- pThr187-p27 was detected by the 71--7100 polyclonal antibody (PcAb) (Zymed Laboratories, San Francisco, CA, USA) and by the sc-16324 PcAb (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). These Ab\'s were raised against a short peptide corresponding to the portion of human p27 containing phosphorylated Thr-187, in order to detect only phospho-p27 and to be unreactive with \"plain\"-p27. The 71--7100 Ab was previously employed in immuno-precipitation experiments by Western blot, \[[@B6],[@B9],[@B10]\] and to immunostain neoplastic and degenerative human tissue \[[@B7],[@B8]\]. \"Plain\" p27 protein levels were detected with the K10125 monoclonal Ab (McAb) from (Transduction Laboratories, Lexington, Ky, USA), and with the rabbit PcAb (C-19) (Santa Cruz Biotechnology). These antibodies were previously shown to share the same staining pattern \[[@B11]\]. MIB-1 McAb from Novocastra (Newcastle upon Tyne, UK) was used to stain proliferating cells and as control of antigenic preservation and of successful antigenic retrieval \[[@B12]\]. Tissues ------- A wide range of normal, dysplastic and neoplastic tissues was obtained from surgical specimens. At least five samples from each type of normal tissue were processed (Table [1](#T1){ref-type="table"}). Dysplastic diseases included five cases of colonic adenoma, five cases of low- and five cases of high grade- squamous intraepithelial lesions (SIL) of the uterine cervix. Several carcinomas were also evaluated, including different tumour types in which p27 down regulation had previously been described, such as invasive squamous cell carcinoma (ISCC) of the oral cavity (n = 7) \[[@B13]\], of the lung (n = 10) \[[@B14]\] and of the uterine cervix (n = 9) \[[@B15]\]; ductal cell carcinoma of the breast (n = 12) \[[@B16]\]; invasive adenocarcinoma of the colon (n = 6) \[[@B17]\] and of the prostate (n = 5) \[[@B18]\]; papillary (n = 6) and anaplastic (n = 5) thyroid carcinoma, \[[@B19]\], glioblastomas (n = 5) \[[@B20]\], and choriocarcinoma (n = 2) \[[@B21]\]. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### pThr187-p27, \"plain\"-p27 and MIB-1 expression in normal tissues. ::: Tissue type Phospho-p27 \"plain\"-p27 MIB-1 ------------------------------------------------------------- ------------- --------------- ------- Normal squamous epitheliumskin, tonsil, larynx, cervix. Parabasal layer +++ \- ++++ intermediate \- ++++ \+ Granular layer \- +++ \- *Germinal centersTonsil, lymphnode, apendix Mantle cells* \- ++++ \- Centroblasts +++ \- ++++ Centrocytes \- +++ \- *BowelCrypt cells Deep* +++ \- ++++ superficial \- ++++ \- Placenta trophoblast +++ \- ++++ syncytiotrofoblast \- ++++ \- *Kidney* +/- +++ +/- *Lung* +/- +++ +/- Thyroid +/- +++ +/- *Prostate* +/- +++ +/- Key: ++++ \>80% of positive cells. +++ 50--80% of positive cells ++ 10--50% of positive cells \+ \<10% of positive cells +/- Only occasional staining \- Negative staining ::: Immunostaining techniques ------------------------- Xylene dewaxed and alcohol rehydrated paraffin sections were placed in Coplin jars filled with a 0.01 M tri-sodium citrate solution, and heated for 3 minutes in a conventional pressure cooker \[[@B15]\]. After heating, slides were thoroughly rinsed in cool running water for 5 minutes. They were then washed in Tris-Buffered Saline (TBS) ph 7.4 before incubating overnight with the specific Ab, diluted as follows: anti-pThr187-p27 (Zymeed) 1:1000; anti-pThr187-p27 (Santa Cruz) 1:200; anti-p27 (Transduction) 1:4000; anti-p27 (Santa Cruz) 1:50; anti-Ki-67 1:50. After incubation with the primary Ab, tissue sections were covered with biotinylated anti-mouse or anti-rabbit immunoglobulins, followed by peroxidase labelled streptavidine (LSAB-DAKO); the signal was developed by using diaminobencidine (DAB) chromogen as substrate. Immunostaining controls ----------------------- pThr187-p27 antibody specificity was controlled by Western blot analysis of MDA MB 468 (breast cancer) and NPA (thyroid papillary cancer) cell lines. These were lysed (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 0.1% Triton X-100, 5 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L Na~3~VO~4~, and 1 mmol/L phenyl methyl sulfonyl fluoride and protease inhibitors) and 20 to 30 μg of proteins were electrophoresed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred onto nitrocellulose membranes. The membranes were first blocked and then incubated with the primary antibody as it follows: anti-pThr187-p27 (Zymeed), 1:2000 and anti \"plain\" p27 (Transduction), 1:3000 for 1 hour at room temperature. To confirm equal loading, membranes were immunoblotted with monoclonal anti b-tubulin antibody (1:1000, Santa Cruz). After three washes, filters were incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies (1:2000; Amersham, Arlington Heights, IL, USA) for 1 hour at room temperature. Detection of immunocomplexes was performed with an enhanced chemiluminescence system (ECL, Amersham). pThr187-p27 immunostaining specificity was assessed by several control experiments performed in parallel, in which the primary Ab was either replaced by a similarly diluted normal rabbit serum, or adsorbed with increasing concentrations of its phospho and dephosphopeptides (up to 0.14 mg mL^-1^). Quantitative study and statistical analysis ------------------------------------------- In the neoplastic cases examined, labelling indices for pThr187-p27, \"plain\" p27 and Ki67/MIB-1 were determined. Adjacent sections were used and counting was performed in similar areas; quantitative analysis performed with a computerised analyser system (Ibas 2000, Kontron, Zeiss) was used to score the nuclei of individual cells for expression of these proteins. As already described, nuclear boundary optical density and Ab threshold were adjusted for each case examined \[[@B15]\]. A minimal threshold was established by counting at least 1000 cells per sample and the results were expressed as a percentage of the total cell population. Statistical analysis was performed by means of SPSS Inc. package. The range of expression of pThr187-p27, \"plain\" p27 and Ki67/MIB-1 for each neoplastic type is reported in table [2](#T2){ref-type="table"}. The relationships among these were analyzed by calculating the nonparametric Spearman R coefficient. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Range of positive cells for pThr187-p27, \"plain\"-p27 and MIB-1 expression in different type of carcinomas ::: Histotype pThr187-p27 \"plain\" p27 MIB-1 ------------------------------------- ----------------- ------------------- ----------- ISCC cervix n = 9 45--60 5--30 50--78 ISCC oral n = 7 40--70 5--15 35--80 ISCC lung n = 10 15--60 5--70 30--70 Thyroid, papillary carcinoma n = 6 1--3 5--60 2--4 Thyroid, anaplastic carcinoma n = 5 23--45 5--11 41--55 Breast carcinoma n = 12 3--55 5--70 5--80 Colonic carcinoma n = 6 30--45 5--80 45--80 Prostate carcinoma n = 5 2.5--10 30--75 4--15 Glioblastoma n = 5 30--50 10--27 50--63 Chorioncarcinoma n = 2 45--55 3--7 60--80 ::: Double Immunofluorscence (IF) staining and Laser Scanning Confocal Microscopy (LSCM) ------------------------------------------------------------------------------------ Tissue sections from both normal and neoplastic specimens were also stained using double IF labelling for pThr187-p27 and \"plain\" p27, according to previous studies with minor modifications \[[@B22]\]. The primary anti pThr187-p27 (Zymeed) was incubated (1/1000) for 1 hour, followed by incubation with swine anti rabbit HRP (1/200) (Dako) and Cy3 tyramide amplification (Perkin Elmer Life Sciences). To avoid cross-reactivity due to residual HRP, sections were incubated with 0.3% H~2~O~2~for 1 hour. The primary mouse McAb anti p27 (1/4000) was detected by overnight incubation at 4°C, followed by goat anti mouse HRP (1/200, DAKO). After washing in TBS, sections were incubated with fluorescein isotiocyanate (FITC) tyramide amplification (Perkin Elmer Life Sciences) and mounted with Vectashield-DAPI mounting medium (Vector). Control sections in which the second primary Ab was omitted were included to ascertain destruction of peroxidase activity. Single IF stained sections as well as colorimetric immunostaining were used as controls of signal specificity. Slides were examined with a Leica TCS SP2 UltraSpectral LaserScan Confocal microscope. FITC was excited at 488 and detected with a bandpass 500 to 550 nm. Cy3 was excited at 514 nm and detected with a bandpass 580 to 655 nm. Series of images were processed with the Leica confocal soft package. Confocal images were captured and imported into Adobe Photoshop 7 (Adobe Systems, Mountain View, CA) and processed with an eMac personal computer. Results ======= pThr187-p27 expression in normal tissues ---------------------------------------- Similar results were obtained with both anti pThr187-p27 Ab\'s, with only proliferating tissue compartments being stained. This pattern overlapped to that of Ki67/MIB-1, whereas it was different to that shown by both Mc and Pc Ab\'s directed to \"plain\" p27 (Table [1](#T1){ref-type="table"}). Stratified squamous epithelium showed the same staining pattern in skin, tonsil, larynx and uterine cervix. In analogy to the Ki67/MIB-1 staining, pThr187-p27 was expressed by parabasal cells (Figure [1A](#F1){ref-type="fig"}); on the contrary, nuclei of the more superficial layers showed intense \"plain\" p27 nuclear labelling, as already described \[[@B13],[@B15]\]. In small intestine and colon (Figure [1B](#F1){ref-type="fig"}), in analogy to MIB-1, pThr187-p27 showed positivity in nuclei of deep crypt cells. On the contrary, the upper half of the crypts was stained by \"plain\" p27 \[[@B17]\]. In germinal centres of normal lymph nodes, tonsil and appendix the expression of the phosphorylated form of p27 was clearly detectable (Figures [1A](#F1){ref-type="fig"} and [1C](#F1){ref-type="fig"}). Within the germinal centres, pThr187-p27 showed a signal distribution similar to that of the Ki-67 protein; the outer rim of centroblast and mitotic cells were strongly positive, whereas centrocytes were less frequently labelled. This pattern of expression was opposite to the one identified for \"plain\" p27: negative staining for centroblasts and strong staining in most centrocytes, mantle cells and interfollicular small lymphocytes \[[@B11]\]. Trophoblastic villi of placenta showed pThr187-p27 intense staining only in the cytotrophoblastic layer and not in the syncytiotrophoblastic layer (Figure [1D](#F1){ref-type="fig"}). On the contrary \"plain\" p27 showed intense staining only in the syncytiotrophoblast overlying the villus, as already described \[[@B21]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### p27 expression was detected by the pThr187 Ab p27 only in the proliferative compartments of normal tissues. In tonsil (A) both parabasal squamous cells and lymphoid germinal cells were stained. In intestinal epitehlium only the nuclei of deep crypt cells were stained (B). Within the germinal centres, the outer rim of centroblasts and mitotic cells were strongly positive for pThr187-p27, whereas centrocytes were less labelled (C). Trophoblastic villi of placenta show pThr187-p27 intense staining only in the cytotrophoblastic layer and not in the syncytiotrophoblastic layer (D). ::: ![](1472-6890-5-3-1) ::: In other normal tissues, comprising breast, lung, kidney, pancreas, prostate, thyroid and parathyroid, \"plain\" p27 was expressed by the vast majority of cells, whereas pospho-p27 single labelled cells could be made out by meticulous scrutiny; in these tissues also MIB-1 staining was sporadic. pThr187-p27 expression in dysplastic and neoplastic tissues ----------------------------------------------------------- As examples of dysplastic lesions, the expression of the pThr187-p27 was assessed in low-and in high grade squamous intraepithelial lesions (SIL) of the uterine cervix and in colonic adenoma. According to the relationship between phospho-p27 expression and proliferation, in low grade SIL the dysplastic basal and parabasal cells were in most instances positive for the expression of both pThr187-p27 (Figure [2A](#F2){ref-type="fig"}), and Ki-67; these were instead negative for \"plain\" p27 whose staining was confined to the intermediate and superficial squamous cells, as described \[[@B15]\]. In high grade SIL, the expression of phospho-p27 correlated well with the extent of the dysplastic cell population (Figure [2B](#F2){ref-type="fig"}), whereas \"plain\" p27 reactivity was restricted to very superficial layers displaying squamous differentiation. In colonic adenoma pThr187-p27 positive cells were randomly located throughout the crypts and in contrast to that seen in normal mucosa, also superficial cells were stained. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The relationship between phospho-p27 expression and proliferation was evident both in low- (A) and high- (B) grade SIL, with its expression correlating well with the extent of the dysplastic cell population. Intense staining for phospho-p27 was also observed in the neoplastic cells of colonic (C) and lung (D) adenocarcinoma, in cervical squamous invasive carcinoma (E) and in choriocarcinoma (F) ::: ![](1472-6890-5-3-2) ::: A wide range of different tumours was examined, in order to assess whether staining for phospho-p27 may yield diagnostic information in addition to those provided by \"plain p27\" (Table [2](#T2){ref-type="table"}). As a general rule, the expression of the two forms of p27 was alternative and dependent on the degree of tumour differentiation, recapitulating the pattern featured by normal and dysplastic tissues; phospho-p27 strongly labelled aggressive tumours, whereas \"plain p27\" staining was only retained by well differentiated tumours. Thus, poorly differentiated and highly proliferating adenocarcinoma of the breast, colon and prostate were strongly labelled by the pThr187-p27 Ab, (Figure [2C--D](#F2){ref-type="fig"}) whereas \"plain p27\" staining was only retained by well differentiated neoplasms. The above staining differences between the two forms of p27 were also evident in squamous cell carcinomas. Poorly differentiated neoplasms, composed of nests of small undifferentiated cells with high MIB-1 index and minimal keratinisation, showed intense staining for pThr187-p27 (Figure [2E](#F2){ref-type="fig"}) whereas \"plain p27\" labelled those neoplasms showing abundant keratin, squamous pearl formation and low mitotic activity. Similarly, anaplastic thyroid carcinoma showed pThr187-p27 staining stronger than papillary carcinomas, while in this latter \"plain p27\" was prevalent (Table [2](#T2){ref-type="table"}). An intense pThr187-p27 expression was also found in choriocarcinoma (Figure [2F](#F2){ref-type="fig"}), in which the level of \"plain-p27\" staining was instead low. The Spearman\'s correlation coefficient for continuous variables revealed a positive and a significant correlation between pThr187-p27 staining and MIB-1 expression (Spearman R = 0.88; p \< 0,001). On the contrary both pThr187-p27 and \"plain\" p27 (Spearman R = -0.61; p \< 0,001) and MIB1 and \"plain\" p27 (Spearman R = -0.55; p \< 0,001) showed significant inverse correlations. Immunostaining controls ----------------------- Western blot analysis of MDA MB 468 (breast cancer) and NPA (thyroid papillary cancer) cell lines revealed a single anti-pThr187-p27 band, whose molecular weight (27 kDa) corresponded to that showed by \"plain\"-p27 (Fig. [3A](#F3){ref-type="fig"}). pThr187-p27 immunostaining specificity was confirmed by: (i) the disappearance of the signal when the primary Ab was replaced by a similarly diluted normal rabbit serum; (ii) the progressive signal quenching due to the competitive inhibition between the increasing concentrations of the phosphopeptide and the phosho-p27 Ab. (Fig. [3 B--C](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Western blot analysis of MDA MB 468 (breast cancer) and NPA (thyroid papillary cancer) cell lines revealed a single anti-pThr187-p27 band, corresponding to that shown by the regular p27 antibodies. (A). pThr187 staining of parabasal squamous cells (B) abolished by absorption of the antibody with the immunizing peptide. ::: ![](1472-6890-5-3-3) ::: ### Double IF & LSCM studies Tissue sections from both normal and neoplastic specimens were also stained using double IF labelling for pThr187-p27 and \"plain\" p27 and analyzed by LSCM. The use of an ultra sensitive detection system based on tiramide signal amplification for IF staining, revealed a more precise pattern of protein expression and distribution, slightly different to results above shown of regular colorimetric IHC. In proliferative compartments of normal tissues the cells frequently showed simultaneous expression of the two forms of p27; in these cells, pThr187-p27 was more localized as an inner rim along the nuclei membrane, whereas \"plain\" p27 was more centrally located in the same nuclei with an intermediate area of colocalization (Fig. [4A](#F4){ref-type="fig"}). Similarly neoplastic nuclei also showed simultaneous expression of the two p27 forms, frequently displaying high amounts of pThr187-p27 throughout the whole nucleoplasm, colocalizing with \"plain\" p27. (figure [4B](#F4){ref-type="fig"}). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Coexpression of the forms of p27 in lymphoid germinal center (A) and in glioblastomas (B). Simulataneous detection of p27 by both regular (green) and pThr187 (red) antibodies. Signal generated by the two antibodies Yellow areas (arrows) showed the colocalitation of the signal generated by both antibodies. ::: ![](1472-6890-5-3-4) ::: Discussion ========== p27 is an approved marker for quiescent cells, extensively used in histopathology and cancer research. However here we showed that an unusual p27 expression pattern may be obtained when only its portion phosphorylated on threonine 187 is stained. By using a specific phosphorylation site antibody, p27 expression may be detected in the proliferative compartments of squamous and intestinal epithelia, of lymph node germinal centres, of trophoblastic villi, of cervical and colonic pre-invasive lesions and of several carcinomas types. These results are similar to those reported in vitroby Montagnoli et al, who also detected p27 expression in proliferating cells by the pThr187-p27 Ab \[[@B6]\]. The same antibody, here, employed to stain a wide range of normal, dysplastic and neoplastic tissues yielded a pattern similar to that of Ki-67 (Spearman R = 0.88; p \< 0,001), underlining the close association between cell proliferation and the expression of that portion of p27 targeted for degradation. Since pThr187-p27 Ab should react with cells from late G1 through G2-M, whereas MIB-1 stains throughout the entire cell cycle, it is also conceivable that the pThr187-p27 Ab stained a lower percentage of cells than MIB-1/Ki-67 in both normal (Table [1](#T1){ref-type="table"}) and neoplastic tissue. (Table [2](#T2){ref-type="table"}) In this study we showed that p27 expression does not feature only one pattern, but that two different staining may be obtained by the use of either regular or pThr187 antibodies; in fact, when the overall data relative to the expression of p27 by both antibodies in tumours were analysed, a significant inverse correlation between the two forms was found (Spearman R = -0.61; p \< 0,001). This data reflects the different relationship between each form of p27 and tissue differentiation:aggressive tumours were strongly labelled by phospho-p27 Ab, whereas plain-p27 was prevalent in well differentiated tumours. Thus the ratio between the two forms of p27, more than the total protein amounts, could yield information on tumour differentiation and behaviour. Since cdk2 activity is low in differentiating cells and p27 is only present in its un-phosphorylated form, it is reasonable that squamous superficial layers, upper half of the intestinal crypts, lymphoid mantle cells and syncytiotrophoblastic cells only react with the regular antibodies. More intriguing is the search for the reasons explaining why in proliferating cells p27 is detected by the pThr187-Ab and missed by the regular antibodies. The possibility of an aberrant cross-reactivity by the pThr187-Ab was excluded both by Western blot analysis and by phosphopeptide adsorbtion. Montagnoli et al, who also confirmed antibody specificity by biochemical assays, showed that p27 expression may be better detected by using both antibodies than either alone and that there is a portion of p27 that is only recognizable by the anti-phospho Ab \[[@B6]\]. Thus, in proliferating cells anti-pThr187 Ab may be more efficient than the regular p27 antibodies, being protein levels low and mainly present in their phosphorylated form. To test this hypothesis, the sensitivity of the p27 detection by the regular antibodies was increased by the use of an ultra sensitive detection system based on tiramide signal amplification and observed at high resolution by laser scanning confocal microscopy. The results obtained by this approach were slightly different to those obtained by colorimetric IHC, as p27 was simultaneously detected by both antibodies in the same nuclei of the proliferating compartments; in particular the signal generated by both antibodies showed specific colocalitation by double immunofluorescence. Conclusion ========== In this study we have reported the following observations: (1) pThr187-p27 Ab selectively stains proliferating cells; (2) this staining does not identify the \"plain\" p27 protein, expressed by quiescent cells. Therefore, the pThr187-p27 antibody is a useful tool to study p27 in vivo regulation. Indeed, the pattern of expression observed in this study, strongly suggest that the anti-pThr187-p27 Ab identifies proliferating cells. Considering the importance of the different biological functions of p27: regulator of cell growth, cell differentiation, contact inhibition, apoptosis, protection against immunological aggression, and protection against environmental stress, among others, the combined use of both \"plain\" and pThr187 p27 antibodies will be a useful tool to better characterize the biology of these conditions. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= GT conceived the study and wrote the manuscript; JCM carried out the LSCM study; AC scored the immunostaining; AI coordinated the study; MR performed the peptide experiments; PZ evaluated the tumour pathological features; IM participated in its design and coordination and helped to draft the manuscript; MLM carried out the double immunostainings; DC carried out the Western blot analysis; EAP performed the statistical analysis. LP participated in the design of the study and evaluated the results. 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/1472-6890/5/3/prepub> Acknowledgements ================ We are grateful to Ms M A Ollacarizqueta and Ms D Gomez Donaire for their excellent technical assistance with LSCM studies and double IF staining respectively. This study was co-financed by PRIN and by Italia- Spagna Azioni Integrate grants from the Ministry of Università e Ricerca Scientifica (MIUR) and from Regione Camparia	PubMed Central	2024-06-05T03:55:53.947725	2005-2-23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554775/", "journal": "BMC Clin Pathol. 2005 Feb 23; 5:3", "authors": [ { "first": "Giancarlo", "last": "Troncone" }, { "first": "Juan C", "last": "Martinez" }, { "first": "Antonino", "last": "Iaccarino" }, { "first": "Pio", "last": "Zeppa" }, { "first": "Alessia", "last": "Caleo" }, { "first": "Maria", "last": "Russo" }, { "first": "Ilenia", "last": "Migliaccio" }, { "first": "Maria L", "last": "Motti" }, { "first": "Daniela", "last": "Califano" }, { "first": "Emiliano A", "last": "Palmieri" }, { "first": "Lucio", "last": "Palombini" } ] }
PMC554776	Background ========== Rates of DNA sequence evolution vary among taxa and among genes, and the causes of this variation are many. In some cases, generation time has been shown to be correlated with rates in plants. For example, annual plants can sometimes have higher rates of DNA evolution than perennials \[[@B1]\]. In one study, it was shown that long-lived woody grasses exhibit slower rates than short-lived herbaceous ones \[[@B2]\]. However, a more extensive set of 33 phylogenetically independent comparisons failed to find a generation time effect for plants in general \[[@B3]\]. A useful method for distinguishing among the potential causes of rate variation is to separately examine nonsynonymous rates (r~N~) and synonymous rates (r~S~). For example, when r~N~increases relative to r~S~, relaxation of purifying selection is a possible explanation. However, when r~S~increases, but the r~N~/r~S~ratio is not greatly affected, then an increase in the mutation rate is a possibility. An example of this is Plantagomitochondrial DNA \[[@B4]\]. However, a decrease in DNA repair efficiency could also explain such a change. In addition, population processes, such as genetic drift could play a role. Reduced effective population size (N~e~) can increase the fixation rate of neutral and slightly deleterious mutations. Thus, if slightly deleterious mutations are common, both r~S~and r~N~are expected to be higher when N~e~is low \[[@B5],[@B6]\]. One expectation of this drift-based hypothesis is that r~N~will vary from protein to protein, as each protein will have different functional constraints and thus a different proportion of slightly deleterious mutations. In contrast, r~S~is expected to be similar among proteins when the cause is a change in mutation rate or repair efficiency\[[@B7]\]. During founder-effect speciation, genetic drift can be expected to increase the substitution rate, even at silent sites. This has been called the speciation-rate hypothesis \[[@B8]\]. For example, speciation rate has been used to explain the difference in non-coding DNA substitution rates between the sister genera Utriculariaand Pinguiculain the plant family Lentibulariaceae \[[@B9]\]. Rates can also vary if the underlying mutation rate varies \[[@B10],[@B11]\] or if DNA repair is impaired \[[@B12],[@B13]\]. Although rates themselves are hard to measure, the number of synonymous (d~S~) and nonsynonymous (d~N~) substitutions can be measured and are used to compare rates and calculate rate ratios. Epifagus virginiana, a nonphotosynthetic plant, has an increased rate of sequence evolution for plastid DNA in general \[[@B14]-[@B17]\] and relative rates tests of the plastid rps2gene indicate a significant increase for both d~N~and d~S~. This suggests that purifying selection is at least partially relaxed [and]{.underline} that there has been an increase in the rate at which mutations are entering the population in this species, due to increased mutation rate or lax DNA repair. MatK, another plastid gene, is characterized by a partial relaxation of purifying selection in the clade containing Epifagus, Orobancheand Boschniakia\[[@B18]\]. In this paper, we explore rate variation in E. virginianaand 38 of its relatives for three plastid genes: rps2, matK, and rbcL. Each of these genes is present in photosynthetic relatives of Epifagus, is accelerated (or even lost) in Epifagus or related parasitic plants. Although plastid encoded, the three genes encode proteins that participate in different processes in the plastid. rps2encodes the ribosomal protein S2 in small subunit ribosome, matKis an intron maturase, and rbcLencodes the large subunit in the CO~2~-fixing enzyme RUBISCO. We ask several questions: When does the rate increase observed in Epifagusbegin, relative to the evolutionary loss of photosynthesis? What are the causes? Relaxation of constraint? More mutations entering the population? Are these patterns consistent across multiple plastid genes? Results ======= Phylogenies of the Orobanchaceae and relatives were constructed using maximum parsimony (MP) and maximum likelihood (ML). The MP analysis discovered four most parsimonious trees of 3817 steps, with CI = 0.6275, CI (excluding uninformative characters) = 0.5118, and RC = 0.3888. The strict consensus tree was unresolved as to the position of Schwalbearelative to the Alectra-Orobancheclade, the Bartsia-Melampyrumclade and the Castilleja-Pedicularisclade. It was also unresolved concerning the relationships among the outgroups Mimulus, Kigelia, Hemimeris, Verbascum, Antirrhinumand Veronica. The ML analysis found two trees, with -ln likelihood values of 24525.88663. The strict consensus of these trees was unresolved, but in a different place, regarding the position of the Cistanche-Epifagusclade. When the MP consensus and the ML consensus were combined into a semistrict consensus tree, a completely resolved tree resulted. This tree is shown in Fig. [1](#F1){ref-type="fig"}. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### MP/ML consensus tree with MP bootstrap values ::: ![](1471-2148-5-16-1) ::: All three gene trees exhibited statistically significant rate heterogeneity (p \< 0.0005), as assessed by the Kishino-Hasegawa (K-H) test \[[@B19]\]. Synonymous and nonsynonymous branch lengths for each of the three genes are shown reconstructed in Fig. [2](#F2){ref-type="fig"}. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Nonsynonymous and synonymous rates of change in three genes.Nonphotosynthetic taxa are named in bold. (A) rps2nonsynonymous branch lengths. (B) matKnonsynonymous branch lengths. (C) rbcLnonsynonymous branch lengths. (D) rps2synonymous branch lengths. (E) matKsynonymous branch lengths. (F) rbcLsynonymous branch lengths. Taxa with rbcLpseudogenes are identified with Ψ. Uncertain pseudogene status is indicated by \"Ψ?\" \[20, 26\]. ::: ![](1471-2148-5-16-2) ::: The correlation analyses show that there is a higher correlation of synonymous evolution across genes than nonsynonymous evolution (Fig. [3](#F3){ref-type="fig"}). They also show that within rps2and rbcLsynonymous and nonsynonymous evolution is poorly correlated, but in matK, the correlation is better (Fig. [4](#F4){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Correlations across each possible pair of genes for synonymous and nonsynonymous rates.(A) rps2versus matK. (B) rps2versus rbcL. (C) matKversus rbcL. All nonsynonymous comparisons involving rbcLpseudogenes are indicated with a hollow triangle. ::: ![](1471-2148-5-16-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Correlation plots of synonymous and nonsynonymous rates within each gene.(A) rps2rates. (B) matKrates. (C) rbcLrates. In 4C, the Epifaguspseudogene has been excluded and the other pseudogenes are indicated with a hollow triangle. ::: ![](1471-2148-5-16-4) ::: Some of the non-photosynthetic plants, (Epifagus, Cistanche, and the Orobanchespecies) have increases in both synonymous and nonsynonymous rates. Rates are not, however, increased in Boschniakia, Harveya, Hyobanche, Lathaea, Alectra orobanchoides, and Striga gesnerioides. Synonymous rates vary markedly among taxa. For example, the branches leading to Epifagusare at least two or three times as long as those for most of the photosynthetic taxa. Despite this among-taxon variation, the pattern for each gene is strikingly similar (Fig. [2D, E, F](#F2){ref-type="fig"}). For example, sister taxa (e.g. Veronicaand Antirrhinum, Euphrasiaand Panetucellia/Tozzia, Epifagusand Cistanche, O. fasciculataand O. corymbosa) usually show an identical pattern of who is faster than whom. This indicates that the causes of synonymous rate variation are affecting the whole plastid genome in a similar way. There is even more extensive variation in nonsynonymous rates, both among taxa and among genes. This is not surprising because these genes have different functions and some of the taxa are photosynthetic while others are not. The scale bars in Figs. [2A, B](#F2){ref-type="fig"}, and [2C](#F2){ref-type="fig"} show the overall differences in nonsynonymous rates. MatKis much more rapidly evolving than the other two genes for nearly all taxa, but Epifagus\' rbcLpseudogene has a similar rate. When looking at taxa across genes, there is much less consistency than with the synonymous rates. There are some big differences, such as the branch lengths for Striga, Cycniumand Schwalbea, and the Euphrasiaspecies. Overall the picture is more complex, as could be expected if there are many causes of variation, differing from taxon to taxon and gene to gene. The pattern of matKrate variation is very similar in the synonymous and nonsynonymous figures. This fits well with the fact that it is less constrained overall, as can be seen by comparing the scale bars in Figs. [2A, B](#F2){ref-type="fig"}, and [2C](#F2){ref-type="fig"}. Rates were compared by using two categories at a time and testing for significant differences using likelihood ratio tests. These tests are summarized in Table [2](#T2){ref-type="table"}. In general, it was found that the nonphotosynthetic plants have higher synonymous and nonsynonymous rates of change, although when tested separately, it was found that Harveya, Hyobancheand Boschniakiado not have higher synonymous rates. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Specimens used for DNA sequencing, with GenBank accession numbers for rps2, matK, and rbcL sequences. ::: GenBank accession numbers ------------------------------- ------------------------------- ------------------- ---------- Alectra orobanchoides U48741 AF489960 AF026819 Alectra sessiliflora U48742 AF051977 AF026820 Antirrhinum majus U48766 AF051978 L11688 Bartsia alpina U48751 AY849600 AF190903 Boschniakia hookeri U48757 AF051979 AF026817 Boschniakia strobilacea U48758 AF051980 AF26818 Castilleja lineariifolia U48739 AF051981 AF026823 Cistanche phelypaea AY849597 AF056149 AY849862 Cycnium racemosum U48745 AY849601 AF026826 Epifagus virginiana EPFCPCG EPFCPCG, AF051982 EPFCPCG Euphrasia disjuncta AY849598 AY849602 AY849863 Euphrasia spectabilis U48752 AY849603 AY849864 Harveya capensis AF055142 AF489961 AF026829 Harveya purpurea U48749 AF051984 AF026830 Hemimeris sabulosa U48765 AF051985 AF123668 Hyobanche atropurpurea AY849599 AF051986 AF026831 Hyobanche sanguinea U48750 AF051987 AF026832 Kigelia africana U48764 AF051988 AF102648 Lathraea clandestina U48755 AF051989 AF026833 Lindenbergia phillipinensis AF055151 AF051990 AF123664 Melampyrum sylvaticum AF055148 AF051991 Melampyrum lineare AF026834 Melasma scabrum U48743 AY849604 AF190904 Mimulus aurantiacus AF055154 AY849605 AF026835 Nicotiana tabacum Z00044 Z00044 Z00044 Orobanche caryophyllacea AF055145 AF051992 AY582187 Orobanche cernua AF055147 AF056147 U73968 Orobanche corymbosa U48760 AF051993 U73969 Orobanche fasciculata AF055143 AF051994 U73970 Orobanche hederae AF055146 AF051995 AF078682 Orobanche ramosa U48761 AF056148 U73971 Parentucellia viscosa U48753 AY849606 AY849865 Paulownia tomentosa AF055255 AF051997 L36447 Pedicularis foliosa U48740 AF489959 AF026836 Schwalbea americana AF055150 AF051998 AY849866 Seymeria pectinata AF055141 AF051999 AF026837 Striga asiatica U48746 AF052000 AF026838 Striga gesnerioides U48747 AF489963 AF026839 Tozzia alpina U48754 AF052001 AF026843 Verbascum blattaria U48763 Verbascum thapsus AF052002 L36452 Veronica arvensis U48768 AF052003 Veronica persica L36453 Taxonomic authorities, localities and voucher information can be found in \[25\], with the exception of Euphrasia disjunctaFernald & Wiegand, Hyobanche atropurpureaBolus., and Veronica persicaPoir. ::: Purifying selection is relaxed in the nonphotosynthetic plants for all three genes. The test values are as follows. matK: 2×LR = 10.67, df = 1, p = 0.001; rbcL: 2×LR = 31.3, df = 1, p = 2.2 × 10^-8^; rps2: 2×LR = 8.56, df = 1, p = 0.00343. Another way to describe the difference in the pattern of synonymous and non-synonymous rates is to say that the former are more correlated across genes. This can be seen in Fig. [3](#F3){ref-type="fig"}, which shows plots comparing two genes at a time. In comparisons including rbcL nonsynonymous rates, the data point from the Epifaguspseudogene has been excluded. Its unconstrained evolution is not typical of \"nonsynonymous\" change and its position on the plot made it an extreme outlier with an enormous influence on the regression line. For rps2and rbcL, the synonymous plots are more highly correlated, whereas for matK, which is relatively unconstrained, they are about the same. Discussion ========== The dramatic rate increase observed in Epifagus\[[@B14]\], with branches 5--10 times as long as other taxa, can now be seen to have begun earlier in the history of the Orobanchaceae. It is shown to be composed of increases in both synonymous and nonsynomymous rates. The general pattern is that many of the non-photosynthetic plants, such as Epifagus, Cistanche, and the Orobanchespecies, have increases in both synonymous and nonsynonymous rates, indicating that both (1) selection is relaxed, and (2) there has been a change in the rate at which mutations are entering the population in these species. However, rate increases are not immediate upon loss of photosynthesis, since we do not see increases in Boschniakia, Harveya, Hyobanche, Lathaea, Alectra orobanchoides, and Striga gesnerioides. This pattern is similar to that found using smaller data sets \[[@B15],[@B20]\]. Separate analyses of synonymous and nonsynonymous rates give us some insight into potential mechanisms. The speciation rate hypothesis predicts that more speciose clades should have a faster r~s~(and therefore larger d~s~) than a species-poor sister group. This was suggested as a cause of rate variation in non-coding DNA in the Lentibulareaceae \[[@B9]\]. In this study, not all genera have been sampled, and those that have are often represented by one or two species. In addition, some genera may not be monophyletic (for example, Orobancheis not). Thus, accurate numbers of species cannot be assigned to individual branches or clades. However, a few things can be noted. Euphrasia, with \~170 spp., is clearly more speciose than its sister group, with 3 spp. It has a somewhat faster d~s~. However, the Schwalbealineage, with a single species, has a fairly high d~s~. Its position is not certain, but its sister group is probably the Bartsia-- Melampyrumclade (\>300 spp.), the Castilleja-- Pedicularisclade, (\>700 spp.), or the union of the two. These groups have do not have dramatically higher d~s~values; in fact the Castilleja-- Pedicularisclade\'s value is slightly lower. Differences in generation time may play some role in the observed rate variation. However, as was found previously \[[@B15]\], the pattern is not clear. Since most of the genera sampled in this study contain both annuals and perennials, it is likely that most branches on the tree actually represent a combination of annual and perennial evolutionary history. However, there are some intriguing details that might merit further study. The clade containing Bartsia, Euphrasiaand Melampyrumcontains mostly annuals \[[@B21]\] and has some high d~s~values, as one would expect from a generation time effect. However, both Euphrasiaand Melampyrumcontain almost exclusively annuals and have very different rates. Likewise, the large clade containing Boschniakiaand Epifaguscontains mostly annuals and has an overall high d~s~. The perennials Boschniakiaand Cistanchehave lower d~s~than their sister taxa, which also supports the generation time hypothesis, but there is as much variation among categories (perennial, annual) as between categories. Conclusions =========== The distinctive pattern of rate increases in Orobanchaceae has at least two causes. It is clear that there is a relaxation of constraint in many (though not all) non-photosynthetic lineages. However, there is also some force affecting synonymous sites as well. At this point, it is not possible to tell whether it is generation time, speciation rate, mutation rate, DNA repair efficiency or some combination of these factors. Clearly, generating additional data from nuclear and mitochondrial genes would help us to more clearly distinguish among these hypotheses. Some of the above-mentioned hypotheses (generation-time, speciation rate) would be expected to affect nuclear and mitochondrial genomes in a similar fashion, whereas factors affecting mutation rate or efficiency of DNA repair would not, as these process involve different, though perhaps overlapping, sets of enzymes in each of the three genomes \[[@B22]-[@B24]\]. Methods ======= Sampling -------- We sampled 15 photosynthetic and 16 nonphotosynthetic Orobanchaceae, and eight outgroup taxa. The specimens used and their GenBank accession numbers are given in Table [1](#T1){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### P-values of the likelihood ratio tests. ::: rps2 matK rbcL --------------------- --------------- --------------- -------------- -------------- --------------- ----------- ------------ -------------- --------------- r~N~ r~S~ ratio (ω) r~N~ r~S~ ratio (ω) r~N~ r~S~ ratio (ω) photo. vs nonphoto. 1.4 × 10^-11^ 0.0014 0.0034 0 0.077 (NS) 0.001 0 2.3 × 10^-8^ 2.2 × 10^-8^ photo. vs OEC 0 6.3 × 10^-10^ 7.2 × 10^-8^ 0 7.6 × 10^-14^ 0.0038 0 0 2.7 × 10^-11^ photo. vs HHB 0.41 (NS) 0.0066 0.24 (NS) 1.4 × 10^-6^ 4.5 × 10^-9^ 0.23 (NS) 0.076 (NS) 0.0011 8.1 × 10^-5^ The two categories at left were compared for nonsynonymous rate differences (r~N~), synonymous rate differences (r~S~) or rate ratio (ω) differences. \"Photo.\" refers to photosynthetic branches, \"nonphoto.\" refers to nonphotosynthetic branches, \"OEC\" refers to Orobanche, Epifagusand Cistanchebranches, and \"HHB\" refers to Harveya, Hyobancheand Boschniakiabranches. ::: Amplification and sequencing ---------------------------- We amplified and sequenced rps2as in \[[@B15]\], matKas in \[[@B25]\], and rbcLas in \[[@B26]\]. A total of 15 new sequences were generated for this study, including 3 rps2, 7 matK, and 5 rbcLsequences. Alignment --------- The rps2alignment was simple, containing only two small indels. For matKand rbcL, a search for the best alignment was conducted using Clustal X and a variety of alignment parameters. Alignments were evaluated according to the following optimality criterion: whichever alignment yields the MP tree(s) with the highest consistency is considered the best alignment\[[@B27]\]. For alignment assessment, MP analyses with and without indel characters were used. When used, indel characters were generated with the program GapCoder \[[@B28]\](available from <http://www.tufts.edu/vet/richlab/young/GapCoder>), which uses the simple indel coding method of Simmons and Ochoterena \[[@B29]\]. The rescaled consistency (RC) index \[[@B30]\] of the resulting parsimony analyses was used to assess alignment optimality, with one exception: very low gap opening penalties (GOP), such as 3 or less were excluded. These low GOP values lead to inflated RC values, due to the large numbers of gaps, which reduce the treelength and the homoplasy. rps2had just one small indel and was aligned by eye. For matK, the optimal computer alignment was generated using GOP = 5 and gap extension penalty (GEP) = 1. Transitions were weighed the same as transitions. The RC from the analysis with indel characters included was 0.3693. The RC without indel characters was 0.3759. The alignment was then adjusted by eye. This final alignment yielded RC values of 0.3878 (indel characters included) and 0.3763 (indel characters excluded). For rbcL, the optimal computer alignment was generated using GOP = 5 GEP = 3. Transitions were weighed the same as transitions. The RC from the analysis with indel characters included was 0.4679. The RC without indel characters was 0.4437. The alignment was then adjusted by eye. This final alignment yielded RC values of 0.4635 (indel characters included) and 0.4499 (indel characters excluded). Phylogenetic analyses --------------------- For rps2, positions homologous to positions 48--660 of the Nicotianagene were used. For matK, the entire gene was used. For rbcL, positions homologous to Nicotianagene positions 5--1325 were used. The three genes were then combined into a single data set. PAUP\* 4.0b8 \[[@B31]\] was used to conduct a MP heuristic search, including indel characters, and using Nicotiana tabacumas the outgroup taxon, TBR branch swapping and 100 random addition replicates. Bootstrap analyses were conducted with the same settings, except with only 40 random addition orders. 500 bootstrap replicates were performed. ML analyses excluded indels. Using the hLRT method of the program ModelTest 3.06 \[[@B32]\], the ML model of GTR+G was selected as the best evolutionary model for the combined data set. Base frequencies (A = .298, C = .177, G = .203) and substitution rates (A-C = 1.51, A-G = 2.11, A-T = 0.264, C-G = 0.788, C-T = 2.62, G-T = 1) were obtained from the MP trees. Among-site variation was included in the model, based on a gamma distribution with four categories. A heuristic search was conducted, similar to the MP search, but with only 10 random addition replicates, each limited to the examination of 5000 rearrangements. ### Rates of DNA change Overall rate heterogeneity was assessed using the K-H test as implemented in PAUP, using the same ML analyses, except that the starting tree was a neighbor-joining tree and the analysis was limited to 40 rearrangements. Nonsysnonymous and synonymous changes were reconstructed on branches using the codon-based likelihood model of Muse and Gaut \[[@B33]\], as implemented in HYPHY for MacOS, ver. 0.95 beta \[\[[@B34]\] 2004\], available at <http://www.hyphy.org>. At least nine of the rbcL \"genes\" are probably pseudogenes. These are indicated in Figure [2C](#F2){ref-type="fig"}. Seven of these have already been discussed elsewhere \[[@B20]\]. The Orobanche caryophyllaceaeand Cistanche phelypaea\"genes\" have internal stop codons and thus are probably also pseudogenes. There may also be other pseudogenes with intact ORFs, making their pseudogene status less obvious \[[@B35]\]. Once a pseudogene is formed, it is no longer constrained for a protein function, so synonymous and nonsynonymous changes can no longer be formally defined. Moreover, changes that would have been synonymous and nonsynonymous are now expected at equal rates. Thus, by including these sequences in the tests, we get additional evidence that constraint is relaxed in nonphotosynthetic plants. Therefore, even for the pseudogenes, we have still calculated the synonymous and nonsynonymous rates separately, assuming a reading frame based on alignment to the other genes in the data set, and have indicated the pseudogenes in Fig. [2C](#F2){ref-type="fig"}. Rate increases were compared among categories of taxa (such as photosynthetic and non-photosynthetic), using d~N~, d~S~, and the d~N~/d~S~ratio (ω) in likelihood ratio tests \[[@B36]\]. These tests were conducted using HYPHY and the category assignments of the branches are those shown in Figure [1](#F1){ref-type="fig"}. In addition, a previous study \[[@B15]\] indicated that some nonphotosynthetic branches might not have rate increases. Thus, two subsets of the nonphotosynthetic branches were tested: (1) Orobanche, Epifagusand Cistanchebranches and (2) Harveya, Hyobancheand Boschniakiabranches. Each of these tests used the data set from a single gene and compared two nested hypotheses: H~1~: the photosynthetic and non-photosynthetic branches share a single value (for one of the parameters d~N~, d~S~or ω). H~2~: the photosynthetic and non-photosynthetic branches have two separate values. If the tree has a significantly higher likelihood under H~2~, that is taken as evidence that the nonphotosynthetic branches have higher rates. Scatter plots and correlation tests were used to examine the degree of correlation between synonymous and nonsynonymous sites within a gene, and also to see if either class of sites was correlated between genes. Authors\' contributions ======================= NDY and CWD conceived of and designed the study together. NDY did the sequencing, data analyses and drafted the manuscript. CWD provided the genomic DNA samples and provided the conducive laboratory environment, both physical and intellectual, as well as many suggestions for the manuscript. Acknowledgements ================ We thank R. Olmstead, K. Kirkman, R. Wyatt, J. Armstrong, J. Alison, A. Batten, M. Weatherwax, K. Steiner, L. Musselman, W. Wetschnig, G. Sallé, C. Morden, and J. Palmer for plants or DNAs used in this study; A. Wolfe for some of the rbcL sequences and DNAs; S. Kovalsky-Pond, J. Leebens-Mack and J. Lyons-Weiler for suggestions on analyses; NSF for financial support (DEB-9120258, DBI-9604814) to CWD.	PubMed Central	2024-06-05T03:55:53.950127	2005-2-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554776/", "journal": "BMC Evol Biol. 2005 Feb 15; 5:16", "authors": [ { "first": "Nelson D", "last": "Young" }, { "first": "Claude W", "last": "dePamphilis" } ] }
PMC554777	Background ========== \"At some stage in the past the waterways of Africa were, from the fishes\' point of view, accessibly interconnected.\"P. H. Greenwood (1983) With estimated numbers of about 1,000 and 500 species respectively the assemblages of cichlid fishes of East African lakes Malawi (LM) and Victoria (LV) are, by far, the most species-rich species flocks \[[@B1]-[@B6]\]. In Lake Tanganyika (LT), Africa\'s oldest lake, \"only\" about 200 to 250 cichlid species occur, but they are phenotypically, behaviorally and genetically more diverse \[[@B7]-[@B9]\]. Remarkably, almost 100% of the species of all of these flocks are endemics and the three East African Great Lakes do not have a single cichlid species in common \[[@B10]\]. While in LT at least twelve eco-morphologically distinct tribes can be clearly distinguished phylogenetically \[[@B7],[@B9],[@B11]\], the species flocks of LM and LV are entirely comprised of cichlids assigned to only one of these tribes, the Haplochromini \[[@B1],[@B4],[@B10],[@B12]\]. Additionally, several less species-rich flocks of haplochromines are found in smaller lakes in East Africa \[[@B10]\], some of which have been combined with the LV radiation into a \"superflock\" of closely related species \[[@B4],[@B12],[@B13]\]. Not all haplochromines are lacustrine, however, and some 200 species inhabit rivers and occur in northern-, eastern-, southern- and central-Africa but are virtually absent from West Africa \[[@B10],[@B12]\] (Fig. [1](#F1){ref-type="fig"}). Traditionally, it was believed that riverine haplochromines seeded the cichlid radiations in all East African Great Lakes \[[@B8],[@B10]\]. Molecular-based phylogenies indeed uncovered haplochromine lineages that are ancestral to the species flocks of LM and LV (see e.g., \[[@B1],[@B4],[@B9],[@B14]\]. In contrast, the primary radiation of LT\'s cichlid assemblage was found to predate the origin of haplochromine lineages \[[@B9],[@B15],[@B16]\] suggesting a close phylogenetic affinity of the ancestor(s) of the haplochromines to other tribes in LT \[[@B9]\]. However, only a relatively small fraction of the genetic and geographic diversity of the haplochromines has been included in previous phylogenetic and phylogeographic studies. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Distribution of the major haplochromine lineages in Africa with special emphasis on the East African lakes(according to our phylogenetic and phylogeographic analyses and references \[4, 12, 13, 34\]). Note that the color scheme is carried throughout this publication. ::: ![](1471-2148-5-17-1) ::: That the haplochromines have a particularly strong propensity for explosive speciation among cichlids is best illustrated by their unparalleled species-richness and diversity. With the exception of two LT lineages (Lamprologini: \~70--100 species; Ectodini: \~25--30 species), discrete adaptive radiations of non-haplochromines are comprised by about a dozen species at most. Thus, which evolutionary novelties might be causally related to the explosive speciation that distinguish the haplochromines from all other cichlid lineages remains a crucial question for the understanding of the explosive patterns of cichlid evolution in East Africa. Haplochromines furthermore represent prime examples for parallel evolution, and it is particularly the lacustrine haplochromine species flocks that independently evolved morphologies and color-patterns that are convergent between species of different lakes \[[@B2],[@B17],[@B18]\]. In order to gain a deeper understanding of the adaptive radiations of cichlids in general and the formation of East African cichlid species flocks in particular, several crucial questions still remain to be answered: (i) What evolutionary lineages from which geographic ranges make up the diversity of haplochromines? (ii) Which were the founding lineages of the lacustrine adaptive radiations and were they riverine generalists? (iii) Can particular behavioral and/or morphological key-innovations be identified that might be causally related with the diversification of haplochromines? With the aim of addressing these questions, we conducted the most extensive phylogenetic and phylogeographic study of haplochromine cichlids so far, analyzing a portion of up to 2,000 bp of the mitochondrial genome of about 100 species. We included representatives of relevant cichlid tribes from LT \[[@B7],[@B9],[@B15]\] as well as members of all major riverine and lacustrine haplochromine lineages and all but one haplochromine genus (following \[[@B12]\]). Results ======= The neighbor-joining analysis based on 304 complete mitochondrial control region sequences (Fig. [2](#F2){ref-type="fig"}) could not resolve the phylogenetic relationships between haplochromine lineages and the LT lineages with convincing bootstrap support. However, this analysis, which primarily aimed to provide a basis for the selection of taxa for the second set of analyses, already indicated the existence of a monophyletic clade that is comprised by the Tanganyikan Tropheini, sister-group to a clade consisting of the LM cichlids plus several East African riverine and lacustrine lineages and the representatives of the LV region superflock \[[@B4]\]. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### 50% majority-rule bootstrap consensus tree of 304 cichlid fish specimens based on 862 bp of the mitochondrial control region(neighbor-joining, HKY85 model with gamma substitution correction, 5,000 replicates). Relevant bootstrap values are depicted on the respective branches. The branches colored in red indicate the taxa that were chosen for the phylogenetic analyses combining the control region with sequences of the NADH dehydrogenase subunit II gene (see Fig. 3; for Ctenochromis oligacanthusfrom GenBank no control region sequence was available). The colors of the boxes that indicate the major clades refer to Figs. 1 and 3, the labels of the clades correspond to Table 1 \[see [Additional file 1](#S1){ref-type="supplementary-material"}\]. Note that Haplochromis bloyeti(marked by an asterisks) had a control region sequence identical to H. sp.1533 of \[25\], which was collected in the Malagarasi River, and grouped -- together with other fishes from the Malagarasi area and from the Lake Edward/George region -- into their group VII. Likewise, our H. sp.Tanzania I (marked by a circle) was identical to H. sp.1738 of \[25\], which was collected in Lake Chala and clustered with other taxa from Tanzania into their group VI. In addition to Haplochromis gracilior(endemic to Lake Kivu), which was recently identified as close relative of the Lake Victoria superflock \[4\], we found another sister group to the superflock. This lineage includes Haplochromis paludinosusthat occurs in the Malagarasi, as well as undescribed species from Tanzania and Lake Edward (see also Fig. 3). Like Haplochromis graciliorfrom Lake Kivu, all these taxa have the diagnostic character state \'Adenine\' in position 630 of the control region alignment and root to the Lake Victoria superflock through the central rift valley haplotype \[4\], corroborating the view that Lake Kivu is the main reservoir from which the Lake Victoria superflock evolved \[4\]. ::: ![](1471-2148-5-17-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Maximum likelihood phylogeny of the haplochromine cichlids\[general time-reversible model with gamma correction\] based on 100 taxa. Numbers above the branches represent Bayesian posterior probabilities obtained with Mr. Bayes, numbers below the branches represent maximum-likelihood bootstraps (100 replicates, obtained with PAUP\). Boulengerochromis microlepisand Oreochromis tanganicae, two tilapiine cichlids in LT, were used as outgroup taxa \[9, 15, 16, 63\]. In accordance to previous studies \[9, 15\] we find that within the LT species flock the Eretmodini (Ere.) are placed as sister group to the Lamprologini (Lampr.) plus several LT tribes (\"Tanganyika\") including the Orthochromisassemblage from the Malagarasi plains, and the haplochromines sensu lato. The latter clade combines four distinct lineages, a Congolese/South-African- (CSA; \~150 species), the Pseudocrenilabrus- (3 species), the Astatoreochromis-lineage (3 species), and the modern haplochromines (\~1,800 species). The modern haplochromines combine the LT Tropheini, the species flock of LM, several riverine lineages as well as the LV region superflock according to \[4\]. The haplochromines sensu latoare characterized by their breeding behavior; true egg-spots (ocelli) are likely to have evolved in the ancestor of the Astatoreochromis-lineage and the modern haplochromines. By contrast, the Malagarasi River Orthochromisare biparental caregivers \[34\] providing behavioral support for our molecular-based classification that excluded these fish from the haplochromines sensu lato. We note that several genera are polyphyletic and major taxonomic revisions will be required in the future to take our phylogenetic results into consideration. For example, Orthochromisof the Malagarasi River plains form a clade outside the remaining haplochromines in close affinity to the LT Ectodini (see also \[9\]) whereas Orthochromis polyacanthusand O. stormsi, which share derived features \[12\], fall -- in accordance to their distribution -- into the Congolese/South African clade. Other polyphyletic genera are Astatotilapia, Ctenochromis, and Haplochromis. The grey arrows next to some species names refer to the pictures on the right, the asterisk symbol marks the ancestor of the haplochromines sensu lato, the circle symbol marks a \"piebald\" (\"orange blotched\") form as found in the modern haplochromines only. ::: ![](1471-2148-5-17-3) ::: In the maximum likelihood tree of the dataset including 100 taxa (Fig. [3](#F3){ref-type="fig"}) the LT Eretmodini were placed as sister group to the LT Lamprologini, followed by a clade comprised by the two representatives of the Limnochromini (Limnochromis auritus, Triglachromis otostigma) plus Cyphotilapia frontosa, and all remaining taxa. Among these, a clade that includes four LT tribes (Cyprichromini, Ectodini, Limnochromini, Perissodini) plus the Orthochromisspecies from the Malagarasi drainage, which had so far been considered to belong to the Haplochromini, was recovered as sister group to the remaining haplochromine representatives. The latter, the haplochromines sensu lato, clustered into four distinct groups, with a Congolese/South-African lineage (CSA) sister to the Pseudocrenilabrus-, the Astatoreochromis-lineage, and the \'modern haplochromines\' (i.e.a clade comprised by the LT Tropheini sister to the LM representatives, several East-African riverine lineages and the members of the LV region superflock). Bayesian inference revealed the same branching order for the different lineages. In the strict consensus topology of the 78,617 most parsimonious trees (unweighted tree length: 4400; tree not shown), the Eretmodini were again resolved as sister group to the Lamprologini, the Limnochromini plus C. frontosa, four LT tribes plus the Malagarasi Orthochromisand the haplochromines sensu lato. Here, the Astatoreochromis-lineage was resolved as most ancestral lineage, and as sister group to a clade comprised by the Pseudocrenilabrus- and the CSA lineage, and the modern haplochromines. Also, the neighbor-joining analysis recovered a tree (not shown) with the Eretmodini as sister-group to the Lamprologini, the Limnochromini plus C. frontosa, the four LT tribes including the Malagarasi River Orthochromisspecies, and the haplochromines sensu lato. In the neighbor-joining tree, the Pseudocrenilabrus-lineage occupied the most ancestral branch in the haplochromines sensu lato, followed by the Congolese/South-African clade, the Astatoreochromis-lineage and the modern haplochromines. A Shimodaira-Hasegawa test \[[@B19]\] revealed that there are no significant differences between the topologies obtained with the different algorithms (P \< 0.05). Similarly, in the four-cluster likelihood mapping analysis \[[@B20]\] none of the three possible alternative branching orders among the four main lineages of the haplochromines sensu latoreceived support greater than 50%. The dating of the major cladogenetic events (Fig. [4](#F4){ref-type="fig"}) found an age of 2.4 MYA (1.22 -- 4.02 MYA) for the most recent common ancestor of the four lineages of haplochromines, and about 1.8 MYA (0.66 -- 3.78 MYA) for the most recent common ancestor of the modern haplochromines. This latter value was smaller in all resampling replications pointing to a younger age of the modern haplochromines compared to the remaining three lineages of haplochromines sensu lato. The most recent common ancestor in the CSA-clade was estimated to have lived about 2.0 MYA (1.15 -- 3.89 MYA), the split of the CSA lineage from the common ancestor with the Pseudocrenilabrus-, Astatoreochromis-, and modern haplochromine lineage was dated to about 2.4 MYA (1.22 MYA -- 4.02 MYA). The test for shifts in the probabilities of speciation in the haplochromines sensu latoaccording to \[[@B21]\] suggested increased rates of lineage diversification (p~c~\< 0.01) along three branches in the maximum likelihood, the Bayesian inference and the neighbor-joining trees: (i) the branch leading to Astatoreochromis-lineage plus the modern haplochromines; (ii) the branch leading to the modern haplochromines; and (iii) the branch leading to the LV region superflock (see Fig. [4c](#F4){ref-type="fig"} for the maximum likelihood tree). In the maximum parsimony strict consensus tree an increased rate of lineage diversification was found: (i) for the branch leading to the modern haplochromines; and (ii) for the branch leading to the LV region superflock. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### The \"out of Tanganyika\" scenario of haplochromine evolution in Africa. (a) Several haplochromine lineages independently left Lake Tanganyika and colonized large parts of Africa viapast and present river connections. Some of these lineages seeded cichlid radiations in distant lakes. The phylogeographic scenario is in agreement with palaeo-geological reconstructions of the evolution of the East African Rift region. LT is the oldest of the rift lakes. Its central basin began to form between 9 and 12 MYA, the northern (8-7 MYA) and the southern basin (2--4 MYA) began to fill at later periods \[76\]; deepwater conditions exist since about 5--6 MYA \[78\]. LM (2--4 MYA) and LV (750,000 years) are considerably younger. (b) Proposed lake level of Lake Tanganyika during the last four million years \[76, 77, 79\] indicating major low- and high-stands. (c) Chronogram of the haplochromine evolution in Africa as reconstructed with r8s \[72, 73\] based on the maximum likelihood topology. The size of each clade represents its species number. The modern haplochromines are a recent and rapidly speciating lineage. Our molecular clock calibration suggested about 2 MYA (1.15 -- 3.89 MYA) for the most recent common ancestor in the Congolese/Southern African lineage and ca. 2.4 MYA (1.22 -- 4.02 MYA) for their split from the common ancestor with the Pseudocrenilabrus-, Astatoreochromis-, and modern haplochromine lineage. This lies in the range of the proposed high lake-level stand of LT between the minima at 3.5 MYA and 1.1 MYA (650--700 m below present level) \[76, 77\] making an overflow through the Lukuga valley possible, thus opening the connection between LT and the Congo drainage. The asterisks mark nodes with a significant burst of lineage diversification (p~c~\< 0.01) \[21\]. Aa\... Astatoreochromis alluaudi, CSA\... Congolese/South African lineage, Hb\... Haplochromis bloyeti, LMF\... Lake Malawi species flock, LVS\... Lake Victoria Region Superflock, MO\... Malagarasi Orthochromisassemblage, Pm\... Pseudocrenilabrus multicolor, Pp\...Pseudocrenilabrus philander, Ps\... Pseudocrenilabrus-lineage, Tr\... Tropheini. ::: ![](1471-2148-5-17-4) ::: The maximum parsimony and maximum likelihood character state reconstructions revealed that the characteristic maternal mouthbrooding behavior, where only the females incubate their fry in their buccal cavities, evolved in the common ancestor of the CSA lineage, the Pseudocrenilabrus-, the Astatoreochromis-lineage, and the modern haplochromines (see asterisk in Fig. [3](#F3){ref-type="fig"}). The true haplochromine-like egg-spots \[[@B10],[@B22],[@B23]\] are likely to have evolved in the common ancestor of the Astatoreochromis-lineage and the modern haplochromines (Fig. [3](#F3){ref-type="fig"}). Some members of the CSA lineage also show yellow or reddish markings on their anal fin. However, these markings do not represent real haplochromine-like egg-spots, which are characterized by an inner yellow or orange ring and an outer transparent and colorless ring \[[@B10],[@B22],[@B23]\]. Instead, the more homogenous markings seen in the CSA lineage might be viewed as an intermediate character state in the evolution of the species-specific egg-spots as found in the Astatoreochromis-lineage and in the modern haplochromines. The mapping of riverine versuslacustrine lifestyle onto the maximum likelihood topology suggested that the ancestor of the modern haplochromines was riverine (Fig. [5](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Maximum parsimony reconstruction of habitat type(lake versusriver) using MacClade (the maximum likelihood reconstruction with Mesquite revealed analogous results) based on the maximum likelihood topology (see Fig. 3). The ancestor of the modern haplochromines (marked by a circle) is likely to have been a riverine species. This implies that also the Tanganyikan Tropheini originated from a riverine ancestor and re-colonized the lake where they presently form an abundant group in the rocky littoral zones. ::: ![](1471-2148-5-17-5) ::: Discussion ========== Phylogeny and evolutionary origin of haplochromines --------------------------------------------------- Mitochondrial DNA (mtDNA) sequence data have a long and successful history in the study of East African cichlid evolution (see e.g.,\[[@B1],[@B4],[@B9],[@B17],[@B24],[@B25]\]). Limitations with mtDNA have only been encountered when focusing on the phylogeny among extremely closely related species due to the possibility of the persistence of ancestral polymorphism (see e.g.,\[[@B26]\]), or because of hybridization events (see e.g.,\[[@B27]-[@B29]\]). Nevertheless, mtDNA sequences proved to be particularly suitable for the reconstruction of the East African cichlid phylogeny at the tribal level and for tribal assignments \[[@B9],[@B15]\]; for phylogenetic reconstruction within older tribes \[[@B30],[@B31]\]; and for phylogeographic analyses \[[@B4],[@B32]\]. Also, because of the extremely fast rate of lineage formation in cichlids, nuclear and even some mitochondrial genes \[[@B1]\] are too slowly evolving to contain phylogenetic information (reviewed in \[[@B6]\]). The different phylogenetic algorithms, with which we analyzed our data, revealed largely congruent results. In all analyses, and in agreement with previous results \[[@B9]\], we found that the Eretmodini are placed as sister group to the substrate spawning Lamprologini -- with an estimated number of up to 100 species the most species-rich tribe in LT -- plus several LT tribes and all haplochromine representatives (see Fig. [3](#F3){ref-type="fig"} for the maximum likelihood tree). The molecular phylogenies thus corroborate that all haplochromines are ultimately derived from LT cichlids and that their ancestor(s) are likely to have left LT secondarily. We consider all species that belong to the monophyletic group descending from this ancestor (asterisk in Fig. [3](#F3){ref-type="fig"}), to being haplochromines sensu lato. These are further divided into four distinct groups, a Congolese/Southern African lineage (CSA), the genera Pseudocrenilabrus(Ps.) and Astatoreochromis(As.), and the modern haplochromines (MH). While the respective monophyly of these four lineages was supported by high bootstrap values and Bayesian posterior probabilities, our analyses could not unambiguously resolve the exact relationships between these four lineages \[maximum likelihood and Bayesian inference (see Fig. [3](#F3){ref-type="fig"}): (CSA, (Ps., (As., (MH)))); maximum parsimony: (As., ((Ps., CSA), (MH))); neighbor joining: (Ps., (CSA, (As., (MH))))\]. An evaluation of these alternative hypotheses by means of a Shimodaira-Hasegawa test \[[@B19]\] and a four-cluster likelihood mapping analysis \[[@B20]\] indicated that none of these alternative branching orders receives significantly more support than the others. This suggests that the four lineages of the haplochromines sensu latoevolved almost contemporaneously from a common ancestor. This is further supported by the observation of relatively short branches interrelating these four lineages and the generally low bootstrap values and Bayesian posterior probabilities supporting the corresponding relationships proposed by the different algorithms. The CSA lineage is composed of several widespread and moderately species-rich groups of the Congo drainage and Southern Africa and consists of two main clades: A clade with a species from the Congo drainage (Ctenochromis oligacanthus) ancestral to Southern African genera (Serranochromis, Sargochromis, Pharyngochromis) was resolved as sister group to a clade comprised by solely Congo drainage taxa (Orthochromis, Haplochromis sp. nov., Cyclopharynxand Thoracochromis). Within the Pseudocrenilabrus- and Astatoreochromis-clades, branch lengths were relatively short and a more detailed phylogeographic sampling would be necessary to resolve the relationships between the different geographic lineages. The modern haplochromines consist of species flocks of an unparalleled diversity. They include the endemic LT tribe Tropheini (\~25 species) sister-group to a clade comprised by the entire species flock of LM (\~1,000 species) and several East African riverine and lacustrine lineages (\~200 species) plus the LV region superflock (\~600 species). With approximately 1,800 species this -- here phylogenetically defined -- monophyletic lineage makes up about 7% of all known teleost fish. Astatotilapia, Thorachochromis, and Orthochromisare polyphyletic genera ----------------------------------------------------------------------------- Our phylogenies show that several genera are in fact polyphyletic, and major taxonomic revisions will be required in the future to take our phylogenetic results into consideration. For example, and in agreement with previous studies (see e.g., \[[@B4],[@B14],[@B33]\]), Astatotilapiaemerges as polyphyletic genus, with representatives assigned to both the East African riverine clade in the modern haplochromines and the LV region superflock. The genus Thoracochromis, represented in our analysis by T. brauschi, has also been shown to be polyphyletic before \[[@B14]\], with T. brauschifrom the Congo drainage as a more ancestral lineage, and T. petroniusand T. pharyngealisfrom the Nile drainage with affinities to the LV region superflock (note that the Nile River Thoracochromisof \[[@B14]\] are consequently listed as Haplochromisin \[[@B34]\]). The placement of T. brauschias sistergroup to T. petroniusand T. pharyngealisplus the remaining representatives of the LV region superflock in the AFLP based phylogeny of \[[@B14]\] seems to contradict our mtDNA based results in which T. brauschiwas identified as member of the CSA lineage. However, the reported branching order did not receive considerable bootstrap support in \[[@B14]\]. Also, the choice of Astatoreochromis alluaudi(mislabeled as Astatotilapia alluaudiin \[[@B14]\]) as single outgroup species seems problematic, as our present analyses (see above) and former results \[[@B4]\] indicate that A. alluaudiand not T. brauschiis more closely related to the modern haplochromines (and, thus, also to the LV region superflock). Further analyses including nuclear DNA sequence data and more taxa assigned to the genus will be necessary to address this problem. Based on the phylogeny it is apparent that the Orthochromislineage, which is confined to the Malagarasi River system and two isolated rivers, East of LT \[[@B35]\], is not part of the radiation of haplochromine cichlids. This is further supported by the breeding behavior of these fish: While the Malagarasi River Orthochromisare biparental caregivers \[[@B35]\], the haplochromines sensu latoare all maternal mouthbrooders. Thus, the Orthochromis-species from the Malagarasi area, a group of exclusively riverine fish, should be placed into its own tribe. The name Orthochromisis, however, also used for riverine species from the Congo drainage. In our analyses, Orthochromis polyacanthusand O. stormsifrom the Congo River system fall -- according to their distribution -- into the CSA-clade leaving also the genus Orthochromispolyphyletic (see also \[[@B9]\]). We suggest to using Orthochromisfor the CSA lineage representatives, since O. polyacanthuswas the first species of the genus to be described \[[@B36]\], and Schwetzochromini (as tribe name) and Schwetzochromis(as genus name) for the Malagarasi area species, since this name was repeatedly used for some species of that complex (see e.g., \[[@B9],[@B34],[@B35]\]). Phylogeography and phylochronology ---------------------------------- We note that the application of a molecular clock for estimating divergence times has the potential of not being without problems for several reasons (see e.g., \[[@B37],[@B38]\]). However, a molecular-clock-based time estimate does surely provide an approximate framework for phylogeographic inferences. Our phylogeographic scenario (Fig. [4a](#F4){ref-type="fig"}), which is derived from the maximum likelihood phylogenetic and molecular clock analyses, suggests that several lineages independently left LT to colonize surrounding river systems and consequently other lakes in East Africa. The molecular clock calibration based on the chronogram generated with r8s \[[@B39]\] (Fig. [4b,c](#F4){ref-type="fig"}) yielded about 2.4 MYA (1.22 -- 4.02 MYA) for the split of the CSA lineage from the common ancestor of the haplochromines sensu lato, and about 2 MYA (1.15 -- 3.89 MYA) for the first branching events within the CSA clade. The spread of Congo drainage taxa into southern river systems occurred at a later stage, most likely at the relatively shallow watershed between upper branches of the Congo River and the Zambezi River -- a scenario that is also supported by the placement of Serranochromis sp.(from Lake Mweru in the upper Congo) as sister group to the Zambezi/Southern African genera Sargochromisand Pharyngochromisin our phylogenies. However, further sampling in that area would be necessary to reconstruct the southward spread of the CSA lineage. At essentially the same time as the CSA lineage, the ancestors of the Pseudocrenilabrus-and the Astatoreochromis-lineage diverged from their common ancestor. Despite their large distributional ranges -- they also colonized the LV (both lineages) and LM (Pseudocrenilabrus) region -- the genera Astatoreochromisand Pseudocrenilabrusnever underwent considerable speciation. The three described Astatoreochromisspecies occur in the LV region including Lakes Edward and George (A. alluaudi), in the rivers Rusizi and Lukuga (A. straeleni), and in the Malagarasi River (A. vanderhorsti). The three species of Pseudocrenilabrusoccur from the Nile system to the LV region (P. multicolor), in Eastern- and Southern Africa including LM (P. philander), and in the central Congo basin (P. nicholsi). All analyzed representatives are relatively closely related suggesting a recent spread of these lineages in East Africa. However, we did not include P. nicholsi, which is morphologically different from P. multicolorand P. philanderand would -- if it really belonged to the Pseudocrenilabrus-lineage -- represent the only haplochromine in the Congo drainage that is not a member of the CSA lineage. The most recent common ancestor of the modern haplochromines was dated to have existed about 1.8 MYA (0.66 -- 3.78 MYA) (Fig. [4](#F4){ref-type="fig"}). This ancestral lineage forms the crucial phylogenetic and biogeographic link between the species flocks of all three East African Great Lakes, and its discovery documents the existence of much earlier hypothesized fish-accessible waterways between these waterbodies \[[@B40],[@B41]\]. Apparently, the Malagarasi River (and possibly the Rusizi) played a major role for the dispersal of these fishes, since many modern haplochromine lineages occur in these drainages and in lakes South-Eastern and North of LT exclusively, which argues against the view that LM haplochromines originated from Zambezi River stocks \[[@B41]\]. Whether or not Lake Rukwa has ever acted as link between the faunas of LT and LM \[[@B41]\] cannot be answered by our data. Lake Rukwa seems to have overflowed at its maximum levels into LT several times. However, Lake Rukwa has also become very shallow in recent geological times and it might have dried up completely \[[@B41]\] eradicating its original fauna. At present, Lake Rukwa harbors haplochromines that belong to the East-African riverine clade in Figs. [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}\[[@B4],[@B25]\]. Our analyses also recovered another closely related lineage to the LV region superflock in the East-African riverine clade, in addition to Haplochromis graciliorform Lake Kivu \[[@B4]\]. This lineage includes H. paludinosusthat occurs in the Malagarasi (which was already suggested by \[[@B33]\]), as well as undescribed species from Tanzania and Lake Edward (Figs. [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}). It is, however, unclear by which waterway haplochromine cichlids once colonized Lake Kivu. The flow of the Rusizi, presently from Lake Kivu into LT with the Panzi falls as strong barrier for fish migration, might actually have been reversed before the uplift of the Virunga volcanoes north of Lake Kivu as suggested by deposits of fossil LT mollusks and fluviatile sands in the upper Rusizi valley \[[@B41],[@B42]\]. This connection could possibly explain how haplochromines of LT origin were able to colonize Lake Kivu about 1.5 million years ago (Fig. [4](#F4){ref-type="fig"}). Evolutionary key-innovations of haplochromines ---------------------------------------------- One of only few synapomorphies of the haplochromines sensu latois the particular type of cranial apophysis for the upper pharyngeal bones \[[@B12]\]. The distinctive organization of the pharyngeal apophysis, a second set of jaws that is functionally decoupled from the oral ones \[[@B43]\], is characteristic to all cichlids and has been interpreted as prominent feature that -- because of its adaptability -- contributes to the cichlids\' evolutionary success \[[@B2],[@B3],[@B10],[@B43]\]. It is, however, not evident how the relatively minor morphological modification of part of that structure in the haplochromines \[[@B12]\] might function as an evolutionary key-innovation. Interestingly, however, all haplochromines sensu latoare maternal mouthbrooders with the females alone incubating the eggs in their buccal cavities \[[@B10],[@B12]\]. Mouthbrooding, which is regarded as an adaptation to predation pressure \[[@B44]-[@B46]\], has evolved several times independently and in diverse behavioral modes in cichlids \[[@B10],[@B22],[@B47],[@B48]\]. The characteristic maternalmouthbrooding behavior displayed by haplochromines is believed to being a derived character state \[[@B35],[@B46],[@B47]\]. Mouthbrooding strongly limits the number of eggs and fry that can be raised and might have led to generally much smaller population sizes, which has, for example, population genetic implications on fixation of alleles, and might result in smaller effective population sizes. Furthermore, mouthbrooding species may be considered to being promising colonizers of new habitats, since only a single mouthbrooding female is necessary for the founding of a new population. An eminent feature of several female mouthbrooding cichlid genera is the occurrence of egg-spots on the anal fins of males. In some species also females show such ovoid markings, but these are smaller and much less conspicuous than in males. Also, some species of the modern haplochromines, e.g., some deep-water lineages of LM, have lost their egg-spots secondarily. In mimicking real eggs to attract females, these egg-spots function as natural releasers \[[@B22],[@B23]\], or intra-specific sexual advertisement \[[@B44]\], apparently serving to ensure a greater fertilization success of the eggs by bringing about greater proximity of the female\'s mouth to the male\'s genital opening. Based on the molecular phylogeny, we could trace the origin of the characteristic egg-spots (ocelli) \[[@B10],[@B23]\] to the common ancestor of the Astatoreochromis-lineage and the modern haplochromines. There are other cichlid species in which males display yellow or red marks on their pelvic, dorsal or anal fins, but only in these lineages true egg-spots on the males\' anal fins with a yellow, orange or red center and a colorless/transparent outer ring \[[@B10],[@B22]\] are found. Interestingly, the branch leading to the Astatoreochromis-lineage and the modern haplochromines is the one with a pronounced potential for an increased rate of speciation (see Fig. [4c](#F4){ref-type="fig"}). Based on the character state reconstructions (Fig. [5](#F5){ref-type="fig"}) it seems likely that this ancestor was riverine. Thus, it may be concluded that the egg-spots first evolved in a haplochromine cichlid that inhabited a turbid riverine environment, where these conspicuous markings would seem to be particularly effective and necessary for intra-specific communication. Another innovation that further distinguishes the exceptionally species-rich modern haplochromines from all other cichlids is the occurrence of numerous color morphs, often accompanied by sexual color dimorphism. Inter- and intra-specific polychromatism combined with maternal mouthbrooding involving egg-spots as releasers can be hypothesized to being permissive for sexual selection through female choice and, hence, the haplochromines\' propensity for species formation, as sexual selection is probably a major causal factor in the origin of isolating mechanisms and the maintenance of reproductive isolation \[[@B18],[@B49]-[@B53]\]. These distinctive features of the modern haplochromines, that have arisen just in their ancestor, in combination with the numerous ecological niches that are provided by the large East African lakes might thus have induced a considerable increase of the haplochromines\' evolutionary potential. The importance of large waterbodies for the evolution of the modern haplochromines is reflected by the fact that these cichlids only radiated in lakes (and species number rather correlates to the size, but not to the age, of a lake), whereas the riverine lineages are all species-poor albeit often widespread (Figs. [1](#F1){ref-type="fig"}, [3](#F3){ref-type="fig"}). Replicate adaptive radiations of the \'modern haplochromines\' -------------------------------------------------------------- A common feature of many adaptive radiations is that their founders are believed to have had a more generalist\'s lifestyle, while adaptive radiations themselves are defined by being composed of highly specialized species with narrower niche widths \[[@B54],[@B55]\]. Theory predicts that generalists more likely have better dispersal abilities and are expected to be able to adapt readily to novel environmental settings \[[@B55]\]. A generalist ancestor scenario fits well with the diversification of haplochromine cichlids. Generalist riverine species of the genera Astatoreochromis, Astatotilapia, Pseudocrenilabrus, and Haplochromis(e.g., bloyeti), are ancestral to the adaptive radiations of the Tropheini of LT, and/or the radiations of LM and the LV region superflock. These genera are widely distributed and not confined to Eastern Africa, and they are the only ones that could inhabit the waterways that -- over geological time spans -- connected the lakes of Eastern Africa. The phylogeny presented here (Fig. [3](#F3){ref-type="fig"}) reveals that modern haplochromines gave rise to several major adaptive radiations; the most prominent ones are those of LM and LV. Interestingly, it uncovers that also the radiation of the Tropheini from LT \[[@B30]\] must now be considered as an additional radiation of the modern haplochromines, corroborating the much older perception that LT accommodates several independent species flocks \[[@B56]\]. It is further suggested by mapping the fishes\' lifestyle onto our molecular phylogeny that the highly specialized Tropheini are descendents of a river-living species. This implies that the ancestor of the Tropheini successfully re-entered the lake habitat and evolved into the presently dominant group in the rocky littoral zone of LT. Thus, this lineage of modern haplochromines managed to occupy \"empty niches\" in an apparently \"full\" ecosystem, as all remaining tribes, which now account for about 200 species, had already been established when the ancestor of the Tropheini secondarily entered LT (Figs. [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}). The observation that these fish underwent an independent adaptive radiation in LT underlines the haplochromines\' propensity for speciation. In an apparent contrast to most other known examples of adaptive radiations \[[@B55]\] is the finding that the generalist ancestors of the haplochromine species flocks were derived from already highly diverse and specialized LT endemics (Fig. [3](#F3){ref-type="fig"}). Therefore, specialization may not be an \"evolutionary one-way street\", but rather some lineages have reversed their level of specialization, i.e., generalists arose from highly specialized lineages, yet, apparently retained their high propensity for speciation and level of evolvability (see \[[@B57]\]). The faunal revolution of LT\'s radiation of cichlids was thus not confined to the lake habitat itself (see also \[[@B8],[@B9],[@B58]\]), but it effectively involved large parts of Africa via the intermediate step of repeatedly evolving generalist riverine lineages -- in much the same way as the adaptive radiation in LV produced haplochromine species that secondarily colonized surrounding rivers \[[@B4]\]. Our phylogeny of haplochromines provides strong support for replicate adaptive radiations in East African cichlids. The concept of replicate radiations, in which the same sequence of adaptations to ecological niches evolved repeatedly in lineages that inhabit similar environments, has been developed based on sympatric species pairs of fishes in postglacial lakes and on the Anolislizard ecomorphs on different islands of the Greater Antilles \[[@B59]-[@B61]\]. Our inclusive phylogenetic and phylogeographic study shows that similar ecological types of cichlids in the different East African lakes evolved independently (see also \[[@B2],[@B17]\]), yet it also shows that the convergent ecotypes in the species flocks of LM, LV, Lake Kivu as well as in the Tropheini \[[@B2],[@B10],[@B12],[@B13],[@B17]\] arose from the same ancestral phenotype in the ancestor of the modern haplochromines. We suggest that a combination of behavioral (maternal mouthbrooding) and morphological innovations (egg-spots, color polymorphisms, pronounced sexual dichromatism) as well as ecological opportunities (after the colonization of large lakes) might have predestined this particular lineage to give rise to these replicate adaptive radiations. It has been noted before that lineages of LT origin have left the lake secondarily (there are, for example, about five lamprologine species that are found in the Congo and Malagarasi Rivers) \[[@B9],[@B58]\]. Here, we show that the entire haplochromine diversity has its origin in LT corroborating the view that ancient lakes not only preserve biodiversity but also act as biodiversity hotspots, genetic reservoirs and cradles from which new lineages evolve \[[@B4],[@B8],[@B9]\]. What remains to be answered is where the LT cichlids originated and to what extent a proposed and meanwhile desiccated Pliocene lake in the Congo plains \[[@B41],[@B42]\] was the source of the ancient LT lineages, pushing back even further the onset of replicate adaptive radiations in East African cichlids. Conclusion ========== Our phylogenetic analyses that include representatives of all major haplochromine lineages show that all haplochromines are derived from Lake Tanganyika cichlids. While the Orthochromisspecies of the Malagarasi area apparently do not belong to the radiation of the haplochromine cichlids and should be placed into a new tribe, we defined four new lineages within the haplochromines sensu lato: A clade combining Congolese and South-African genera (CSA-lineage), the Pseudocrenilabrus-, the Astatoreochromis-lineage, and the exceptionally species-rich modern haplochromines. The ca. 1,800 species of modern haplochromines are comprised of the entire haplochromine species flocks of Lake Malawi and the Lake Victoria region, some 200 riverine and lacustrine species, as well as the Tanganyikan Tropheini, which are likely to have evolved from a riverine ancestor and secondarily colonized Lake Tanganyika. This proposed \"out of Tanganyika\" scenario of haplochromine evolution is in agreement with the geological and palaeo-geological history of East Africa. Based on a character-state reconstruction from this new phylogeny, we were able to discover the evolution of several key-innovations that arose in the lineage leading to the modern haplochromines. These character reconstructions suggest that a combination of behavioral (maternal mouthbrooding) and morphological characteristics (egg-spots, color polymorphisms, pronounced sexual dichromatism) as well as ecological opportunities (after the colonization of large lakes) might have predestined this particular lineage to give rise to replicate adaptive radiations and, therefore, be causally related to the extraordinary success of these particular cichlid fish radiations. Methods ======= Specimen information and DNA methods ------------------------------------ For this study, a total of 304 specimens were analyzed. We combined all available GenBank entries from previous studies \[[@B4],[@B9],[@B15],[@B25],[@B62]\] with 180 newly determined DNA sequences. Table 1 \[see [Additional file 1](#S1){ref-type="supplementary-material"}\] lists specimen information, geographic origin of the specimens, names of collectors, and GenBank accession numbers. When available, voucher specimens have been deposited at the Royal Museum for Central Africa, Tervuren, Belgium. Sample preparation, polymerase chain reaction (PCR) amplification and DNA sequencing have been performed as described elsewhere \[[@B9]\] for both mitochondrial DNA segments the complete non-coding control region and the entire NADH Dehydrogenase Subunit II (ND2) gene. Forward and reverse sequences have been assembled using the computer programs Sequence Navigator (Applied Biosystems, USA) and Sequencher (GenCodes, USA). Phylogenetic reconstruction and hypotheses testing -------------------------------------------------- The complete sequences of the mitochondrial control region (895 bp) and the ND2 gene (1,047 bp) were aligned using the computer program Clustal W \[[@B63]\]; alignments have been further adjusted by eye. Up to 34 gaps had to be included in the control region alignment, which were coded as indels. Due to missing data on the 5\'-end of the control region in about one quarter of the taxa, a terminal section of 34 bp has been excluded from the phylogenetic analysis, leading to an alignment of 1,908 bp for the combined dataset. For phylogenetic reconstruction we performed maximum likelihood, maximum parsimony and neighbor-joining methods in parallel using the computer program PAUP\ 4.0b10 \[[@B64]\]. Two taxa belonging to tilapiine cichlids were included as outgroup based on previous phylogenetic analyses using mitochondrial DNA \[[@B9],[@B15],[@B16]\], nuclear DNA \[[@B65]\] and SINE insertion patterns \[[@B66],[@B67]\] demonstrating that the Tilapia-lineages are ancestral to all remaining LT tribes (but excluding Tylochromis, which is ancestral to the Tilapiini). We did not include representatives of three LT tribes -- the Bathybatini, Trematocarini and Tylochromini -, as these ancient lineages were shown to have existed before the primary radiation of mouthbrooders in LT \[[@B9],[@B15],[@B67]\]. After an initial neighbor-joining analysis including the control region sequences of all 304 specimens, we reduced the dataset to 100 taxa based on the obtained topology. This reduction was necessary to allow computational feasibility for maximum likelihood and maximum parsimony analyses. The optimal model of molecular evolution for the maximum likelihood analysis was determined in a likelihood-ratio test running the computer program Modeltest v3.06 \[[@B68]\]. For the heuristic maximum likelihood search of the combined dataset we used the general-time-reversible model of molecular evolution, with a gamma shape parameter of 0.7937 and a proportion of invariable sites of 0.3426. Due to the many closely related taxa in the dataset, maximum parsimony analyses were completed for 10^10^rearrangements. For neighbor joining, we used the HKY model and conducted a bootstrap analysis with 5,000 replicates. Bootstrap analyses for maximum likelihood were performed with 100 replicates and for maximum parsimony with 1,000 replicates. We also applied Bayesian inference of phylogeny with Mr. Bayes 3.0b4 \[[@B69]\] running four Metropolis Coupled Monte-Carlo-Markov-Chains in parallel for 250.000 generations, using the general-time-reversible model with gamma correction (six types of substitutions), and excluding 5 % of the trees as burn-in. The obtained topologies from the different phylogenetic algorithms were evaluated by means of a nonparametric Shimodaira-Hasegawa test \[[@B19]\] under a resampling-estimated log-likelihood as implemented in PAUP\* \[[@B64]\]. To estimate the support for distinct internal branches critical for our interpretations, we performed a four-cluster likelihood mapping analysis with the program PUZZLE 5.0 \[[@B20],[@B70]\] in which we grouped the taxa of the haplochromines sensu latointo four groups according to the four lineages that were recovered from the phylogenetic analyses (CSA-, Pseudocrenilabrus-, Astatoreochromis-lineage, and modern haplochromines). To test whether or not shifts in the probabilities of speciation occurred on certain branches of the obtained phylogeny, we quantitatively tested the fit of this tree to a Markovian null model in which the probability of speciation is equally distributed along branches. Therefore, we calculated the cumulative probability p~c~\[[@B21]\] for relevant branches based on the different trees. We then mapped lifestyles (riverine versuslacustrine), breeding behavior, and the occurrence of true egg-spots on the maximum likelihood tree using MacClade 4.0 (Sinauer, Sunderland, MA) for maximum parsimony character state reconstructions and Mesquite \[[@B71]\] for maximum likelihood reconstructions. To tentatively date the major cladogenetic events in the haplochromines, we constructed a chronogram based on a maximum likelihood tree with constraint molecular clock (see above for search parameters). Therefore, we used the computer program r8s \[[@B72]\] applying the local molecular clock method and an optimization via the truncated newton method \[[@B73]\]. Confidence intervals were assessed by means of a bootstrap approach. We simulated 25 bootstrap matrices with Mesquite and, for each matrix, constructed a maximum likelihood tree (general-time-reversible model; model parameters, gamma shape correction and proportion of invariable sites being estimated from each matrix; rearrangements limited to 1,000). The resulting trees were then analyzed with r8s as described above, and in addition with the minimum and maximum values of geological datings separately, in order to define upper and lower bounds. As calibration points, we used an age estimate for the LM species flock of about one million years \[[@B74],[@B75]\], the maximum age of 200.000 years for the LV region superflock \[[@B1],[@B4],[@B25]\] as well as the time window for the Lukuga connection between LT and the Congo system (between the minima at 3.5 MYA and 1.1 MYA) \[[@B76],[@B77]\]. An escape of LT lineages into river systems draining into the lake was possible at any time, whereas the only connection to the Congo system, the Lukuga, was available at periods of high lake level stands only. The Lukuga channel, the only outlet of LT, was dry when first seen in 1874 by Cameron, but four years later the lake overflowed. Since then, the Lukuga was repeatedly flooded. Intermittent connections with the Congo River system in the late Pliocene/early Pleistocene were suggested on the basis of large lake level fluctuations in LT during periods with increased precipitation \[[@B41],[@B76]-[@B79]\]. The resulting molecular clock rate fits well with previously used rates in East African cichlids \[[@B4],[@B75]\]. List of abbreviations used ========================== As., Astatoreochromis; bp, base pairs; CSA, Congo/South Africa; LM, Lake Malawi; LT, Lake Tanganyika; LV, Lake Victoria; MH, modern haplochromines; mtDNA, mitochondrial DNA; MYA, million years ago; ND2, NADH Dehydrogenase Subunit II; Ps., Pseudocrenilabrus. Authors\' contributions ======================= WS, EV, and AM designed the study and were involved in sampling. WS and TM carried out the molecular work and the analyses. All authors contributed to the preparation of the manuscript. They read and approved the final version. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Table 1 -- Specimen information, geographic origin and GenBank accession numbers of all taxa included in this study. This table lists the species and tribe names, geographic origin, source of specimens, names of collectors and collection numbers (if available), and GenBank accession numbers for both mitochondrial gene segments. The taxa that are included in Fig. [3](#F3){ref-type="fig"} are marked by a circle, specific taxon labels in Fig. [3](#F3){ref-type="fig"} are depicted in the \"Label\" column. The clade names according to Figs. [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"} are shown in the last column. Tribe names are according to \[[@B7]\]. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This paper is dedicated to the memory of the eminent African ichthyologist L. De Vos (1957--2003). We thank R. Abila, C. Katongo, I. Kornfield, R. Paul, T. Reuter, U. Schliewen, E. Schraml, L. Seegers, O. Seehausen, J. Snoeks, C. Sturmbauer, and L. De Vos for providing specimens and/or sharing unpublished sequences; K. Lezzar and J.J. Tiercelin for discussion; A. McArthur and M. J. Sanderson for technical assistance; M. Barluenga, P. Bunje, G. Fryer and an anonymous reviewer for valuable comments on the manuscript. This work was funded by the European Union (Marie Curie fellowship) and the Landesstiftung Baden-Württemberg to W. S.; a grant from The Belgian Federal Office for Scientific, Technical and Cultural Affairs and support through the Ecological Genetics Research Network of the Fund for Scientific Research-Flanders to E. V.; and grants of the Deutsche Forschungsgemeinschaft to A. M.	PubMed Central	2024-06-05T03:55:53.953098	2005-2-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554777/", "journal": "BMC Evol Biol. 2005 Feb 21; 5:17", "authors": [ { "first": "Walter", "last": "Salzburger" }, { "first": "Tanja", "last": "Mack" }, { "first": "Erik", "last": "Verheyen" }, { "first": "Axel", "last": "Meyer" } ] }
PMC554778	Background ========== Schistosoma mansoni, the African blood fluke and etiological agent of intestinal schistosomiasis, is endemic in numerous countries in Africa, the Middle East, the Caribbean and northeastern South America. The life cycle of S. mansoniinvolves parasitism of both humans and aquatic snails of the genus Biomphalaria. Cercariae, the infectious larvae, emerge from the snails into lakes and fresh water streams, where they initiate human infection by direct penetration of the skin. Within the infected person, the worms develop into male and female adults within the portal system blood vessels and mesenteric veins of the intestines. Eggs released from the female parasite into the blood traverse the intestinal wall and are passed out with the feces. Among the tropical diseases, schistosomiasis ranks second only to malaria in terms of morbidity and mortality \[[@B1]\] and has proved refractory to control by the more conventional public health approaches. No vaccine is yet available. Mobile genetic elements (MGEs) represent a major force driving the evolution of eukaryotic genomes \[[@B2]-[@B4]\] and play an important role in the establishment of genome size \[[@B5]\]. One of the major categories of MGEs is the long terminal repeat (LTR) retrotransposable element, i.e. the LTR retrotransposons and the retroviruses \[[@B6]\]. These elements are of interest for their potential for horizontal transmission, as well as their ability to shed light on phylogenies of their host organisms when solely vertically transmitted. The genomes of schistosomes, blood flukes of the phylum Platyhelminthes, are estimated at \~270 megabase pairs (MB) per haploid genome \[[@B7]\], arrayed on seven pairs of autosomes and one pair of sex chromosomes \[[@B8],[@B9]\]. Both the evolution and size of this genome may be highly influenced by mobile genetic elements. Indeed, more than half of the schistosome genome appears to be composed of, or derived from, repetitive sequences, to a large extent from retrotransposable elements \[[@B10],[@B12]\]. Mobile genetic elements colonizing the genome of S. mansoniare of interest both for their potential in developing tools for schistosome transgenesis and for their influence on the evolution and structure of the schistosome genome \[[@B13],[@B14]\]. Previously characterized schistosome MGEs include SINE-like retroposons \[[@B15],[@B16]\], long terminal repeat (LTR) retrotransposons \[[@B12],[@B17],[@B18]\], non-LTR retrotransposons \[[@B10],[@B11]\], and DNA transposons related to bacterial IS1016 insertion sequences \[[@B19]\]. Boudicca, the first LTR retrotransposon characterized from the genome of S. mansoni\[[@B20]\] belongs to the gypsy-like retrotransposons, one of three highly divergent groups of LTR retrotransposons: the Gypsy/Ty3group, the Copia/Ty1group and the Pao/BELgroup \[[@B21]\]. Although active replication of schistosome retrotransposons has not been established, transcripts encoding reverse transcriptase (RT) and endonuclease are detectable \[[@B10],[@B11],[@B22]\], as is RT activity in parasite extracts \[[@B23]\], suggesting that at least some of these elements are actively mobile within the genome. Indeed, actively replicating MGEs have been described from other platyhelminths as RNA intermediates \[[@B24]\] and DNA transposons \[[@B25],[@B26]\]. Furthermore, the schistosome retrotransposons characterized so far are highly represented within the genome with copy numbers of up to 10,000 \[[@B10],[@B20]\]. It has been suggested that the Pao-like elements exhibit a host range limited to insects and nematodes \[[@B27]\]. More recently, however, Pao-like sequences have been reported from vertebrates including the teleost fishes Takifugu rubripesand Danio rerio\[[@B28]\]. Here we have characterized a new Pao-like element from the genome of S. mansoni, which we have named Sinbadafter the mariner-explorer Sinbad from the classical Persian/Arabic tales of the \"1001 Arabian Nights\" (e.g., \[[@B29]\]). (Sinbad roved through near Eastern countries where schistosomiasis remains endemic even today \[[@B30]\].) Further, we investigated the phylogenetic distribution of Pao-like elements related to Sinbadand report that there is a discontinuous distribution of these elements throughout the Ecdysozoa, Deuterostomia, and Lophotrochozoa that suggests horizontal transmission and/or efficient elimination of Pao-like mobile genetic elements from some host genomes. Results ======= A LTR retrotransposon in BAC 33-N-3 ----------------------------------- BLAST analysis indicated the presence in BAC 30-H-16 of a reverse transcriptase (RT)-encoding sequence with identity to Paoand other Pao-like retrotransposons including Ninjaand MAX(not shown). Using a probe based on an RT encoding segment of the end sequence of BAC 30-H-16, we identified 14 positive clones in the S. mansoniBAC library \[[@B31]\]. DotPlot analysis of a 7,531 bp portion of one of the positive BACs, 33-N-3, revealed the presence of two identical, direct repeat sequences of 386 bp separated by \~5.5 kb of intervening sequence, suggesting the presence of an LTR retrotransposon of 6,287 bp in length. This dot matrix is presented in Figure [1](#F1){ref-type="fig"}, with a map predicting the size and general domain structure of the new element provided below the matrix (both matrix and map share the same size scale). The direct repeats appeared to be LTRs, and included the promoter initiation motifs CAAT (positions 347--350) and TATA (positions 111--114 and 216--219), transcriptional signals for RNA polymerase II. The LTRs begin with TGT and end with TCA. These motifs (TGN/NCA), known as the direct inverted repeats (DIR), are common to LTRs of many retrotransposons and retroviruses \[[@B32]\]. BLAST searches of GenBank revealed that this retrotransposon closely resembled the elements Paoand Ninja, followed by other Pao/BELtype retrotransposons. We have termed this new retrotransposon Sinbad. The coding region between the two LTRs of Sinbadwas disrupted by several stop and frameshift mutations (as has been seen in many other retrotransposons (e.g., see Ref. \[[@B32]\]), although the reverse transcriptase, retroviral protease, and gag-like domains of Sinbadwere clearly evident. The sequence of the copy of Sinbadfrom BAC 33-N-3 has been assigned GenBank accession AY506538. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Pustell DNA matrix (DotPlot) of 7,531 bp of Schistosoma mansonigenomic DNA sequenced from BAC clone 33-N-3, revealing two identical 386 bp repeat sequences flanking 5,515 bp of unique sequence. A schematic representation of the 6,287 bp retrotransposon encoded by the sequence is shown below the matrix, with the positions of the LTRs and several domains (CHB, Cys-His Box; PR, protease; RT, reverse transcriptase; RH, RNaseH; IN, integrase) labeled. The position of the probe employed in library screening and Southern hybridization is indicated by a red box above this schematic representation. Both matrix and schematic are to scale. ::: ![](1471-2148-5-20-1) ::: Pao-like nucleoprotein, protease and reverse transcriptase ------------------------------------------------------------ Inspection of the region downstream of the 5\'-LTR of Sinbadrevealed the presence of an ORF encoding retroviral gagand pol-like proteins. A multiple sequence alignment of some of the key structural and enzymatic domains is presented in Figure [2](#F2){ref-type="fig"}, with the Sinbadsequence and orthologous regions from Pao, Roo, BEL, MAXand Ninja. The Cys-His box is a highly conserved cysteine and histidine based motif of the nucleocapsid protein (part of the gagpolyprotein) of retroviruses and retroviral like elements \[[@B33]\]. Whereas many other retroviral and retrotransposon families exhibit Cys-His boxes based on a single or double motif of three cysteine and one histidine residues, Pao-like elements are characterized by a distinctive triple Cys-His box \[[@B21],[@B27]\], with zinc finger motifs of CX2CX3-4HX4C, CX2CX2-4HX4-5C, and CX2-4CX3HX4H. Sinbadalso exhibits the latter type, hallmark triple Cys-His box motif (Fig. [2](#F2){ref-type="fig"}, panel A), although neither Sinbadnor Paoshows a doublet HH in the middle of the third zinc finger motif, another characteristic of this group of retrotransposons \[[@B32]\]. Notably, Tas, a Pao/BELlike element from Ascaris lumbricoidesdoes not share this characteristic triple Cys-His box \[[@B34]\], and though Suzufrom Takifugu rubripesexhibits a triple Cys-His box, its third zinc finger motif exhibits the structure CX4CX6HHX3C\[[@B28]\]. As illustrated in Figure [2](#F2){ref-type="fig"}, panel B, Sinbadexhibited a protease domain motif ALLDSGS-X98-LIGCD, typical of the LLDXGand LIGprotease motifs conserved in Pao-like retrotransposons \[[@B27]\]. The usual active site tripeptide motif in retroviral aspartic proteases is DTG, with a full conserved sequence of LLDTG, complemented by another site, a highly conserved G preceded by two hydrophobic residues, often I or L, which loops around to interact with the LLDTG \[[@B35]\]. Whereas the Gypsy-like and Copia-like elements exhibit DTG at the active site, Sinbadhas DSG, as do two other Pao-like elements, Rooand MAX. Other Pao-like elements have even more divergent catalytic domains: DCG for Kamikaze, GDG for Yamato, and DNG for Moose\[[@B27]\]. Since only Thr and Ser include the alcohol groups required for catalysis \[[@B35]\], the non-DT/SG motifs, including the DDG and DEG of Paoand Ninjalikely represent inactivating mutations in non-functional copies of the retrotransposons. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Multiple sequence alignments of key domains of the nucleocapsid protein and protease of the Sinbadretrotransposon and related elements. A. Amino acid alignment of the Cys-His box region of the nucleocapsid protein of Sinbadand five other Pao-like elements. Sinbadshares the triple Cys-His box motif of these elements (underlined). B. Amino acid alignment of the protease domain of Sinbadand five other Pao-like elements. Sinbadshares the LLDXG+ LIGprotease motifs conserved in Pao-like elements (underlined). Identical and chemically similar residues are boxed and shaded. ::: ![](1471-2148-5-20-2) ::: Nucleotides 2761 to 3375 of the Sinbadsequence from BAC 33-N-3 encoded a RT domain, a conceptual translation of which was aligned with the RT domain from six other elements, Pao, Ninja, Roo, BEL, Max, and Saci-1. A frameshift apparent in the ORF was resolved by inserting a N at the frameshift site, position 2761. The seven blocks of conserved RT residues of Pao-like elements, as modified by Abe et al. \[[@B27]\] from the blocks described by Xiong et al. \[[@B21]\], are annotated in green in the alignment (Figure [3](#F3){ref-type="fig"}). The Pao-like retrotransposons presented in Figure [3](#F3){ref-type="fig"} all exhibited the RT active site motif YV/MDD, in block 5, a motif conserved in the RT of many other retrotransposons, including the gypsyfamily \[[@B32]\]. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Multiple sequence alignment of deduced amino acid residues of the reverse transcriptase (RT) domain of Sinbadand six other Pao-like elements. Numbered blocks delineated by green brackets correspond to the seven conserved blocks of RT residues as described by Xiong et al. \[21\]. Identical and chemically similar residues are boxed and shaded. ::: ![](1471-2148-5-20-3) ::: RNAse H and Integrase of Sinbad --------------------------------- An RNaseH domain spanning \~300 amino acid residues was located carboxyl to RT, in which the conserved active site motif DAS was apparent \[see [Additional file 1](#S1){ref-type="supplementary-material"}\]. At its COOH-terminus, the Sinbadpol included an integrase (IN) domain of \~260 amino acids in length. Integrase mediates integration of a DNA copy of the viral genome into the host chromosome. Integrase is composed of three domains, the amino-terminal zinc binding domain, a central catalytic domain, and a carboxyl terminal domain that is a non-specific DNA binding domain \[[@B36]\]. A multiple sequence alignment of the IN zinc binding and central catalytic (DDE) domain of several informative BEL/Pao-like retrotransposons including MAX, Saci-1, Pao, Ninja, Roo, Suzu, BEL, and Tasas well as Sinbadis presented in Figure [4](#F4){ref-type="fig"}. All three domains were apparent in the Sinbadsequence. The NH~2~-terminal zinc-finger region of Sinbadincluded two conserved Cys residues and one His residue characteristic of other zinc finger motifs of IN (Figure [4](#F4){ref-type="fig"}). A second His expected here was replaced by Asn in this copy of Sinbad. The catalytic active site DDE motif of Sinbad\'s integrase displayed the residue spacing of D(62)D(49)E. The IN of non-Pao/BELretrotransposable elements, for example, Copia, exhibit a DD(35)E motif \[[@B36]\]. However, the IN of BEL/Paolike elements is unusual in that there is an expanded number of residues between the second D and E conserved residues, with DD(45)E for Paoand DD(53)E for BEL. Sinbadconformed to this BEL/Pao-like paradigm with a spacing of DD(49)E. Saci-1, also from S. mansoni, shows DD(49)E, although the IN domain of these two elements exhibited only 52% identity. The carboxy terminal domain of IN of Sinbadextended about 135 amino acids beyond the E residue of the catalytic domain \[see [Additional file 1](#S1){ref-type="supplementary-material"}\]. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Multiple sequence alignment of deduced amino acid residues of the integrase (IN) domain of Sinbadfrom Schistosoma mansoniand eight other Pao-BEL family retrotransposons. The position of the active site residues are indicated with asterisks above and bold face letters (D, D or E) below, as are the key Cys (C) and His (H) residues of the zinc-finger motif. Identical and chemically similar residues are boxed and shaded. ::: ![](1471-2148-5-20-4) ::: As noted, the IN of Sinbadexhibited identity to Saci-1from S. mansoni, and indeed these Pao-like retrotransposons from S. mansonishare substantial identity in deduced amino acid sequence and in structural organization \[[@B37]\]. This similarity extended to several other domains including the Triple Cys-His box region of Gag, 32% identical (23/71, Fig. [2A](#F2){ref-type="fig"}); PR,32% identical (36/111, Fig. [2B](#F2){ref-type="fig"}); and RT, 45% identical (106/236, Fig. [3](#F3){ref-type="fig"}). Whereas these levels of sequence identity confirmed a close relationship between Sinbadand Saci-1, they also demonstrated that Sinbadand Saci-1are distinct retrotransposons. Finally, Sinbaddid not appear to encode an envelope protein, the retroviral gene product necessary for extracellular existence and infection \[[@B38]\]. Sinbad, a new Pao/BELclade retrotransposon, is closely related to Pao and Ninja --------------------------------------------------------------------------------------- The RT domain of Sinbadwas aligned with that of 19 Pao/BELretrotransposon family elements, and with RT from informative Gypsy-like elements, from HIV-1, and Copiausing ClustalW. Bootstrapped trees were then assembled using the neighbor joining method and Njplot. Copiawas employed as the outgroup to root the tree. The phylogenetic tree confirmed that Sinbadbelonged to the Pao/BELfamily of LTR retrotransposons (Figure [5](#F5){ref-type="fig"}), and revealed that its two closest relatives were the Saci-1element from S. mansoniand an unnamed element from D. rerio, the zebrafish (BK005570). Sinbadalso grouped closely with Paoand Ninja. Sinbadis clearly distinct from the Gypsy-like retrotransposons, including Gulliverof Schistosoma japonicumand Boudiccaof S.mansoni. Sinbadis also clearly distinct from HIV-1, representative of vertebrate retroviruses, and from Copia, representative of the Ty1/Copiagroup of LTR retrotransposons. Among the 20 BEL/Paofamily elements represented in the tree, it was possible to distinguish several subfamilies. First, the outlying subfamily was a clade including Suzu(from T. rubripes) and an unnamed element from zebrafish. These are the only two elements that we have observed in this subfamily, and both occur in fish genomes. The other two branches of these retrotransposons include Pao, on the one hand, and BELon the other. Moreover, two subfamilies of elements were apparent within each of the Paoand BELbranches. For the Paobranch, one sub-family included Pao(from B. mori), ninja(from D. simulans) and an unnamed element from Anopheles gambiae(XP\_3092181). These subfamily elements were all from insect genomes. The other subfamily included Sinbad, Saci-1and the D. rerioelement BK005570; this subfamily has elements from schistosomes (Phylum Platyhelminthes) and fish. On the BELbranch of the tree, the first subfamily includes elements solely from nematode genomes -- Tas(A. lumbricoides), several Cerelements from C. elegans, and an unnamed element from C. briggsae(BK005572). The other branch included BELitself (from D. melanogaster), Kamikaze(B. mori), MAX(D. melanogaster) and Moosefrom A. gambiae. Members of this fifth subfamily occurred only in insect genomes. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Phylogenetic tree based on Clustal X alignments of the reverse transcriptase domains of several Pao-like and non-Pao-like elements, drawn using the neighbor joining algorithm. The names of elements, followed by host species names, in parentheses, are provided. Size bar reflects phylogenetic divergence in genetic distance units. Bootstrap values were drawn from 1,000 trials. ::: ![](1471-2148-5-20-5) ::: In addition, a phylogram of IN sequences was assembled from 14 Pao/BELfamily retrotransposons. The tree displayed the same general topography of branches as the RT-based phylogram and supported our suggestion that there are (at least) five discrete sub-families of BEL-Paofamily retrotransposons: Tas-like, BEL-like, Pao-like, Sinbad/Saci-1-like, and Suzu-like (not shown; tree available from corresponding author). In similar fashion to the RT based tree, Sinbadand Saci-1were closely related to each other and to the IN from the unnamed Pao-element from zebrafish (BK005571). Copies of Sinbadinterspersed throughout the schistosome genome ---------------------------------------------------------------- Southern hybridization analysis of S. mansonigDNA, S. japonicumgDNA and BAC 33-N-3 confirmed the presence of Sinbadin the S. mansonigenome but indicated it was absent from the genome of the related schistosome, S. japonicum(Figure [6](#F6){ref-type="fig"}). BamH I was expected to cut three times within Sinbad, whereas Hind III, which cleaves the BAC 30-H-16 copy of Sinbad, was not predicted to cut within the sequence of the BAC 33-N-3 copy. The probe did not contain restriction sites for BamH I or Hind III. The hybridization signals from the two S. mansonigDNA lanes (Hind III or BamH I digested) were strong and dispersed, with a band of \~2.6 kb in the Hind III digest. The smeared pattern of hybridization indicated that a number of copies of Sinbadwere interspersed throughout the genome of S. mansonirather than being localized at a discrete locus. By contrast, the probe did not hybridize to the gDNA of S. japonicum. Additional blots with larger amounts (30 μg) of S. japonicumgDNA, digestion with BamH I instead of Hind III, and exposure of the film for longer periods failed to yield any signal from S. japonicumgDNA (not shown), indicating that Sinbadwas absent from this schistosome species. Strong hybridization signals were evident in the positive control lanes of digests of BAC 33-N-3. Densitometric analysis of the hybridization signals indicated the presence of 50 to 60 copies of Sinbadper S. mansonihaploid genome, based on four separate estimates comparing the signal in each of the genomic DNA lanes to the signal in each of the 33-N-3 BAC lanes (comparison of lane 1 with lane 4, comparison of lane 2 with lane 5, comparison of lane 1 with lane 5, and comparison of lane 2 with lane 4). (These estimates assumed that BAC 33-N-3 included only one copy of Sinbad.) ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Southern hybridization of Schistosoma mansoniand S. japonicumgenomic DNAs, and S. mansoniBAC clone 33-N-3 BAC DNA to a Sinbadretrotransposon-specific gene probe. Lane 1, S. mansoniDNA (30 μg) digested with Hind III; lane 2, S. mansoniDNA (30 μg) digested with BamH I; lane 3, S. japonicumDNA (20 μg) digested with Hind III; lane 4: BAC 33-N-3 (0.8 μg) digested with Hind III; and lane 5, BAC 33-N-3 (0.8 μg) digested with BamH I. Molecular size standards in kilobase pairs (kb) are indicated at the left. ::: ![](1471-2148-5-20-6) ::: Copy number was estimated by two additional methods. First, upon screening the 23,808 clones of the BAC library of Le Paslier et al. \[[@B31]\] that represents a \~8-fold coverage of the haploid S. mansonigenome, approximately 0.7% to 1.0% of the clones were positive, indicating a copy number for Sinbadof \~20 to 30 copies (not shown). Second, the bioinformatics approach of Copeland et al. \[[@B20]\] was used to compare these estimates with reference copy number estimates of other mobile genetic elements and genes reported previously. BLASTn searches were undertaken using the nucleotide sequences of these reference genes and the complete sequence of Sinbad(Table [1](#T1){ref-type="table"}). Because the construction of the BAC library involved partial digestion of the genomic DNA with Hind III \[[@B31]\], genes without Hind III sites will be underrepresented in the BAC end sequences. Accordingly, since sequenced BAC ends from this library constitute a large proportion of the genomic S. mansonisequences in the public domain, we used only genes containing Hind III sites as reference sequences. As shown in Table [1](#T1){ref-type="table"}, the number of hits for Sinbad, 38, was higher than the number of hits for the single-copy cathepsin D gene (0 hits) but lower than that for the multiple-copy 28S ribosomal RNA gene (157 hits) (\~100 copies; Ref. \[[@B7]\]) and for three high copy number retrotransposons Boudicca(100 hits, 1,000--10,000 reported copies), SR2(102 hits, 1,000--10,000 copies), and SR1(104 hits, 200--2,000 reported copies). In overview, all three methods were in reasonably close agreement, and together they indicated that approximately 50 (range \~20--100) copies of Sinbadreside in the genome of S. mansoni. Based on copy numbers estimated for other schistosome retrotransposons (see \[[@B13]\]), we consider that Sinbadis not a high copy number element. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Estimation of gene copy number of the Sinbad LTR retrotransposon in the genome of Schistosoma mansoni. ::: Gene GenBank Accession Length(bp) Number of hits (Expect 0.000001) Reported copy number Key references ----------------------- -------------------------- ---------------- -------------------------------------- -------------------------- -------------------- Cathepsin D, Intron 4 AY309267 (nt 3213--4849) 3926 0 1 66 *Sinbad* AY506538 6288 38 \~50 This study 28S rRNA Z46503 1694 157 100 67 Boudicca AY662653 5858 100 1,000--10,000 20 SR2 AF025672 3913 102 1,000--10,000 62 SR1 U66331 2337 104 200--2,000 61 Saci-2 BK004069 4946 107 85--850\* 37 Saci-1 BK004068 5980 133 70--700\* 37 \estimated solely by the gene index bioinformatics approach of DeMarco et al. \[37\], whereas the other copy numbers listed here were determined by hybridization and/or other analyses. ::: Sinbad-like elements transcribed in developmental stages of S. mansoni* -------------------------------------------------------------------------- BLASTn analyses were undertaken using the full length of Sinbadas the query sequence and the GenBank EST database of non-human, non-mouse sequences. The database includes more than 130,000 EST sequences from six developmental stages of S. mansoni-- egg, miracidium, cercaria, germball (= sporocyst), schistosomulum, and mixed sex adults \[[@B39],[@B40]\]. Significant hits were found to ESTs from all of these six developmental stages. Of these, the hits with highest similarity to Sinbad, CD111741, CD060185, CD163413, CD062550, CD156994, and CD156946, exhibited contiguous ORFs spanning each EST without frameshifts or stop mutations. Positive ESTs spanning most or all of the LTR, gag, PR, RT, RH and/or IN regions were located in most of these six developmental stages, indicating that Sinbad-like elements are actively transcribed in all or most developmental stages of S. mansoni. Discontinuous distribution of Sinbad-like elements ---------------------------------------------------- In order to examine the phylogenetic distribution of Sinbad-like retrotransposons, we examined numerous complete and partial genomes, including prokaryotes, plants, fungi, animals, and lower eukaryotes \[[@B41]\]. The genomes were searched using tBLASTn with the amino acid sequence corresponding to the region of Sinbadspanning from the Cys-His box to the conserved protease catalytic domain (bp 1588--2236) \[see [Additional file 1](#S1){ref-type="supplementary-material"}\] as the query. To minimize the likelihood of spurious positives, we lowered the E-value for significance from 10 to 0.001; this corresponded to a bit score of 40 or above. Although it is more stringent than that of the BLAST default, this cutoff point was employed because it is permissive enough to detect both Sinbad-like elements and members of the Pao/BELfamily at large. No significant hits were found in any of the plant, fungal, or protist genomes examined, or in the 275 bacterial and 21 archaean genomes searched. All of the nematodes examined were positive. Of the insects, the other branch of the Ecdysozoa, Drosophila melanogasterand Anopheles gambiaecontained Sinbad-like elements, whereas Drosophila pseudoobscuraand Apis melliferadid not. Of the vertebrates, Danio rerioand Takifugu rubripescontained Sinbad-like sequences, whereas Homo sapiens, Mus musculus, Rattus norvegicus, Canis familiaris, Sus scrofa, Gallus gallus, and Bos taurusdid not. Interestingly, although most higher chordates examined were free of Sinbad-like elements, the tunicates Ciona intestinalisand Ciona savigny, were positive for over 100 hits of sequences highly similar to the Sinbadsearch sequence (up to an E-value of 4e^-22^). In addition, the echinoderm Strongylocentrotus purpuratus, a non-chordate deuterostome, was positive for the Sinbadsearch sequence. These findings are summarized in a tree-of-life style illustration, based on the tree presented in Pennisi \[[@B42]\], and drawn in the style of the taxonomic relationship diagrams used at NCBI \[[@B43]\]. (This diagram is not a phylogram, and displays broad relationships among major taxa only; although relationships are in correct branching order, branch lengths are not to scale.) Genomes with regions of significant similarity to Sinbadare marked with a \"+\" symbol and those without are indicated with a \"-\" symbol. The results of a search of dbEST corroborated and expanded these findings, revealing nine non-Schistosomaorganisms with Sinbad-like sequences: C. intestinalis, Molgula tectiformis(tunicate), S. purpuratus, D. melanogaster, Bombyx mori, Salmo salar, Xenopus laevis, and Trichinella spiralis. E-values and accession numbers for the top match for each organism are provided in Table [2](#T2){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Organisms other than schistosomes with significant EST matches to Sinbad. ::: Organism Accession number BLAST score (bits) Expect value --------------------------------------------------- ---------------------- ------------------------ ------------------ Ciona intestinalis(tunicate) BW308116 89 9 e^-17^ Molgula tectiformis(tunicate) AU283942 87 5 e^-16^ Srongylocentrotus purpuratus(purple sea urchin) CD295138 83 7 e^-15^ Drosophila melanogaster(fruit fly) BI583252 75 2 e^-12^ Bombyx mori(silk worm) CK529741 59 1 e^-7^ Salmo salar(Atlantic salmon) CB500934 58 2 e^-7^ Xenopus laevis(African clawed frog) BJ073921 45 0.002 Trichinella spiralis(parasitic nematode) BG520200 45 0.002 ::: Discussion ========== Sinbad-- a novel Pao/BELfamily LTR retrotransposon from the genome of S. mansoni ---------------------------------------------------------------------------------------- Although several LTR retrotransposons have been characterized previously from the genome of S. mansoni, including Boudicca, Saci-1, Saci-2, Saci-3and the fugitive\[[@B17],[@B20],[@B37]\], the Sinbadretrotransposon characterized here is a novel retrotransposon and it is discrete from these other elements. Sequence identity, structure, and phylogenetic relationships indicate that Sinbadis a member of the Pao/BELfamily of retrotransposons. The hallmark structures included a triple Cys-His box zinc finger domain in the Gag polyprotein, protease with the active site tripeptide DSG, RT domain that included a YVDD active site motif, RNAseH with DAS at the active site, and an integrase domain with a DD(49)E spacing of the active site aspartic acid and glutamic acid residues. The YVDD motif of RT, a version of the F/YXDD consensus motif of Gypsy-like LTR retrotransposons, is shared by Paoand BEL. Bowen and McDonald \[[@B32]\] reported that the Cer7-Cer12series of elements from C. elegansdisplayed YVDN at this site. Whether the Asn could replace Asp as the carboxy-residue of this conserved tetrapeptide with retention of enzyme activity remains to be determined by biochemical analysis, although mutation of either aspartate in YXDD of retroviral RT (HIV-1 or Moloney murine leukemia virus) inactivates the polymerase \[see \[[@B44]\]\]. The LTRs of Sinbadin BAC 33-N-3 are identical in sequence, and appeared to contain a putative promoter for initiation of transcription by RNA polymerase II. Along with conservation of most residues contributing to the active sites of the retrotransposon enzyme domains, these structural characteristics suggested that Sinbadis active or had been transpositionally active in the recent past. Several other features also indicated that Sinbadis transpositionally active. Numerous transcripts spanning enzymatic domains and LTRs of Sinbad, from at least six developmental stages of S. mansoni, have been sequenced \[[@B40]\], and of these, the ESTs most closely resembling Sinbadare composed entirely of contiguous open reading frames, suggesting non-mutated copies. On the other hand, potentially inactivating mutations, including stop codons and frameshifts, suggested that the BAC 33-N-3 copy of Sinbadwas incapable of autonomous retrotransposition. If active copies are present, functional proteins coded by these copies could have been used in the recent past to mobilize the 33-N-3 Sinbadcopy in trans, as recorded for other retrotransposons \[[@B45]-[@B47]\], explaining the presence of identical LTRs. Indeed, Frame et al. \[[@B28]\] noted that mutated copies framed by similar LTRs are common in BELlike elements in C. elegans, implying recent transposition. The LTRs of Sinbad, at 386 bp in length, were substantially shorter than those of Saci-1, \~840 bp \[[@B37]\], but longer than those of Gypsy-like LTR retrotransposons from schistosomes, the fugitive, Gulliverand Boudicca. Whereas Sinbadand Saci-1are clearly closely related, dissimilar LTRs and the low amino acid identity of the most highly conserved domains (35 to 52%) confirmed they are distinct retrotransposons. Sinbadcan be added to the catalog of mobile genetic elements characterized from the schistosome genome, where retrotransposons appear to have proliferated and flourished and contributed significantly to its relatively large size (270 MB; \~14,000 protein-encoding genes) \[[@B13],[@B20],[@B40]\]. The colonization of the genome of S. mansoniby Sinbadand Saci-1and that of S. japonicumby the related Tiaoelement \[[@B48]\] represents the first demonstration of infection of a Lophotrochozoan taxon by Pao/BELfamily LTR retrotransposons. The presence of Sinbad, Saci-1, and Tiaoin two species of Schistosomasuggests that an ancestral schistosome was already host to the ancestors of these elements. (Though Tiaois a Pao/BELfamily retrotransposon, and is therefore predicted to be detected in low-stringency BLAST searches, as in Figure [7](#F7){ref-type="fig"}, the absence of a positive signal on the genomic Southern hybridization suggests that it is not particularly closely related to Sinbad.) ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Phylogenetic illustration of species and higher taxa for which data are available concerning Pao-like elements. Species for which genomes have been sequenced and are available for whole genome BLAST searches in GenBank are enclosed in ovals. These genomes were tBLASTn searched using a deduced amino acid sequence from Sinbad(from the Cys-His Box through the protease domain) as the search sequence. Genomes with sequences significantly similar (E ≤ 0.001) to Sinbadare identified by a green \"+\" symbol, and those negative for Sinbad-like sequences with a red \"-\" symbol. Other species, with not yet fully sequenced genomes, shown to include Pao-like sequences (through EST searches or other means) are shown in smaller font and unenclosed, and are also marked with a green \"+\". This diagram is based on a tree of life style diagram in Pennisi \[42\] and reflects broad relationships between taxonomic groups only. It is not a phylogram -- stem lengths do not represent phylogenetic distances. ::: ![](1471-2148-5-20-7) ::: A Sinbad/Saci-1subfamily of Pao-BELlike LTR retrotransposons ------------------------------------------------------------------ Whereas the sequence and deduced structure of the three signature Pao-like elements, Paofrom the silk moth B. mori, Tasfrom the human roundworm Ascaris lumbricoidesand BELfrom D. melanogasterhave been known for about a decade, the Pao/BELfamily is not as well understood or apparently as widespread as the other two major families of LTR retrotransposons, the Copia/Ty1and the Gypsy/Ty3families. However, at least three branches of the Pao/BELfamily have become apparent -- branches represented by Pao, BEL, and Suzu(from T. rubripes) \[[@B27],[@B28],[@B32],[@B49]-[@B51]\]. Using the new sequence information from Sinbad, and some related elements, we have been able to investigate the intra-family relations of the Pao/BELelements more thoroughly. Our findings, based on phylogeny of RT, and supplemented by phylogeny of IN, indicated the presence of at least five sub-families of Pao/BELelements. The majority of the sub-families may have a restricted host range; the Tassubfamily occurred only in nematodes (these elements may be endogenous retroviruses because they appear to include envgenes), the BELsubfamily only in insects, the Paosubfamily only in insects, and the Suzusubfamily only in fishes. By contrast, the Sinbad/Saci-1subfamily is known from schistosomes and zebrafish. Phylogenetic range of Sinbad-like retrotransposons ---------------------------------------------------- The Pao/BELretrotransposons are known only from animals, a less extensive distribution than those of the Copia/Ty1or Gypsy/Ty3groups that include elements known from fungi and/or plants as well as animals. The ostensible absence of these elements from prokaryotes, lower eukaryotes, fungi and plants suggests that ancestral Pao-like elements appeared after the differentiation of the Animalia. Though the number of sequenced entire genomes of animals is small, the distribution of Pao/BELLTR retrotransposons within these few genomes displays a topography that we would not expect to be the result solely of vertical transmission alone (Fig. [7](#F7){ref-type="fig"}). Sinbad-like sequences were found in D. melanogaster, but not in D. pseudoobscura, nor in A. mellifera, even though close relatives are found in other insects such as B. moriand A. gambiae, and even in species as phylogenetically distant as D. rerio(a fish) and S. mansoni(a platyhelminth). Further, the distribution among chordates is enigmatic. Of the vertebrate whole genomes searched, only two, T. rubripesand D. rerio, were positive for Sinbadlike elements. The human, mouse, rat, cow, chicken, pig and dog genomes were devoid of Sinbad-like matches. Since the genomes of lower chordates and a non-chordate deuterostome were positive for Sinbad-like sequences, progressive radiation would be expected to give rise to similar sequences in these vertebrates. Feschotte \[[@B19]\] reported a similarly patchy distribution for the MerlinDNA transposons; Merlinlike elements are abundant, for example, in anopheline mosquitoes but are absent from D. melanogaster, D. pseudoobscura, and A. mellifera. Also, they are present in some vertebrate genomes but not others. Merlin-like elements are also present in schistosome chromosomes. This type of distribution suggests that either the vertical lineage of the elements has been curtailed by the extinction of these elements from several genomes, or that horizontal transmission has taken place. Genomes need to restrain the uncontrolled proliferation of mobile genetic elements, especially retrotransposons, and indeed some eliminate mobile sequences more efficiently than others \[[@B5],[@B52]\]. Goodwin and Poulter \[[@B53]\] have shown that Ngaroelements have been lost from certain genomes, as evidenced by the presence of small, corrupt fragments serving as fossil sequences. Similarly, especially in view of the low number of Sinbadcopies, Pao-like elements may have followed a course of progressive radiation followed by elimination from the Sinbad-negative genomes. However, if this were the case with Pao-like elements, relic sequences could be expected in at least some of the Sinbad-negative genomes. Their absence from mammalian and avian genomes favors the alternative explanation, that the current range reflects horizontal transmission. What might have been the origin of the Pao/BELradiation within the Animalia? Felder et al. \[[@B34]\] suggested that a common ancestor of Tasand Paomay have undergone a horizontal transmission event between the Insecta and Nematoda, followed by the eventual differentiation of these elements, including the gain or loss of env. Of the sub-families of Pao/BELelements apparent in the RT-based phylogram (Figure [5](#F5){ref-type="fig"}), the Tassubfamily includes retrotransposons with an envelope encoding gene (specifically Tasfrom A. lumbricoidesand Cer7 from C. elegans). The acquisition of an envelope protein by an ancestral Tasor Tas-like element would have enabled its extracellular existence and facilitated its horizontal transmission and infection of other hosts \[[@B38]\]. Interestingly, the deuterostomes bearing Sinbad-like sequences included a sea urchin, tunicates, pufferfish, zebrafish, the Atlantic salmon, and the African clawed frog X. laevis(Figure [7](#F7){ref-type="fig"}). These are aquatic species and, moreover, all are known from coastal or brackish waters at the interface of freshwater and marine systems. The secondary hosts of S. mansoni, snails of the pulmonate genus Biomphalaria, are also aquatic, as are the larval (miracidium and cercaria) stages of S. mansoniwhich enter and exit the snail. It will be of interest to determine whether or not Pao-like elements are present in this snail host, from which numerous RT-encoding sequences already have been reported \[[@B54]\]. Also of potential relevance is that the genomes of both X. laevisand S. mansonicontain Pao-like elements and that X. laevisis the secondary host of the trematode parasite Tylodelphys xenopi\[[@B55]\], a fluke closely related to the human schistosomes. Both T. xenopiand another human schistosome, Schistosoma haematobium, use snails of the genus Bulinusas intermediate hosts. An aquatic lifestyle is an obvious relationship that links all of the deuterostome hosts of Sinbad-like elements. This aquatic, in comparison to a terrestrial, existence may have facilitated transmission of infectious particles of the Tas-like ancestors of Pao, Tas, BEL, Suzu, Sinbad, and relatives. Alternatively, schistosomes may have acquired a Tas- element directly from Ascaris lumbricoides, an exceedingly common human parasite and the host of Tas. A. lumbricoidesoccurs in the intestines of infected people, as do schistosome eggs, so direct transmission of a mobile genetic element from roundworm to schistosome could have been facilitated by their physical proximity within the human intestines. Conclusion ========== A Pao/BELlike LTR retrotransposon named Sinbadis interspersed within the genome of the blood fluke, S. mansoni. About 50 copies of this element appear to reside in the S. mansonigenome. Analyses of the phylogenetic distribution of Pao/BEL-like retrotransposons indicated that Pao/BEL-like elements are present only within phyla of the Animalia, and not in prokaryotes, fungi or plants. Further, the analyses indicated that there are at least five discrete sub-families of the Pao/BELclade of LTR retrotransposons, and that the distribution of these retrotransposons among the Ecdysozoa, Lophotrochozoa and deuterosomes has been influenced by horizontal as well as vertical transmission. Methods ======= Screening the bacterial artificial chromosome library ----------------------------------------------------- Le Paslier et al. \[[@B31]\] described the construction and characterization of a bacterial artificial chromosome (BAC) library of the Schistosoma mansonigenome. The library, constructed in the plasmid vector pBeloBac11 with genomic DNA (gDNA) from cercariae of a Puerto Rican strain of S. mansonipartially digested with Hind III, consists of 23,808 clones, about 21,000 of which are estimated to contain inserts ranging from 120 to 170 kb, providing \~8-fold coverage of the schistosome genome. Numerous BAC end sequences determined from randomly selected clones from this library are in the public domain. Inspection of the end sequence of BAC clone number 30-H-16 indicated identity with Pao-like LTR retrotransposons (not shown). Because the retrotransposon sequence was located at the end of the BAC, the clone was unlikely to contain the entire Pao-like element. Given that retrotransposons can be expected to be present in multiple copies in the host genome, we screened the library with a probe based on the end of BAC 30-H-16 in order to locate an entire copy of the retrotransposon. The gene probe was obtained by PCR amplification of a fragment of BAC 30-H-16 using the primers 5\'-CGCGGATCCAAGAGAAAAACCTTGATAGAC and 5\'-CCGGAATTCCTGTCGAAGATAAAAGAGC, was cloned into pBluescript and its identity confirmed by sequencing (Accession AY871176). This probe spanned residues 2457 to 2823 of the BAC 33-N-3 copy of the new retrotransposon (see below). The cloned insert was labeled with digoxygenin (DIG) and employed to screen the BAC library, as described \[[@B20]\], represented as high-density clone arrays on nylon membranes. Positive clones were cultured as described \[[@B31]\] and the presence of sequences with identity to the novel retrotransposon in the positive clones was confirmed by PCR (primers as above) or by colony hybridizations \[[@B56]\] to the DIG labeled probe. One positive clone, BAC 33-N-3, was investigated further by sequence analysis. BAC plasmid DNA was isolated from bacterial cultures using the PhasePrep BAC DNA purification system (Sigma). Analysis of the insert of 33-N-3 was accomplished after subcloning BamH1 fragments of the BAC into pNEB 193 (New England Biolabs, MA), sequencing the inserts of the sub-clones, and also by direct sequencing of BAC 33-N-3. Automated nucleotide sequencing, using ABI BigDye Terminator chemistry (ABI, Foster City, CA) and an ABI Prism 3100 sequencer, was undertaken using primers specific for the probe and subsequently with gene specific primers at Tulane University and at Davis Sequencing (Davis, CA). Sequence analysis and alignments -------------------------------- Contigs of the sequences were assembled using SeqMan (DNAstar, Inc., Madison, WI). Repeat sequences were identified with a Pustell style dot matrix \[[@B57]\] using the DotPlot3 program (Ramin Nakisa, Imperial College, London, UK) \[see \[[@B58]\]\] and the Pustell DNA Matrix function in MacVector (Accelrys). Amino acid alignments were accomplished with MacVector and ClustalW \[[@B59]\] using sequences from GenBank or using conceptual translations of nucleic acid sequences. Open reading frames were located and conceptually translated using MacVector. Sequences of the following retrotransposons were used in the multiple sequence alignments based on gag, protease, and reverse transcriptase: Ninja, T31674; Pao, S33901; MAX, CAD32253; Roo, AAN87269; BEL, AAB03640; and Saci-1, BK004068. Sequences of the following retrotransposons were used in the multiple sequence alignment based on Integrase: Saci-1, DAA04498;Pao, S33901; Ninja, T31674; Roo, AAN87269; Suzu, AF537216, BEL, AAB03640, Tas, Z29712, and MAX, CAD32253. Parasite DNAs, Southern hybridization, densitometric estimation of copy number ------------------------------------------------------------------------------ Genomic DNAs of cercariae of a Puerto Rican strain of S. mansoniand of adults of a Chinese (Anhui Province) strain of S. japonicumwere extracted using the AquaPure Genomic DNA Purification system (Bio-Rad, Hercules, CA). S. mansonigDNA (30 μg/lane) and 33-N-3 BAC DNA (800 ng) were digested with Hind III and BamH I restriction enzymes, and S. japonicumgDNA (20 μg/lane) was digested with Hind III. Digested gDNA and BAC DNA were size fractionated by electrophoresis through a 0.8% agarose gel, transferred to a nylon membrane (Zeta-Probe GT, Bio-Rad) by capillary action \[[@B60]\], and UV-light cross-linked to the membrane. Southern hybridization analysis to the DIG-labeled probe (above) was performed as described \[[@B20]\]. Chemiluminescent signals were detected using X-ray film (Fuji). Densitometric analysis of Southern hybridization signals was accomplished using the Versa-Doc gel documentation system (Bio-Rad) and Quantity-One software (Bio-Rad). Densitometry values for signals evident in the gDNA and BAC DNA lanes were used to estimate the copy number for the new retrotransposon, Sinbad, according to the formula \[(A/B) × C\]/E= F. This formula was derived from two equations: (A/B) × C= Dand D/E= F, where Awas the number of copies of Sinbadin the BAC 33-N-3 lane, Bwas the density volume of the 33-N-3 lane in units of optical density per mm^2^, Cwas the density volume of the S. mansonigenomic DNA lanes in units of optical density per mm^2^, Dwas the total number of copies of Sinbadper genomic DNA lane, Eis the number of haploid genomes in the gDNA lane, and Frepresented the copy number of Sinbadper haploid S. mansonigenome. The insert of 33-N-3 was estimated to be 145 kb in length and assumed to contain only a single copy of the retrotransposon. Other copy number estimations ----------------------------- In addition to the densitometry-based estimate, estimates of the copy number of the Sinbadretrotransposon also were obtained by a comparative bioinformatics approach \[[@B20]\] wherein BLAST analysis of the bacterial artificial chromosome (BAC) -end database of S. mansonigenomic sequences targeted more well-characterized retrotransposable elements from S. mansonifor which copy numbers had been reported. These included the BoudiccaLTR retrotransposon \[[@B20]\] and the non-LTR retrotransposons SR1and SR2\[[@B61],[@B62]\]. The NCBI database was searched by BLAST using the sequences of these mobile genetic elements and some other genes of S. mansoni, all of which included at least one Hind III site. Specifically, the Advanced BLAST function was used, set to search only the S. mansonisequences in the GSS database (Limit by Entrez Query: \<Schistosoma mansoni\[organism\]\>), and with the E value at 0.000001. The E value (Expect value) reflects the probability of obtaining a match purely by chance. Scores at or below this stringent cutoff E value of 10^-6^were counted as positive. This exceptionally stringent cutoff value was used to minimize the chance of counting other Pao-like elements in the total copy number of Sinbad. Since the formula for E is based not only on the bit scores of the local alignment of each pair of sequences, but also on the lengths of the subject and query \[see \[[@B63]\]\], no additional correction was made for the length of the query sequence. Phylogenetic analysis of Pao-like elements -------------------------------------------- Sequences for phylogenetic analysis comparing the RT region of several different retrotransposons were prepared by trimming sequences from the large single polyprotein of each retrotransposon to just the conserved domains of RT (see \[[@B21],[@B27]\]). Pol sequences presented in Xiong et al. \[[@B21]\] and Abe et al. \[[@B27]\] were trimmed exactly to the stretch of sequence shown by these authors to represent the RT domain. Other elements were aligned with these sequences and likewise trimmed to obtain just the RT domain. For some elements, nucleotide sequences were analyzed for open reading frames and translated before being trimmed to include just the 7 conserved blocks of the RT domain. Alignments were accomplished using Clustal X \[[@B64]\], after which bootstrapped trees (1,000 repetitions) were prepared using the neighbor joining method \[[@B65]\] and drawn with Njplot. The accession numbers for sequences included in the phylogenetic analysis are as follows: Ty3, S53577; Tas: Z29712; Suzu, AF537216; Sinbad, AY506538 (an N was inserted at position 2761 to a resolve a frameshift and generate a single ORF) Saci-1, DAA04498; Roo, AAN87269; Ninja, T31674; Moose, AF060859; Max, CAD32253; Kamikaze, AB042120; HIV-1, P04585; Gypsy, GNFFG1; Gulliver, AF243513; Copia, OFFCP; BEL, AAB03640; Cer7, AAB63932, Cer8, CAB04994, Cer9, CAB1647, and Cer11, AAA82437, two uncharacterized Anopheles gambiaeretrotransposons, XP\_309281 and XM\_308737, an uncharacterized Caenorhabditis briggsaeretrotransposon, AC084491, and two uncharacterized Danio rerioretrotransposons, BX537152 and BX005079 \[see [Additional file 2](#S2){ref-type="supplementary-material"}\]. Two additional sequences were either not in the database or were composites made to reconstruct sequences more closely resembling non-mutated forms of the retrotransposons. The sequence representing Paowas a reconstruction prepared by Abe et al. \[[@B27]\], from accession numbers S33901, AB042118, and AB042119; the sequence representing Boudiccawas a composite of translated cDNA sequences introduced in Copeland et al. \[[@B22]\], AY308018, AY308019, AY308021 and AY308022 \[see [Additional file 2](#S2){ref-type="supplementary-material"}\]. Screening entire or partial genomes for Sinbad ------------------------------------------------ A panel of fully or partially sequenced entire genomes was searched by BLAST for elements exhibiting sequence similarity to Sinbad. The deduced amino acid sequence encoding the region from the Cys-His Box through to the protease domain (encoded by nucleotides 106 to 753 of Sinbad\[Y506538\]) was employed as the query to search each genome individually using tBLASTn. The genomes searched in this way were as follows: Homo sapiens, Mus musculus, Rattus norvegicus, Takifugu rubripes, Danio rerio, Bos taurus, Gallus gallus, Sus scrofa, Canis familiaris, Anopheles gambiae, Apis mellifera, Drosophila melanogaster, Drosophila pseudoobscura, Brugia malayi, Caenorhabditis elegans, Caenorhabditis briggsae, Strongylocentrotus purpuratus, Ciona intestinalis, Ciona savigny, Giardia lamblia, Plasmodium falciparum, Plasmodium yoelii, Plasmodium berghei, Cryptosporidium parvum, Eimeria tenella, Theileria annulata, Toxoplasma gondii, Dictyostelium discoideum, Entamoeba histolytica, Leishmania major, Trypanosoma brucei, Trypanosoma cruzi, Arabidopsis thaliana, Avena sativa, Glycine max, Hordeum vulgare, Oryza sativa, Triticum aestivum, Zea mays, Lycopersicon esculentum, Schizosaccharomyces pombe, Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces bayanus, Saccharomyces castelli, Saccharomyces kluyveri, Saccharomyces kudriavzevii, Neurospora crassa, Magnaporthe grisea, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus terreus, Candida albicans, Coccidioides posadasii, Gibberella zeae, Coprinopsis cinerea, Cryptococcus neoformans, Ustilago maydisand Encephalitozoan cuniculi. In addition, 275 eubacterial and 21 Archaean genomes were searched \[see [Additional file 3](#S3){ref-type="supplementary-material"}\]. Genomes with matches with E values less than 0.001 (corresponding approximately to bit scores greater than 40) were considered positive for Sinbad-like elements. GenBank accession numbers ------------------------- Sequences of the SinbadLTR retrotransposon have been assigned accession numbers AY506537, AY506538, AY645721, AAT66412, and AY871176. Other sequences introduced here been assigned GenBank Third Party Annotation accession numbers; BK005570 (Danio rerio), BK005571 (D. rerio), BK005572 (Caenorhabditis briggsae), BK005573 (Anopheles gambiae), BK005574 (D. rerio). Abbreviations ============= MGE, mobile genetic element; ORF, open reading frame; EST, expressed sequence tag; gDNA, genomic DNA; LTR, long terminal repeat; RT, reverse transcriptase; PR, protease; IN, Integrase; CHB, Cys-His box; BAC, bacterial artificial chromosome; MB, megabase pairs Authors\' contributions ======================= CSC carried out the sequence analyses, sequence alignments, phylogenetic studies, other bioinformatics analyses, and Southern hybridizations, participated in cloning, sequencing and design of the study, and, together with PJB, drafted the manuscript. VHM and MEM participated in cloning and sequencing. BHK contributed to the design of experiments and analyses. PJB participated in the design and coordination of the study, and drafting the manuscript. All authors read and approved the final version of the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 \"Annotated Sinbadsequence\". Nucleotide and deduced amino acid sequence of the entire Sinbadretrotransposon in BAC clone 33-N-3. Hallmark features of the retrotransposon are identified in colored highlights as described in the key at the bottom of the figure. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 \"RT domain sequences of new and consensus elements used in the phylogenetic analysis\". Deduced amino acid sequences of the RT domains used in the phylogenetic analysis from newly characterized elements, uncharacterized elements found within genome survey sequences, and elements for which consensus sequences were used. Accession numbers for the source sequences of each element are listed, as well as references where applicable. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 \"Prokaryotic genomes negative for Sinbadlike elements\" Table of prokaryotic genomes indicated by whole genome analysis to be devoid of Sinbadlike elements. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ Schistosome parasites were supplied by Dr. Fred Lewis through NIAID-NIH supply contract NO1-A1-55270. We thank Dr. Philip LoVerde for provision of the BAC library and the anonymous reviewers for helpful suggestions. This investigation received financial support from the Ellison Medical Foundation (Infrastructure Grant award ID-IA-0037-02). PJB is a recipient of a Burroughs Wellcome Fund Scholar Award in Molecular Parasitology.	PubMed Central	2024-06-05T03:55:53.958163	2005-2-23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554778/", "journal": "BMC Evol Biol. 2005 Feb 23; 5:20", "authors": [ { "first": "Claudia S", "last": "Copeland" }, { "first": "Victoria H", "last": "Mann" }, { "first": "Maria E", "last": "Morales" }, { "first": "Bernd H", "last": "Kalinna" }, { "first": "Paul J", "last": "Brindley" } ] }
PMC554779	Background ========== There is wide variation in the quality of care provided by primary care practices to individuals with chronic illnesses \[[@B1]\]. Individual doctor attitudes and interest have been demonstrated to influence patient outcomes in some instances \[[@B2],[@B3]\] and it has been argued that specific attitudes are more predictive of behaviour than general attitudes\[[@B4]\]. For chronic diseases, in line with the distinction proposed by Katz \[[@B5]\], these doctor attitudes may be separated into perceptions of knowledge, skills and personal preferences. The importance of these specific doctor attitudes on patient outcomes is however largely unknown. There is a marked trend to larger partnerships in primary care practices and more flexible working practices. It is likely, therefore, that continuity of care will continue to fall, and that patient experience of care of a particular condition will be based on contact with more than one general practitioner \[[@B6]\]. Thus practice-based rather than individual general practitioner (GP) attributes and attitudes are likely to become increasingly important. The extent to which GP attitudes to specific chronic conditions cluster within practices, is however currently unknown. There is evidence that where attitudes within a group are shared this enhances the influence of individual attitudes on behaviour \[[@B7]\]. Thus, on the basis of this observation, if attitudes are shared by general practitioners within practices, these group-based attitudes are then more likely to influence GP behaviour and the quality of care provided by the clinician. In the next few years there is likely to be considerable reorganisation of the way in which epilepsy care within primary care is delivered, with GPs taking on a more active role in providing care. Information on how individual general practitioners view and value their role in providing epilepsy care is considered as important \[[@B8]\]. However, what may be more important is whether or not these views are shared within the practice and if these attitudes influence the quality of care provided by the practice. If this is the case, then taking GP attitudes in a given practice into account will be crucial in deciding how primary care services for people with epilepsy are best organised and improved. In this paper, data from a completed community-based study on people with epilepsy are used to examine the following questions: 1\. To what extent do individual general practitioner attitudes to the care of people with epilepsy cluster within practices? 2\. Do general practitioner attitudes predict how people with epilepsy rate the quality of the general practitioner care of their epilepsy? Methods ======= General practitioners and adults with epilepsy taking part in an intervention study in Greater Manchester provided information for this study. The results of the intervention study (a prompt and reminder card for general practitioners to complete, held by patients or placed in their medical records and used opportunistically over the course of a year) have been reported \[[@B9]\]. No group differences in patient rated outcome measures were found for the intervention study \[[@B9]\]. Ethical approval was obtained in the 4 areas of Greater Manchester from where patients were approached. The patients who consented to participate in the study had their medical records examined to extract data on recording of clinical information about epilepsy and other markers of quality of care. Patients were also sent questionnaires for self-completion. These included both a generic quality of life measure- the EUROQOL 5D\[[@B10]\] and a disease specific quality of life and quality of care measure, the \"Living with Epilepsy\" questionnaire which has been psychometrically tested and shows good reliability and validity \[[@B11]\]. Self-rated seizure frequency (included in the \"Living with Epilepsy\" questionnaire) was based on the response to a question \"How many epileptic attacks have you had in the past year\" with the 3 response categories being \"None\", \" Less than one a month\" and \"One or more a month\". General practitioners completed a 17-item GP epilepsy attitudes questionnaire at the end of the study. Responses to items such as \"I feel comfortable changing the type of anti epileptic drugs in my patients\" were scored using a Likert scale. The attitudes scale was developed and validated for a previous study and results reported in an earlier paper \[[@B12]\]. Statistical analysis -------------------- Mean practice scores for each item on the GP attitudes questionnaire were computed and significant factors underlying the grouping identified (using eigenvalues \>1.2 and the Scree test.). Clustering of responses on the attitudes questionnaire were examined using the intra-class correlation coefficient both for individual items as well as for a mean score of responses to items loading on each of the main factors. Finally linear regression analysis with the patient-rated quality of care provided by the practice as the dependent variable and GP attitudes and other patient derived measures (such as seizure frequency, age, gender, other long term illness) as independent variables was carried out (using aggregated GP attitude and patient scores). Intervention group was adjusted for. P values of 0.05 and 95% confidence intervals were used to assess significance. Results ======= 1255 patients consented to participate in the study and 975 patients filled in final questionnaires. 199 GPs from 82 practices consented to participate in the study. Responses were obtained from 115 GPs (60% of total) from 64 practices (83% of total). 29 practices had a single respondent. These practices were excluded from the analysis of attitude clustering. In this study 54% of individuals were seizure-free in the previous year (\"controlled\" seizures) and 46% had reported a seizure in the previous year (\"uncontrolled\" seizures). Factor analysis and clustering of GP attitudes ---------------------------------------------- Factor analysis with varimax rotation was undertaken on aggregate GP responses. Four factors had an eigenvalue of above 1.2. Three of these factors were selected after the scree test. Both the Kaiser-Meyer-Olkin measure of Sampling adequacy test (0.778) and Bartletts test for sphericity (Chi-square 413.7, Df 120, p \< 0.0001) suggested that factor analysis was appropriate for this data set. Using guidelines for identifying significant factor loadings based on sample size from Hair et al.\[[@B13]\] a cut-off of 0.65 was used. Individual items within each factor were used to generate mean factor scores. These mean factor scores were normally distributed. Responses to the 11 questions that were included in the first three factors were further examined. The aim was to detect if significant clustering of responses to these items occurred within practices. The average cluster size was 2.74. The results of the factor analysis and clustering of attitudes for the two main factors are listed in Table [1](#T1){ref-type="table"}. The other two factors were excluded. The first excluded factor was not clinically meaningful with only two disparate items loading on it (\"epilepsy care straightforward\", \"epilepsy patients viewed as being well-informed\"). The second excluded factor explained less than 10% of variance and only had one item loading on it (\"self-perceived knowledge of epilepsy\"). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Two main extracted factors, significant factor loadings and intraclass correlation coefficients (ICC) for general practitioner attitudes within practices ::: Factor loading^1^ ICC^2^ --------------------------------------------------------------------------------------------------------- ------------------- ---------- Factor 1: \"Primary care responsibility\"(Eigenvalue 3.98, 24.9% of variance explained)-mean scores 0.40 \"Not too time pressured to take on epilepsy care\" 0.785 0.37\\ \"GP has primary responsibility for organising follow up care\" 0.769 0.13 \"Epilepsy care not too difficult to organise\" 0.767 0.19\* \"Epilepsy care not a specialist responsibility\" 0.732 0.34\\ \"Epilepsy care should be based in general practice\" 0.684 0.44\\ \"Annual structured review should be carried out in primary care\" 0.657 0.10 Factor 2: \"Medication skills\"(Eigenvalue 2.74, 17.1% of variance explained) 0.35 \"Comfortable adjusting dose of medication\" 0.724 0.31\\ \"GP responsible for adjusting treatment if more fits\" 0.718 0.25\* \"Comfortable adjusting type of medication\" 0.655 0.17 p \< 0.05, \\ p \< 0.01 ^1^(based on mean GP scores per practice) ^2^(based on individual GP scores in practices with \>1 respondent) ::: Do GP attitudes predict how patients rate the quality of GP care of their epilepsy? ----------------------------------------------------------------------------------- Data from 60 practices where both patient and general practitioner data were available were used in the linear regression analysis. As the data were obtained at the end of an intervention study, the intervention group was also included as an independent variable. The results of this analysis are given in Table [2](#T2){ref-type="table"}. Significant predictors of patient-rated quality of GP care were patientseizure frequency and patient-rated \"shared decision making\" and GP-rated score on \"epilepsy care being a primary care responsibility\" (Factor 1). Recording of clinical information about epilepsy was not a significant predictor of patient-rated quality of GP care. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Linear regression analysis: GP and patient predictors of patient rated satisfaction with GP care of epilepsy ::: Unstandardized Coefficient Standardized Coefficient t value Sig. 95% Confidence intervals for B ---------------------------------------------------------------------------- ---------------------------- -------------------------- --------- ---------- -------------------------------- ---------- ----------- *Patient measures* Age -.012 .006 -.19 -1.80 .079 -.025 .001 Gender -.4 .258 -.19 -1.55 .128 -.921 .120 Long term health problems other than epilepsy -.141 .255 -.05 -.55 .583 -.655 .373 Anxiety score .051 .028 .24 1.80 .078 -.006 .108 Depression scores -.048 .041 -.17 -1.16 .255 -.131 .036 Ease of talking to GP about epilepsy .392 .348 .13 1.128 .266 -.309 1.109 GP takes views of epilepsy into account (\"shared decision making\")\* .931 .390 .31 2.38 .022 .144 1.719 Seizure frequency\* .306 .148 .26 2.07 .044 .008 .604 *GP measures* *Epilepsy as primary care responsibility\ (factor 1) .154 .069 .29 2.23 .031 .015 .294** Medication skills (factor 2) -.033 .059 -.07 -.564 .576 -.152 .086 Data adjusted for intervention group r^2^= 0.635, adjusted r^2^= 0.525, standard error = 0.224 \* p value \<0.05 ::: Some further bivariate analyses were also undertaken. Recording of clinical information about epilepsy by GPs was not significantly associated with the GP-rated score on epilepsy care being a primary care responsibility but was associated with seizure frequency. Discussion ========== In this study two main factors (\"epilepsy viewed as a primary care responsibility\" and \"medication skills\") were found to underlie GP attitudes to the care of people with epilepsy. Responses to questions constituting these factors demonstrated a high and significant level of clustering within practices. The main factor that accounted for the largest proportion of variance, general practitioner-rated \"epilepsy viewed as a primary care responsibility\", significantly predicted patient-rated quality of care. Patient-rated shared decision-making and seizure frequency were other significant predictors of patient-rated quality of GP epilepsy care. Recording of clinical information by GPs about epilepsy was not associated with GP attitudes to epilepsy care but was related to patient seizure frequency. In this study general practitioner attitudes to the care of people with epilepsy were found to cluster within practices to a considerable extent. This has not previously been shown in the U.K. A recent Dutch study \[[@B14]\] showed that GPs working in the same partnership showed more resemblance in overall attitudes to patient care and behaviour than GPs not working in the same partnership and hypothesised that social processes in partnerships and local circumstances may be particularly relevant. The present study has quantified these intra-practice GP similarities in terms of attitudes to one specific chronic condition. Moreover the results of this study also demonstrate that certain general practitioner attitudes predict patient-rated quality of care provided by the practice. The high level of clustering of GP attitudes and the effect of these attitudes on patient-rated outcomes in terms of quality of care, may have important implications in determining the effectiveness of practice level interventions in primary care. These results suggest, firstly, that when planning educational interventions, changing GP attitudes within practices should also be a key aim and, secondly, to focus on changing attitudes for the practice as a whole rather than simply for individual general practitioners. In addition, for practice level intervention studies (especially those using patient-rated quality of care as an outcome measure) an estimate of clustering of doctor attitudes as well as estimates of clustering of patient responses when carrying out power calculations should be incorporated to avoid making a Type 1 error. There is relatively little information of the relationship of GP attitudes to patient ratings of the quality of GP epilepsy care. Existing evidence suggests that GPs with a special interest in a particular condition improves outcomes \[[@B2]\]. The results of the present study extend these findings by highlighting the importance of specific attitudes (accepting a key role in management) rather than perceptions of specific skills (skills in medication management) in predicting patient rated quality of care. The results of a multilevel analysis examining patient and doctor predictors of patient satisfaction from the Netherlands \[[@B15]\] suggested that most of the variance in \"patient satisfaction\" scores was at the patient level (age, morbidity and previous negative experience with the GP being the main predictors) with only 5--10% of the variance in \"patient satisfaction\" being at the doctor level. However in that Dutch study \[[@B15]\], specific GP attitudes were not included as predictors and the \"patient satisfaction\" score was a composite score incorporating measures of accessibility, availability, humaneness of the GP and information provision. Patient-centred communication skills are known to be associated with improved patient satisfaction \[[@B16]\] and our analysis indeed found that patient-rated shared decision making (\"GP took my views into account\") was another significant predictor. Patient ratings of the quality of care do vary according to whether individuals have controlled or uncontrolled seizures. Individuals with controlled seizures rate the quality of care provided higher than individuals with uncontrolled seizures. However why the ratings of care provided are higher is not clear as individuals with controlled and uncontrolled epilepsy differ from each other in other characteristics that may influence quality ratings apart from seizure frequency (e.g. depression scores, social functioning). At practice level GP attitudes are not related to mean practice patient seizure frequency. Although it is likely that individual GP attitudes will be influenced by whether an individual patient has controlled or uncontrolled seizures it is not possible to empirically demonstrate this relationship, as nearly all general practitioners will see a mix of individuals with \"controlled\" and \"uncontrolled\" seizures in a given year. Their attitudes to the care of people with epilepsy will be influenced by this spectrum of epilepsy severity (and often to a greater extent by other factors including significant events with individual patients). In terms of limitations of the results, some practices did not consent to take part in this intervention study and not all GPs who participated completed questionnaires. However there were no significant differences between practices that participated and did not participate in terms of size, average deprivation or training status \[[@B9]\]. Moreover responses to the GP questionnaire were received from over 80% of practices that participated and 60% of the doctors that participated. Aggregate scores were used when doctor and patient views were analysed. This will reduce variability and may result in a loss of statistical power. However given that many different patient-doctor encounters are likely within a given year this approach was the most pragmatic. Although the results were obtained at the end of an intervention study that may have influenced attitudes one of the groups in the study was a control group and no significant differences in GP attitudes between groups was found. Furthermore, results on GP attitudes in the present study were very similar to those found in a previous survey using this scale \[[@B12]\]. Conclusion ========== Specific general practitioner attitudes to the care of people with epilepsy are significantly associated with patient-rated quality of epilepsy care and cluster within practices. It is important to take these findings into consideration when planning interventions and services. General practitioners need to have good knowledge and skills in the management of epilepsy and should be aware of and utilise current guidelines for good clinical epilepsy care \[[@B17]-[@B19]\] to fully utilize medical and surgical expertise in managing epilepsy. Recognising and addressing general practitioner attitudes to the care of people with epilepsy may be important in ensuring these goals of good epilepsy care are met. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= AT designed and ran the study, undertook the analysis and wrote the manuscript. MR was involved in the design and running of the study and edited the paper. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2296/6/9/prepub> Acknowledgements ================ The original study was funded by the Department of Health. AT has been subsequently funded by a Health Foundation mid-career fellowship. We thank all the practices, GPs and the patients who took part and are grateful for suggestions on statistical analysis provided by Dr. Gordon Harold, Dr Kerenza Hood and Professor Ian Russell. Professor Ann Jacoby, Professor Ian Russell, Dr Chris Roberts, Mrs Elaine Porter, Ms Sonia Wall were involved in the original intervention study.	PubMed Central	2024-06-05T03:55:53.964127	2005-3-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554779/", "journal": "BMC Fam Pract. 2005 Mar 1; 6:9", "authors": [ { "first": "Ajay K", "last": "Thapar" }, { "first": "Martin O", "last": "Roland" } ] }
PMC554780	Background ========== Many persons with schizophrenia report chronically disturbed sleep \[[@B1],[@B2]\]. Independent of the phase of illness, sleep disturbance documented by polysomnography include: difficulties falling asleep, awakening too early and being unable to go back to sleep, a preference for being awake during the evening, reduced deep or slow-wave sleep (the most restorative stage of sleep), and short REM latencies \[[@B3]-[@B6]\]. In addition to being a source of distress, various forms of sleep disturbance have also been linked to heightened levels of thought disorder \[[@B7]\] and symptoms of excitement \[[@B8]\] and may portend relapse \[[@B9]\]. While sleep disturbance appears widespread in schizophrenia and is related to clinical features, less clear is whether it is also related to impaired coping that characterizes the disorder. Is poor sleep quality another factor that uniquely contributes to the difficulties persons with schizophrenia experience coping with stressors, and sustaining relationships and work? There appear to be several reasons to hypothesize that it may. Impaired sleep may make it difficult for persons with schizophrenia to cope with stressors \[[@B10]\]. Persons with schizophrenia and impaired sleep might feel, for instance, especially exhausted and highly inclined to avoid stressors and have difficulty seeing the positive aspects of daily challenges. Consistent with this, at least one study finds that damaged sleep quality in schizophrenia relates to longer periods of time spent in bed \[[@B2]\]. With low energy level and need for more time in bed, it seems a matter of intuition that persons might have great difficulty sustaining interpersonal relationships and adapting to the demands of a work setting. Additionally, beyond its effects on coping, we might expect persons with impoverished sleep, irrespective of their symptom level, to have less social and vocational satisfaction than those with better sleep. Indeed, one study indicates that sleep quality is associated with quality of life for patients with schizophrenia \[[@B11]\]. To examine relationships between sleep quality, coping and quality of life, we simultaneously assessed these domains along with symptom level in a group of patients with chronic schizophrenia and schizoffective disorder in a post acute phase of illness. To assess sleep quality we used the Pittsburgh Sleep Quality Index (PSI \[[@B12]\]), a standardized self-assessment tool that gauges sleep quality during the past month. We chose the PSI because it has been successfully used in several studies of persons with schizophrenia \[[@B2],[@B11],[@B13],[@B14]\]. To assess coping we used the Ways of Coping Questionnaire (WCQ), a widely used instrument that measures the relative degree of preference for a number of coping strategies and which is sensitive to the maladaptive coping preferences common to schizophrenia \[[@B15]\]. To assess symptoms and quality of life, we used two instruments that are the gold standard in schizophrenia research: the Positive and Negative Syndrome Scale (PANSS \[[@B16]\]) and the Quality of Life Scale (QLS \[[@B17]\]). We predicted that, controlling for levels of positive and negative symptoms, poorer sleep quality would predict poorer quality of life overall, a greater preference for avoidant coping, and a lesser preference for coping by positive reappraisal. Some doubt the abilities of persons with schizophrenia to report their own sleep patterns accurately. However, a study comparing polysomnography with subjective measures of sleep found that they were highly correlated in a group of patients with chronic schizophrenia \[[@B18]\]. Similarly, others found that persons with severe mental illness can generally report the quality of their lives as well as those with other non-psychiatric illnesses \[[@B19],[@B20]\]. In addition, we wanted to know if actigraphy is an effective way of demonstrating sleep problems in the post-acute phase of schizophrenia. To address this question, we used actigraphy to monitor sleep patterns in a subset of this patient population. Wrist actigraphy consists of monitoring locomotor activity with a motion sensor slightly bigger than a wrist-watch that is worn on the non-dominant wrist. It is a common method used to assess sleep \[[@B21]\]. Actigraphy is valid and reliable for evaluating sleep patterns in insomnia, in diagnosing circadian rhythm disorders, and in assessing sleep in subjects who are unlikely to tolerate polysomnography \[[@B22]\]. There are small studies that suggest the feasibility of using actigraphy in assessments of sleep disturbance in patients with schizophrenia \[[@B23],[@B24]\]. Methods ======= Participants ------------ All subjects met the Structured Clinical Interview for DSM IV (SCID \[[@B25]\]) diagnosis DSM-IV-TR \[[@B26]\] criteria for schizophrenia or schizoaffective disorder. All were in the post acute phase of illness; with no hospitalizations or medication changes for at least one month. Most were patients with chronic schizophrenia who were in a VA day treatment setting. All patient-subjects participated in an informed consent procedure and signed informed consent forms for this research. A tabulated summary of the participants\' characteristics is in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Subject symptoms and demographics ::: Subject Characteristics Mean (S.D.) ------------------------------ ------------- Subjects Enrolled 29 Men 27 Women 2 Age 48 (7) Education 12 (2) Age at first hospitalization 25 (9) Number of hospitalizations 12 (6) SCID diagnosis Schizophrenia 23 Schizoaffective disorder 6 PANSS components Positive 19 (5) Negative 19 (6) Cognitive 19 (5) Emotional 13 (4) Excitement/Hostility 9 (3) Medication (CPZ equiv.) 755 (752) ::: Instruments ----------- ### Actigraphy Sensors (Model \# 24.000, Ambulatory Monitoring Inc, Ardsley, NY) slightly bigger than a wristwatch were worn by subjects on their non-dominant wrists for 21 days. Actigraphic logs of the non-dominant arm have a high correlation with gross locomotor activity \[[@B27]\]. Actigraphy is validated and reliable for evaluating sleep patterns \[[@B22]\]. The data from each actigraph was processed by the software included with the actigraphic monitors. ### Pittsburgh Sleep Quality Index (PSI) The PSI is an effective instrument used to measure the quality and patterns of sleep. It differentiates \"poor\" from \"good\" sleep by measuring seven subscales: Subjective Sleep Quality, Sleep Latency, Sleep Duration, Habitual Sleep Efficiency, Sleep Disturbances, Use of Sleeping Medication, and Daytime Dysfunction over the last month. The client self-rates each of these seven areas of sleep by answering nine questions. Scoring of answers is based on a zero to three scale, and a score of three reflects the negative extreme on the Likert Scale. A global sum of \"5\" or greater indicates a \"poor\" sleeper. The PSI has internal consistency and a reliability coefficient (Cronbach\'s alpha) of 0.83 for its seven components. Numerous studies using the PSI have supported high validity and reliability. ### Positive and Negative Syndrome Scale (PANSS) The PANSS is a 30 item rating scale completed by clinically-trained research staff at the conclusion of chart review and a semi-structured interview. For the purposes of this study the five PANSS factor analytically derived components are used: Positive, Negative, Cognitive, Excitement and Emotional discomfort \[[@B28]\]. ### Quality of Life Scale (QLS \[[@B29]\]) The tool is a 21 item scale completed by clinically trained staff after a semi-structured interview and chart review that assesses quality of life. Items are scored on a 7-point scale with higher ratings representing higher levels of satisfaction. Items tap a range of essential aspects of psychosocial interactions incorporating four subscales: Interpsychic Foundations; Occupational Functions; Commonplace Objects; and Activities and Interpersonal Relationships. For the purposes of this study we used the sum of all items as an index of overall quality of life. Interrater reliability for this instrument has been reported to range between 0.85 and 0.93 \[[@B15]\]. ### Ways of Coping Questionnaire (WCQ \[[@B30]\]) The WCQ is a self-report instrument that asks participants to call to mind a recent stressor and then rate how often they have used 66 different behaviours to cope with that stressor. Scale scores are additively derived from individual items and divided by a total score to provide a relative score. This relative score reflects participants\' relative preferences among a set of discrete coping strategies. Relative scores are generally preferable because, among other things, they control for response bias. For the purposes of this study we calculated the relative scores for two subscales: \"Escape Avoidance,\" and \"Positive Reappraisal.\" Escape Avoidance describes wishful thinking and behavioural efforts to actively escape or avoid the problem and includes items such as: \"I refused to believe it had happened\" and \"I wished the situation would go away or be over with.\" Positive Reappraisal describes efforts to create positive meaning by focusing on personal growth and includes items such as: \"I changed or grew as a person in a good way\" and \"I found new faith.\" Internal consistency assessed using Cronbach\'s alpha have been reported to range from 0.61 to 0.79. Procedures ---------- Following informed consent participants were administered the PSI. PANSS, QLS, and WCQ were completed within the previous month for another study of the effects of personality on function in schizophrenia. Diagnosis was determined using the SCID. Ratings of sleep quality were elicited by a research assistant blind to the results of the PANSS, QLS and WCQ. We fitted a subset of patients with actigraphs that they wore for 21 days. Statistical analysis -------------------- The statistical analyses were Pearson correlations and Mixed Models analyses in SAS^®^software. We also repeated the correlations for patients with schizophrenia separate from the patients with schizoaffective disorder. Results ======= Mean scores ± SD on the tests were: Quality of Life total 51.73 ± 16.16; Pittsburgh Sleep Quality Index 11.6 ± 4.57; Ways of Coping Questionnaire subscales: \"Escape Avoidance,\" 0.138 ± 0.062 and \"Positive Reappraisal\" 0.125 ± 0.079. There was no correlation between QLS and \"Escape Avoidance\" scores, but a modest positive correlation between QLS and \"Positive Reappraisal\" (r = 0.421, p = 0.021). To examine the associations between sleep and quality of life we next performed partial correlations, using the PSI total score to predict QLS total score and QLS subscores with age and positive and negative component scores as covariates. Similarly, we used PSI subscores to predict QLS total score. Results revealed that poor sleep was related to poor quality of life (total PSI vs total QLS, r = -0.493; p = .022). The PSI accounted for 24% of the variance in QLS. Total PSI did not correlate with any of the QLS subscores. The PSI subscores: \"Subjective Sleep Quality\", \"Sleep Duration\", and \"Sleep Disturbances\" correlated with total QLS at p \< 0.05 but none were significant after making the Bonferroni correction for multiple comparisons. To examine the associations between sleep and coping style we next performed partial correlations, using the PSI total score to predict WCQ Escape Avoidance, and WCQ Positive Reappraisal score with age and positive and negative component scores as covariates. Results revealed that poor sleep was related to a reduced preference for Positive Reappraisal (r = -0.594; p = 0.0012). Sleep quality was found to be unrelated to Escape Avoidance. The PSI accounted for over 37 % of the variance in coping. Patients with schizoaffective disorder had worse sleep as measured by the PSI than did patients with schizophrenia (13.7 ± 4.4 versus 9.7 ± 4.7; t-test, p = 0.03). The two groups did not differ in total scores of the QLS or WCQ. The correlations between total PSI and total QLS, and between total PSI and Positive Reappraisal for patients with schizophrenia were significant (r = -0.516; p = 0.039 and r = -0.645; p = 0.006 respectively). The same correlations were not significant for patients with schizoaffective disorder. Seven participants with schizophrenia accepted the activity monitors and were willing to wear them continually for up to 3 weeks. Seven patients completed actigraphic monitoring, and the actigraphic results are in Table [2](#T2){ref-type="table"}. Actigraphy verified that participants with schizophrenia had less overall sleep and more interrupted sleep than published community norms. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Sleep measures for subject subset and controls\* ::: Sleep Measure Means Patients Controls Units --------------------------- -------------- -------------- ----------- Participants 7 \>200 Sleep Duration 1359 ^c^ 1440 min/d Sleep Proportion 25 33 \% of d \# of Long Awake Episodes 10.2 ^a^ 6.7 ea d Mean Sleep Episode 28 ^b^ 60 min Longest Sleep Episode 105 ^c^ 231 min \*Controls data provided by Ambulatory Monitoring ^a^= p \< .05; ^b^= p \< .005; ^c^= p \< .0005 ::: Discussion ========== The results are consistent with the hypothesis that poor quality sleep that typically characterizes schizophrenia may have a powerful impact on both patients\' perception of their quality of life and their ability to cope with stress. Independent of age and symptoms, poorer sleep quality predicted poorer quality of life and greater difficulties appraising stressors in a positive light. This may suggest that among the deficits accompanying schizophrenia, poor sleep is often underappreciated. Impaired sleep may erode the ability of schizophrenic patients to cope with the routine stress associated with work and interpersonal relationships. Furthermore, chronic sleep deprivation may contribute to anergy that impairs attendance and work performance. Since the found associations are independent of symptom level they may be trait variables. These findings are consistent with polysomnographic studies of sleep in schizophrenia which show that many sleep deficits that are not dependent on the acuity of illness \[[@B3]-[@B7]\]. The findings are also consistent with a prior study of sleep quality and quality of life in patient with schizophrenia \[[@B11]\]. We had an insufficient number of participants to confirm a correlation between PSI subscores and total Quality of Life. The negative relationship between complaints of poor sleep quality and Preference for coping by Positive Reappraisal remained significant when the confounding effects of symptom acuity and age were partialled from the correlation matrix. One interpretation is that chronically disturbed sleep may erode both the ability to find positive meaning and the desire to achieve personal growth. Given the correlative nature of the data analysis, however, it is not possible to infer causality. It may be that poor coping or poor quality of life lead to greater difficulties sleeping or that another unmeasured variable may account for the relationships. We plan future longitudinal studies that may uncover if sleep changes typically precede or follow changes in either coping or quality of life. There are other limitations to this study; most participants were male and in their 40s. Further experiments will be needed which include females and males in earlier phases of illness. The association of disturbed sleep and both quality of life and coping may hold for community controls as well. Conclusion ========== These findings may have important clinical implications. If poor sleep quality is indeed a critical factor in quality of life and coping impairments in schizophrenia, clinicians will need to focus on and aggressively treat sleep problems. Specific treatments could include training in sleep hygiene with a focus on regular waking and sleep times, avoiding naps, morning bright light, evening melatonin, or other hypnotic agents. Improved sleep may lead to improved ability to cope with stress, and increased energy. These would improve the quality of life and coping in patients with schizophrenia. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= JH participated in the design and coordination of the study and performed the statistical analysis and helped to draft the manuscript. PL conceived of the study and participated in its design and helped to draft the manuscript. AM participated in the design of the study and helped to draft the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-244X/5/13/prepub> Acknowledgements ================ This research was supported by Veterans Administration Merit Grants to Drs Mayeda and Lysaker.	PubMed Central	2024-06-05T03:55:53.966786	2005-3-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554780/", "journal": "BMC Psychiatry. 2005 Mar 3; 5:13", "authors": [ { "first": "John R", "last": "Hofstetter" }, { "first": "Paul H", "last": "Lysaker" }, { "first": "Aimee R", "last": "Mayeda" } ] }
PMC554781	Background ========== Protein-protein interactions are fundamental to biological processes and their analysis is essential for the understanding of cellular pathways. Given the complexity and the dynamic range of the proteome, estimated at 10^7^proteins, the elucidation of protein interactions requires the development of comprehensive, high-throughput proteomic methods that allow quantification of multiple proteins simultaneously \[[@B1],[@B2]\]. The development of protein microarrays represents an attractive new high-throughput technology platform. It involves the printing of ordered arrays of biomolecules onto a solid surface in miniaturized format that allows for the simultaneous determination of multiple interactions using small amounts of samples within a single experiment. The basic principles for highly sensitive \"microspot\" ligand-binding assays were described by Ekins \[[@B3],[@B4]\] who proposed the \"ambient analyte theory\" and showed that microspots containing small amounts of capture molecules were able to detect low analyte concentrations with very high accuracy and sensitivity. Since then, miniaturized protein arrays are emerging as one of the most powerful proteomics tools but their application is far more complex \[[@B5]\] than the DNA microarrays (reviewed in \[[@B6]-[@B8]\]) due to structural complexity and heterogeneity of proteins, including their post-translational modifications. Binding of the proteins onto the solid surface of an array must maintain tertiary structure sufficient for functions such as receptor-ligand binding or antibody reactivity. Chemically derivatized microarray surfaces \[[@B9],[@B10]\] or the use of mAb \[[@B11],[@B12]\] have been shown to maintain protein functionality, thus increasing the potential for successful application of microarray technology in proteomics. The study of leukocyte membrane protein interactions provides a particular need because of the large number of interactions yet to be defined \[[@B13],[@B14]\] and a technical challenge as these interactions are often of very low affinity with K~D~in the range 1--200 μM \[[@B15],[@B16]\]. Although weak, these interactions are important in the context of leukocytes interacting with other cells as illustrated by all the functional data on the interaction of CD8 with MHC Class II (K~D~= 200 μM) \[[@B17]\]. The proteins involved usually contain folded domains, the most common type belonging to the immunoglobulin superfamily (IgSF) \[[@B13]\]. Such domains often interact through large faces of the proteins and require proper folding \[[@B18],[@B19]\]. When measuring low affinity interactions, misleading results can be obtained from unfolded or aggregated materials which are not really a problem when dealing with high affinity interactions such as with cytokines and their receptors, or between proteins and linear epitopes such as lectins and carbohydrates. In addition many leukocyte surface proteins are heavily glycosylated and the oligosaccharides, even if not directly involved in binding, may be important in maintaining biologically active proteins \[[@B20]\]. Thus, in applying the protein microarray technology to the study of leukocyte surface protein interactions, it is imperative that the proteins are expressed in eukaryotic systems to ensure correct disulphide bond formation and post-translational modifications. In this study we chose a well characterized interaction between CD200 (previously called OX2) and its receptor CD200R (reviewed in \[[@B21]\]) as a model system to devise a high throughput protein array method for characterization of the interactions between leukocyte surface proteins. CD200 is a widely distributed membrane protein with two extracellular IgSF domains and a short cytoplasmic region unlikely to signal. It interacts with a receptor (CD200R) expressed mostly on myeloid cells, which also has two extracellular IgSF domains but a longer cytoplasmic region with several tyrosine residues that can be phosphorylated \[[@B22]\]. Functional analysis suggests that the leukocyte CD200 protein can mediate a down-regulatory signal to myeloid cells through the inhibitory CD200R. Thus the CD200 null mice have an increased susceptibility to autoimmune disease induction and myeloid cells expressing CD200R are more activated \[[@B23]\]. CD200 and a viral homologue found in Kaposi sarcoma virus, when expressed at the cell surface, gave inhibition of production of inflammatory cytokines from activated macrophages \[[@B24]\]; and targeting the CD200-CD200R interaction with agonistic mAb or CD200-Fc fusion proteins in vivoameliorates autoimmunity in disease models \[[@B25],[@B26]\]. Protein arrays can be divided into two major classes: \'forward phase\' if the analytes are captured from solution; or \'reverse phase\' if the analytes are bound directly to the solid phase \[[@B27]\]. In forward phase protein microarrays, a bait molecule such as an antibody is immobilized onto a solid support to capture the analytes which can be proteins in purified form, or in complex solutions such as cell lysates \[[@B12]\] or tissue samples \[[@B27],[@B28]\]. The bound analytes are detected either by direct labelling or via a secondary antibody. In reverse phase arrays, the analytes (typically purified proteins or cell lysates) are directly immobilized on the solid phase and antibodies or interacting proteins are applied in solution phase. The analytes can be labelled directly or detected using tags and signal amplification. We have used the forward phase approach in the mapping of antigenic epitopes of hCD200R where different antibodies were immobilized on epoxy coated glass slides, incubated with the hCD200R analyte and detected with fluorescently labelled anti-CD200R antibodies. We have applied reverse phase arrays to three different purposes: -to test the reactivity of the fluorescently labelled mAb with directly immobilized hCD200R protein, -to map epitopes located near the ligand binding site using arrayed mutant hCD200R recombinant proteins and detection with fluorescently labelled mAb that block ligand-receptor interactions and -to detect the low affinity binding of immobilized CD200R to the multivalent CD200 ligand presented on fluorescently labelled beads. Our study extends the use of protein microarrays to the detection of transient cell surface protein interactions, which are of lower affinity than the reported cytokine arrays \[[@B29],[@B30]\]. Results and discussion ====================== Quantitative binding of DX147, DX136 and OX108 mAb to human CD200R ------------------------------------------------------------------ Purified, soluble recombinant hCD200R protein, engineered with domains 3 and 4 of rat CD4 as an antigenic tag (hCD200R-CD4d3+4) \[[@B31]\] was directly immobilized at different concentrations on epoxy-coated glass slides, in a reverse phase array as illustrated schematically (Fig. [1A](#F1){ref-type="fig"}). The hCD200R array was tested for reactivity with three different mAb: DX147, and two previously reported \[[@B31]\] mAb DX136 and OX108 able to block ligand binding. Controls on the arrays included recombinant mouse CD200R-CD4d3+4 protein (mCD200R) \[[@B31]\] and rat CD4d3+4. Figure [1B](#F1){ref-type="fig"} and [1C](#F1){ref-type="fig"} illustrate the strong and specific binding of all three fluorescently labelled mAb to hCD200R, as demonstrated by minimal reactivity with rCD4 (rat CD4d3+4) and lack of cross-reaction with mCD200R. DX147 gave the strongest labelling with linear binding from 0.08 to 20 μg ml^-1^mAb, reaching the upper limits of detection under the optimized voltage settings (65,000 units of green fluorescence) at 40 μg/ml (Fig. [1B](#F1){ref-type="fig"}). Binding of DX136 was linear over the full range of concentrations with a maximum binding of 48,000 units. OX108 bound more weakly, reaching a maximum value of 32,000 units. Sensitivity of detection, defined as two-fold binding above background, was estimated as the lowest hCD200R-CD4d3+4 concentration tested (0.08 μg ml^-1^) for DX136. A three-fold signal to noise ratio was achieved for DX147 at that concentration suggesting that sensitivity of detection was closer to 0.05 μg ml^-1^. For OX108, the limit of sensitivity was estimated at 0.3 μg ml^-1^. Thus DX147, DX136 and OX108 were able to detect 0.5 pg, 0.8 pg and 3.0 pg of hCD200R per spot respectively estimating a spot volume of 10 nl. The limit of sensitivity achieved was therefore between 8 and 50 attomol, assuming a molecular weight of 60,000 for hCD200R-CD4d3+4 protein. The amounts of human and mouse CD200R-CD4d3+4 and rCD4d3+4 protein on the microarray spots were similar as visualized by the red fluorescence of OX68 mAb recognising the CD4 tag present in each of the recombinant proteins (Fig. [1B](#F1){ref-type="fig"} and [1D](#F1){ref-type="fig"}). This indicates that the amount of protein detected is proportional to the amount arrayed in each spot and is highly reproducible. The limit of sensitivity of protein detection with Alexa 647-OX68 was approximately 0.3 μg ml^-1^, corresponding to 50 attomol of CD200R-CD4d3+4 proteins. Although not all molecules will be in a proper orientation for equal access to both anti-CD200R and anti-CD4, as illustrated in Fig. [1A](#F1){ref-type="fig"}, our results suggest that on average, there is a good correlation between the amount of specific antibody bound and the amount of protein arrayed. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Quantitative binding of mAb to hCD200R-CD4d3+4.(A). Scheme illustrating a reverse phase microarray in which purified CD200R proteins were immobilized and then screened with fluorescent mAb specific for hCD200R or for the antigenic rCD4 tag (OX68) of the hybrid recombinant protein.(B). Typical microarray shows binding of OX68 mAb (red) to control proteins or the overlapping binding of hCD200R mAb and OX68 (yellow) to immobilized human CD200R.(C). Shows the green fluorescence intensity of each spot for all four replicates (mean ± SEM).(D). Shows red fluorescence intensity due to binding of OX68 mAb using data in left panel for DX147 (similar levels were found with the other mAb). Serial two-fold dilutions of purified, soluble, recombinant human and mouse CD200R-CD4d3+4 proteins, and of control rat CD4d3+4 were arrayed onto epoxy-coated microscope slides. Each protein dilution series was arrayed in 3 rows of 4 spots, ranging in concentration from 40 μg ml^-1^(first spot) to 0.08 μg ml^-1^(spot 10), with control spotting buffer containing 0.5 mg ml^-1^BSA in the last two spots. All arrays were performed in quadruplicate and a representative set is shown in (B). Each slide was incubated for 16 h at 4°C with a mixture of hCD200R mAb (DX147, DX136 or OX108) labelled with Alexa-555 (indicated as green fluorescence measured at 532 nm) and rCD4 mAb (OX68, detecting the antigenic tag and allowing for measurement of recombinant protein concentration) labelled with Alexa-647 (red fluorescence measured at 635 nm). At the highest concentrations, the hCD200R spots appear either white (saturating conditions) or yellow, due to the combination of green and red signals given by the specific binding of the Alexa-555-mAb to hCD200R and Alexa-647-OX68 mAb respectively. Quantitative measurements are expressed as mean fluorescence units at 532 nm (green) and 647 nm (red) versus amount of protein arrayed. ::: ![](1471-2091-6-2-1) ::: Orientation via antibody immobilization for epitope mapping on human CD200R --------------------------------------------------------------------------- We used forward phase protein microarrays to define the epitopes of hCD200R recognized by the three mAb introduced in the previous section. Serial dilutions of DX147, DX136 and OX108 mAb and the control CD4 mAb OX68 were directly immobilized on epoxy-coated glass slides as shown (Fig. [2A](#F2){ref-type="fig"}). The mAb arrayed act as capture reagents for hCD200R, used at a concentration of 20 μg ml^-1^. Each capture mAb binds to a different epitope on the hCD200R-rCD4d3+4 recombinant protein, thus orientating the protein on the array in a conformation that permits or restricts access to the same panel of mAb, used as detection reagents (Fig. [2A](#F2){ref-type="fig"}). Visual observation (Fig. [2B](#F2){ref-type="fig"}) and quantitative analysis (Fig. [2C](#F2){ref-type="fig"}) showed that DX147 reacts with an epitope spatially distinct from the more closely related DX136 and OX108 epitopes. As expected, OX68 is the most suitable capture mAb, as it binds the common CD4 tag allowing exposure of the two extracellular domains of hCD200R and binding of all three specific mAb. When the detection mAb is the same as the capture mAb, no significant binding above background is observed (Fig. [2C](#F2){ref-type="fig"}). DX147 binds specifically to hCD200R-CD4d3+4 orientated via OX68 and DX136 (16,021 and 12,570 green fluorescence units respectively at 80 μg ml^-1^of capture mAb), but not via OX108 or via itself. This is an indication that the binding epitopes for DX136 and DX147 are dissimilar. DX136 in turn, binds well to hCD200R-CD4d3+4 immobilized on OX68 (13,954 units of green fluorescence at 80 μg ml^-1^) and to a lesser degree to hCD200R-CD4d3+4 immobilized on DX147 (2,647 units), while not at all to hCD200R captured by OX108 (496 units). These data indicate that the DX136 epitope is spatially distinct from the DX147 epitope, but in close proximity to the OX108 epitope. This conclusion is substantiated by the lack of binding of OX108 mAb to hCD200R-CD4d3+4 captured on DX136. Orientating hCD200R via mAb OX68 allows for specific binding of all three anti-human CD200R mAb, in a linear fashion with limits of sensitivity of about 5 μg ml^-1^of immobilized mAb. The maximum amount of signal was obtained by capturing hCD200R with 80 μg ml^-1^of mAb OX68 and was equivalent to that observed by directly immobilizing approximately 10 μg ml^-1^hCD200R. OX68 is therefore the best mAb for capturing the chimaeric hCD200R-CD4 protein for optimal detection of hCD200R epitopes. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Analysis of mAb reactivity with hCD200R by orientation via antibody immobilization.(A). Scheme of one forward phase microarray in which purified human CD200R protein was immobilized via OX68 mAb and detected with the DX136 hCD200R mAb (green fluorescence).(B). Typical microarray shows binding of the hCD200R mAb (green) and OX68 mAb (red) to hCD200R immobilized via four different capture mAb.(C). Shows the mean fluorescence intensity ± SEM for each spot of all replicates. Serial two-fold dilutions of capture human CD200R mAb OX108, DX136 and DX147 and control rat CD4 mAb OX68 were arrayed onto epoxy-coated microscope slides. Each mAb dilution series was arrayed in quadruplicate of 2 rows of 6 spots, ranging in concentration from 80 μg ml^-1^(first spot) to 0.16 μg ml^-1^(spot 10), with control spotting buffer containing 0.5 mg ml^-1^BSA in the last two spots. The whole array was repeated on the slide for a total of 8 replicates per spot. Each slide was incubated for 2 h with 20 μg ml^-1^of purified recombinant hCD200R-CD4d3+4 protein, prior to incubation with Alexa-555-labeled CD200R mAb (DX147, DX136 or OX108) or Alexa-647 control rCD4 mAb (OX68). Quantitative measurements are expressed as mean fluorescence units at 532 nm (green) and 635 nm (red) versus amount of capture mAb arrayed. ::: ![](1471-2091-6-2-2) ::: Mapping of CD200R antigenic epitopes using mutants -------------------------------------------------- Both CD200R and its ligand, CD200 contain two extracellular IgSF domains. The ligand-receptor interaction is therefore likely to occur in an end-to-end topology, requiring opposing cell surfaces to come into close proximity \[[@B22]\]. Previous studies have shown that the membrane distal N-terminal domain of CD200 is involved in binding its receptor \[[@B32]\]. In a recently published study \[[@B33]\], site directed mutagenesis was employed to map the ligand-binding domain of human CD200R using the structure of a typical Ig V domain, that of the human junctional adhesion molecule 1, JAM1 to predict the positions of out-pointing residues \[[@B34]\]. A panel of mutants was designed so as to target residues likely to be out-pointing from predictions of the beta strands of the N-terminal IgSF domain of hCD200R. The binding sites of CD200 and of the OX108 mAb known to block ligand interaction to the hCD200R mutants, were shown to be on the GFCC\' face of the N-terminal IgSF domain \[[@B33]\]. The same panel of hCD200R-CD4d3+4 mutant proteins (Table [1](#T1){ref-type="table"}) was analyzed by reverse protein microarrays (analogous to Fig. [1A](#F1){ref-type="fig"}) for binding to mAb DX147, DX136 and OX108 in order to map these epitopes. The mutant proteins were expressed by transient transfection in serum-free medium, concentrated and arrayed. Purified human and murine CD200R serving as positive and negative controls were immobilized at concentrations ranging from 0.08 to 40 μg ml^-1^. Lack of binding by a specific mutant or group of mutants is suggestive of the corresponding residues defining the location of the antigenic epitopes. None of the hCD200R mutants tested had lost binding to DX147, confirming unpublished data that this epitope lies, not in the N-terminal domain of CD200R but in the membrane proximal domain. Some of the mutants showed increased binding of mAb over the wild type despite these being normalized with OX68 (e.g. R67, I71K). This presumably reflects variations in epitope availability due to direct immobilization of the mutant proteins on the array, which are less likely to occur in BIAcore studies where mutants were immobilized via OX68 mAb \[[@B33]\]. Thus our data analysis was focused on mutants with major impairment in binding activity. Nine of the mutants showed reduced binding to either DX136 and/or OX108 mAb, relative to wild type hCD200R, as represented graphically in Figure [3](#F3){ref-type="fig"}. One mutant illustrated (E75K) did not affect binding in a significant manner. The position of these mutants is shown on the model of the N-terminal domain of hCD200R, based on a typical IgSF domain of similar size (Fig. [4](#F4){ref-type="fig"}). Panel A shows the mutants affecting CD200 binding, as reported in \[[@B33]\]. These mutants were mostly in the GFCC\' face and in particular the F and C strands. Panel B shows the mutants with reduced binding to DX136 and/or OX108. It is immediately noticeable that mutants within the F and C strands also appear to have lost binding to DX136 and OX108 mAb, suggesting close proximity of these epitopes to the CD200 binding site, in agreement with the ligand blocking activity of these mAb. However, the key residues were not identical as shown by the differing effects of the mutants I71K and R67D. Furthermore, the microarray study confirms the results obtained by individual analysis of each mutant by surface plasmon resonance in terms of residues involved in OX108 binding; the most critical amino acid appears to be R67 located in the B-C loop \[[@B33]\]. The finding of mutants that had apparently gained CD200R mAb binding activity compared to the OX68 mAb recognising the CD4 tag, shows that spotting can have effects not seen by indirect methods such as the BIAcore. However there was very good correlation between available BIAcore data and microarray data. Thus this method provides a rapid high throughput method to identify the rare mutants that affect antigenic activity that can then be characterised further e.g. by BIAcore analysis. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### hCD200R protein mutants tested. Mutant proteins were constructed as described in \[33\], expressed by transient transfection in serum-free medium, concentrated and immobilized on epoxy-derivatized slides. The predicted positions of the residues are located in the modelled V-like N-terminal IgSF domain unless noted otherwise (C domain). ::: \# Mutant Name Predicted Position -------- ----------------- ------------------------ 1 Wild-type 2 D30K\* N-term 3 K40D\* A strand 4 L42E\* A strand 5 E44K\* A strand 6 E44A A strand 7 E44D\* A strand 8 M53K A-B loop 9 N56D B strand 10 P62F B strand 11 I64S\* B-C loop 12 R67D B-C loop 13 I71K C strand 14 T73R C strand 15 E75K C strand 16 R79E C-C\' loop 17 Q81K C-C\' 18 S83D\* C\" strand 19 E97K C\" strand 20 T106D\* D strand 21 D116K\* D-E loop/ E strand 22 A123D E strand 23 Y129D F strand 24 R131E F strand 25 I133K F strand 26 D138K\* F-G loop 27 R143D\* F-G loop 28 H146D G strand 29 Q148E\* G strand 30 L150D\* G strand 31 T156N\* A strand C domain 32 N160D A-B loop C domain 33 A175D\* B-C loop C domain \* Mutants expressed with concentration below sensitivity threshold, for which no antibody binding data could be derived. ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Mapping of antigenic epitopes on hCD200R mutants.The hCD200R mutants described in Table 1 and produced by transient transfection were arrayed (reverse phase) and tested for specific binding to mAb DX147, DX136 and OX108 and for reactivity with OX68 to quantify the relative amount of protein in each sample as described in Methods. Results for a panel of 10 mutants are plotted as percent antibody binding normalized to the wild type, non-mutated hCD200R protein (WT) values. ::: ![](1471-2091-6-2-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Mapping of DX136 and OX108 epitopes on the N-terminal domain of human CD200R.(A). The mutants giving complete or nearly complete inhibition of CD200 binding, determined by BIAcore analysis, are indicated with red circles (\<35% binding compared to non-mutated WT hCD200R protein), whereas those giving partial effects (35--70% binding) are depicted in orange. Data from \[33\].(B). The mutants giving severe inhibition (\<35% compared to WT) of OX108 and DX136 mAb binding, as determined by microarray analysis, are depicted in blue and yellow respectively. Dark green circles represent mutants that severely impaired both OX108 and DX136 mAb binding. Mutants partially affecting DX136 binding (35--50%) are shown in pale yellow. Mutants that severely affect DX136 binding (35% binding or less) but have only a partial effect on OX108 binding (50%) are represented in pale green. Open circles depict mutants that did not affect the binding of CD200, OX108 or DX136 mAb. The CD200R model is based on a typical Ig V domain from the human junctional adhesion molecule-1 (JAM1) \[34\]. The beta sheets are labelled with the GFC face orientated in front and the BED face behind. ::: ![](1471-2091-6-2-4) ::: Reactivity of human, rat and mouse CD200R with multimeric human CD200 --------------------------------------------------------------------- The interaction of human CD200 with its receptor hCD200R is of low affinity, with a K~D~of \~0.5 μM at 37°C and t~1/2~of 7 s \[[@B24]\], typical of the interaction of many leukocyte membrane proteins \[[@B15]\]. Such an interaction could not be detected when immobilized hCD200R was incubated with fluorescently labelled purified monomeric hCD200 protein (data not shown). In order to develop leukocyte membrane receptor-ligand microarray assays, high avidity detection reagents are required. Recombinant hCD200 protein was constructed by linking the extracellular domains of human CD200 with domains 3 and 4 of rat CD4 (CD4d3+4) as an antigenic tag. This construct contains a 19 amino acid sequence at the C-terminus of the protein, which can be enzymatically biotinylated on a specific lysine residue using the E. coliBirA enzyme \[[@B35]\]. Expression of the construct was demonstrated by inhibition of a rat CD4 ELISA using OX68 mAb. The recombinant protein bound the mAb OX104 (mouse anti-human CD200), indicating that it was antigenically active, as assessed by BIAcore analysis (data not shown) and its biotinylation was confirmed by streptavidin binding. The biotinylated hCD200-CD4d3+4 protein or the control CD4d3+4 protein were attached to avidin-coated FITC-fluorescent beads via their biotin tag, thus creating polyvalent CD200 and control reagents. These beads were used to detect specific binding of CD200 to human, mouse and rat CD200R proteins arrayed directly on epoxy-coated glass slides (Fig. [5A](#F5){ref-type="fig"}). Additional controls included rCD4d3+4 protein arrayed on the slide and tested for reactivity with both types of beads. Strong binding of hCD200-beads to all three CD200R proteins was observed (Fig. [5B](#F5){ref-type="fig"} and [5C](#F5){ref-type="fig"}) indicating that the proteins immobilized on the glass surface had retained their capacity to bind ligand with maximum mean values of 63,500 green fluorescence units for rat CD200R (saturating), 43,600 for mouse CD200R and 23,800 for human CD200R. The fluorescence appears granular as one is actually visualizing the small fluorescent beads. Binding was detected at concentrations of receptors ranging from approximately 5 to 40 μg ml^-1^corresponding to 1--8 femtomol per spot on the microarray. The non-specific binding of hCD200-beads to immobilized rCD4 protein was negligible (Fig. [5B](#F5){ref-type="fig"} and [5C](#F5){ref-type="fig"}) and control CD4d3+4 beads did not react with any of the arrayed proteins (data not shown). The multivalent hCD200-beads cross-reacted with rat and mouse CD200R as expected, as BIAcore analysis has shown that hCD200 interacts with human, rat and mouse CD200R with affinity constants within a log of each other \[[@B31]\]. Sensitivity of detection, defined as two-fold binding above background was achieved with concentrations of 5 μg ml^-1^for rat CD200R and 10 μg ml^-1^for human and murine CD200R. This corresponds to 1--2 femtomol of immobilized receptors interacting with the multimeric human CD200 ligand. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Binding of multimeric CD200 ligands to CD200R proteins.(A). Diagram of the reverse phase array depicting immobilized CD200R interacting with the multivalent bead ligand.(B). A representative microarray set showing binding of hCD200 beads to CD200R-CD4d3+4 proteins, but not control rat CD4.(C). Mean fluorescence intensity ± SEM of all four sets is shown versus receptor protein concentration arrayed. Serial two-fold dilutions of purified, soluble, recombinant human, mouse and rat CD200R-CD4d3+4 proteins, and of control rat CD4 were arrayed onto epoxy-coated microscope slides. Each receptor protein dilution series was arrayed in 3 rows of 4 spots, ranging in concentration from 40 μg ml^-1^(first spot) to 0.08 μg ml^-1^(spot 10), with control spotting buffer containing 0.5 mg ml^-1^BSA in the last two spots. Only the first 8 dilutions (2 rows) are shown in (B) and analyzed in (C). All arrays were performed in quadruplicate. All receptors were arrayed on the same slide, which was incubated for 16 h at 4°C with polyvalent human CD200-CD4d3+4 FITC-fluorescent beads. At the highest concentrations, the hCD200R spots appear white (saturating conditions). Quantitative measurements are expressed as fluorescence units at 532 nm (green) versus amount of protein arrayed. ::: ![](1471-2091-6-2-5) ::: Conclusion ========== In order to study interactions of leukocyte membrane proteins using high throughput microarray techniques, it was essential that the proteins be immobilized at low concentrations and in a biologically active form. It is critical that weak interactions between leukocyte membrane proteins be detected without interference by the anomalous binding due to denatured proteins, which is more of a contributing factor in the study of low affinity interactions. We first established that recombinant CD200R proteins could be immobilized directly in reverse phase arrays, in a conformation capable of reacting with three different mAb. We then demonstrated that recombinant mutant hCD200R proteins produced in transient expression systems were present in sufficient amounts to be immobilized directly and tested for reactivity with specific mAb, permitting mapping of epitopes. These data show that high throughput analysis of cell surface proteins can be achieved in reverse phase arrays using recombinant proteins derived from transient transfectants in a non-purified form. We also used forward phase arrays for competitive analysis of antibodies and mapping of their epitopes. This approach is valuable for rapidly screening antibody specificities and assessing protein orientation needed for optimal presentation of immunogenic determinants. We also showed that binding of CD200 ligand to its cell surface receptor can be achieved by increasing the avidity of the reaction via coupling of the biotinylated recombinant CD200 protein to fluorescently labelled avidin coated beads. The fluorescent beads offer an efficient technology for the analysis of low affinity interactions typical of those observed for leukocyte membrane proteins and many other cellular proteins. Methods ======= Materials --------- Monoclonal antibodies (mAb) DX147 (rat IgG1), and DX136 (rat IgG2a) to human CD200R were generously given by DNAX Research Institute (Palo Alto, CA). The mAb OX108 (mouse IgG1) to human CD200R \[[@B31]\] and OX68 (mouse IgG1) to rat CD4 domains 3 and 4 (rCD4d3+4) have been described previously \[[@B36]\]. Recombinant proteins -------------------- The soluble biotinylated forms of human, mouse and rat CD200R were produced as described \[[@B22],[@B31],[@B36]\]. Briefly, the entire extracellular region of human, mouse or rat CD200R was amplified by PCR and cloned in the pEF-BOS-CD4d3+4bio-XB vector \[[@B35]\]. These constructs were then subcloned into the expression vector pEE14, and stably secreting CHO.K1 cell lines were established \[[@B37]\]. Human, mouse or rat CD200R-CD4d3+4 proteins were purified from the tissue culture medium by immunoaffinity chromatography with OX68 mAb-Sepharose 4B that recognizes the CD4 protein tag \[[@B36]\]. Prior to use, the purified CD200R proteins were fractionated by gel filtration on Superdex S-200 (Pharmacia, Uppsala, Sweden) to exclude protein aggregates. The soluble, biotinylated form of human CD200 was produced in an identical fashion, by subcloning the amplified extracellular region of human CD200 \[[@B38]\] using XbaI/SalI digestion to the pEF-BOS-CD4d3+4bio-XB vector \[[@B36]\]. This construct was used to transfect HEK293T cells using the calcium phosphate method. The protein expressed was enzymatically biotinylated and used to generate multivalent binding reagents by coupling to avidin-coated fluorescein isothiocyanate (FITC)-loaded beads (Spherotech Inc., Libertyville, IL) as described previously \[[@B35]\]. Mutants of human CD200R (hCD200R) were prepared as described \[[@B33]\]. Briefly, the mutations were introduced by site directed mutagenesis using PCR and two mutagenic oligonucleotides into a construct comprising the extracellular domains of human CD200R together with domains 3 and 4 from rat CD4 (CD4d3+4) as an antigenic tag. The mutants were transiently expressed in HEK 293T cells using X-VIVO 10 media (BioWhittaker, Nottingham), concentrated about 10 fold and levels of expressed protein quantified by ELISA. This media contains 1 mg ml^-1^BSA so after concentration the final protein concentration is around 10 mg/ml. Antibody labelling ------------------ Purified antibodies were dialysed against PBS prior to labelling with Alexa Fluor 555 or Alexa Fluor 647 fluorescent amine-reactive dyes using the Molecular Probes Monoclonal Antibody Labelling Kits (Cat. No. A-20186 and A-20187) and according to the manufacturer\'s instructions (Molecular Probes, Invitrogen Ltd.). Labelling reactions were carried out using 100 μg of IgG and yielded labelled proteins ranging in concentration from 1 to 4 × 10^-6^M. The degree of conjugation was estimated at 2--4 moles of dye per mole protein. Labelled antibodies were stable for up to 2 months at 4°C. Preparation of microarrays -------------------------- Protein solutions to be arrayed were prepared in 96 well plates and 12 μl aliquots were transferred to single wells of Genetix 7020, 384-well plates (Genetix Ltd, New Milton, UK). Concentrations tested ranged from 0--80 μg/ml and all dilutions were performed in Protein Array Spotting Solution (Genetix) with the addition of 0.5 mg/ml BSA and 0.02% NaN~3~. A QArrayMini microarray printer (Genetix) was used to apply the protein solutions onto epoxy-coated microscope slides using 300 μm solid tipped tungsten microarraying pins (Genetix). Preliminary experiments established the printing conditions with fluorescently labeled OX68 mAb. Of several types of slides tested, the epoxy-coated ones were the best in terms of spot morphology, cost and reproducibility and were used in all subsequent experiments. Most array designs were performed using 8 pins to obtain spots with a 440 μm diameter and centre-to-centre spot spacing of 700 μm in both directions. Source plates were kept at 8°C, and a 65% average internal humidity was maintained. After printing, the slides were left in the arraying chamber for 30--60 minutes under the same conditions. The slides were then washed using the Protein Array Processing Kit (Genetix Ltd; stored at 4°C and the solutions supplemented with 0.02 % NaN3) by inversion for 1 min in Clean Up Buffer (Genetix) to remove unbound proteins and incubation for 30 min in Blocking Buffer. Slides were washed 3 times in PBS, once in H~2~O to remove excess salt and dried using an air brush, and stored at 4°C, with desiccant in a sealed slide box. Preliminary experiments were done by forward phase arrays to establish optimal conditions. OX68 mAb (100 μg ml^-1^ to 20 μg ml^-1^) was immobilized, incubated with rat CD4 at 5 μg ml^-1^ 0.1 mg ml^-1^ BSA, washed and incubated with labeled W3/25 mAb (5 μg/ml) and linearity of detection demonstrated. Specificity was also shown by the fact that OX68 was not reactive with CD4 immobilised on OX68 and vice versa. Labelling of microarrays ------------------------ Slides were placed in hybridization chambers (Corning Incorporated, UK) and the humidification wells filled. LifterSlips, (Erie Scientific, Portsmouth, USA) were placed gently over the marked boundaries of the arrays and the binding reagent (25--70 μl) was introduced with a micropipette. In experiments where CD200R-CD4 hybrid proteins (including the mutant studies) were arrayed, Alexa-555 anti-CD200R antibodies (mAb DX147, DX136 or OX108; 5--10 μg ml^-1^) were added to measure the amount of specific antibody bound and Alexa-647-CD4 mAb (OX68 5--10 μg ml^-1^) was included to assess the amount of hybrid protein present. In experiments where capture antibodies were arrayed (Fig. [2](#F2){ref-type="fig"}), a 2 h incubation with purified protein, such as CD200R-CD4d3+4 or CD4d3+4 at 20 μg ml^-1^, was performed prior to incubation with the detection antibodies. In the experiments detecting CD200 (ligand) binding to immobilized CD200R (Fig. [5](#F5){ref-type="fig"}), arrays were incubated with biotinylated hCD200-CD4d3+4 streptavidin-FITC beads. Incubations with detection reagents were carried out for 16 h at 4°C unless otherwise stated. The slides were immersed upside down in PBS/0.05% Tween-20, washed thrice with copious amounts of PBS/Tween-20, alternating shaking up and down under liquid in the Copeland jar and gentle rocking for 5--10 min each, followed by PBS (twice for 5 min) and a final H~2~O rinse. All washes were at room temperature and repeated following each incubation. After drying the slides with an air brush, the arrays were scanned using a GenePix4000B microarray scanner (Axon Laboratories, Palo Alto, CA) scanner using 532 nm and 635 nm lasers using the GenePix Pro 5.0 (Axon Laboratories) software. The PMT values were 720 and 1000 (532 nm and 635 nm respectively) for Figure [1](#F1){ref-type="fig"}, 780 and 950 for Figure [2](#F2){ref-type="fig"}, 900 and 1000 for Figure [3](#F3){ref-type="fig"} and 850 for Figure [5](#F5){ref-type="fig"} (532 nm only). Data analysis ------------- All samples were tested in quadruplicate and all experiments repeated several times. The amount of antibody or ligand bound to the arrayed proteins and the amount of protein present in each spot were determined by comparing the fluorescence intensities read at 532 and 635 nm. Extraction of spot intensity data was performed using GenePix Pro 5.0 (Axon Laboratories) and ScanArray Express (Perkin Elmer). The background, calculated as the median of pixel intensities from the local area around each spot, was subtracted from the mean pixel intensity within each spot. To graphically represent the data, the values of the background-subtracted signal intensities were plotted against the known concentration of the protein spotted in the array. Sensitivity of detection for each spot was defined as a signal to noise ratio (S/N) of two-fold above background. S/N was calculated as: S/N = (background-subtracted median signal intensity) / (standard deviation of background signal intensity). In the case of the mutant hCD200R proteins generated from culture supernatants of transient transfections, where protein concentration is unknown, the background-subtracted values for both 532 and 635 nm-signal intensities were corrected for internal protein signal by subtracting the corresponding value of a \"mock transfectant\" spot. The corrected values for the red channel (representing the amount of protein assessed from the CD4 content) were normalised to 100% with respect to the wild-type hCD200R transient transfection sample. All hCD200R mutants with red channel values below 50% were assumed to contain insufficient amount of protein and were excluded from the analysis. The green channel background-subtracted, \"mock\"- transfectant corrected values (G) were divided by the corresponding red channel ones (R) to correct for variations in the amount of expressed protein contained in each individual spot (G/R ratio). Finally, the G/R ratio was normalized to 100% with respect to the hCD200R-CD4d3+4 wild-type protein, before graphical representation. List of abbreviations ===================== IgSF, immunoglobulin superfamily; hCD200, human CD200; CD200-CD4d3+4, chimaeric recombinant CD200 protein with rat CD4 domains 3+4; hCD200R, human CD200 receptor; mCD200R, mouse CD200 receptor; rCD200R, rat CD200 receptor; SPR, surface plasmon resonance; WT, wild type. Authors\' contributions ======================= ML set up and developed the protein arrays with DV specialising in the experiments with the multimeric beads and data analysis. Both wrote the paper and DV prepared the figures. DH produced the mutant CD200R proteins and advised on their analysis. MF produced the mAb OX108 and the human, mouse and rat CD200R proteins. NS provided expertise in setting up the system for protein microarrays and their basic analysis. NB devised the initial project, advised throughout and helped with the manuscript writing. Acknowledgements ================ This work was supported by the Medical Research Council (UK) and the Arthritis Research Campaign (UK). The microarray infrastructure established with support from the EPA Cephalosporin Trust. We are grateful to Holly Cherwinski and Joe Phillips (DNAX, Palo Alto) for the gift of purified DX147 and DX136 mAb and advice.	PubMed Central	2024-06-05T03:55:53.968977	2005-3-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554781/", "journal": "BMC Biochem. 2005 Mar 1; 6:2", "authors": [ { "first": "Michelle", "last": "Letarte" }, { "first": "Despina", "last": "Voulgaraki" }, { "first": "Deborah", "last": "Hatherley" }, { "first": "Mildred", "last": "Foster-Cuevas" }, { "first": "Nigel J", "last": "Saunders" }, { "first": "A Neil", "last": "Barclay" } ] }
PMC554782	Background ========== One important goal in bioinformatics is to integrate data from disparate sources of heterogeneous biological information. Data integration allows us to assemble targeted data reagents for bioinformatics analyses, and to discover scientific relationships between data. Most public repositories of biological data focus on deriving and providing one particular type of data, be it biological sequences (e.g., GenBank \[[@B1]\], UniProt \[[@B2]\]), molecular interactions (The Biomolecular Interaction Network Database (BIND) \[[@B3]-[@B5]\], The Human Protein Reference Database (HPRD) \[[@B6]\]), or gene expression (The Stanford microarray database \[[@B7]\]). Integrating these disparate sources of data enables researchers to discover new associations between the data, or validate existing hypotheses. Several recent studies have demonstrated the power of integrative bioinformatics. Using data from genomic sequences and annotations, mRNA expression, and subcellular localization, Mootha et alwere able to use bioinformatics approaches to identify one of the disease genes responsible for Leigh syndrome \[[@B8]\]. In another example of an integrative bioinformatics approach, Stuart et alused existing publicly available data to generate hypotheses about the functional roles of gene sets \[[@B9]\]. These two examples illustrate the potential of querying integrated public data to reveal novel relationships. However, working with publicly available biological data can be challenging due to the volume and complexity of the data types. With the proliferation of massive, publicly available data sets, researchers need a way to readily access this data. Querying distributed data has inherent limitations such as the server resource restrictions of the remote resource, concerns of secure data transmission over the internet, and of course the actual logistics of querying distributed resources. In such an environment, the distributed search space is difficult to process in a high-throughput way, and requires complex queries to tie together the heterogeneous data. Consequently, there is a need for a data integration solution that facilitates search and retrieval in an efficient, flexible, high-throughput manner. Several active solutions are available that attempt to integrate data and that provide the tools to retrieve that data. We have grouped these existing systems into three major categories, based on how the data is stored and integrated: full record, SQL-based, and distributed. Full record systems like SRS \[[@B10]\] and Entrez \[[@B11]\] store the intact record in a table and extract specific fields to index and cross-reference. SeqHound \[[@B12]\] is a powerful system that stores Entrez information (fully annotated sequence and structure information) locally and can be accessed programmatically through application programming interfaces APIs. Much like Entrez and SRS, fully intact records are stored in SeqHound, with specific fields indexed. The major advantages of SeqHound over Entrez is that it is locally installable and provides API access to the data. SeqHound highlights the power and utility of a locally installable warehouse. SQL-based systems implement relational models to store data. This allows SQL-level access to specific parts of the data model, enabling detailed queries on the data for greater specificity of results. The data in relational models are stored as primitive data types as opposed to storing fully intact records that need parsing or processing to access the parts therein. For example, sequences and their annotated biological features can be stored in their own fields in the database, permitting \'substring\' operations to extract parts of the sequence that span a particular feature type using SQL. Systems like EnsMart \[[@B13]\] and DBGET/LinkDB \[[@B14]\] provide data in a relational form, such that the power of SQL is at users\' disposal. EnsMart\'s relational back-end provides users with the ability to construct intricate queries on the data by taking advantage of SQL. Distributed systems implement software to access heterogeneous databases that are dispersed over the internet. JXP4BIGI \[[@B15]\] have created a generalized method to access, extract, transform, and integrate distributed data. The tool acts as a middle-ware for constructing a local instance of a data warehouse. This system is customizable, versatile and uses industry standard data modeling, distribution, and presentation software. BioMOBY \[[@B16]\] is a semantic-based system utilizing ontologies, and a services model to support user queries. TAMBIS \[[@B17]\], like BioMOBY, is also a semantic-based system, and is also service-model driven. These semantic web implementations do not house the data locally, but rather query the original data provider for available services before sending queries to that particular data provider. These systems are quite powerful for interrogating disparate data sources of information. However, a disadvantage is that large queries may take a long time to return or may not be returned at all due to server resource restrictions. As well, the level of data integration is only at the services level, rather than at a field-based level which can provide much better resolution for queries. Atlas is a versatile, flexible, and extensible data warehouse that provides a solution to these challenges. Our approach establishes common relational data models enabling the reuse of each class of data model to store all data of the same type. For example, a single interaction data model is used to store information from any of the interaction data sets such as BIND, MINT, EBI IntAct \[[@B18]\], Database of Interacting Proteins (DIP) \[[@B19]\], and HPRD. Instances of these data models, once populated by the source data, can then be interrogated using the developed retrieval APIs. These APIs encapsulate the SQL calls used for fine granular access to the data. Furthermore, ontological information stored in these databases captures the relationships between the many data types. Finally, tools are developed that capitalize on the API methods, to facilitate application specific demands of end-users, ranging from simple queries of specific data types, to complex queries that infer molecular interactions across species. Atlas then, is designed for use by a wide audience from biologist to software developer. Construction and content ======================== The Atlas system is made up of five main parts: 1) the source data, 2) the ontology system, 3) the relational data models, 4) the APIs, and 5) the applications (see Figure [1](#F1){ref-type="fig"}). The following sections outline the Atlas architecture in detail. Source data ----------- We categorize the Atlas data sources into four main groups: \'sequence\', \'molecular interactions\', \'gene related resources\', and \'ontology\' (Figure [1](#F1){ref-type="fig"}). Currently, the data sources that fall into these categories are: \'sequence\', GenBank, RefSeq \[[@B11]\], and UniProt ; \'molecular interactions\', HPRD, BIND, DIP, IntAct, and MINT; \'gene related resources\', Online Mendelian Inheritance in Man (OMIM) \[[@B20]\], LocusLink \[[@B11],[@B21]\], Entrez Gene \[[@B22]\], and HomoloGene \[[@B11],[@B23]\]; and \'ontology\', NCBI Taxonomy \[[@B11],[@B24]\], and Gene Ontology \[[@B25],[@B26]\]. Table [1](#T1){ref-type="table"} lists each of the sources of data incorporated into Atlas, and provides URLs where those sources can be found. Note that GenBank refers to the integrated records from the International Nucleotide Sequence Database Collaboration (GenBank \[[@B11]\], DDBJ \[[@B27]\], and EMBL \[[@B28]\]). Relational data models (schema design) -------------------------------------- This section describes the composition of the data models of the source data included in Atlas. The data models we present here are implemented in MySQL \[[@B29]\], an open source relational database management system (RDBMS). As such we only provide Data Definition Language (DDL) files that are compatible with MySQL. Currently there are no plans to port these to other RDBMS systems. ### Ontology Ontologies serve to define the concepts and relationships both within a system and between systems. This vocabulary of concepts and relationships is representative of a given expert domain of discourse such as sequences, gene annotations, and taxonomy. In Atlas, ontologies are categorized into two classes: Atlas defined ontologies and external ontologies. The Atlas defined ontologies are used to represent the concepts and relationships found specifically within Atlas, as well as to characterize concepts and relationships implicitly defined by the GenBank Sequence Feature data model. External ontologies include such things as NCBI Taxonomy for organism classification, Gene Ontology for gene annotations enabling categorization of biological features based on function, process, and cellular component, and the Proteomics Standards Initiative Molecular Interaction Standard (PSI-MI) controlled vocabulary \[[@B30]\]. The Atlas internal ontologies contain definitions of terms such as identifier types like accession numbers, GI numbers, PSI-MI terms and identifiers, PubMed identifiers, file format types like XML, relationship terms, and concepts like GenBank Sequence Features and Feature Qualifiers, Sequencing Techniques. This part of the Atlas ontology consists of three tables: Ontologywhich include terms and definitions, Ontology\_typethat defines ontology source and category, and Ontology\_Ontologywhich stores term-term relationships. Foreign key constraints are used to ensure data integrity. In contrast to these tightly integrated ontologies, two other external vocabularies are instantiated as independent MySQL databases: GO and NCBI Taxonomy. These ontologies, unlike the others, do not implement foreign key enforcements to the other database modules. As a result, when ontology terms are updated, references to deleted terms deemed to be invalid are kept in the system until such time a full data set reload is performed. The Atlas internal ontology exists largely to help describe Sequence Features as they exists in the GenBank Sequence Feature model, as this is the primary data source for features. Neither the Open Biological Ontologies (OBO) \[[@B31]\] relationship terms, nor the Sequence Ontology (SO) \[[@B32]\] relationship terms suited our needs as a feature ontology. We utilize the basic relationships similarly found in OBO and SO, such as \'is-a\', \'part-of\', and \'inverse-of\' but we also define more specific terms such as \'is-synonym-of\', \'refers to PubMed\', \'feature-includes-qualifier\', and \'gene-contains-promoter\'. By defining these specific relationships, we simplify the ontology tree into a flatter structure that is simple to query. In addition, subject-predicate-object triples are not explicitly defined in the internal ontology, but rather are assigned at loading-time as the GenBank Sequence Feature data is parsed and stored into the database. The relationship terms are not necessarily complete, but sufficient for our needs, and as new relationships are encountered, these are added accordingly. For example, we mapped all 66 GenBank feature keys to an entry in our Ontology table, which has enabled us to do feature-level queries for any type of feature in GenBank, or genomes we annotated in-house. We caution the reader that it is generally understood that not all GenBank features have the same informational value, nor quality of information. However, to capture the maximum amount of information, we chose to extract and store all annotated features. With the locations of the features stored in Atlas, sub-sequences of features can be extracted in a high-throughput manner using SQL, the APIs, or the toolbox applications. This is particularly useful, for example, in extracting features like non-coding RNAs from complete genomes, or regions spanning a particular gene of interest. We are actively integrating selected external ontologies, and expanding our internal ontologies. Plans for ontology integration include the National Library of Medicine (NLM) MeSH term and the Microarray Gene Expression Data (MGED) ontology \[[@B33]\]. We are evaluating the option of adopting frame-based ontology representations, and existing ontologies such as TAMBIS Ontology (TaO) \[[@B17],[@B34]\]. In the near future, we will release the Atlas ontology in other formats such as GO flat file, RDF/XML, and OWL. A complete list of the ontologies is available on the Atlas website, and we provide the MySQL dumps for these: <http://bioinformatics.ubc.ca/atlas/ontology/>. ### Sequence model The schema for sequences is organized into three main parts: sequence, which stores the sequence string and associated meta-data such as sequencing technique and molecule type; sequence identifiers, for which all identifiers that appear in the records are stored (see Figure [2](#F2){ref-type="fig"}); and annotated sequence features, for which feature keys, qualifier keys and values and feature locations are stored. Though output of features into General Feature Format Version 2 (GFF2) \[[@B35]\] is supported, the Featuretable, one will note, does not explicitly contain source or type fields. This information is stored in other tables and can be pulled together dynamically as a GFF2 record is being constructed. For example, the BioID\_typetable contains the database source information in its db\_source field and the internal Atlas Ontologytable\'s term field which represents the feature type used in the GFF2 output. However, to reflect the fact that features in such output are now reconstructed from the Atlas system, we prefix the original source type with \'Atlas:\', such as in \'Atlas:GenBank/RefSeq\'. The reader will note that there are two different Ontology tables in Atlas. A more detailed explanation for the motivation for having two different kinds of Ontology tables is described in the previous Ontology section. However, in the context of sequence features, it is the internal Atlas Ontologytable that is relevant. The sequence string is stored in the Sequencetable. Additional fields for: sequencing technique, tech, such as expressed sequence tags (ESTs); molecule type, moltype, such as DNA, RNA, protein, and nucleic acid; sequence length, length; the NCBI taxonomy identifier, taxonid; and the definition line, defline, are also stored in the Sequence table. Fields such as taxonid, tech, and moltype can be used separately, or in combination to produce customizable queries that return highly specific sets of data. Sequence identifiers, as with all other external identifiers, are managed through a layer of abstraction by associating them with internal identifiers within Atlas, which act as primary keys. Having a single internal identifier for a sequence allows us to relate all other identifiers found in the record to each other. In addition, ontologies for all types of identifiers currently found in GenBank ASN.1 data files, as well as relationships between these identifiers are modeled in the Bioidand Bioid\_Bioidtables, respectively. As mentioned above, sequence features are also modeled in Atlas. For details please refer to the Ontology section below. ### Molecular interactions For molecular interaction data, we developed a relational model compliant with the PSI-MI. Adopting a common interaction data model allowed us to unify data from different sources, and allows us to develop a set of common interaction retrieval APIs. Currently, HPRD, BIND, DIP, IntAct and MINT are included as interaction data sources. BIND, DIP, MINT and IntAct release their data in PSI-MI format. HPRD is releasing data in both PSI-MI standard format, and their own XML format. At the time of this publication, BIND released data as indexed flat files, ASN.1, XML, and PSI-MI format (level 2). The Atlas interaction model consists of four major entities: Interactor, Interaction, Experimentsand Dbxref. Interactorholds information about one of the interacting members in an interaction, such as the interactor\'s name, taxonomy, sequence, molecular type, features, subcellular localizations, and external identifiers. An Interactionconsists of one or more interactors, and one or more experiments. Experimentstores information about the experiments used to identify interactions. Finally, Dbxrefis used to crosslink the external identifiers such PubMed id, RefSeq accession, HPRD id, BIND id, and Ontology id, for example (see Figure [2](#F2){ref-type="fig"}). As an additional note, the Featuretable in the Interaction database is mainly used to store protein features involved in the interactions. We will release a PSI-MI level 2 compliant version of the Interaction model, and API upon public release of the level 2 specification. ### Gene related resources We integrated OMIM, LocusLink, Entrez Gene, HomoloGene and the annotation part of GO into the Atlas system in order to provide gene-related information. Conveniently, the OMIM and LocusLink data sources provide flat file tables which could be imported directly with the MySQL import function. Entrez Gene will eventually replace LocusLink, however in order to maintain a smooth transition and backward compatibility, we are maintaining populated relational models for both Entrez Gene and LocusLink until LocusLink is officially retired. Integration between HomoloGene and Sequence is achieved by relating the taxonomic, protein sequence and gene identifiers with Atlas\' Bioidtable. This allows us to integrate these databases and provide linkage between, for example, orthologous genes present in different interaction scenarios (see Utility of the Atlas system). Application programming interfaces ---------------------------------- There are two classes of APIs in Atlas: loader and retrieval. Components of Atlas for which we have developed our own relational models, such as the Biological Sequences component, or the Molecular Interactions component, each have their own set of loader APIs. The loader APIs used to build the loading applications, populate instances of the relational models in the Atlas databases. Though most end-users will never need to use the loader APIs, they are critical to the implementation of the Atlas loading process, and are provided to the software development community. The other class of APIs are the retrieval APIs. These APIs serve to retrieve the data stored in Atlas. They are required for developing custom retrieval applications such as the Atlas toolbox applications. The loader API for Biological Sequences is implemented in C++ as it relies heavily on the NCBI C++ Toolkit \[[@B36]\] to parse the ASN.1 data. The Biological Sequence retrieval API, on the other hand, is provided in all three languages: C++, Java, and Perl. The Java and Perl APIs return sequences as BioJava SimpleSequence and BioPerl Bio::Seq objects, respectively. The loader and retrieval APIs for Molecular Interactions are provided in Java. Though retrieval APIs are not supported in all languages, further development in Perl and C++ will be added if our user community requests them. Please refer to Figure [1](#F1){ref-type="fig"} for a mapping of data modules to currently supported programming languages. The project is also open source and other developers are encouraged to contribute. All the transactions between the APIs and the database are specified by the numerous SQL statements which are all defined within the majority of the API methods. ### Application programming interface architecture The API is constructed using object-oriented methodologies, employing objects to represent everything from low-level database connections to high-level data structures, and their access methods. This is illustrated in Figure [3](#F3){ref-type="fig"}. Common in the design of the C++, Java, and Perl APIs, are a set of APIs written for MySQL database connectivity which handles the opening and closing of MySQL connections, as well as managing the execution of the SQL statements themselves. All subsequent APIs that interact with the Atlas database extend from this set of APIs. Both the data loader and the retrieval utilities share a common class responsible for low-level data transformations. This class includes methods that facilitate conversions between two internal Atlas identifiers, such as bioid\_id to ontology\_id, or methods that convert internal Atlas identifiers to externally referenced public identifiers, such as GenBank accession numbers, or GI numbers. Inheriting this shared identifier conversion class benefits both the loader APIs and the retrieval APIs, by providing them with the necessary tools to integrate information. The Biological Sequences component of Atlas manages common identifiers, and hash maps in the Seqclass. This class is inherited by both the SeqLoadclass and SeqGetclass, which define the loader methods, and retrieval methods, respectively. Another feature of the Biological Sequences API, is its ability to control stream output based on molecule types. API users simply specify which molecule type to filter by, through calls to higher-level retrieval methods, and SeqGetwill then handle the logistics of stream management. Similarly with the Molecular Interactions component of Atlas, the InteractionDbclass is inherited by the InteractionLoadclass and the InteractionGetclass, respectively defining the loader and retrieval methods which manipulate the data in memory. Our Java interaction APIs, for example, are tightly coupled to our interaction data model with classes representing all the major schema objects such as Interaction, Feature, Dbxref, and Experiment. The APIs are works in progress and we continue to develop and improve them. We are considering even more tightly coupled API development by using XML schema code generators such as JAXB. All the source code is provided under the GNU General Public License (GPL), and therefore any developer can model future API development on numerous functions we have already implemented. Applications ------------ ### Toolbox The Atlas toolbox is a collection of applications that use the C++ API to perform common sequence and feature retrieval tasks. The applications are standard Unix command-line based tools that follow a command-line option-based interface for parameter entry. These are end-user applications and do not require any programming ability to use them. We have developed toolbox applications for sequence retrieval from accession and GI numbers, retrieval of sequences from all organisms beneath a given node in the NCBI taxonomy tree, retrieval of features given accession and GI numbers, retrieval of sub-sequences corresponding to specific features identified by qualifiers and their values, and retrieval of a set of interactions associated with a molecule given the accession number of an Interactor. Besides being useful tools, the toolbox applications\' source code provides good examples of application development using the APIs. Software developers wishing to use the APIs can use these toolbox applications as a starting point for their own custom applications (see Table [2](#T2){ref-type="table"}). ### Data loaders Data loaders are provided in Atlas to facilitate the parsing and loading of the source datasets into their respective Atlas database tables. Two main classes of loaders are currently supplied in the Atlas package: sequence loaders and interaction loaders. Though other types of data are loaded into Atlas, their loading is trivial as MySQL database dumps of these datasets are already provided by the data providers. The first class of loaders is the sequence-based loaders. Within this class there are two applications provided: seqloader and fastaloader. The seqloader performs the majority of the sequence loading from GenBank and RefSeq datasets. These datasets have long been represented as ASN.1 (binary/text) by the NCBI \[[@B37]\], and are compact and well defined for storing of structured data. The seqloader was built using the NCBI C++ Software Development Toolkit \[[@B36]\] which was designed to specifically parse the ASN.1 sequence data, extracting such things as the sequence, associated identifiers, features of the sequence and related publications. There are, however, instances where sequence data is missing from the ASN.1 records. In these situations, we obtain the missing records from the NCBI Entrez system in the form of Fasta records. The fastaloader application is then used to update the sequence field in Atlas with the sequences from the Fasta records. The second class of loaders is interaction-based loaders. These loaders are exclusively implemented in Java. The datasets loaded by this class of loaders include BIND, HPRD, MINT, IntAct and DIP. All the interaction loaders are designed to parse the data in the way that best deals with that particular source data\'s structure and content (mostly XML). The interaction data is loaded using a common interaction object model, and the interaction loading APIs provide a flexible and extensible framework for future interaction data loading efforts. Currently, we are developing a PSI-MI level 2 data loader. Besides these classes of loaders, there is also a Java based loader that parses and loads UniProt sequence data. In addition, scripts are used to load datasets for which MySQL dumps, or tab-delimited database dumps are provided. This is handled using the MySQL import function, and eliminates the need to devise special parsers and loaders. GenBank and RefSeq are checked daily for incremental updates from the NCBI. Accession numbers are used to maintain the integrity of the data. New accession numbers reflect new records and will be inserted into the database. Updated sequences or records with same root accession number and patched annotations will replace existing records in the database. When new releases of GenBank/RefSeq are made available, all databases are purged and reloaded to remove retired records and to maintain referential integrity. ### Web tools Though we encourage the use of Atlas as an in-house repository, it can also act to serve the wider internet community. We provide a publicly available web interface to the Atlas databases to demonstrate some of its functionality. This offers basic access to GenBank, RefSeq, NCBI Taxonomy, Atlas Ontologies, BIND, HPRD, MINT, IntAct and DIP. Web interfaces to the Atlas toolbox applications: ac2gi, ac2seq, ac2tax, feat2seq, gi2ac, gi2feat, gi2seq, gi2tax, tax2seq, techtax2seq, tech2seq are available. In addition, interacting partners for proteins identified by accession numbers or GI numbers can be retrieved from any of the four interaction databases stored in Atlas. These web tools can be found at: <http://bioinformatics.ubc.ca/atlas/webtools/>. Utility of the Atlas system =========================== The Atlas data warehouse offers maximum flexibility of data retrieval and integration. Users can access data in Atlas at the SQL, API and end-user application levels. Routine, pre-defined queries can be accessed through the APIs in Java, C++, and PERL (see API section, above), enabling developers to incorporate these queries in their software applications. Most of these queries have been used to build the Atlas toolbox, a set of end-user applications that run on the Unix command-line (Table [2](#T2){ref-type="table"}). Included in the toolbox are common utilities for converting GenBank ASN.1 sequences to file formats supported by the NCBI Toolkit \[[@B1]\] such as XML, GenBank Flat File, and FASTA. In addition, information regarding features that are annotated on sequence records can be exported as General Feature Format Version 2 (GFF2). The recently developed General Feature Format Version 3 (GFF3) is not currently supported in Atlas, to allow its specification time to stabilize. However, its support in Atlas is planned in future releases. In the following sections, we illustrate use-cases of the system at the SQL, API and toolbox levels with specific biological themes in mind. Single record queries --------------------- Single record queries are the simplest use case of the system. Users can input a GenBank or RefSeq accession number and/or GI number into the ac2seq and gi2seq toolbox applications to retrieve the relevant sequence record in Fasta, GenBank or ASN.1 format. Features on a particular sequence can also be retrieved independently with GenBank or RefSeq accession numbers and/or GI numbers. The single record queries can also be performed in batch mode where the user supplies a list of accession numbers or GI numbers and all data pertinent to the list of identifiers is then retrieved. Genome annotation ----------------- Atlas provides tools to generate data reagents for genome analysis as well as a data model for storing biological features that have been annotated on the sequences. Coupled with Pegasys \[[@B38]\] and Apollo \[[@B39]\], the Atlas system is an essential part of our annotation platform (see Figure [4](#F4){ref-type="fig"}). Atlas functions simultaneously as a data reagent generator for sequence alignment analysis, a storage system for annotations that are to be submitted, and a data transformation tool that can convert Apollo-compatible data to NCBI submission tool compatible data. Atlas provides users with the ability to generate custom sets of data to use as reagents. For example, using tax2seq, users can input a specific node of the NCBI taxonomy tree using its scientific name, or its NCBI taxonomy id and retrieve all nucleotide and amino acid sequences from organisms in the tree rooted at that node. This has special utility in genome analysis where specific sets of data from close relatives of the genome of interest enable comparative genomic methods for functional annotation. Furthermore, this type of taxonomy querying can be combined with the \'tech\' field in the NCBI data model to produce sequences derived from different sequencing techniques such as expressed sequence tags (EST), genome survey sequence (GSS), sequence tagged sites (STS), high throughput genomic (HTG), etc. Compiling these specific data sets allows the user to perform more directed sequence similarity searches, for example, that yield more specific hits. Using the sequence data structure to model existing annotations in sequence records, Atlas can be used to store additional annotations created in Sequin \[[@B40]\] and Apollo \[[@B39]\]. We have built a GAME XML \[[@B41]\] loader that stores annotations exported from Apollo. When used for this purpose, Atlas serves as a holding bay for sequences that can be submitted to DDBJ, EMBL, or GenBank in a relational form that can be mined in the interim using the multi-level query system provided by the Atlas APIs (see Figure [4](#F4){ref-type="fig"}). Additionally, the annotations stored from a GAME XML \[[@B41]\] file are exportable in GFF2, or Sequin Feature Table Format \[[@B42]\] for use with NCBI submission tools like tbl2asn \[[@B42]\]. Inference of protein-protein interactions ----------------------------------------- Deriving new associations from the information extracted from Atlas has proven to be particularly useful in developing a prototype system that infers interactions across species, detailed in \"Ulysses -- an Application for the Projection of Molecular Interactions across Species\" (Kemmer D: in preparation, from the Wasserman and Ouellette laboratories). Given that the data for protein-protein interactions found within model organisms can be extremely sparse, Ulysses employs homology information to help bridge the gaps in the interaction data by projecting known interactions in one species onto other species for which those interactions are not known, and subsequently inferring potentially novel interactions in those species. Ulysses is able to perform its analyses and inferences, in part, by capitalizing on the integration, offered by Atlas, of HPRD, BIND, and HomoloGene. Atlas makes it possible to retrieve interactions for one species known to occur in another species, by integrating these datasets under one query space, and by providing the API and tools which make such queries simple. As an example, in both the MINT and DIP databases, protein C-C chemokine receptor type 3 (SwissProt accession number P51677) was found to interact with protein Small inducible cytokine A24 precursor (SwissProt accession number O00175) in human (MINT interaction 14962; DIP interaction 10472E). Although referenced by different publications (\[[@B43]\], \[[@B44]\]), both interactions are likely to be the same. With corroborating evidence for these seemingly synonymous interactions, it can be claimed with more certainty that two proteins do indeed interact. Furthermore, homologs for both sequences can be found in mouse and rat through HomoloGene. Though these homologs are not found to be interacting partners in either mouse or rat, it is reasonable to speculate that such interactions exist in both these organisms. Disease-gene associations ------------------------- The Atlas system is also being used to determine yeast orthologs of genes that are implicated in human disease (Hieter P: in preparation). The inference being that human genes for which there are yeast orthologs represent essential genes which are candidates for human disease agents. Compiling the reagents for this custom database was straightforward using the Atlas tools. It takes advantage of the linkage between sequence identifiers, Taxonomy, HomoloGene, and OMIM. Discussion ========== We have built a data warehouse of biological information with the goal of providing high-throughput, flexible access to the data by means of SQL queries, API-level queries, and end-user application-level queries. Our goal was to create a system that serves as a platform through which information from many sources of data can be interrogated, enabling biologists and computer scientists to easily carry out queries necessary for their research. The data warehouse facilitates complex queries on local instances of GenBank, RefSeq, UniProt, HPRD, BIND, NCBI Taxonomy, HomoloGene, Gene Ontology, OMIM, Entrez Gene, and LocusLink. With previously disparate data now unified in a relational model, SQL can be used to retrieve this consolidated information at once. Though Atlas can act to serve data publicly over the internet, its simple setup enables anyone or any institution to easily serve their own customized data warehouses to their own local users. Installing Atlas in-house to serve local users gives the data provider full control over the data they serve. Giving users access to the system on a high-bandwidth internal network offers convenience and high-performance for large queries, such as retrieving all human ESTs. Such data is then more readily retrieved with lower latency and higher bandwidth than attempting to retrieve the same data over the internet. One of the important strengths of the Atlas architecture is that it allows data integration at two levels. The first level uses a common data model to integrate similar types of data from different sources (e.g., GenBank or UniProt, and BIND or HPRD). The second level uses the APIs, ontologies, and tools to cross-reference disparate types of data. For example, consider the task of retrieving all amino acid sequences, and from all organisms found within the taxonomic tree rooted at a given taxonomic node (e.g., vertebrata), from the RefSeq database. With a single call to the taxonName2Sequences method, the user can accomplish this task. Within these API methods are SQL statements which first retrieve the taxonid from the Taxonomydatabase. Then using a recursive method, the taxon identifiers for all organisms beneath that given taxon node, are returned. All amino acid sequences for each of these taxon identifiers are then retrieved using taxonId2Sequences (see API documentation \[[@B45]\] for more details). Uniting disparate sources of data is a useful exercise that highlights the challenges that the data itself presents. Any changes to the source data structure often requires software code changes in order to properly parse the new data format. Failure to do so often leads to the inability to load at least some of the information, if not all. Furthermore, the quality of the original data may often be imperfect as much of this data is curated manually, and hence is subject to data entry errors. Everything from missing data to improperly spelled key terms can impede the loading process. For this reason, it is essential to devise a system that is robust enough to handle unforeseeable exceptions. Policies on how to handle such exceptions are important to define and implement. We try to adhere to the careful logging of incorrect entries that we find during the loading process, and to promptly report these to the data providers for remediation. This is especially important when the specifications of the data are already strictly defined, yet are not followed, or are being misinterpreted. Semantic inconsistencies may arise due to differences in the interpretation of biological concepts and data, and differences in how such information is mapped into an integration system. That is to say, two systems may contain different data for the same semantic entity. For example, two interaction databases containing localization data for the proteins stored within, may indicate conflicting localization information for a given protein if the set of experimental evidences, used to determine localization, were different between the two systems. Such conflicts between data source providers pose challenges during the integration process as decisions need to be made to resolve the conflict. We continue to evaluate methods of resolving such conflicts. One simple solution is to store the information from all sources as is, and also annotate that information with the source from which it came, so as not to have any information loss. In this way, users can decide on which source they believe and poll the data accordingly. Another solution, which is not as clear cut, would be to selectively merge data, pruning those facts we determine to be incorrect (perhaps based on some measure of consensus between multiple systems), thus leaving only one instance of a factoid in our database. However, as it would not necessarily be our goal to judge the correctness of data, this is perhaps a task better left to users of our system. Comparison with other systems ----------------------------- Several other systems are available which have similar goals and provide good solutions to the problem of data integration. We have chosen to discuss Atlas in the context of three other systems: Entrez \[[@B11]\], SeqHound \[[@B12]\] and EnsMart \[[@B13]\]. The Entrez system, produced by the NCBI, provides \"an integrated database retrieval system that enables text searching, using simple Boolean queries, of a diverse set of 20 databases\". This web-based system is extremely extensive in the scope of data it provides, and in fact many of the Atlas data sources originate from NCBI (GenBank, RefSeq, HomoloGene, Taxonomy, OMIM, Entrez Gene, and LocusLink). The Entrez resources can be found on the NCBI website \[[@B46]\]. In contrast to Entrez, Atlas warehouses the data locally, obviating the need for low-throughput, internet-based queries. Also, additional data sets like HPRD, DIP, MINT and BIND, not currently available through the Entrez interface, have been added to Atlas. SeqHound \[[@B12]\] is a database of biological sequences and structures, developed by the Blueprint Initiative \[[@B47]\]. SeqHound also stores information on OMIM, LocusLink, and Gene Ontology. SeqHound and Atlas warehouse similar data types. SeqHound provides some different data than Atlas (most notably MMDB). For interaction data, SeqHound utilizes the BIND database. In contrast, Atlas stores interaction data from a number of sources including BIND, HPRD, MINT, DIP, and IntAct. Atlas then, is a more comprehensive repository of interaction data. The major difference between SeqHound and Atlas is in their architectural design. SeqHound stores full records and indexes specific fields which are extracted upon loading. In contrast, Atlas provides relational models for all data sources. This allows SQL-level access to specific parts of the data model. The data in the Atlas relational models are stored as primitive data types as opposed to storing whole records that need parsing or processing. For example, sequences and their annotated biological features can be stored in their own fields in the database, permitting \'substring\' operations to extract parts of the sequence that span a particular feature type using SQL. Other systems like EnsMart \[[@B13]\] and the UCSC genome browser \[[@B48]\] have also adopted fully relational models. These systems also provide SQL access over the full data model, and allow arbitrarily complex queries similar to Atlas. EnsMart is a software system designed by EMBL-EBI \[[@B49]\] and the Sanger Institute \[[@B50]\] which produces and manages automated annotations. The focus of EnsMart is slightly different than Atlas in that its \'core\' data is fully sequenced eukaryotic genomes. While information on these genomes is extremely rich in EnsMart and well-integrated using relational models, Atlas attempts to provide a much more extensive source of sequence information. This enables researchers interested in bacteria, viruses, plants or humans to access the system and sources of integrated data with equal facility. The Atlas system is designed to be locally installed and is not a data provider per se, but rather an engine that should be accessed \'in-house\'. As with any locally-installable system of this nature, significant time and hardware resources are needed to make the system functional. The utility of the Atlas system will far outweigh the setup time required to get it up and running. Currently, API access to Atlas is limited to the users at the UBC Bioinformatics Centre, University of British Columbia, however the web tools are available worldwide. Future work ----------- When working with sources of data from different data providers (for example UniProt and RefSeq), it is advantageous to create mappings from one data source to the other to prevent redundancy and to make associations between proteins to map annotations from one source to the other. We are investigating the idea of an identifier consolidation that can resolve mRNAs and proteins from different sources that are referring to the same protein product to a single identifier. We will constantly monitor and adjust any change of the data sources. In the near future, we will provide support for a PSI-MI level 2 release, and complete the migration of LocusLink to Entrez Gene. In addition, we are expanding Atlas to include other sources of data. We are currently adding MEDLINE, dbSNP and pathway data to support an integrative genomics and clinical informatics initiative, currently underway in our laboratory. With Atlas in hand, we are also working on an integration project that superimposes co-expression networks derived from microarray experiments and protein-protein interaction networks, to estimate the utility of co-expression networks in inferring protein interactions. Conclusion ========== Atlas is a data warehouse that enables high-throughput, flexible and complex queries on biological data. The system integrates sequences, molecular interactions, taxonomy and homology, and functional annotations on genes. The system functions as data infrastructure to support bioinformatics research and development. Atlas is currently being used in genome annotation projects, disease-gene association projects and inference of molecular interactions. We are releasing Atlas to the scientific community in the hope that it will foster creative ideas for how to make novel associations between disparate sources of data using existing public data sets. Availability and requirements ============================= Atlas is available from the UBC Bioinformatics Centre, University of British Columbia. The Atlas package can be downloaded from the Atlas website at: <http://bioinformatics.ubc.ca/atlas/> The Atlas package contains the Atlas source code and represents the core of the project. The package is distributed under the GNU General Public License. Atlas is designed to run on Unix based systems. Please consult the user manual (available with the distribution) for detailed configuration, compilation and installation instructions. Additional packages are also provided at the website listed above. These packages include a snapshot of the NCBI C++ Toolkit (CVS version 20040505), a MySQL dump of sample data, and additional documentation. The NCBI C++ Toolkit, that is provided, is required only for those users who wish to build the loader applications or for those that require the utilities that convert ASN.1 format to GBFF, EMBL, and XML formats, etc. Those setting up the database will need to install MySQL Server 4.x. Atlas has been tested, specifically, with MySQL Server versions 4.0.9, 4.0.18 and 4.0.20, running on either Linux or AIX. The Atlas sequence-related binaries (toolbox applications and loader applications) are developed in C++ and therefore a C++ compatible compiler, such as the one included with the GNU GCC suite of tools, should be installed before attempting to build these binaries. We have tested the build process with GNU GCC versions 2.95.3, 2.96, 3.1 and 3.2. In addition, MySQL Client version 4.x and particularly its runtime library, libmysqlclient.a(so), is required. MySQL Client versions 4.0.14 and 4.1.0-alpha were tested. Details on the configuration and use of this library are outlined, in more detail, in the Atlas manual. For users that require Atlas tools that are based on Java, such as the loading and retrieval tools for LocusLink, BIND, HPRD, and HomoloGene datasets, a compatible Java interpreter must be installed. The API has been tested with J2SE 1.4.1 and J2SE 1.4.2. The Atlas Java API also requires BioJava version 1.4pre, or higher. For those using the Perl based Atlas tools, a compatible Perl interpreter must be installed. BioPerl version 1.4 must also be installed. Perl version 5.6.1 has been tested. Each of the packages have their own minimum system requirements. Specific memory, hard disk space and CPU requirements for each package are listed in the manual. As a general guideline, it is essential to have a generous amount of available memory, especially if one anticipates processing large sequences in memory. Another important factor is the amount of available hard disk space. The amount of sequence data to be loaded into Atlas will largely determine your disk space requirements. The Atlas database requires a minimum of 50 GB (RefSeq), plus adequate space for satellite databases. The satellite databases include such things as GO, LocusLink, HPRD, BIND, MINT, and DIP, which are relatively smaller datasets. Note that sequence data can greatly exceed these minimum estimates and the requirements should be carefully planned. Authors\' contributions ======================= SS was the architect of the system, developed the C++ APIs and wrote the first draft of this manuscript. YH was the database administrator responsible for schema design, data integrity and maintenance. TX contributed the Java APIs. MMSY contributed the PERL APIs. JL developed the C++ APIs, the toolbox and the user manual. BFFO was the principal investigator, conceived of the project and guided its development. JL, YH, MSSY and BFFO all contributed to the writing of this manuscript. Acknowledgements ================ TX is supported by CIHR grant \#MOP-53259, Juergen Kast and BFFO. We thank Miroslav Hatas and Graeme Campbell for systems administration support. We thank Michael E. Smoot, for his contributions and enhancements to his Templatized C++ Command Line Processor \[[@B51]\]. Special thanks to Joanne Fox and Graeme Campbell for their help in reviewing and editing this paper. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Architecture of the Atlas data warehouse. The data integrated in Atlas are first downloaded as data files from the public repositories shown in the Data Sourcepanel. These data files are then parsed and loaded into the MySQL relational databases using the Atlas loaders. The Atlas Databasespanel shows the databases grouped by biological theme. These groups are sequences (green), molecular interactions (yellow); genes and functional categorization (blue); and ontologies (orange). For each database the available data retrieval methods are marked as SQL (S), C++ Atlas API (C), Java Atlas API (J), and Perl Atlas API (P). The Retrievalpanel shows the flexible, layered architecture of the interfaces to the databases. Data can be accessed directly using the MySQL client with SQL statements, through the APIs in C++, Java, and Perl, and through the end-user applications implemented in the Toolbox. The APIs can also be used to implement web-based tools, or standalone applications. ::: ![](1471-2105-6-34-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Atlas database schema. There are four major functional groups. Biological Sequences: includes instances of GenBank sequences, RefSeq sequences, and UniProt sequences; Molecular Interactions: includes instances of BIND, HPRD, DIP, IntAct and MINT; Gene Related Resources: includes instances of OMIM, Entrez Gene, and LocusLink, and HomoloGene; and Ontology: includes instances of Taxonomy, Atlas internal ontologies, Gene Ontology, and PSI-MI ontologies. ::: ![](1471-2105-6-34-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Atlas API architecture. MySqlDb, Seq, SeqGet and SeqLoad classes/modules (grey) are available in all three languages: C++, Java, and Perl. The SeqLoad and Seqloader modules are created in C++ only as these are tightly coupled to the NCBI C++ Toolkit. All other classes are available in Java. Applications share the common modules SeqLoad, SeqGet, InteractionLoad, and InteractionGet which provide the methods necessary for loading and retrieval operations, to and from the databases. These modules employ additional classes (not shown) that are representative of the major data model components such as Sequence, Interaction, Interactor, and Dbxref, for example. ::: ![](1471-2105-6-34-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Using Atlas in genome annotation.Atlas facilitates genome annotation at multiple levels: creation of data reagents, storage of annotations, and data transformation for submission. Here we show a schema of our genome annotation process that integrates Pegasys, Apollo, NCBI tools and Atlas into a comprehensive platform. Data reagents for sequence alignment are compiled using the Atlas toolbox applications. Computational analyses are run through the Pegasys system which outputs GAME XML for import into Apollo. Annotations are saved in a GAME XML which are then imported into Atlas using the GameLoader. At this step, the biological features created in the annotation process are stored in the Atlas Featuretables, exactly the same way a GenBank sequence record containing annotations are stored. These annotations can then be retrieved using the Atlas toolbox application ac2feat and exported in GFF2 or Sequin Feature Table Format for import into the NCBI submission tools for validation, and submission to GenBank. ::: ![](1471-2105-6-34-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Data sources included in Atlas. ::: Atlas Data Source Summary Table \* ------------------------------------ --------------------------------------------------------------------- ----------------- ------------------ ---------------------- Data Source URL Data Format Atlas Update Update Mechanism GenBank Sequence <ftp://ftp.ncbi.nih.gov/ncbi-asn1/> ASN.1 Daily Incremental GenBank Sequence <ftp://ftp.ncbi.nih.gov/ncbi-asn1/> ASN.1 Release Reload GenBank Refseq <ftp://ftp.ncbi.nih.gov/refseq/> ASN.1 Daily Incremental GenBank Refseq <ftp://ftp.ncbi.nih.gov/refseq/> ASN.1 Release Reload NCBI Taxonomy <ftp://ftp.ncbi.nih.gov/pub/taxonomy/> Delimited Text Release Reload HomoloGene <ftp://ftp.ncbi.nih.gov/pub/HomoloGene/> Delimited Text Daily Reload OMIM <ftp://ftp.ncbi.nih.gov/repository/OMIM/> Delimited Text Daily Reload Gene <ftp://ftp.ncbi.nih.gov/gene/> Delimited Text Daily Reload LocusLink <ftp://ftp.ncbi.nih.gov/refseq/LocusLink/> Delimited Text Daily Reload UniProt <ftp://ftp.uniprot.org/pub/databases/uniprot/knowledgebase/> XML Bi-weekly Reload HPRD <http://www.hprd.org/download/> XML Release Reload MINT <http://mint.bio.uniroma2.it/mint/> XML Release Reload DIP <http://dip.doe-mbi.ucla.edu/dip/Download.cgi> XML Release Reload BIND <ftp://ftp.blueprint.org/pub/BIND/current/bindflatfiles/bindindex/> Delimited Text Release Reload GO <http://www.godatabase.org/dev/database/archive/latest/> MySQL dump Release Reload \* up-to-date information about data sources and statistics are available from the Atlas website <http://bioinformatics.ubc.ca/atlas/> ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Atlas toolbox applications. ::: Application Function Input Output ----------------- --------------------------------------------------------------- ---------------------------------------------------------------- --------------------------------------- Sequence ac2seq Retrieve sequences given an accession Nucleic acid or Protein Accession Number(s) Sequences in Fasta format feat2seq Retrieve sub-sequences that span features Feature type (and qualifier) Sequences in Fasta format gi2seq Retrieve sequences given a GenInfo identifier GenInfo Identifier(s) (GI Number(s)) Sequences in Fasta format gi2seqentry Retrieve sequences given a GenInfo identifier GenInfo Identifier(s) (GI Number(s)) GBFF, EMBL, GFF, FTABLE, ASN.1, GBSEQ tax2seq Retrieve sequences by taxonomy NCBI taxon identifier or scientific name of taxon Sequences in Fasta format tech2seq Retrieve sequences by sequencing technique Sequencing technique (eg EST, GSS, etc.) Sequences in Fasta format techtax2seq Retrieve sequences by taxonomy and sequencing technique Sequencing technique and NCBI taxonid/scientific name of taxon Sequences in Fasta format Loader fastaloader Fasta sequence data loader Sequences in Fasta format seqloader ASN.1 sequence data loader GenBank/RefSeq ASN.1 records Feature ac2feat Retrieve features GenBank Accession number (s) Features in GFF or FTABLE format gi2feat Retrieve features GenInfo Identifier(s) (GI Number(s)) Features in GFF or FTABLE format Taxonomy ac2tax Retrieve taxonomy given an accession number GenBank Accession number (string) NCBI taxon identifier (integer) gi2tax Retrieve taxonomy given a GenInfo identifier GenInfo Identifier (integer) NCBI taxon identifier (integer) ID Converters ac2gi Convert an accession number to a GenInfo identifier GenBank Accession number (string) GenInfo Identifier (integer) gi2ac Convert a GenInfo identifier to an accession number GenInfo Identifier (integer) Accession number (string) tax2gi Retrieve GenInfo identifiers associated with taxon identifier NCBI taxon identifier (integer) GenInfo Identifier (integer) :::	PubMed Central	2024-06-05T03:55:53.972715	2005-2-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554782/", "journal": "BMC Bioinformatics. 2005 Feb 21; 6:34", "authors": [ { "first": "Sohrab P", "last": "Shah" }, { "first": "Yong", "last": "Huang" }, { "first": "Tao", "last": "Xu" }, { "first": "Macaire MS", "last": "Yuen" }, { "first": "John", "last": "Ling" }, { "first": "BF Francis", "last": "Ouellette" } ] }
PMC554783	Background ========== Epidemiological studies report that alcoholism as defined by the Research Diagnostic Criteria (RDC) affects almost 10--15% of the general population in the USA and 1--5% in Europe \[[@B1]\]. Drinking behaviour is clearly partly determined by cultural and psychological factors, but genetic factors also play an important part as shown by family, twin and adoption studies \[[@B2]\]. The overall heritability for alcoholism has been estimated to be around 50% to 60% \[[@B3]\]. However there are almost certainly multiple aetiological subtypes of alcoholism, which will eventually be shown to have heterogeneous genetic and cultural components \[[@B2],[@B4]\]. Many genetic association studies of alcoholism have sought to identify candidate susceptibility genes, but few linkage studies have been undertaken so far. Two genome-wide linkage studies have been performed on US populations. One was carried out on a well defined population of south western USA American Indians \[[@B5]\] and the other on a large sample of families by the Collaborative Study of the Genetics of Alcoholism (COGA) \[[@B6],[@B7]\]. In the native American Indian study two chromosomal regions provided suggestive evidence for linkage. One was on chromosome 11p close to the DRD4 dopamine receptor and the tyrosine hydroxylase genes and the other on chromosome 4p near the β1 GABA receptor gene. Three loci in the ADH cluster on chromosome 4 also gave evidence of linkage on two point but not multi-point linkage analyses \[[@B5]\]. On the other hand the COGA study, the multi-point linkage analysis provided suggestive evidence of linkage on chromosome 1 and 7 with more modest evidence for a locus on chromosome 2. In addition, there was suggestive evidence for a protective locus on chromosome 4 near the ADH gene cluster \[[@B6]\]. This study \[[@B6]\] implicated chromosome 1 in two distinct regions on 1p21-35. Two-point analysis of affected sib-pairs showed a significant increase of allele sharing for two adjacent markers D1S532 and D1S1588. The multipoint linkage analysis reported a lod of 2.93 for D1S1588. A second region near D1S224, 60 cM apart from the first locus, had a multipoint lod score of 1.65. In the COGA replication set of families \[[@B7]\], linkage near the marker D1S224 with a maximum multipoint lod score of 1.6 was reported. In the combined COGA sample a LOD score of 2.6 was reported near markers D1S2614 and D1S1588. Re-analysis of COGA data \[[@B8]\] for linkage to an alcoholism-related phenotype consisting of alcoholism and depression in the combined COGA samples found a maximum lod of 5.12 near the markers D1S1648 and D1S1588. Furthermore, the region on chromosome 1p near D1S1588 and D1S1631 was also identified as demonstrating possible linkage to the \"low level of response to alcohol\" phenotype with a maximum lod score of 2.0 \[[@B9]\]. Linkage was also supported in the COGA dataset of an endophenotype characterized by a later age of onset of regular drinking and higher harm avoidance to a region near D1S518 on 1q \[[@B10]\]. Mouse linkage analyses have also shed light on the genetic determinants of alcohol consumption. The extensive syntenic homology between the mouse and human genomes enables predictions about which human loci are syntenic with mouse alcohol related loci. Buck and co-workers, who studied several mouse alcohol related phenotypes, predicted that genes related to physical dependence on ethanol may localize to human chromosome regions 1q21-43, 2q11-32, 5p15, 5q14-21, and 9p24-22, 10q23-26 \[[@B11]\]. The purpose of the current study was to test the hypothesis that the positive linkage reported on human chromosome 1p21-23 by the COGA study and elsewhere on chromosome 1 could be replicated in a United Kingdom sample of families multiply affected by alcoholism. Results ======= Selection criteria for the families eliminated all but 9% of the families contacted from all sources. Press contacts were found to be the most productive source of suitable families willing to participate in the study, accounting for over 50% of subjects recruited. There appeared to be no bias in the affection status of subjects recruited from the different sources \[[@B12]\]. The affection status of all the subjects is summarized in table [1](#T1){ref-type="table"}. The individuals interviewed were 297 and 176 subjects were genotyped for the purpose of this study. Alcohol consumption and severity of dependence indices measured using the severity of alcohol dependence questionnaire (SADQ) confirmed that ADS subjects were the most severely affected, the next level of lesser severity of alcoholism was defined by the Research Diagnostic Criteria of Alcoholism (RDCA) and then by the Heavy Drinking category (HD). The mean maximum regular lifetime consumption of the HD subjects (including the RDCA and ADS subjects, who are all HD) is 138 units/week. 80% of HD subjects reported a period of at least one month during which they were drinking in excess of 50 units/week for males or 35 units/week for females, which are the amounts above which the Royal College of Psychiatrists advises that drinking is likely to be harmful (Royal College of Psychiatrists, 1986). Almost half the HD subjects (49%) gave a history of a one month or longer period during which they were drinking in excess of 150 units/week for males, and 100 units/week for females. The mean SADQ score for ADS subjects was just below 30 (SD 14.2). The range for this rating scale was from 0 to 60. Stockwell and co-workers \[[@B13]\] suggested that scores above 30 indicate severe dependence. In our sample, 40% of the ADS subjects were severely dependent according to this definition. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Proportion of the individuals for each affection status category and their mean age (TTLifelong abstainers; SDSocial Drinkers; HDHeavy drinkers; RDCAResearch Diagnostic Criteria Alcoholism; ADSAlcohol Dependence Syndrome) ::: Affection Status Family Sample -------------------- ------------------ ---- ------------------------------------------------ Number Genotyped \% Mean age ± SD (yrs) in all cases and relatives TT 7 4 58.2 ± 13 SD 43 24 50.4 ± 17.1 HD 22 13 41.5 ± 14.1 RDCA 50 28 41.5 ± 13.5 ADS 54 31 42.1 ± 11.9 ::: HD affection model ------------------ Assuming dominant transmission, two-point heterogeneity LODS (HLOD) for linkage to the HD affections status gave a score of 1.8 near the marker D1S1653. The three-point multipoint analysis produced an HLOD of 1.8 with the markers D1S1653 and D1S1677. Analyses with the same markers assuming recessive transmission produced an HLOD of 1.1 with a two-point analysis and an HLOD of 1.5 with a three-point analysis. The model-free (MFLINK) analysis produced an MLOD of 1.5 for both the two-point and three-point analyses. Three-point analyses of D1S1677 and D1S1679 produced an HLOD of 1.7 assuming recessive transmission and a MALOD of 1.3 for the model-free three point analysis. The marker D1S1675, on the short arm of chromosome 1, produced an HLOD of 1.2 with both a two-point analysis and a three-point analysis in combination with D1S3723 assuming dominant transmission, Model-free analyses of D1S1588 produced a MALOD of 1.5 and 1.2 using two-point and three-point analyses, respectively. Figure [1](#F1){ref-type="fig"} shows the two-point linkage HLOD results for the HD category under both the dominant and the recessive model and also the model-free MFLINK analyses. The HD category is a cumulative definition which includes all the more severely affected categories for linkage analysis as well as those at the borderline of becoming alcoholic. The HD category is justified because drinking at this level is well recognised to be a predictor of future alcoholism, an observation that has also been incorporated in another genome linkage study of alcoholism which also employs a similar definition \[[@B14]\]. We have consistently used this affection status category in all our previous linkage analyses and it has not been applied in an arbitrary manner that could have inflated any lod scores. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Two-point linkage analysis for the Heavy Drinking (HD) category under the dominant and the recessive model (HLOD) and model-free analysis (MALOD) ::: ![](1471-2156-6-11-1) ::: RDCA affection model -------------------- Assuming recessive transmission the marker D1S1679 was linked to RDC alcoholism with a two-point HLOD of 1.0 and a three-point HLOD of 1.6 with the markers D1S1677 and D1S1679. The MLOD two point MFLINK score with D1S1677 was 0.9 and this increased to 1.4 with a three-point model-free analysis. For the RDCA level of affection status the highest MALOD score of 1.8 was found near the telomere of chromosome 1p near the marker D1S1588 under the model free analysis. Figure [2](#F2){ref-type="fig"} shows two-point linkage analysis for the RDCA category under the dominant and recessive models and also the model-free analyses. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Two-point linkage analysis for the RDC alcoholism (RDCA) category under the dominant and the recessive model (HLOD) and model-free analysis (MALOD) ::: ![](1471-2156-6-11-2) ::: ADS affection model ------------------- The marker D1S1591 gave the highest maximum admixture lod (MALOD) of 1.00 with the model-free method of analysis for the ADS category. The results of MFLINK multipoint analysis for this and the other affection status models are shown in table [2](#T2){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### MFLINK two-point MALODs for the HD, RDCA and ADS diagnostic categories. In bold MALOD score value ≥1. ::: Markers HD RDCA ADS --------- --------- --------- ------- D1S1591 0 0 1 D1S2134 0.1 01 0.2 D1S1665 0.2 0.5 0 D1S532 0.5 0.4 0.4 D1S1728 0.1 0.4 0.4 D1S551 0.4 0.8 0.5 D1S1588 1.5 1.8 0 D1S1631 0 0.3 0.01 D1S3723 0.2 0.3 0.04 D1S1675 0.8 0.4 0.4 D1S1595 0.7 0.3 0.3 D1S1653 1.5 0.3 0.3 D1S1677 0.4 0.5 0.06 D1S1679 0.8 1 0.5 ::: Conclusion ========== The COGA study first reported evidence of a locus linked to alcoholism on chromosome 1p with the markers D1S532 and D1S1588 linked to Alcohol Dependence \[[@B6]\]. Curtis and coworkers, in their reanalysis of the initial COGA dataset computed a MALOD lod score of 1.75 with the marker D1S1588 \[[@B15]\]. The COGA group repeated the analysis in a new set of families and the lod score at D1S1588 was 1.6 in this replication data set. The combined analysis of the two COGA samples gave a lod of 2.6 \[[@B7]\]. The COGA study also conducted a regression method of linkage so as to include unaffected and discordant pairs. D1S1588 then gave a peak lod score of 2.9. Our own results of a lod of 1.8 at the region near D1S1588 offers support but not clear confirmation for the COGA findings. A second region on chromosome 1p, near D1S224, approximately 60 cM proximal to D1S1588, gave a multipoint lod score of 1.7 in the COGA study \[[@B6]\]. The current British study gave a MALOD score of 1.2 over this region with the marker D1S1675 under the HD model, and a MALOD of 1.2 with the marker D1S3723 for the RDCA category. Dick et al \[[@B10]\] who analysed the COGA data have suggested that there is a locus on the long arm of chromosome 1 near the marker D1S518 linked to the endophenotype of a late age of onset of regular drinking and high harm avoidance. In a mouse recombinant inbred strain linkage study, a quantitative trait locus for physical dependence on alcohol was mapped to the murine chromosome 1 which is a region syntenic with human chromosome 1q21-43. In the present UK linkage analyses a three point HLOD score of 1.8 was found with the combined markers D1S1653 and D1S1677 for the HD phenotype. The positioning of the lod reported by Dick \[[@B10]\], the mouse genetic study and the UK linkage study are all compatible with a second locus for alcoholism which is on the proximal part of the long arm of chromosome 1 at 1q21.3-24.2. In the UK linkage study the two regions on chromosome 1 are implicated with two different affection status categories. We could speculate that the two different loci on chromosome 1 could be mediating an effect on alcoholism through two loci that have different types of effect on susceptibility. The most interesting and probably important finding from the COGA linkage study is that the stratification of families that have members with both alcoholism and depression maximizes the lod to 5.12 on chromosome 1p near the markers D1S1648 and D1S1588 \[[@B14]\]. Without stratification based on clinical phenotype in the combined COGA sample a LOD score of 2.6 was obtained. These findings provide a strong indication that the 1p locus near D1S1588 is mediating its effect on alcoholism through a genetic susceptibility for depression and anxiety. A recent genome wide scan for quantitative-trait loci carried out by Fullerton and colleagues \[[@B16]\] identified a locus on chromosome 1p, D1S2868, linked to neuroticism. The authors speculated that this locus influences traits genetically related to neuroticism and maps near the locus, D1S1588, identified by Nurnberger et al. \[[@B14]\] for the endophenotype of alcoholism and depression. It is a generally accepted clinical fact that it is very difficult to diagnose whether depression or anxiety are predisposing to alcoholism or resulting from it. It is likely that both effects occur simultaneously. The twin and family study data to date, strongly supports the hypothesis that alcoholism both causes and augments pre-existing depression and anxiety \[[@B17]-[@B19]\]. The resolution of this issue at a biological and psychosocial level is a high priority for psychiatry as a whole because of the very high population prevalence of these comorbid disorders in most populations. The full understanding of these linkage findings in alcoholism may help us unravel basic aetiological mechanisms and help create new treatment and preventive strategies based on a fuller understanding of genetic and environmental factors involved in comorbidity. It would seem justified to start allelic association studies on chromosome 1 in order to fine map susceptibility genes for patients comorbid for depression, anxiety and alcoholism. A positive finding would gain support from the prior linkage studies of both alcoholism and affective disorders and vice versa. The presence of locus heterogeneity for genetic effects on depression, anxiety and alcoholism will require relatively large case-control studies of alcoholics with adequate clinical assessment to detect alcoholics with and without comorbid disorders. Methods ======= a) Family Sample ---------------- Prior to commencing the study, ethical permission for this research project was obtained from the University College London Medical School Clinical Investigations Panel which has been updated in 2003 with multicentre research ethics committee (MREC) approval. Information stored on computer was registered under the 1984 Data Protection Act. Caucasian families multiply affected by alcoholism, and suitable for linkage studies, were ascertained with the following selection criteria. \(a) Presence of two or preferably more cases of alcoholism as defined by the RDC. \(b) Large families, preferably with two or more generations willing and able to participate. \(c) Evidence of unilineal inheritance of alcoholism in the parental generation, which meant that families were excluded where both parents were affected. \(d) Willingness of as many family members as possible, especially affected individuals, to participate in the research project. The families were identified from hospital records, clinicians, advertisements in the media and other contacts \[[@B12]\]. The families were all white Caucasian in origin. The subjects were diagnosed using the following interview schedules: the Schedule for Affective Disorders and Schizophrenia -- Lifetime version (SADS-L) which provides an RDC diagnosis of alcoholism \[[@B20]\], a \"lifetime modification\" of sections 1 and 3 of the Clinical Alcoholism Interview schedule \[[@B21]\], the Lifetime Drinking History \[[@B22]\] and a lifetime modification of the Severity of Alcohol Dependence Questionnaire (SADQ) \[[@B13]\]. Heavy Drinking (HD) was defined as drinking in excess of the Royal College of Psychiatrists recommendations, i.e. more than 14 units per week for females or 21 units per week for males, for over one month (Royal College of Psychiatrists, 1986). Diagnoses were made at three levels and these were employed in the linkage analyses in a hierarchical manner. All subjects fulfilling criteria for the Alcohol Dependence Syndrome (ADS) also fulfilled the criteria for RDC alcoholism (RDCA) and HD. All subjects meeting criteria for RDCA by definition also met the criteria for HD. Individuals who were drinking regularly below these limits, or who drank infrequently, were classified as social drinkers (SD), these individuals were considered as unaffected in the linkage analyses. Subjects who had never drunk alcohol, or had only one or two drinks in a lifetime, were considered to be lifelong abstainers (TT) and were also considered as unaffected. In order to check the RDCA, HD, SD and TT assessments, the interview schedules were examined by two independent psychiatrists. Where discrepancies arose between two raters, a consensus diagnosis was reached by process of joint discussion and consideration of the data. b) Laboratory procedures ------------------------ Fifty nanograms of total genomic DNA was extracted from venous blood samples and amplified by Polymerase Chain Reaction (PCR) with oligonucleotide primers using standard methodology \[[@B23]\]. Short Tandem Repeat markers from the Research Genetics Set 9 genome screening panel and key markers from the COGA study were typed. A M13 tail was added to one of the oligonucleotide primers which was used to hybridise with a complementary oligonucleotide pre-labelled with infra red dyes that fluoresce at either 700 or 800 nm. The resulting images from argon laser scanning allow a highly detailed visualisation of genetic polymorphisms \[[@B24]\]. Gel electrophoresis and pattern visualization was performed using LI-COR Model 4200 automated fluorescent DNA sequencers \[[@B25]\]. Ten markers on 1p21-35 were genotyped: D1S1591, D1S2134, D1S1665, D1S1728, D1S532, D1S1588, D1S551, D1S1631, D1S3723 and D1S1675. Four markers on 1q were genotyped: D1S1595, D1S1653, D1S1677 and D1S1679. Marker order and intermarker distances were as compiled in the Ensembl database [http://www. ensembl.org](http://www. ensembl.org). Positions of the markers are shown in table [3](#T3){ref-type="table"}. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Marker location and type of polymorphism <http://www.ensembl.org> ::: Markers Mb Type ------------- -------- ---------- D1S1591 39 TETNUC D1S2134 48 TETNUC D1S1665 73 TETNUC D1S532 77 TETNUC D1S1728 81 TETNUC D1S551 82 TETNUC D1S1588 92 TRINUC D1S1631 105 TRINUC D1S3723 107 TETNUC D1S1675 114 TETNUC D1S1595 153 TETNUC D1S1653 155 TETNUC D1S1679 159 TETNUC D1S1677 161 TETNUC ::: Genotypes were read blind to diagnostic information. Tests for Mendelian inheritance of marker data were performed and inconsistent genotypes were repeated or omitted. c) Linkage analysis ------------------- The linkage analysis was performed using the classical lod score method and using likelihood-based model-free analysis carried out with the MFLINK program \[[@B26]\]. For lod score analyses, the programs MLINK and LINKMAP were used from the FASTLINK package \[[@B27]\]. Three affection models were used: ADS, RDCA and HD. Each affection model was analysed assuming dominant transmission, recessive transmission and using the model-free method. For dominant models the frequency of the abnormal allele was set to 0.02 and for recessive models to 0.2. The penetrance for normal and abnormal genotypes respectively were set to 0.02 and 0.5 for ADS, to 0.04 and 0.7 for RDCA and to 0.2 and 0.9 for HD. These penetrance values were chosen to produce models approximately consistent with prevalence data from previous epidemiological research of 0.04, 0.06 and 0.2 for ADS, RDCA and HD respectively \[[@B28],[@B29]\]. Two-point and three-point analysis was carried out for all affection definitions and transmission models. Classical linkage analysis was carried out under the assumption that locus heterogeneity might be present, yielding an HLOD statistic. Authors\' contributions ======================= IG carried out the genotyping and produced the manuscript CCHC collected the multiplex family sample and participated in the design of the study WK participated in genotyping AD participated in genotyping AM participated in genotyping DC performed the linkage analysis HMDG conceived the study and participated in its design and coordination All authors read and approved the final manuscript Acknowledgements ================ We are grateful to Prof. G. Edwards, Dr N. Gunaratna and Dr. GK Shaw for assistance in identifying families. The research has been funded by the University of London, the Brewer\'s Society, the Wellcome Trust (Senior Fellowship in Clinical Science to HMDG), the UK Medical Research Council, the Clinical Research and Development Committee of University College London Medical School, and the Sir Samuel Scott of Yew\'s Trust.	PubMed Central	2024-06-05T03:55:53.979105	2005-3-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554783/", "journal": "BMC Genet. 2005 Mar 1; 6:11", "authors": [ { "first": "Irene", "last": "Guerrini" }, { "first": "Christopher CH", "last": "Cook" }, { "first": "Wendy", "last": "Kest" }, { "first": "Audrey", "last": "Devitgh" }, { "first": "Andrew", "last": "McQuillin" }, { "first": "David", "last": "Curtis" }, { "first": "Hugh MD", "last": "Gurling" } ] }
PMC554784	Background ========== Current statistics indicate that approximately 64.5% of US adults can be considered overweight while 30.5% can be classified as obese \[[@B1]\]. Obesity is also linked to a variety of chronic diseases, and is associated with approximately 300,000 deaths each year and annual economic costs of over \$117 billion \[[@B2]\]. According to the latest Behavioral Risk Factor Surveillance Survey (BRFSS) data, obesity and overweight continue to be major pubic health concerns, and reports indicate that no state had met the Healthy People 2010 objective of reducing obesity to 15% \[[@B3]\]. As a result, there is need for a strong evidence base for prevention and treatment strategies. Systematic reviews can offer the most reliable sources of information on which to develop guidelines and base treatment policy. However, such reviews need to contain a high proportion of all relevant evidence, which relates both to the need to find all trials and the need to analyze data on all participants \[[@B4]\]. To improve the quality of reporting for randomized controlled trials (RCTs) in the overweight and obesity literature, as well as any intervention trial, investigators are encouraged to publish reports in clear and unambiguous language, accounting for all events that occurred during the conduct of the investigation, and providing an accurate and thorough description of subjects who were selected, excluded, withdrawn or did not complete the study. Failing to report essential variables, such as subject characteristics, does not allow readers to adequately judge the validity or applicability of the study results, and may influence the interpretation of findings \[[@B5]\]. In response to the need for quality reporting, a panel of clinical investigators, epidemiologists, biostatisticians, and journal editors published a statement called the Consolidation of the Standards of Reporting Trials (CONSORT), which is designed to improve the standard of written reports of RCTs \[[@B6]\]. The CONSORT statement includes a checklist of 21 items and a flow diagram that can be used by authors to mark the page of the manuscript in which each of the items is addressed. In addition, the flow chart provides a detailed description of the progression of subjects through the intervention trial, from the number of potentially eligible participants for inclusion in the study to the number of intervention subjects in each group who completed the trial \[[@B6]\]. Using a similar format, we adapted the CONSORT form to examine the frequency of explicit reporting of subject characteristics as they relate to enrollment, allocation, and follow-up for weight loss studies published in the diet and exercise literature. Methods ======= For the present study, we used information collected from a meta-analysis of the diet and exercise literature. The primary purpose of the meta-analysis was to statistically integrate and analyze published research studies on the effects of diet restriction only, diet restriction and exercise, or exercise only on weight loss, body composition, fat distribution, metabolism, and aerobic fitness. We examined the frequency of explicit reporting of subject characteristics (i.e., age, gender, co-morbid disease states, medication use (in addition to intervention drug), race/ethnicity, and postmenopausal status). We also examined whether or not initial and final sample size was reported and if so, we recorded if the final sample size was reported by gender. Search strategy. With the assistance of a clinical medical librarian, we developed search strategies, key words, and check tags to begin our initial search for relevant articles to include in the meta-analysis. We identified studies from a computerized search of the MEDLINE database (US National Library of Medicine) from 1966 -- 2003, manual searches of bibliographic references of relevant published articles, and extensive cross-referencing and manual searches of 92 review articles. Overall study inclusion criteria -------------------------------- Subject types. We required that intervention trials must study overweight or obese (BMI ≥ 25 kg/m^2^) adult participants (≥ 18 years old). Ambulatory patients who were kept on wards exclusively for study purposes were also included. However, studies including pregnant women, patients with serious medical conditions, military personnel, and trained or professional athletes were excluded. Intervention types. For inclusion, trials were required to meet each of the following criteria: 1) active intervention trials with one group assigned to a weight-loss program involving energy restriction, exercise (aerobic or weight training) or both energy restriction and exercise; 2) outcome measures included weight loss; however, weight loss was not required to be a primary outcome; 3) the minimum duration of the intervention for weight loss must be greater than one day; and, 4) the article must be published in the English language. Case studies, crossover trials (due to possible carryover effects of weight reduction), and exploratory studies were excluded. We did not require studies to have been conducted in the United States. Pharmacotherapy, hormonal therapy, or surgical treatment studies, which did not include a separate group receiving a weight loss intervention (i.e., diet and/or exercise) were not included in the review process. Initial screening process. We independently screened all records resulting from our MEDLINE search strategies and manual searches by examining the titles and abstracts. We rejected articles if we could determine from the title and/or abstract that the study did not meet our selection criteria. If we could not determine whether or not to reject the article or if there was disagreement among reviewers, we retrieved the full text of the article for further evaluation. Quality control measures ------------------------ To control for abstraction bias, we trained five graduate research assistants to extract information from articles, using sample articles. Reliability checks were completed on the first several sample articles before graduate students were allowed to code independently. On a monthly basis thereafter, inter- and intra-rater reliability checks were completed to control for drift. All research assistants were required to attend weekly meetings with the principal investigator and co-investigators to discuss coding issues and to resolve coding disagreements. To control for potential selection biases after our initial screening procedure, we selected or rejected studies based on an examination of the research design and methodology. Acceptance or rejection for inclusion in the meta-analysis was not based on the study\'s outcome. We required coders to list specific reasons for all excluded studies. In addition, we tried to enhance data reporting for studies with missing covariate data by contacting authors by email or letter. Data entry quality control measures included the design of screens to match the coding sheet, automatic range limits placed on items entered, and queries to check for outliers. Values greater than two standard deviations from the mean were checked against the article. In addition, a random sampling of 58 articles (\~25%) was pulled to check for accuracy. Standardized coding sheet for data abstraction. A separate checklist was devised based on CONSORT recommendations and quality indicators suggested by other authors \[[@B6]-[@B10]\]. Specific subject information was extracted from the meta-analysis database to examine if investigators reported age, race/ethnicity, and gender of their study subjects. Coders also recorded whether or not investigators reported the subjects\' health status and presence of co-morbidities (i.e., diabetes, cardiac problems, cancer, or hypertension). Whether or not investigators reported medication use by subjects, other than the intervention drug, was also recorded. In addition, we tracked reporting of the postmenopausal status of subjects in investigations which included women 45 years or older. When analyzing the reporting quality for initial and final sample size separated by gender, only studies that included a group(s) consisting of both men and women were selected. Statistical analysis -------------------- The frequency of reporting in the journal articles for the selected variables was obtained by performing a proc frequency using SAS (Version 8.2, Cary, NC). Results are presented as totals. Results ======= Of the 604 randomized clinical trial articles that were reviewed, 231 (38%) met eligibility criteria for the present study. Figure [1](#F1){ref-type="fig"} displays the frequency of articles included in the analysis by year of publication. Of these, 123 (53%) articles were randomized studies without a control group and 108 (47%) were randomized controlled trials. Seventy-six articles (33%) were dietary intervention only, 60 (26%) were exercise intervention only, and the remaining 95 articles (41%) included both exercise and dietary intervention for weight loss. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Frequency of randomized trials published by year. ::: ![](1471-2288-5-9-1) ::: Contribution of journals ------------------------ For this analysis, the [American Journal of Clinical Nutrition]{.underline} contributed the most articles that met the eligibility criteria (47), followed by the [International Journal of Obesity]{.underline} (32), [Medicine & Science in Sports & Exercise]{.underline} (24), [Journal of the American Medical Association]{.underline} (11), [Metabolism]{.underline} (11), [New England Journal of Medicine]{.underline} (7), [Archives of Internal Medicine]{.underline} (6), and [Journal of the American Dietetics Association]{.underline} (6). In addition, 52 journals had 5 or fewer articles in this analysis. Subject characteristics ----------------------- Of the 231 articles that met eligibility criteria for the present study, subjects\' age was not reported in 25 (11%) articles. Investigators of ten (4%) different studies failed to report the gender of their study participants. Race and/or ethnicity of study subjects were not reported in 199 (86%) studies. In addition, 78 (34%) studies failed to report the health status (i.e., presence or absence of co-morbidities) of their study subjects at baseline. Further, medication use was not reported in 213 (92%) of the articles (Figure [2](#F2){ref-type="fig"}). For postmenopausal status, 89 articles were included in the overall analysis with only 7 (8%) studies that did not report the postmenopausal status of their female subjects. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Proportion of selected subject characteristics not reported in randomized trials. Values reported are means. Co-morbid disease status included the reporting of diabetes, cardiac diseases, cancer, hypertension, or any other metabolic disease. ::: ![](1471-2288-5-9-2) ::: Sample size and attrition ------------------------- Initial sample size was not reported in 14 (6%) studies while final sample size was not reported in 133 (58%) of the 231 eligible articles. When reporting initial and final sample size by gender, 68 articles were included in the analysis. Of the eligible articles, 14 (21%) did not report initial sample size by gender while 47 (69%) failed to report final sample size by gender (Figure [3](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Proportion of articles that did not report initial and final sample size by group or gender in randomized trials. Values reported are means. ::: ![](1471-2288-5-9-3) ::: Discussion ========== The present study evaluated the quality of reporting of RTs and RCTs in the diet and exercise literature for weight loss, focusing exclusively on subject characteristics, including age, gender, race, health status, medication use, postmenopausal status, and attrition. Transparent reporting of subject data is important in the scientific literature so that readers can efficiently evaluate outcomes in RTs and RCTs. Inadequate reporting creates numerous difficulties with interpretation and can lead to biased conclusions. For example, the effects of diet and/or exercise can vary based upon an individual\'s age. Schwartz et al. 1991\[[@B11]\] compared the effects of 6 months of endurance exercise (4 days/wk, 45 minutes/day, 85% of heart rate reserve) in older (Mean = 67.5 yrs, SD = 5.8 yrs) versus younger (Mean = 28.2 yrs, SD = 2.4 yrs) on body weight and composition. They found that older men had a 2.5 kg reduction in body weight, a 2.3% decrease in percent body fat, and a 2.4 kg decline in fat mass compared to no changes in the younger individuals. The findings suggest that subjects\' age is an important determinant in the response to exercise. However, despite the difference in response to exercise based on the age of subjects, we found that 11% of authors failed to report their subjects\' ages within their published report. Reporting gender of the study participants is also important as men and women respond differently to diet and/or exercise treatments. For example, Donnelly et al. \[[@B12]\] reported that following 16 months of verified, supervised aerobic exercise at 45 minutes per day, 5 days per week resulted in a decrease in body weight of 6% for the men compared to no change in the women. It also has been shown that individuals from different races and/or ethnic background may respond differently to diet and/or exercise. Jakicic et al. \[[@B13]\] reported that resting energy expenditure was lower in African-American women (Mean = 7279 kJ/d, SD = 825 kJ/d) compared to Caucasian women (Mean = 7807 kJ/d, SD = 854 kJ/d) even after correcting for body weight and lean body mass. The authors concluded that such differences might partially explain the smaller weight losses typically seen in African-American women when compared to Caucasian women enrolled in a weight loss program. In a recent meta-analysis on walking and resting blood pressure in adults, the investigators reported that only 13% of their included studies reported information on race \[[@B5]\]. In the current investigation 86% of authors failed to report the race/ethnicity of the subjects, which highlights the need for caution when extrapolating findings from one population to another. In the present study, co-morbid health conditions of study subjects were only marginally reported (66%) in the literature. It is well known that certain disease states, such as diabetes and cancer, affect metabolism \[[@B14]-[@B17]\], which can influence the effect of diet and exercise on body composition. Closely related to the need for reporting health conditions of study participants is the issue of medication use. Davis et al. \[[@B18]\] determined the effects of taking an antihypertensive medication (i.e., atenolol or chlorthalidone) compared to a placebo group combined with weight loss. They found that at 6 months those taking chlorthalidone lost (6.9 kg) the most weight compared to either the placebo (4.4 kg) or atenolol (3.0 kg) groups. They speculated that the group taking chlorthalidone might have reduced appetite or increased fat mobilization due to the volume depletion and increases in serum and urinary catecholamines. However, despite the profound effects that medication may have on body weight and composition 92% of authors failed to report medication use in our study. Without an adequate description of subject characteristics, readers are unable to judge the comparability of study groups. Menopause is associated with decreases in lean mass and increases in fat-mass \[[@B19]-[@B22]\]. Therefore, post-menopausal status and the associated decline in estrogens, androgens, and other hormones may significantly influence the effects of dietary/exercise interventions on body composition. It appears from the present study the majority of investigators (92%) of dietary/exercise studies report post-menopausal status of their subjects. Attrition may threaten the internal and external validity of the scientific literature as well as the efficacy of a specific dietary/exercise intervention \[[@B23],[@B24]\]. For example, failure to report the number of dropouts and completers prevents the reader from calculating attrition rates for different experimental conditions, which can result in an overestimate of treatment effectiveness. In the present study, we found that end of study sample size was not reported in the majority (58%) of studies. Without knowledge of the number of subjects who were lost to follow-up, readers are unable to judge the effectiveness of a clinical treatment or ascertain whether or not a research finding has practical significance. Conclusion ========== In the present critical appraisal of the methodological quality of weight loss studies, we found major shortcomings in the reporting of subject characteristics as they relate to enrollment, allocation, and follow-up in trials that evaluated diet and exercise interventions. Many studies did not report variables that may explain some of the variance in outcomes. These findings are consistent with those of similar studies, which indicate inadequate reporting of subject characteristics \[[@B5],[@B25]\] and reveal that poor adherence to published standards of reporting is common \[[@B26]\]. Clearly, additional attention should be paid to ensure compliance with reporting standards for diet and exercise intervention studies. List of abbreviations ===================== Consolidation of the Standards of Reporting Trials (CONSORT), randomized controlled trials (RCTs), randomized trials (RTs), kilojoules per day (kJ/d), standard deviation (SD), kilograms (kg), years (yrs) Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= CAGmade substantial contributions to the conception and design, acquisition of data, analysis and interpretation of data, and drafting and revising the manuscript for important intellectual content. EPK, JDL, BWBand GHmade substantial contributions to the submitted manuscript by assisting with the acquisition of data, analysis and interpretation of data, drafting of the manuscript, statistical analyses, and critical revisions. JEDmade substantial contributions to the manuscript by helping with the conception and design, critical revisions of the manuscript, and obtaining funding. All authors read and approved the final manuscript. Funding source ============== Supported by National Institutes of Health NIH DK56303, Joseph E. Donnelly, Ed.D., principal investigator Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2288/5/9/prepub> Acknowledgements ================ Preliminary results were accepted as an abstract at the North American Association for the Study of Obesity (NAASO) conference held in November 2004, Las Vegas, NV. We gratefully acknowledge the assistance of Katrina DuBose, Ph.D. for her help in the acquisition of articles.	PubMed Central	2024-06-05T03:55:53.981774	2005-2-23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554784/", "journal": "BMC Med Res Methodol. 2005 Feb 23; 5:9", "authors": [ { "first": "Cheryl A", "last": "Gibson" }, { "first": "Erik P", "last": "Kirk" }, { "first": "James D", "last": "LeCheminant" }, { "first": "Bruce W", "last": "Bailey" }, { "first": "Guoyuan", "last": "Huang" }, { "first": "Joseph E", "last": "Donnelly" } ] }
PMC554785	Background ========== Cigarette smoking is a source of worldwide misery \[[@B1],[@B2]\] and revenue for corporations and governments. Landmark tobacco regulatory efforts in the United States include the 1992 Synar Amendment, requiring States to establish and enforce prohibitions on the sale and distribution of tobacco products to persons under 18 years of age \[[@B3]\]. The 1998 Master Settlement Agreement (MSA) prohibited numerous marketing practices, especially those likely to entice youth \[[@B4]\]. The six corporate signatories also promised an endless series of payments to the settling States, currently \$8 billion annually. The United States has endorsed the World Health Organizations\' 2003 Framework Convention on Tobacco Control (FCTC) \[[@B5]\]. The FCTC enumerates well-studied, politically safe measures such as taxation; limiting youth access; regulating the content, packaging, advertising, and sales of tobacco products; and educating the public about risks. Fire-safe cigarettes \[[@B6]\] are now required in New York. Several states and many municipalities now require smoke-free workplaces. The recently defeated \[[@B7]\] 2004 Family Smoking Prevention and Tobacco Control Act, S2461 \[[@B8]\], would have established Food and Drug Administration (FDA) regulation of cigarettes for the first time. Although average citizens could easily have taken \"FDA regulation\" to suggest the extensive power that the FDA holds over pharmaceutical products\[[@B9]\], tobacco regulations would have been weaker. The defeated Act sought publication of brand-specific ingredient information; forbade adding children\'s favorite flavorings to cigarettes, further restricted teen sales and advertising; and allowed the FDA to regulate cigarettes\' nicotine delivery. The FDA would not have been able to ban or eliminate nicotine from cigarettes, nor could the FDA ban a class of tobacco products. Although modestly effective at preventing and ending tobacco use, such measures will not achieve the Healthy People 2010 (HP2010) goal of 12% adult smoking prevalence \[[@B10]\] on schedule \[[@B11]\], if ever. While cigarettes should never again afflict more than a quarter of the United States population, tobacco regulation will abate the current level of carnage only very slowly. A complimentary strategy called \"harm reduction\" proposes that smokers could improve their health by frequently substituting less hazardous tobacco products, such as \'smokeless cigarettes,\' chewing tobacco, and newer smokeless tobacco products for cigarettes \[[@B12]-[@B14]\]. \'Smokeless cigarettes\' are neither smokeless nor cigarettes, but complicated devices that release a mixture of chemicals from a heated tobacco substrate and can emit more carbon monoxide than a cigarette \[[@B15]-[@B18]\]. For individuals who are determined to inhale nicotine, these devices are probably no worse than cigarettes. However, for individuals who would have quit instead of switching to a smokeless cigarette, the health cost may be large. The Institute of Medicine has called for extensive research to quantify harms and patterns of use of these \"potential reduced exposure products\" (PREP) \[[@B19]\]. In contrast, chewing tobacco is clearly safer than smoking cigarettes \[[@B20],[@B21]\], exposing users to as little as 2% of the risks of smoking \[[@B22]\]. The newest smokeless tobacco products are discrete enough to use in almost any social setting. Smokeless tobacco products are much less popular than smoking in the United States for a variety of reasons, including restrictions on advertising. However, the most important limitation of smokeless tobacco may be absorption through the nicotine through systemic veins rather than the pulmonary vasculature. All current pharmaceutical nicotine products share this limitation. Some tobacco control advocates suggest a third strategy: national policies encouraging competition between safer nicotine products and cigarettes \[[@B20],[@B23]-[@B34]\]. Typically, this involves a \"level playing field\" with similar regulations for pharmaceutical grade nicotine delivery systems and cigarettes. Advocates cite evidence that health risk correlates with exposure to tobacco\'s combustion and curing by-products \[[@B35]-[@B38]\], with nicotine replacement products posing the least risk \[[@B39],[@B40]\]. There are different ways to level this playing field. At one extreme, the FDA could increase regulation of cigarettes to match that of nicotine products. This is politically challenging, as the FDA discovered in the 1990\'s \[[@B41]\], The Family Smoking Prevention and Tobacco Control Act divided both the tobacco industry and the public health community. Altria, corporate parent of Philip Morris, was the only tobacco company to endorse this legislation. Calling a similar bill the \"Marlboro monopoly act\" \[[@B42]\], critics suspect that Altria hoped to discourage competition, for instance by encumbering smaller producers with regulations, by prohibiting relative safety claims sought by chewing tobacco manufacturers \[[@B43]\], or by discouraging new products with stringent new standards \[[@B44],[@B45]\]. Other critics believe that the Act safeguarded certain cigarette marketing and legal defense strategies \[[@B9]\], or paved the way for the company\'s unproven \'smokeless cigarettes,\' potentially ushering in another generation of suffering. At another extreme, the FDA could regulate other nicotine products more permissively, as cigarettes have been regulated. The FDA has that authority, but would face criticism for facilitating a common addiction. For instance, the FDA could approve non-prescription nicotine nasal sprays and slow acting inhalers for indefinite use by adults. These nicotine replacement therapies have excellent safety records and low potential for addicting non-smokers, although a few ex-smokers become addicted. The widespread misconception that nicotine prohibition is practical and desirable makes it politically difficult for the FDA to pursue this path. Discussion ========== Problems with nicotine prohibition ---------------------------------- A recent Lancet editorial called for the criminalization of tobacco products \[[@B46]\]. However, a litany of centuries-old problems with prohibition provides a strong argument for permissive nicotine regulation as an alternative or essential prerequisite to prohibition. Many people will pay high prices to a coalition of suppliers and governments to obtain tobacco cigarettes that efficiently deliver nicotine, a drug perceived to provide some benefits, in spite of substantial immediate and long-term risks of smoking. While comprehensive public smoking restrictions are possible, and some companies may exit the tobacco trade, traditional tobacco control and litigation may not provide additional large public health gains in the United States, and prohibition is a practical impossibility. The first problem is that historically, smokers accept personal and public hazards that make the dangers we associate with tobacco look quaint. After Christopher Columbus failed to control his crew\'s tobacco use, monarchs from England to China tried to contain the weed by execution, disfigurement, exile, and onerous taxation \[[@B47]\]. Tobacco trade drained monarchs\' wealth, compromising national security, and smoking accidents incinerated whole cities. Tobacco use spread anyway. The second problem is that risk-tolerant smokers are no small group. Smoking persists where Mormon and Islamic prohibitions discourage it \[[@B48],[@B49]\]. If 70% of smokers in the USA want to quit \[[@B50]\]. then 30% of smokers, more than 6% of the adult population, do not want to quit. The third problem is a common genetic predisposition to nicotine addiction \[[@B51]\]. The modifiable risk factors we associate with smoking initiation and persistence -- parent behavior, peer pressure, role models, advertising, accessibility, repeated exposure, and perceived norms -- were irrelevant to Christopher Columbus\' crew and the first tobacco users across Eurasia. Risk factor modification will not alter genetically predisposed users\' fascination with their first tobacco products. The fourth problem is that so much money is involved. When state and local taxes raised New York City cigarette prices to US\$7 per pack, tax revenues and black marketing both increased \[[@B52]\]. During the invasion of Iraq, stressed US soldiers reportedly paid up to US\$50 per pack \[[@B53]\]. The black market for tobacco in Colorado prisons may achieve a 45,000% markup \[[@B54]\]. This industry makes large profits selling a simple product. If liability claims bankrupt corrupt companies, new suppliers will fill the void and try to avoid predecessor\'s mistakes. Black markets will undermine the benefits of high taxation or prohibition. The fifth problem is that government taxation compromises tobacco control efforts. Historically, States willingly trade citizens\' health for wealth. Seventeenth century monarchs who opposed smoking relented as tobacco tax revenue accumulated. The States demonstrated the same perverse values with petitions to protect their MSA payments when an Illinois court threatened Altria with a US\$12 billion bond \[[@B55]\]. The States will predictably protect the MSA corporate signatories from new competitors, for instance with taxes targeting generic brands. Tax revenues diminish prospects for even slowing the growth of tobacco sales in Africa, Eurasia, and South America, and prohibition in those areas is currently impossible. The sixth problem is the lack of a popular mandate for prohibition. Tobacco control advocates in the United States are rightfully pleased with smoking\'s declining prevalence, reduced teen smoking, rising cigarette prices, spreading restrictions on public smoking, and fire-safe cigarette initiatives. However, none of this demonstrates popular support for prohibition. Citizens will balk at limiting the supposedly personal choice to smoke in private. Midwesterners have already rejected modest cigarette tax hikes \[[@B56]\]. Furthermore, most taxes and MSA payments subsidize programs that benefit non-smokers \[[@B57]\]. Prohibition would eliminate that subsidy and require non-smokers to pay for enforcement, a very unlikely prospect. The seventh problem is that nicotine use could have a favorable risk-benefit profile for some informed users. Nicotine causes a mild euphoria without intoxication, in contrast to more tightly regulated drugs: it does not destroy relationships as intoxicating drugs routinely do. Many smokers may use nicotine to treat various problems \[[@B58]-[@B60]\] including depression \[[@B61]\], attention deficits \[[@B62]\], other mental illness \[[@B63]\], symptomatic systemic diseases \[[@B64]\], or to control weight \[[@B65]\]. Nicotine users may perform some tasks better, especially those involving vigilance and rapid visual cue processing \[[@B66],[@B67]\]. There is uncertainty regarding many of these benefits \[[@B68]\]. Nevertheless, expected benefits are politically hard to withhold. If some of these benefits are real, nicotine prohibition may not even be desirable. Nicotine accounts for very few of the long-term hazards of smoking. A smokeless tobacco proponent has likened nicotine\'s risks to the risks of consuming caffeine \[[@B54]\]. Fetal exposure causes placental constriction and reduced birth size, alters brain development in disturbing ways, and may increase susceptibility to later nicotine addiction \[[@B69]-[@B76]\]. Nicotine might contribute to sudden infant death syndrome \[[@B69],[@B77]-[@B79]\], destruction of connective tissue \[[@B80]\], modulation of immune function \[[@B81]\], prevention of apoptosis \[[@B82],[@B83]\], and alcohol or other substance abuse \[[@B84]-[@B86]\]. Fatal nicotine poisoning is quite unlikely \[[@B87]-[@B89]\]. Given that smokeless tobacco users experience only about 2% of the risks of smoking \[[@B22]\], and that inhaled nicotine is similarly benign in animal models \[[@B90]-[@B92]\], it is very unlikely that inhaled nicotine could account for even one-tenth of the harms of smoking. In summary, however desirable tobacco prohibition may be, it is hopelessly impractical -- unless smokers, governments, and producers have an equally satisfying alternative. Stifling innovation ------------------- Legal and regulatory pressures have prevented the development of meaningful alternatives to cigarettes. Directed to approve drugs as \"safe and effective\" for specific indications, and to ignore tobacco, the FDA has had little reason to approve chronic, addictive nicotine. Although the FDA approves more dangerous drugs for specified indications, without an indication there is no benefit to weigh against any nicotine risk. Consequently the FDA requires prescriptions for slow acting inhalers and nasal spray because of a small risk of inconsequential addiction in ex-smokers. Unfortunately, seemingly risk averse regulation of nicotine forces public health policies to rely on difficult smoking prevention, cessation, use restrictions, and treatment. The net effect is a risky public health policy, and very slow development of new nicotine delivery systems \[[@B32]\]. Another unfortunate side effect has been that companies put tobacco into any device that they do not want regulated, particularly, \'smokeless cigarettes,\' even if the device would be safer without tobacco. In permissive nicotine regulation, the FDA\'s missing indication is to improve nicotine addicts\' safety. As prescription methadone substitutes for illegal, immediately incapacitating heroin, non-prescription nicotine could substitute for legal, slowly injurious cigarettes. Tort threats have also delayed product improvement efforts within the industry. In 1963 an industry lawyer, anticipating condemnation in the 1964 Surgeon General\'s report, suggested competing on safe nicotine delivery, but was overruled \[[@B93]\]. Product liability and regulatory issues pushed tobacco companies into pointless projects including filters, light cigarettes, \"smokeless cigarettes\", nicotine-free cigarettes, and fabricating a controversy over the health risks of smoking. Haddon matrix ------------- In the mid 1960\'s, motor vehicle accidents were also taking a terrific health toll in the United States. While alternatives to driving existed, no one expected private automobiles to disappear. Instead, systematic efforts transformed traffic safety. Haddon\'s matrix illustrated how complimentary strategies could work together to reduce the morbidity and mortality of driving \[[@B94]\]. Table [1](#T1){ref-type="table"} shows a matrix with two axes representing time and objects. Most injury control efforts fall into one of the nine cells, although some cells are empty. Before an accident, typical preventive efforts remove or modify unsafe drivers, vehicles, and road conditions. During an accident a vehicle\'s design and environmental safeguards may prevent or limit injuries. After an accident, prompt medical attention limits the morbidity and mortality of the injuries that still occur. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Haddon\'s Matrix for Reducing Traffic Accident Injuries ::: -------------------------------------------------------------------------------------------------- Driver/Passenger Vehicle Environment --------------------- -------------------------- ---------------------- -------------------------- Before Accident Licensing\ Running lights\ Road design\ Stops, tickets, arrests\ Antilock brakes\ Road maintenance\ Drug, alcohol screening\ High traction tires\ Road lighting, marking\ Physician advice Vehicle inspections\ Traffic law\ Impact avoidance Police patrols During Accident \-\-\-- Bumpers\ Deformable barriers\ Seat belts\ Fences Airbags\ Crumple zones\ Fuel containment After Accident \-\-\-- Fuel containment Telecommunications\ Emergency stabilization\ Emergency transport\ Trauma centers -------------------------------------------------------------------------------------------------- ::: For tobacco control, smoking replaces accidents as the focal event (Table [2](#T2){ref-type="table"} -- anticipated strategies are italicized) \[[@B95]\]. As with accidents, cells contain only partially effective interventions. Many people experiment with tobacco in spite of efforts to prevent tobacco use. During smoking, smokers quit infrequently in spite of warnings and medications. More effective smoking cessation products may appear \[[@B96],[@B97]\]. but some nicotine use will persist. In the center cell, hazardous cigarettes easily dominate all available nicotine replacement products \[[@B98]\]. Available and anticipated harm reduction products may be safer, but addictive nicotine would be safer still. After injury, detection of disease may be slow, and treatments for tobacco-related diseases are seldom curative. The matrix highlights the problems of incomplete prevention, unsafe nicotine sources, and poor treatment options, and demonstrates why permissive nicotine regulation could benefit public health: when prevention fails, results are bad. Making nicotine addiction as safe as possible would make prevention failures less disastrous. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Haddon\'s matrix adapted to tobacco control ::: ---------------------------------------------------------------------------------------------------------- Potential user Nicotine delivery device Environment ---------------------- ------------------------ ------------------------------ --------------------------- Before Addiction Addiction education\ Warning labels\ Youth sales restrictions\ Disease education\ Labeling regulations Taxation\ Counter-advertising Black market policing\ Prohibition During Use Disease education\ Filters\ Taxation\ Cessation advertising\ Low tar cigarettes\ Prohibition Bupropion\ Smokeless tobacco\ Nortiptylline\ Smokeless cigarettes\ Varenicline\[105\],\ Content regulation\ Rimonabant \[97\] Nicotine replacement\ Addictive inhalers After Injury Disease awareness\ Nicotine replacement\ Disease screening\ Cessation advice Addictive inhalers Disease treatment\ Cures ---------------------------------------------------------------------------------------------------------- ::: Proposal -------- For the reasons outlined above, the health benefits of addictive pharmaceutical grade nicotine products would likely outweigh the harms. If so, the FDA could improve public health by regulating nicotine much as the government has regulated cigarettes. A permissive nicotine regulatory policy would allow sales of pharmaceutical grade nicotine delivery systems to adults without a prescription. The FDA could suggest warnings appropriate for classes of products and delivery system constituents. Manufacturers would accurately label contents, and would be legally liable for undisclosed harms caused by the delivery system and constituents other than nicotine, as with any pharmaceutical product. The FDA could prohibit inherently risky delivery systems, and would undertake a full, traditional review of nicotine systems that give users a faster or higher peak arterial level of nicotine than cigarettes. The FDA would also review additives intended to provide antidepressant or other effects familiar to smokers \[[@B99],[@B100]\]. Youth marketing and access would be illegal, as with cigarettes. The Drug Enforcement Agency would have no jurisdiction over non-intoxicating nicotine products, just as it has no interest in tobacco. Most public and workplace restrictions on cigarette smoking would be irrelevant to other nicotine delivery systems. States could tax cigarettes and nicotine at different rates, in theory recovering expenses related to each product. This tax policy would discourage cigarette use, encourage switching to nicotine, and maintain some State revenue as cigarette sales decline. A satisfying, safe, legal, and affordable alternative to cigarettes would discourage black markets. The FDA and Federal Trade Commission could permit advertising of nicotine as an alternative to smoking, and monitor relative harm claims. Nicotine manufacturers would pay for monitoring of adverse health effects from their products until remaining health questions are answered. The FDA could implement a permissive policy in a series of simple, informative steps. First, the FDA could immediately approve non-prescription sales of the existing nicotine spray and inhaler, and similar competitors, with restraints consistent with the Synar amendment, the MSA, and the FCTC. In particular, it should require disclosure of known nicotine risks, such as addiction, invite comparisons to smoking risks, and forbid marketing to minors. Data collected during this first step will inform subsequent steps. Economists have shown that the price of nicotine gum and patches affects demand for cigarettes \[[@B101]\]. This means that some smokers will substitute less expensive but very slow acting nicotine replacement products for cigarettes. Non-prescription access to more addictive and competitively priced nicotine should increase this substitution, especially if addicts can purchase a one day supply of about 20 mg. Clinical trials and post-marketing studies of non-prescription use of the spray and inhaler would begin to answer questions about nicotine and pregnancy, substance abuse, and heart disease. Surveillance studies would determine whether clean nicotine options lead more people to smoke, to smoke longer, or to use nicotine during pregnancy. While the Institute of Medicine recommends years or decades of study to quantify the risks of \"smokeless cigarettes\", the most important remaining uncertainties about nicotine might be answered in a few years. Data collected during this first step should document the relative safety of addiction to pharmaceutical nicotine versus cigarettes, the market share of each product, and the increase in total nicotine use. Analyses using these data will predict the consequences of more permissive regulation \[[@B102]\]. If experience with the current nicotine spray and inhaler is reassuring, the FDA can take a second step, to approve clearly addictive nicotine inhalers for non-prescription use. These fast acting inhalers will deliver nicotine in a powder \[[@B103]\] or aerosol to the alveoli, as pulmonary inhalers deliver steroids and beta agonists. Fast acting nicotine inhalers would be subject to the same marketing requirements and surveillance described above. Several trends could develop that encourage smokers to switch to addictive nicotine inhalers, while limiting recruitment of non-smokers. Smokers will appreciate the healthier alternative to cigarettes, especially as they develop smoking related illnesses, while warning labels will discourage casual experimentation by non-smokers. Smoke-free workplace regulations would spread with less controversy, as fast acting inhalers give smokers a reasonable alternative. Employers might voluntarily forbid smoking to reduce health care and workman\'s compensation costs; reduce workplace fires; and increase productivity by eliminating both smoking breaks and withdrawal symptoms. Physicians might strongly encourage smokers to switch for their own health, primarily, but also for the health of family members. The government might permit more aggressive advertising of nicotine inhalers to further undermine cigarette smoking. Governments will probably find nicotine taxation irresistible, especially if their tobacco revenue declines. Nicotine taxation will raise the cost of experimenting with inhalers beyond some teenager\'s means. Communities might press smokers to switch so that fire departments, health care providers, and research funding agencies could shift resources to many other pressing problems. Ultimately, palatable inhaled nicotine products could finally allow governments to ban tobacco cigarettes. The public health benefits of these policy shifts could be substantial. We can estimate the long term relative public health burdens of different policies as the product of the risks posed by a delivery system, relative to cigarettes, and the fraction of the adult population using it. Using this formula, the current burden is more than 20% use × 100% risk = 0.2. Healthy People 2010 calls for 12% use × 100% risk = 0.12, about half of the current burden. If nicotine accounted for a surprising 10% of smoking risks, eliminating smoking by quintupling the prevalence of inhaled nicotine use -- universal addiction -- would cause 100% use × 10% risk = 0.1, a slight improvement over HP2010. A slightly less pessimistic scenario would be that all past and present smokers become inhaled nicotine addicts, but their risk is only twice that of smokeless tobacco users. This scenario is no more than 70% use × 4% risk = 0.028, about a quarter of the HP2010 burden. The most likely scenario is that nicotine risks are about 2% of smoking risks, and that policy changes will slowly shift the probabilities that smokers will attempt and succeed in quitting, that smokers will switch to nicotine indefinitely or use both nicotine and cigarettes, that ex-smokers relapse to nicotine use, and that non-smokers begin using nicotine. If the risks of inhaled nicotine are this low and if at least one smoker can switch to addictive inhaled nicotine for every 50 ex-smokers and non-smokers who start using nicotine, then public health will benefit from permissive nicotine regulation. One public health risk is that more people might begin or continue smoking cigarettes in the belief that inhaled nicotine will provide an easy escape path, but suffer irreparable harm before switching. We should quantify these probabilities through surveillance of the public\'s behavior and health consequences following each step toward more permissive nicotine regulation. Although S2461 was defeated, the continuing carnage will motivate similar legislative proposals. Tobacco policies must preserve the possibility of permissive nicotine regulation. Bad legislation could solidify the position of cigarettes, delay safer products, prohibit disclosure of relative harms, or otherwise interfere with market forces that ought to benefit nicotine addicts \[[@B104]\]. The FDA must retain the option to unleash real competition against a disastrous status quo in the tobacco industry. Summary ------- Nicotine use will remain common indefinitely. Pharmaceutical grade nicotine is the safest known substance that could replace cigarettes, but inherently addictive products are required to compliment traditional tobacco control policies effectively. The FDA could apply restrictions, similar to those on cigarettes, to an increasingly addictive portfolio of nicotine products, requiring honest portrayals of absolute and relative risks, and expect a significant reduction in cigarette smoking and related illness with modest recruitment of new nicotine addicts at each step. A brief and focused research effort could solidify the already substantial justification for such permissive nicotine regulation. Meanwhile, federal legislation must not obstruct free market ideals of competitive innovation and informed consumption. Competing interests =================== The author(s) 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/1471-2458/5/18/prepub>	PubMed Central	2024-06-05T03:55:53.983679	2005-2-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554785/", "journal": "BMC Public Health. 2005 Feb 24; 5:18", "authors": [ { "first": "Walton", "last": "Sumner" } ] }
PMC554786	Background ========== Felsenstein \[[@B1]\] was the first to introduce an evolutionary model for DNA sequences, which allows for unequal nucleotide frequencies (see also \[[@B2]\]). His F81 model allows for substitutions at a rate proportional to the frequencies of the ending nucleotides. It is considered the simplest rate matrix for accommodating variable nucleotide frequencies and is therefore the starting point for the consideration of more complex models with frequency variation (e.g., the HKY model of Hasegawa et al. \[[@B3]\]). Goldman and Whelan \[[@B4]\] described new variants of these F81-based models (their +gwF (generalized weighted frequencies) models; e.g., JC+gwF for Jukes and Cantor \[[@B5]\], and K2P+gwF for Kimura \[[@B6]\]). At the heart of their +gwF variants was a new free parameter (f) to accommodate the frequencies of the starting, as well as ending, nucleotides in the evolutionary process: ![](1471-2148-5-21-i1.gif) where q~ij~refers to the substitution rate from nucleotide ito j, π~i~and π~j~correspond to their equilibrium base frequencies, and s~ij~is the exchangeability between the two. In the +gwF variants, the substitution rate becomes more dependent on the ending nucleotide as fdecreases from 1 to 0, with f= 0 for the classic F81-type models. This study starts with a population genetics model to derive equations that link weak selection, genetic drift, and mutation to the fparameter and evolutionary rate matrices of the +gwF variants. These theoretical derivations lead to an expected value of f= 0.5. However, as illustrated with simulations, the fparameter is complex and thus its biological interpretation must be considered with caution. Results ======= Derivation of the rate matrix for the weak selection model ---------------------------------------------------------- The nearly neutral model of molecular evolution states that most DNA mutations of longer-term evolutionary consequence are under weak selection and are therefore prone to drift \[[@B7],[@B8]\]. For a diploid population of size N, a neutral mutation has a probability of 1/2Nof becoming fixed in the population. However, because of drift, even slightly deleterious mutations can become fixed, but at a probability of less than 1/2N. Advantageous mutations have higher fixation probabilities than neutral mutations. In the nearly neutral model, the distribution of alleles is determined by an equilibrium of selection, drift, and mutation. Consider a number of sites under identical evolutionary constraints and with a bias in nucleotide distribution. Assume that weak selection and drift are the causes of this bias; e.g., as for the codon usage biases in micro-organisms and Drosophila\[[@B9],[@B10]\]. In our model, some nucleotides confer a slightly higher fitness onto the organism than do others, regardless of their position, and these can become fixed in the population through drift and/or selection. Here, we also assume that selective advantages are additive for the two alleles of the diploid organism \[[@B11],[@B12]\]. Let the selective advantages of the four nucleotides be given by s~A~, s~C~, s~G~, and s~T~. The differences between these selection coefficients will be very close to zero, since no strong selection is expected. Consider a mutation from nucleotide ito j, with a selective advantage of s= s~j~- s~i~(a selective disadvantage exists when sis negative). For a population of size Nand an effective size of N~e~, Kimura \[[@B11]\] showed that the fixation probability in this population is given by: ![](1471-2148-5-21-i2.gif) when s≠ 0. For s= 0, we have P(s) = 1/2N. This approximation is valid for small values of s, which is the case here. The substitution rate from nucleotide ito jis proportional to P(s~j~- s~i~): q~ij~= 2Nμ~ij~P(s~j~- s~i~), (3) where μ~ij~is the mutation rate from i to j. For different iand j, μ~ij~can vary because of unequal transition versus transversion rates (for example). Furthermore, let us assume that the mutation rate is the same for either direction of substitutions between iand j. This assumption is necessary to maintain the widely used condition of time reversibility in the evolutionary process, which thereby keeps the following derivations tractable \[[@B1],[@B13]\]. We then have: ![](1471-2148-5-21-i3.gif) Since q~ij~/q~ji~can be written as a function evaluated at s~j~divided by the same function evaluated at s~i~, evolution is time reversible according to this model with: ![](1471-2148-5-21-i4.gif) Here, cand c\' are constants with c\' = -l/4N~e~log c, which will be chosen to make the equilibrium frequencies sum to one. The substitution rates can now be approximated as: ![](1471-2148-5-21-i5.gif) Given an exchangeability of s~ij~= μ~ij~, this equation reduces to equation (1) with f= 0.5 and an adjustment factor of: ![](1471-2148-5-21-i6.gif) This adjustment factor is close to one for moderate ratios of π, with a horizontal tangent around π~j~/π~i~= 1 and a slight bending downwards when deviating from this value (Fig. [1](#F1){ref-type="fig"}). Thus, a value of f= 0.5 is suggested for the +gwF variants according to these derivations of the weak selection model. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### *Adjustment factor as a function of the ratio of π\'s. The adjustment factor is given by ![](1471-2148-5-21-i7.gif) (equation (7)). ::: ![](1471-2148-5-21-1) ::: Evolutionary simulations ------------------------ Evolutionary simulations were conducted to examine the effects of violating certain assumptions in the above model of weak selection. Unless otherwise noted, these simulations were based on the K2P+gwF model with f= 0.5 and k= 2 (for the transition/transversion ratio). Simulations consisted of four sequences of length 10,000 and relied on a symmetric rooted phylogeny with all branch lengths equal to 0.10 expected substitutions per site under the model in question \[i.e., ((seq1:0.10, seq2:0.10):0.10, (seq3:0.10, seq4:0.10):0.10)\]. Violations of the weak selection model were incorporated in the simulations by: (1) heterogeneous sequences with sites drawn from different equilibrium base frequencies; (2) populations in disequilibrium due to changing N~e~; and (3) an accelerated C to T substitution rate. Estimates of ffor the simulated sequences were made with the K2P+gwF model. Forty simulations were run for each test condition, with the results for the festimates summarized as their means and twice their standard errors. In the first set of simulations, six categories of sites with different equilibrium distributions were considered (Table [1](#T1){ref-type="table"}). The festimates for the simulations with each category alone were not significantly different from 0.5 (i.e., the value under which the sequences were generated). In contrast, for the simulated heterogeneous sequences (i.e., those composed of equal numbers of sites from two or three different categories), their values of fvaried significantly in either direction from 0.5. Analogous results were obtained for the simulations of homogeneous and heterogeneous sequences under the HKY model (with f= 0.0 instead of 0.5). Thus, the value of fcan vary considerably when heterogeneous sequences are analyzed with a +gwF model. Here, such deviations are a consequence of using a single rate matrix to analyze sequences that were derived from two or three different ones. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Starting equilibrium base frequencies and results for the simulations with either homogeneous or heterogeneous sequences (i.e., those with sites from single versus multiple categories, respectively). ::: Categories π~C~ π~G~ π~T~ Bias^b^ f^c^ ------------ ------ ---------- ---------- ---------- --------- ------------- -------------- A 0.10 0.40 0.30 0.20 0.154 0.50 ± 0.01 0.00 ± 0.01 B 0.30 0.30 0.30 0.10 0.105 0.50 ± 0.01 0.00 ± 0.01 C 0.30 0.20 0.20 0.30 0.029 0.50 ± 0.02 0.00 ± 0.03 D 0.40 0.20 0.20 0.20 0.078 0.49 ± 0.01 0.01 ± 0.01 E 0.20 0.40 0.20 0.20 0.078 0.51 ± 0.01 -0.01 ± 0.02 F 0.20 0.20 0.40 0.20 0.078 0.50 ± 0.02 -0.01 ± 0.01 A+B 0.20 0.35 0.30 0.15 0.074 0.43 ± 0.01 -0.11 ± 0.02 A+C 0.20 0.30 0.25 0.25 0.015 0.34 ± 0.03 -0.16 ± 0.03 B+C 0.30 0.25 0.25 0.20 0.015 0.24 ± 0.02 -0.33 ± 0.03 A+B+C^e^ 0.23 0.30 0.27 0.20 0.016 0.39 ± 0.04 -0.13 ± 0.04 D+E+F^e^ 0.27 0.27 0.27 0.20 0.010 0.68 ± 0.03 0.29 ± 0.04 ^a^Expected nucleotide distribution. ^b^Nucleotide bias, as information content measured in bits: . ^c^Mean ± twice the standard error of the estimate. ^d^f= 0:0 for these simulations with the HKY model. With f= 0:0, the HKY+gwF variant is reduced in these simulations to its more standard F81 based model. ^e^The heterogeneous sequences in these simulations were of length 9,999, rather than 10,000, since the latter is not a multiple of 3. ::: In the second set of simulations, N~e~was kept constant until the time of the most recent common ancestor for the four simulated sequences. Then, N~e~was either left unchanged or was suddenly changed by a certain factor. The latter was done by replacing the rate matrix derived from equation (4), resulting in new equilibrium frequencies of the nucleotides. When N~e~was kept constant, the selective pressures and drift were left unchanged, thereby maintaining the same starting equilibrium frequencies throughout the phylogeny. Thus, the corresponding festimates did not significantly differ from 0.5 (Fig. [2](#F2){ref-type="fig"}). In contrast, increases in N~e~lowered the value of fas the efficiency of selection was increased relative to drift \[[@B4]\]. Correspondingly, the evolutionary process became more dominated by the ending nucleotide. This increasing dominance can be expected to continue until a new equilibrium is restored (which occurs on a longer time scale than that in these simulations). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Two situations where fis affected by deviations from the model. (A) The effect of a change in N~e~on the value of f. This change in N~e~occurs in the most recent common ancestor of the four simulated sequences. Population ratio refers to its N~e~after versus before this change. (B) The effect of an increased C to T substitution rate. Categories A, B, and C are defined in Table 1. ::: ![](1471-2148-5-21-2) ::: In the third set of simulations, an acceleration in the C to T substitution rate was incorporated, thereby modeling an increase in their mutation rate due to the deamination of methylated C\'s in CpG pairs \[[@B14]\]. The introduction of this bias resulted in significant deviations of fin either direction from 0.5, even though their sequences were simulated in equilibrium (Fig. [2](#F2){ref-type="fig"}). Thus, the value of fcan vary considerably when the rates for reciprocal mutations are unequal. Discussion ========== This study illustrates how selection, drift, and mutation within a population can be linked to the fparameter and rate matrices of the +gwF variants for sequence evolution. Our weak selection model relies on the fixation probabilities of mutant alleles with additive genie selection and equal mutation rates for reciprocal substitutions. What is now needed are additional studies that link other population genetics models to the +gwF variants \[[@B9]\]. For example, the population genetics models of Li \[[@B15]\], which focus on allele frequency distributions and different modes of selection and mutation, could be studied for their connections to the fparameter and +gwF rate matrices. Collectively, the three sets of simulations highlight that the fparameter is complex and can be influenced by a number of different factors \[[@B4]\]. This complexity limits its biological interpretation and the use of its expected value of 0.5 as derived for the weak selection model. Correspondingly, in many +gwF analyses, f*ill need to be estimated as a free parameter rather than fixed beforehand. Goldman and Whelan \[[@B4]\] focused on amino acid sequences, where they found that the +gwF models provided better fits to the majority of their protein data sets. They also analyzed two rather small nucleotide data sets for which the general reversible model (REV) outperformed the +gwF variants. As noted by them, the REV model provides enough free parameters to cover the effects of a +gwF analysis. Thus, given sufficient data, this model will consistently outperform the simpler +gwF variants, since it can always accommodate more of the evolutionary process by virtue of its extra parameters. Nevertheless, as widely acknowledged, simpler models have their place, since they allow one to maximize analytical power for more limited data, while minimizing the risk of over-parameterization \[[@B13],[@B16]\]. Thus, as for the JC, K2P, and HKY models, we expect their +gwF variants to remain of interest as part of the hierarchy of simple to complex models for sequence evolution. Authors\' contributions ======================= Both authors contributed to the conception and design of this study and to the writing, reviewing, and final approval of this article. B.K. performed the simulations and parameter estimations. Acknowledgements ================ B.K. thanks the Carlsberg Foundation, the University of Aarhus, and the Danish National Science Research Council (grant number 21-00-0283) for their support. Both authors also thank the Department of Zoology, University of Florida for its support.	PubMed Central	2024-06-05T03:55:53.986660	2005-3-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554786/", "journal": "BMC Evol Biol. 2005 Mar 2; 5:21", "authors": [ { "first": "Bjarne", "last": "Knudsen" }, { "first": "Michael M", "last": "Miyamoto" } ] }
PMC554787	Background ========== It is estimated that 31--55% of caregivers of chronically ill elderly relatives experience depression \[[@B1]\], and depression is likely to be one of the first \[[@B2]\] and most enduring psychological outcomes for caregivers \[[@B3],[@B4]\]. Caregivers\' depression scores have been found to be substantially higher than those of the general population \[[@B1],[@B5],[@B6]\], and higher levels of caregiving stress have been related to greater depression \[[@B7],[@B8]\] and to more depressive symptoms in caregivers \[[@B9]\]. Factors that may be related to stress and depression in caregivers include hours of care, stressful life events, social support, age, and gender. Studies have found that hours of care were significantly related to caregivers\' anxiety/depression and somatic symptoms \[[@B10],[@B11]\], and to their emotional and physical strain \[[@B12]\]. Though stressful life events have been associated with both psychological wellbeing \[[@B13],[@B14]\] and physical symptoms among the general population \[[@B14]\], no studies have examined stressful life events in combination with caregiving stress. Stressful life events might have additional impact on caregivers\' health other than chronic caregiving. Social support may enhance the ability of the individual to cope with events or change the individual\'s cognitive appraisal of events \[[@B15]\]. Quayhagen and Quayhagen found that caregivers who reported needing more social support had lower well-being scores than other caregivers \[[@B16]\]. In other studies, low social support predicted higher perceived burden \[[@B7]\], and adverse social contacts were associated with increased stress \[[@B17]\]. Age has been shown to have indirect effects on depression through its influence on perceived stress, the coping process, and perceived efficacy \[[@B18]\]. Younger caregivers experience more distress than older caregivers \[[@B19],[@B20]\], and they express more subjective burden than older caregivers \[[@B21]\]. Gender has also been shown to have effects on depression. Female caregivers report more distress \[[@B1],[@B22]\] and higher psychiatric morbidity \[[@B5]\] than male caregivers. Tsai et al. have suggested that stress and depression are emotional aspects of coping mechanisms and depression is the outcome of perceived caregiver stress \[[@B23]\]. Stress has in turn been shown to be the strongest predictor of depression in caregivers \[[@B24]\]. Though depression has been associated with caregivers\' physical health \[[@B25],[@B26]\], the data on physical health are less consistent than on psychological health. Some studies have found that caregivers had poorer self-reported health than non-caregivers \[[@B27]-[@B29]\], more chronic illnesses \[[@B30]\], and lower immune function \[[@B31]\]; and they used more health care services and took more prescriptions \[[@B27]\]. Convinsky et al. reported that depression was associated with physical function dependence. Caregivers with functional dependence has 2.53-fold chance to be depressed as compared to those who with functional independence \[[@B32]\]. Other studies, however, have found that caregivers did not use more medical services \[[@B33]\] or rate their physical health as less satisfactory than the general population \[[@B6]\]. Further, as Schulz, Visintainer and Willamson point out, even though some studies have suggested possible effects of caregiving on physical health, the evidence is confounded by sampling bias, inadequacy of measurements, and subjective appraisals \[[@B34]\]. Although the associations between depression and self-esteem and marital satisfaction have been examined extensively, only a few studies have been conducted in the context of caregiving. Caregivers have been shown to have lower self-esteem \[[@B35]\], and this has been associated with depressive symptoms \[[@B10]\]. Caregivers who had higher self-esteem experienced less depression \[[@B36]\]. In one study, depressed caregivers were more likely to experience less marital satisfaction \[[@B37]\]. Also, high levels of marital conflict were associated with high levels of depression in adult daughter caregivers \[[@B38]\]. Finally, spousal caregivers reporting low marital cohesion and satisfaction had more depressive symptoms \[[@B39]\]. A recently developed Theory of Caregiver Stress \[[@B23]\] based on theoretical propositions from the Roy adaptation model \[[@B40]\] suggests that depression is the mediator between perceived stress and self-esteem and marital satisfaction. However, the relationships of depression to other outcomes of caregiving, such as physical function, self-esteem and marital satisfaction, remain unclear. The research reported here therefore explored these relationships. We proposed that hours of care would be the primary source of caregiver stress. Stressful life events, social support, age, and gender were antecedent variables and expected to influence caregivers\' outcomes through caregiver stress. Depression was conceptualized as a mediator between caregiver stress and other outcomes of chronic caregiving (physical function, self-esteem, and marital satisfaction). Thus, a high level of stress was expected to lead to a high level of depression, which in turn would result in lower levels of physical function, self-esteem, and marital satisfaction. Methods ======= Sample ------ Data for the study were obtained from the Americans\' Changing Lives (ACL) Survey: Wave 1, 1986 (N = 3,617), and Wave 2, 1989 (N = 2,867) \[[@B41]\]. The ACL collected longitudinal data on subjects aged 25 years and over in the continental United States. Individuals residing in group homes or institutions were not included. The survey used multistage-stratified probability sampling, with Blacks and elderly (60 years and older) oversampled. We used Wave 2 data to test hypotheses and build a data-derived model. Wave 1 data were then used for model validation. Only individuals with experience in caregiving to a chronically ill aged relative were included in the analyses reported here. This reduced the number of cases available for study to 335 from Wave 1 and 271 from Wave 2. The two samples were not completely independent because 99 cases were included in both waves; therefore, to ensure the independence of samples, these 99 cases were dropped from Wave 1. The final samples from Wave 1 and Wave 2 were thus 236 and 271, respectively. Measures -------- Since the study was a secondary data analysis, indicators of the study variables (hours of care, age, gender, social support, stressful life events, perceived stress, depression, physical function, self-esteem and marital satisfaction) were selected from the Americans\' Changing Lives Survey questionnaires, based on face validity. That is, the questions selected gave the appearance of measuring the content of interest. Exploratory factor analysis, confirmatory factor analysis, and internal consistency tests were then conducted to confirm the underlying structures of established scales and develop outcome measures for the current study. Hours of care were the total hours estimated by the caregiver in the past year, categorized as less than 20 hours, 20 to 39 hours, 40--79 hours, 80--159 hours, and 160 hours or more. Providing more hours of care was expected to indicate more burden of caregiving. Stressful life events were measured by a 12-item checklist of negative or undesirable events, such as being robbed or burglarized, losing a job, being physically attacked, or experiencing the death of spouse, death of a parent, death of a close friend/relative, serious illness, life-threatening illness/accident, divorce/separation, serious financial problem, death of children, and other such events. Respondents were asked to report whether they had experienced any of these events within the past 2 years. A simple score, the stressful life events index, was created by summing the number of events reported by each respondent. A high score reflected more stressful life events. Social support was measured by two items: friends/relatives\' love and care, and their willingness to listen. Alphas reliabilities were .73 and .74 for Wave 1 and Wave 2, respectively. Higher scores indicated greater support from friends/relatives. Demographic data included age, defined as the chronological age of the caregiver, and gender, coded as biological sex identity. Perceived caregiver stress was measured by one item asking how much stress the caregiver felt about caring for or arranging care for the elderly relative. Responses were on a 5-point scale ranging from not stressful to very stressful; a higher score reflected more perceived stress. Other studies have shown that stress was associated psychosocial well-being, such as depression \[[@B42],[@B43]\]; in this study the correlation between perceived caregiver stress and depression was .25 (p \< .001). Depression was measured by the 11-item Center for Epidemiological Studies Depression (CES-D) scale \[[@B44]\], which assesses mood and level of overall functioning in the last 7 days. The CES-D was originally developed as a 20-item unidimensional scale. The shorter 11-item CES-D version contains items on feeling depressed, restless, happy, lonely and sad; feeling that people dislike me; people are unfriendly; I enjoy life (reverse scored); I have a poor appetite; cannot keep going; and everything is an effort. The items are rated on a 3-point scale from \"hardly ever\" to \"most of the time.\" Higher scores indicate higher levels of depression. Based on exploratory factor analysis, three factors of the CES-D scale -- depressed and positive mood, somatic symptoms and interpersonal relations -- were identified as indicators of the latent variable, depression Physical function was defined as consisting of functional health, number of chronic illnesses, and self-rated health. Functional health was measured by asking the caregiver whether the caregiver was bedbound, and whether the caregiver had difficulty bathing, climbing stairs, walking, or doing heavy housework, and the degree of difficulty of these tasks. Higher scores reflected a higher level of physical function. The number of chronic illnesses was the sum of the following: arthritis or rheumatism, lung disease, hypertension, heart disease, diabetes, cancer, circulation problems, stroke, fracture, and urinary incontinence. A low score on this measure indicated high physical function. Self-rated health was measured by a single item that asked caregivers to rate their own health on a 4-point scale ranging from poor to excellent. A high score reflected high physical function. The caregiver\'s self-esteem was measured by five items: \"I take a positive attitude toward self,\" \"I am no good at all,\" \"I see myself as a failure,\" \"I have the feeling of being pushed around in life,\" and \"I perceive myself able to solve problems.\" These items were measured on a 4-point scale ranging from strongly agree to strongly disagree. A higher score indicated higher self-esteem. Marital satisfaction was also measured by five items: \"Overall satisfaction with relationship,\" \"love and affection expressed from spouse or significant other,\" \"spouse treats me well,\" \"thinking about divorce or separation,\" and \"things happened that I can never forget.\" Higher scores indicated more marital satisfaction. Cronbach\'s alphas for all measures were above the acceptable criterion of .70 in both waves except for self-esteem in Wave 1. However, that measure was on the margin of acceptance, at .68. Since Cronbach\'s alpha is a conservative estimate of internal consistency \[[@B45]\], the self-esteem index was retained. Analytic procedure ------------------ Univariate and bivariate analyses were used to examine the descriptive findings. To test the appropriateness of the indicators for each latent variable in both waves, the following procedures were used. First, a single indicator was extracted when applicable (e.g., for social support, self-esteem, and marital satisfaction), and summary scale scores were used as single indicators. This strategy was used to reduce the number of parameter estimations in a complex model; it is considered appropriate when individual factor item loadings in a specific scale are high \[[@B46]\]. Second, for all latent variables with single indicators (i.e., hours of care, stressful life events, social support, age, gender, perceived caregiver stress, self-esteem, and marital satisfaction), the measurements were assumed to be perfect (with 0% error). This conservative estimation was made since increasing measurement errors would induce artificial correlations among the latent variables in the measurement model. Thus, a full factorial loading of 1.0 was assumed for all single indicators in the subsequent latent variables. For latent variables with multiple indicators (i.e., depression and physical health), one factor loading was arbitrarily set to 1.0 to test the relative contribution of the factors. Error variances were not allowed to correlate, but all the latent variables were allowed to correlate with each other. The confirmatory factors analysis indicated that all factor loadings were above 0.4 and significant (p \< .01), and they accounted for at least 16% of the true score variance \[[@B47]\]. The only exception was the \"interpersonal\" factor in depression, with a factor loading of 0.39. Although it was slightly below the required value of 0.4, it was included because it is a well established measure of depression. The factor loading and measurement error for each indicator are shown in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Standardized factor loadings and measurement error variances for the measurement model predicting caregiver stress ::: Latent variable Indicators Factor loading (Measurement error) ----------------------- ----------------------------------- ------------------------------------ Hours of care Hours of care 1.00^a^(.00)^b^ Stressful life events Number of stressful life events 1.00^a^(.00)^b^ Social support Friend/relatives positive support 1.00^a^(.00)^b^ Age Age 1.00^a^(.00)^b^ Gender Gender 1.00^a^(.00)^b^ Perceived stress Perceived caregiver stress 1.00^a^(.00)^b^ Depression CES-D Depressed & positive mood .75^a^(.44) CES-D Somatic symptoms .74 (.45) CES-D Interpersonal .39 (.84) Physical function Functional health .55^a^(.70) Numbers of chronic illness .65 (.57) Self-rated health .77 (.41) Self-esteem Self esteem/mastery index 1.00^a^(.00)^b^ Marital satisfaction Marital satisfaction index 1.00^a^(.00)^b^ Factors and measurement errors were from the completely standardized solution. All factor loadings and measurement errors were significant at .01 level in the preliminary measurement model. ^a^Parameter was fixed to 1.0 in the unstandardized solution. ^b^Parameter was fixed to 0 in the unstandardized solution. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Sample characteristics and comparisons by waves ::: Wave 1 Wave 2 --------------------------------- -------- ------ --------------- ----- ------ --------------- ----- Hours of care \<20 hours 38 16.1 30 11.1 .45 20--39 hours 28 11.9 39 14.4 40--79 hours 35 14.8 48 17.7 80--159 hours 35 14.8 42 15.5 ≥160 hours 100 42.4 112 41.3 Stressful life events .57 (.69) .50 (.64) .13 Social support 7.65 (1.95) 7.91 (1.77) .12 Age, in years 53.56 (16.36) 53.41 (14.43) .91 Gender Male 85 36.0 86 31.7 .31 Female 151 64.0 185 68.3 Perceived caregiver stress Not at all stressful 46 19.5 57 21.0 .75 Not too stressful 62 26.3 78 28.8 Somewhat stressful 77 32.6 76 28.0 Quite stressful 26 11.0 35 12.9 Very stressful 25 10.6 25 9.2 Physical function 1\) Functional health Most severe impairment 7 3.0 9 3.3 .87 Moderately severe impairment 19 8.1 17 6.3 Least severe impairment 18 7.6 23 8.5 No impairment 192 81.4 222 81.9 2\) Number of chronic illnesses 1.26 (1.28) 1.37 (1.34) .36 3\) Self-rated health Excellent 34 14.4 42 15.5 .95 Very good 88 37.3 95 35.1 Good 65 27.5 79 29.2 Fair 38 16.1 45 16.6 Poor 11 4.7 10 3.7 Self-esteem 16.02 (3.11) 16.84 (2.86) .00 Marital satisfaction^b^ -0.12 (3.66) 0.05 (3.61) .67 Depression 16.00 (4.14) 15.15 (3.87) .02 ^a^Statistical significance is determined either by t-test or chi-square. ^b^Score of marital satisfaction is standardized. ::: A covariance matrix derived from data in the Wave 2 sample was analyzed as input data in the process of model testing. Hypothesized models were tested using the maximal likelihood procedure in the LISREL statistics program. The model tests used absolute goodness-of-fit indices (Chi-square \[χ^2^\], the goodness-of-fit index \[GFI\], and the adjusted goodness-of-fit \[AGFI\]) and comparative fit indices (change in Chi-square \[Δ χ^2^\], the relative noncentral index \[RNI\] and the relative normed fit index \[RNFI\]). Values of GFI, AGFI, RNI, and RNFI between 0.90 and 1.00 were considered to indicate a good fit between the model and the data \[[@B48]\]. An exploratory structural modeling method, specification search \[[@B49]-[@B51]\], was then used to develop the data-derived model for the Wave 2 sample. The specification search procedure removed all invalid paths in the hypothesized model and added plausible paths suggested by the modification index. Cross-validation was performed to verify that this data-derived model was valid and stable across samples. In this procedure, the data-derived model was cross-validated by the Wave 1 sample, with both Wave 1 and Wave 2 data sets as input files at the same time. Results ======= Descriptive findings -------------------- The characteristics of the two wave samples are summarized in Table [2](#T2){ref-type="table"}. Less than half the caregivers spent more than 160 hours per year taking care of their chronically ill relative (42.4%, Wave 1 and 41.3%, Wave 2). About half reported no stressful life events in the past 2 years (51.7%, Wave 1 and 57.6%, Wave 2); 38% in Wave 1 and 35% in Wave 2 reported one stressful life event in the past 2 years, and 10.1% and 7.4% in Waves 1 and 2, respectively, reported two or more such events. There was no significant difference in social support between the two samples, although mean scores on social support were slightly higher in Wave 2 (M \[SD\] = 7.91 \[1.77\]) than in Wave 1(M \[SD\] = 7.65 \[1.95). The average age was 53 in both waves. Sixty-four percent of the caregivers in Wave 1 and 68% of those in Wave 2 were female. Respondents in both waves rated perceived caregiver stress similarly: 21.6% and 22.1% in Waves 1 and 2, respectively, reported \"quite and very\" stressful experiences, while over 45% in both waves reported that their experiences were not at all stressful or not too stressful. Caregivers\' physical function and marital satisfaction were also similar in both waves. Approximately 81% of the caregivers in each wave reported no functional impairment. Only approximately 20% of caregivers rated their health fair or poor, and the averages numbers of chronic illnesses were 1.26 and 1.37 for Waves 1 and 2, respectively. Scores on caregivers\' marital satisfaction were -. 12 and .05 for Waves 1 and 2, respectively; the difference was not significant. However, caregivers in Wave 1 had significantly lower self-esteem scores than those in Wave 2 (16.02 vs. 16.84, p \< 0.01). Depression also differed significantly (p \< 0.05). Respondents in Wave 1 reported more depression than those in Wave 2, with mean scores on the 11-item CES-D of 16.00 (SD = 4.14) and 15.15 (SD = 3.87) for Waves 1 and 2, respectively. The hypothesized model ---------------------- When the hypothesized model was tested to determine whether depression mediated the relationship between perceived caregiver stress and caregiving outcomes (physical function, self-esteem, and marital satisfaction), the statistics showed a moderate fit between the model and the data (χ^2^= 237.22; d.f. = 73; GFI = .89; AGFI = .84; RNFI = .85). More hours of care and female gender predicted greater caregiver stress, accounting for 7% of the variance in stress, as shown in Figure [1](#F1){ref-type="fig"}. Greater perceived caregiver stress was associated with higher depression. Higher levels of depression in turn predicted poorer physical function, lower self-esteem, and lower marital satisfaction, accounting for 40%, 40%, and 15% of the variance in physical health, self-esteem, and marital satisfaction, respectively. Depression served as a mediator between perceived caregiver stress and caregivers\' physical function, self-esteem, and marital satisfaction. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The hypothesized model ::: ![](1472-6955-4-3-1) ::: The data-derived model ---------------------- Since the hypothesized model fit the data only moderately well, a data-derived model was built to compare with the hypothesized model. After all the insignificant paths in the hypothesized model had been dropped, paths were added at each step to improve the goodness-of-fit statistics based on the modification index and pre-set assumptions. The resulting data-derived model had a good fit with the data (χ^2^= 147.73; d.f. = 74; p = .00; GFI = .93; AGFI = .90; RNFI = 1.00), close to that of the measurement model, with an insignificant difference (Δ χ^2^= 33.58; Δ d.f. = 34; p = ns). The strength and direction of the relationships among the latent variables are shown by the standardized coefficients in Figure [2](#F2){ref-type="fig"}. The data-derived model accounted for 6%, 10%, 57%, 40%, and 11% of the variance in perceived caregiver stress, depression, physical function, self-esteem, and marital satisfaction, respectively. Hours of care were predicted by age: older caregivers provided more hours of care than younger caregivers. Social support was predicted by gender and depression: males and depressed caregivers tended to have less social support. Being a female caregiver and giving more hours of care made the caregiver more susceptible to perceived stress. Having less caregiver stress and fewer stressful life events reduced the chances of depression. Older age and higher levels of depression tended to result in poorer physical function. Greater depression was also associated with less self-esteem and less marital satisfaction. The data-derived model confirmed that perceived stress mediated the relationships between hours of care, gender, and depression, while depression was the mediator between perceived stress and other outcome variables (physical function, self-esteem, and marital satisfaction). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The data-derived model ::: ![](1472-6955-4-3-2) ::: Cross-validation ---------------- In order to test the robustness of the paths across the samples, the data-derived model was cross-validated by Wave 1 sample. The results are summarized in Table [3](#T3){ref-type="table"}. The validating process showed the data-derived model was not confirmed only in Step 2 when the paths between endogenous variables were constrained (p \< .01). This step showed that some paths between endogenous variables were not confirmed by Wave 1. To investigate the differences between Wave 1 and Wave 2, especially in the Beta linkage, each path was examined individually. The results showed that the differences came from the links between stressful life events and depression, and depression and physical function. That is, the cross-validation procedure confirmed that the data-derived model was stable across two waves of data, except in two paths (stressful life events to depression, and depression to physical function). These two paths need to be further examined. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Summary of cross-validation for the data-derived model ::: Comparison to previous model ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------ ----- ----- ------- ---- ------- Step 0 Factor loadings, path coefficients, factor variance, and covariance were all set to be inequality across group 369.14 148 .00 Step 1 Constrain factor loadings 376.21 152 .00 7.07 4 n.s. Step 2 Constrain factor loadings, and the paths between endogenous variables (beta linkages) 390.92 159 .00 14.71 7 \<.01 Step 3 Constrain factor loadings, the paths between endogenous variables (beta linkages), and the paths between exogenous variables and endogenous variables (gamma linkages) 396.04 163 .00 5.12 4 n.s. Step 4 Constrain factor loadings, path coefficients, and error variance 401.39 169 .00 5.35 6 n.s. Step 5 Constrain factor loadings, path coefficients, error variance, and factor variance 413.63 179 .00 12.24 10 n.s. ::: Discussion ========== The hypothesized model postulated that hours of care would be the most important factor in perceived caregiver stress, and other factors would include stressful life events, social support, age, and gender. Higher perceived caregiver stress was expected to result in more depression, which in turn would lead to poorer health function, lower self-esteem, and lower marital satisfaction. These expectations were only partially supported by the data. The data-derived model suggested that age had an indirect effect on perceived caregiver stress, through hours of care. Stone et al. found that older caregivers tended to assume the role of primary caregiver in attending to their chronically ill relatives \[[@B28]\]. Thus, it is likely that the older caregivers in this sample assumed more hours of care than young caregivers, resulting in more caregiver stress. Age was also found to predict physical function. George and Gwyther noted that spouse caregivers were more susceptible to diminished physical function than other caregivers and this was probably caused by their older age \[[@B33]\]. Stressful life events failed to predict perceived caregiver stress; instead, stressful life events predicted caregivers\' outcomes through depression. One explanation for this might be that stressful life events and caregiver stress work independently in predicting caregivers\' outcomes. One other study found that stressful life events did not influence health outcomes through perceived stress; rather, they were a confounding factor in predicting health \[[@B52]\]. And Stone et al. reported that stressful life events led directly to adverse health outcomes instead of being mediated by perceived stress \[[@B53]\]. Hours of care and perceived caregiver stress were expected to play important roles in caregiver outcomes. However, while hours of care predicted caregiver stress, hours of care was not the only nor the most important determining factor. Further, perceived caregiver stress explained only a small amount of the variance in depression. This is consistent with Pruchno et al\'s finding that caregiving had little impact on depression or the physical health of the caregiver \[[@B25]\]. The present findings support the view that caregivers of chronically ill relatives adapt to the demands of the situation and stabilize or even improve over time \[[@B54]\]. It is possible that the chronic nature of the recipient\'s illness enables the caregiver to adjust to persistent needs and reestablish a balanced life over a period of providing care. Viewed from this perspective, hours of care should not be expected to have a major effect on perceived caregiver stress. It can also be argued that caregivers confront many problems other than caregiving burden, and the impact of chronic caregiving may be diluted by competing daily stressors or stressful life events. Thus, the relationship between hours of care and perceived caregiver stress may not be as clear in long-term caregiving as in short-term caregiving to an acutely ill relative. Unexpectedly, in this study social support had no impact on perceived caregiver stress; but this is not unprecedented: similar results were reported by Lawton et al. \[[@B8]\]. We used friend/relative positive support as the indicator of social support; however, some aspects of social support may be more important than others in reducing caregiver stress. It is also possible that the measure used here was not sensitive enough to detect actual social support. Research has shown that low social support makes people more vulnerable to depression, and that has been clearly demonstrated for the elderly \[[@B55]-[@B58]\]. However, in our study, depression predicted social support rather than the other way around. Depressed persons may withdraw from some aspects of life, including their social network, especially friends and non-nuclear family relatives. The data-derived model showed that, as expected, depression mediated the relationship between perceived caregiver stress and self-esteem and marital satisfaction. However, the relationship between depression and physical function was not confirmed by the cross-validation. This is inconsistent with the findings of Pruchno et al. \[[@B25]\] and Zanetti et al. \[[@B26]\]. One possible explanation is that in this sample, depression and physical function may both have been outcomes of important factors that were not included in the study. Clearly, the relationship between depression and physical function needs to be further examined. The study was limited to the variables in the original Americans\' Changing Lives survey, constricting our choices in operationalizing constructs. Further, the study was cross-sectional and consequently was limited in testing the causal relationships depicted in the model. Although the findings provide preliminary evidence of causal relations among the variables, better examination of causality will require longitudinal data. Conclusion ========== In spite of its limitations, the study shows the importance of psychological mediators in the care of a chronically ill relative. The question of how caregivers manage to avoid adverse outcomes or why some caregivers are at risk for adverse outcomes can be answered in part by understanding the role of depression. Clearly, to avoid adverse outcomes, clinical interventions should target caregivers who are experiencing depression. List of abbreviations used ========================== Americans\' Changing Lives: ACL Center for Epidemiological Studies Depression: CES-D The goodness-of-fit index: GFI The adjusted goodness-of-fit: AGF The relative noncentral index: RNI The relative normed fit index: RNFI Declaration of competing interests ================================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= Author 1, PT, developed the research proposal, carried out the data analysis, interpreted the data and participated in the sequence alignment of the manuscript. Author 2, MMJ, participated in developing the research proposal and interpreting the data. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6955/4/3/prepub> Acknowledgements ================ The data used in this study (Americans\' Changing Lives: Wave 1, 1986, and Wave 2, 1989) were originally collected by Dr. James House and were made available to the authors by the Inter-University Consortium for Political and Social Research. We are grateful to both. Neither the collector of the data nor the Consortium, however, bears any responsibility for the analyses or interpretations here. We also thank Drs. Jeffery Dwyer, Ann Horgas, Marjorie Isenberg, Michael Marsiske and Douglas Baer for their suggestions, and acknowledge the helpful comments of Drs. Anne Scott Stiles, Elaine Souder, Janet Lord, Jason Chang, and Kathy Richards, and Ms. Elizabeth Tornquist. We also acknowledge the helpful comments of Dr. Cornelia Beck, Director of the Alzheimer\'s Disease Center, University of Arkansas for Medical Sciences, and the resources and support available from the Center staff. Work on this manuscript was partially supported by funds from the Building Academic Geriatric Nursing Capacity Scholar Program, John A. Hartford Foundation.	PubMed Central	2024-06-05T03:55:53.988116	2005-2-22	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554787/", "journal": "BMC Nurs. 2005 Feb 22; 4:3", "authors": [ { "first": "Pao-Feng", "last": "Tsai" }, { "first": "Mary M", "last": "Jirovec" } ] }
PMC554971	Background ========== The WHO target of eliminating indigenous measles in Europe by 2007 represents a challenge for public health systems. The requirements for success in this battle are summarised by Gay \[[@B1]\]. Italy, compared with other European countries, is still quite far from meeting these requirements. Here, in contrast with mandatory tetanus, polio, hepatitis B and diphtheria vaccinations, measles vaccination since its initiation in 1976 is only classed as \'Recommended\' and has traditionally been characterised by very low coverage, with a national average in 12--24 months old children of only 56% in 1998 \[[@B2]\], and 76% in 2003 \[[@B3]\], despite intensified and supplementary efforts. A further worrying problem is persistent strong heterogeneity in coverage at the regional level (often also within each region). The substantial measles epidemics in Southern Italy in 2002--2003 \[[@B4]\] tragically underlined these points, also confirmed by routine and serological data \[[@B5]\]. In order to confront this state of affairs Italy is embarking on implementation of a Measles National Elimination Plan \[[@B6]\]. In designing an optimal elimination strategy, it is crucial that planners have at their disposal a clear picture of the regional \"geography\" of the intensity of effort required for measles elimination as a preliminary step for ranking intervention priorities. This is especially in view of the claim that there could be spatial heterogeneity in transmission rates due to the large socio-economical differences existing in the country \[[@B7]\]. However the evaluation of the required elimination effort in terms of critical coverages still largely and necessarily relies on estimates of basic reproduction numbers (also known as basic reproduction ratios) from pre-vaccination data \[[@B8]\]. Here we deal with this pre-vaccination epidemiology of measles in Italy, taking inspiration from two distinct standpoints. The first is a very practical one, i.e. the need to summarise the degree of effort needed for measles elimination in the Italian regions. This is carried out using the fundamental parameters of the basic SEIR (i.e. Susceptible-Exposed-Infected-Recovered) transmission dynamics mathematical model of vaccine preventable diseases\[[@B9]\]: forces of infection (FOI), defined as the per capita annual rate of infection among susceptible individuals), contact or \'Who Acquires Infection from Whom\' (WAIFW) matrices (specifying the rates of transmission of infection between and within age groups) due to contacts arising from its own and from other age groups), and basic reproduction numbers (R~0~, the mean number of secondary infections which would arise from the introduction of a primary infection in a wholly susceptible population). As primarily shown by Anderson & May \[[@B9]-[@B11]\], provided it is possible to estimate them from high quality pre-vaccination data, these parameters allow concise summary of the natural history of a given infection in a given country in the absence of vaccination, e.g. Edmunds et al. \[[@B8]\] for the epidemiology of measles, mumps and rubella (MMR) in Europe. Edmunds et al. \[[@B8]\] have shown that pre-vaccination patterns of measles (and mumps) in European countries were broadly similar, suggesting it may be possible to use parameter values estimated from other countries with good-quality pre-vaccination data to model measles (and mumps) in countries with no or poor infection data. The case of Italy was however more puzzling than that of the other countries. On the one hand they found that the FOI for measles (but also for mumps) computed from Italian national case notifications data was greatly different from other available European FOIs. On the other hand they computed the FOI implicit in the data of Santoro et al.\[[@B7]\] - the sole pre-vaccination measles sero-survey in Italy - finding figures that, at least for the youngest age groups (0--4), were in agreement with those summarising infection experience in European countries with good infection data, \[[@B8]\] - here referred to as \"EURO\" FOIs. They conjectured that Italian data suffered from strong selective under-reporting by age, and concluded: \"It is tempting to dismiss FOI estimates from Italian case notifications data as the serological data are likely to be more robust\". We believe, however, that this conclusion relies more on the generally assumed greater reliability of serological data compared to case reports, rather than on a full demonstration. Indeed, the FOI they estimated from the data of Santoro et al. \[[@B7]\] for school age children (age 5--10) is just 50% of the corresponding EURO FOI, i.e. even smaller than that estimated from case reports. We believe therefore that it would be prudent and worthwhile to try to obtain further insight into this problem. The Edmunds et al. \[[@B8]\] paper provides therefore the motivation for our second, more theoretical, question of whether or not the true FOI acting in Italy during the pre-vaccination era is homologous to the EURO FOI, as they suggest? The implications are relevant especially for the purposes of modelling which may be used to inform policy: is it advisable simply to rely on this EURO FOI, or does prudence dictate that one should consider also other possibilities? In what follows we use the term \"EURO conjecture\" to denote the suggestion of Edmunds et al.\[[@B8]\] that i) the Italian FOI would indeed be essentially homologous to the EURO FOI, and ii) Italian case notifications data simply camouflage this fact thanks to broad selective under-reporting. To shed more light on these issues, we have analysed more deeply Italian case notifications data by looking at patterns at several spatial levels. First we have systematically looked at the structure of the FOIs (and contact patterns, basic reproduction numbers, etc) for all the Italian Regions and Provinces. Since the national datum used by Edmunds et al. \[[@B8]\] to compute the Italian FOI, was obtained by pooling regional data in presence of strong spatial heterogeneity in under-reporting (documented in Williams et al. \[[@B12]\]), a deeper investigation of spatial patterns of infection could reveal the existence of some between regions heterogeneity in age-related transmission rates that could be an indicator of a higher force of infection (or indeed perhaps suggest the presence of selective under-reporting by age). It is indeed quite conceivable that true heterogeneity might exist within Italy as a result of contrasts between North and South in terms, for example, of family size (including that of the extended family), patterns of shared childcare and schooling, impact of climate on time spent indoors and out, etc.; however in the absence of systematic community based investigation such ideas must remain in the realms of speculation. Second, we carried out a systematic time series analysis of measles periodicities in the Italian regions during the pre-vaccination era (Fig [1](#F1){ref-type="fig"} &[2](#F2){ref-type="fig"}). Our feeling here is that only under exceptional circumstances can selective under reporting by age mask true time patterns of incidence. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Measles cases notifications in Italy. The regionally heterogeneous monthly pattern of Italian measles cases reported for the period 1949-1996 (NB Two time series are shown for Friuli as the city of Trieste was not incorporated into the region until its post-war status within Italy was resolved in 1954) ::: ![](1478-7954-3-1-1) ::: Third, we add some results from our modelling work on measles in Italy. The results suggest that i) forces of infection estimated from case reports of measles in all the Italian regions are systematically and significantly lower compared with the EURO FOI with little, or only moderate, between regions heterogeneity; ii) regional periodicities mostly suggest a longer (usually 3 years or more) inter-epidemic period compared to England & Wales, a fact which is consistent with lower transmission (and which, together with the lower FOIs, suggests lower vaccination coverage might suffice to achieve control and elimination); iii) predictions from a mathematical model using the EURO force of infection, even compared with those based on a FOI estimated from Italian case reports, poorly match the 1996/1997 Italian serological data \[[@B13]\]. Methods ======= The geographic analysis of the epidemiology of vaccine preventable diseases is made difficult by complex correlations between local dynamics. A first, though clearly partial, step is characterization of local infection patterns by treating spatial sub-areas as autonomous epidemiological units. Two measures are used here to characterise the regional (i.e. local) \"landscape\" of measles in Italy in the pre-vaccination era: summary parameters from the SEIR model for vaccine preventable disease, and summary periodicities from time series analyses of measles incidence. Age patterns of infection, reproduction numbers and critical vaccination coverage --------------------------------------------------------------------------------- From the pre-vaccination age distribution of cases we computed, for each region, summary equilibrium parameters from the SEIR model: i) forces of infection, FOI; ii) mixing or contact (\"who acquires infection from whom\", WAIFW) matrices; iii) basic reproduction numbers, R~0~; and iv) related critical vaccination coverages, p~c~, the proportion of a population needed to be successfully immunised at age zero with a 100% effective vaccine in order to eliminate the infection, where p~c~= 1-1/R~0~(i.e. whereas in a wholly susceptible population one primary case will on average successfully transmit to R~0~contacts, in the case when the proportion already immune is greater than p~c~transmission will succeed for less than R~0~(1-p~c~) = R~0~(1- \[1-1/R~0~\]) = 1.0contacts) \[[@B9]\]. For comparison purposes we used the same age groups as in Edmunds et al. \[[@B8]\], i.e. 0--1 years, 2--4 yr., 5--10 yr., 11--17 yr., 18+ yr. The basic reproduction numbers \[[@B14]\] were computed for several plausible mixing matrices. Mixing (WAIFW) matrices ----------------------- Mixing matrices (Table [1](#T1){ref-type="table"}) are used in standard infectious disease modelling \[[@B9]\] to summarise age patterns of contacts between susceptible and infected individuals; the generic element β~ij~summarises the risk of acquiring infection for a susceptible individual in age group idue to contacts with infective individuals aged j. When estimating from age-structured data (e.g. serology or case reports), a mixing matrix with mage groups can have at most mdistinct elements \[[@B9]\]. In the simplest type of mixing, i.e. homogeneous mixing, the β~ij~entries are independent of age (β~ij~= βfor all i,j). Other types of mixing matrix are considered in the paper and are listed below. However not all forms of mixing are compatible with a given force of infection. As noted by Anderson & May \[[@B9]\], a mixing matrix can be non feasiblefor the given FOI (i.e. it may yield negative values for some of the βcoefficients) and in such circumstances \"the chosen matrix is inappropriate to the observed age dependence in the FOI\". Hethcote \[[@B15]\] further pointed out that not all feasible mixing matrices are \"acceptable\", in that they could lead to numbers of contacts between individuals outside plausibility bounds; inspection of the ensuing mixing matrices is thus necessary to avoid trivial results. Hethcote also proposed a \"plausibility criterion\", based on a postulated \"preference for assortativeness\", which is simple to use for preferred matrices (type PREF below). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Some of the types of mixing matrices used in the paper. Three of the mixing matrices discussed in Methods: a) fully assortative mixing (RDIAG), b) \"Realistic\" assortative mixing (DIAG) and c) the default mixing matrix (DEF) of Edmunds et al(2000). Succeeding rows and columns represent the age groups 0--1 year, 2--4 years, 5--10 years, 11--17 years and 18+ (e.g. for DIAG β~3~is the element corresponding to contacts between those in the 5--10 year age group and their peers in the same age group) ::: ------------ ------------- ----------- -------- -------- -- -------- -------- -------- -------- -------- -- -------- -------- -------- -------- -------- a\) DIAG b\) RDIAG c\) DEF β~1~ 0 0 0 0 β~1~ β~5~ β~5~ β~5~ β~5~ β~1~ β~1~ β~1~ β~1~ β~5~ 0 β~2~ 0 0 0 β~5~ β~2~ β~5~ β~5~ β~5~ β~1~ β~2~ β~4~ β~4~ β~5~ 0 0 β~3~ 0 0 β~5~ β~5~ β~3~ β~5~ β~5~ β~1~ β~4~ β~3~ β~5~ β~5~ 0 0 0 β~4~ 0 β~5~ β~5~ β~5~ β~4~ β~5~ β~1~ β~4~ β~5~ β~3~ β~5~ 0 0 0 0 β~5~ β~5~ β~5~ β~5~ β~5~ β~5~ β~5~ β~5~ β~5~ β~5~ β~5~ ------------ ------------- ----------- -------- -------- -- -------- -------- -------- -------- -------- -- -------- -------- -------- -------- -------- ::: Matrix types considered ----------------------- ### 1) Fully assortative mixing (matrix DIAG, Table [1(a)](#T1){ref-type="table"}) Individuals of a given age are assumed to mix only with individuals of the same age, yielding a diagonal matrix. Given a specific FOI it is the form of mixing allowing R~0~to achieve its upper bound \[[@B16]\]. ### 2) Realistic assortative mixing (RDIAG, Table [1(b)](#T1){ref-type="table"}) This matrix (termed \"Diagonal\" in Edmunds et al. \[[@B8]\], see also \[[@B17]\]) has the same diagonal elements as the fully assortative one, but some mixing across age groups is also possible (i.e. non diagonal elements are greater than zero). Here mixing across age groups is assumed to be at the same rate (β~5~) as that between adults \[[@B8]\]. ### 3) Default mixing (matrix DEF in Table [1(c)](#T1){ref-type="table"}) This matrix emphasises transmission between school age groups \[[@B8]\]. ### 4) Proportionate mixing (PM) Under PM \[[@B15]\] contacts occur at random, thus implying a larger probability of meeting more socially active individuals. The entries of the PM matrix have \"multiplicative\" form β~ij~= b~i~b~j~j= 1,\...m. ### 5). One-parameter preferred mixing (PREF) The PREF matrix is a single parameter (h) weighted average of proportionate (PM) and fully assortative (DIAG) mixing: PREF= (1 - h) \* DIAG+ h\* PM, 0\<h\<1 \[[@B15]\], representing a contact pattern which can be split into a selective (i.e. non random) component (here mixing with individuals of the same age) and a random one. The PREFmatrix has (m+1) distinct entries: the extra parameter his usually estimated \"ad hoc\". The matrices RDIAG & DEF are defined \"ad hoc\", though they have a behavioural basis, whereas DIAG & PM are limit cases (though in distinct senses), and hence PREF is a weighted average between two limit cases. There is thus no clear relationship between, for instance, RDIAG & DEF on the one hand and PREF on the other. For this reason, we explored all forms that have been reported in the paper. Periodicities of time series of measles incidence ------------------------------------------------- In contrast to the spatially well synchronised biennial England & Wales oscillation, visual inspection of Italian regional data did not suggest clear common patterns of oscillation or hence of the inter-epidemic period. Thus investigation of periodicities in regional measles incidence became necessary to provide satisfactory characterisation of the inter-epidemic period. Though incidence data might be seriously affected by under-reporting, recent work \[[@B12]\] suggests that, as long as we are concerned with the pre-vaccination period, overall under-reporting rates in the Italian regions seem to have remained fairly constant over time. Thus the available time series should nonetheless represent a sufficiently reliable picture of the regional dynamics of measles. The cyclical behaviour in the monthly incidence time series of measles for each Italian region in the pre-vaccination period 1949--1976 was analysed in the frequency domain (first used for childhood diseases by Anderson et al. \[[@B18]\]) with special attention being given to the long term cycle. A drawback of periodogram analysis is the assumption that cyclical components of frequencies depend on the length of the observed series, i.e. that they are integer multiples of 2π/T(T= series length); this is the basis of \"harmonic\" analysis. Such analysis may not identify exactly cyclical components where true frequency falls between two \"harmonic\" frequencies, e.g. it may suggest periodicity of either 3 or 4 observations when true periodicity lies between these values and hence is not identifiable exactly with this procedure. This problem may be overcome with \"non-harmonic frequency domain\" analysis where dependence of periodicities of cyclical components on series length is relaxed. Here the following procedure, as proposed in \[[@B19]\], is used for identifying true cyclical components: after log transformation to stabilise variances, and de-trending, using a deterministic function of time, the true frequency of a cyclical component, denoted by λ, was estimated, as in \[[@B19]\], by minimising, with respect to λ, the quantity ![](1478-7954-3-1-i1.gif) where x~t~denotes the detrended series (a starting value for λwas obtained by examining a non-parametric estimate of the spectral density function; significant non-harmonic functions can be estimated in this way and deleted, if required, in a stepwise manner beginning with the frequency corresponding to the largest spectral density ordinate). The results from the time series analysis were also compared with the prediction from the homogeneous mixing SEIR model that where the sum Kof the expected duration of the latent and infectious states is short compared to the life of the host (as is the case with measles), disease incidence will have a long-term oscillation around its endemic equilibrium with the period given, to an excellent approximation, by ![](1478-7954-3-1-i2.gif)\[[@B9]\], where Ais the average age at infection and Kcan be taken to be 14 days in the case of measles. Analyses described above were carried out for all Italian regions (Valle d\'Aosta, a very small northern Region with few reported cases per year was aggregated with neighbouring Piemonte). Data ---- In addition to published official Italian data on births, and birth and death rates, monthly measles case reports were provided by the Istituto Nazionale di Statistica (ISTAT) for the period from the first available year, 1949, to 1976, together with regional age structured measles case reports from the first available year, 1971. Pre-vaccination FOIs at the regional level were estimated from data for the time window 1971--76, which encompassed about two full three-years long epidemic cycles (inclusion also of the first few years in the post-vaccination window, when vaccine uptake was known to be extremely small, resulted in no significant change). Results: the pre-vaccination landscape of measles in Italy ========================================================== Regional patterns of incidence over time ---------------------------------------- The spectral densities of the pre-vaccination (1949--76) Italian regional time series of measles incidence indicate, besides a well-pronounced annual cycle, the presence of a less pronounced longer term cycle of varying length, which is in contrast with the sharp biennial oscillation in England & Wales. Given the importance of the long term oscillation which is taken as representing the true \"inter-epidemic period\", the non-harmonic estimator was calculated (after de-trending each series using a function of time, and de-seasonalising, using monthly dummies,). Table [2](#T2){ref-type="table"} reports for each region: i) the average age of cases in the pre-vaccination period (A); ii) the non-harmonic estimate T~O~of the period of the long-term oscillation; iii) the length of the inter-epidemic period from the homogeneous SEIR model via formula for Tgiven in the Methods section. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Average age at infection (A~15~) and inter-epidemic period (T) in the Italian regions. Results arising from the time series analysis of regional measles notifications data for the period 1949--76 (N = North, C = Centre, S = South.) ::: **A~15~(years) T from non harmonic estimate (T~O~) (years) T from SEIR model (T) (years)** ---------------------------- ---------------------- --------------------------------------------------- ------------------------------------- Piemonte & Valle Aosta (N) 6.34 2.39 3.08 Lombardia (N) 5.70 2.37 2.94 Trentino (N) 5.75 2.85 2.94 Veneto (N) 5.87 3.21 2.98 Friuli (N) 5.98 2.77 2.98 Liguria (N) 6.92 5.34 3.22 Emilia (N) 6.24 3.33 3.06 Toscana (C) 7.30 2.85 3.29 Umbria (C) 6.81 3.40 3.21 Marche (C) 6.63 3.27 3.16 Lazio (C) 6.34 3.23 3.08 Abruzzo (S) 6.46 3.40 3.13 Molise (S) 6.19 5.88 3.04 Campania (S) 5.64 3.03 2.90 Puglia (S) 5.20 3.79 2.79 Basilicata (S) 5.43 3.74 2.82 Calabria (S) 5.63 3.45 2.90 Sicilia (S) 5.47 3.14 2.85 Sardegna (S) 5.70 3.79 2.92 North 6.09 2.35 3.02 Centre 6.78 3.27 3.22 South 5.65 3.68 2.90 Italy 6.18 2.35 3.06 ::: Values of T~O~in table [2](#T2){ref-type="table"} show that, apart from a few Northern regions with a long term cycle with a period below 3 years (the shortest period, 2.4--2.5 years, being observed in Piemonte and Lombardia), most Italian regions have a three-year, or greater, long term oscillation (though a note of caution must be sounded with regard to the 5 year period observed in Molise, a very small isolated region). In comparison, values of Tpredicted by the SEIR model range from a minimum of 2.8 (Puglia) to 3.3 years (Tuscany). The agreement between Tand T~O~in some cases is not very good: North-Eastern regions show a higher average age at infection, A, compared to Southern regions and yet the shorter inter-epidemic periods in the former compared with the latter, as suggested by the time series analysis, would imply the reverse. Compared to England & Wales there is also a surprising lack of synchronisation between regional cycles in the pre-vaccination period. A cross-spectral analysis suggests limited correlation between pre-vaccination long term cycles; coherencies greater than 0.7 were observed, as expected, only for a few neighbouring regions such as Emilia-Romagna and Marche, Marche and Umbria, Friuli and Trentino, Lombardia and Piemonte, Liguria and Piemonte The structure of the force of infection at the regional level ------------------------------------------------------------- Pre-vaccination age-distributed forces of infections in the Italian regions (we report a sample of results) can be well summarised by 3 clusters, North, Centre, South. These are shown in Fig. [3](#F3){ref-type="fig"} together with the EURO FOI \[[@B8]\] which displays a similar qualitative pattern with age (single-humped, peaking in the \"elementary school\" age group 5--10, etc.), although the \"Italian\" FOIs exhibit surprisingly lower levels amongst pre-secondary school (\< 11 years) age groups (with the exception, perhaps, of the very youngest age group in Southern Italy). Of the three Italian clusters the FOI for the two youngest age groups is highest in the Southern regions, and lowest in the Central regions. Relatively lower FOIs persist in Central regions in the elementary school age group (5--10 y.) compared with those in the North and South which resemble each other. Finally, in the two highest age groups, FOIs are similar, though Central and Northern regions now have marginally higher FOI values than the South. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Measles cases notifications in Italy. Annual age distributions of measles case reports from 1971 (the first year in which notifications were recorded by year of age rather than age band) through to 1986 (10 years after the start of measles vaccination in 1976, albeit at very low coverage). ::: ![](1478-7954-3-1-2) ::: The average age at infection A(computed over the restricted support 0--18 yr. age group, as more robust than the overall average age), that coarsely summarises the force of infection, shows similar spatial patterns. For instance Ais systematically smaller in the South (little above 5 years), around 7 years in the Centre, and takes intermediate values in the North (Table [2](#T2){ref-type="table"}, second column). From the regional FOIs we also computed a national FOI corrected for under-reporting (\'Italy-UR\' in Table [3](#T3){ref-type="table"}) using the under-reporting factors estimated in Williams et al.\[[@B12]\] which suggested great heterogeneity in reporting rates, the higher rates being observed in the Northern Italian regions (around 10--12% in the pre-vaccination era) and the smaller in the South (as low as 2% in Campania!). Conceivably the concurrence of spatial heterogeneity in overall rates of under-reporting with spatial heterogeneity in age-related transmission rates might be a factor responsible for a selective age-bias in the national age distribution even in absence of selective under-reporting by age, because it weights incorrectly the regional cases that are pooled into the national datum. Fig. [3](#F3){ref-type="fig"} suggests however that the quantitative impact of the correction for under-reporting is likely to be rather small. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Measures indicating the size of the task of eliminating measles in the Italian regions. Estimates for basic reproduction numbers (R~0~) and critical age zero routine vaccination coverages (p~c~) for measles elimination in the Italian regions. Results in column pairs correspond to different assumptions on contact patterns. (NA = contact matrix non admissible; \* = contact matrix admissible only after FOI redefined on adult age group; N = North, C = Centre, S = South.) ::: Type of mixing pattern Homogeneous Default Realistic assortative Proportionate *Preferred mixing ε= 0.9)* ------------------------------- ----------------- --------------- --------------------------- ------------------- -------------------------------- --------------- ---------- --------------- ---------- --------------- R~0~ P~C~age 0 R~0~ P~c~age 0 R~0~ P~c~age 0 R~0~ P~c~age 0 R~0~ P~c~age 0 Piemonte & Valle D\'Aosta (N) 11.83 0,92 NA NA 11.8\* 0.92 4.09 0.76 4.30 0.77 Lombardia (N) 13.16 0,92 6.2 0.84 16.1\* 0.94 4.80 0.79 5.18 0.81 Trentino (N) 13.05 0,92 5.4 0.81 16.0\* 0.94 4.50 0.78 4.82 0.79 Veneto (N) 12.77 0.92 5.5 0.82 17.6\* 0.94 4.77 0.79 5.13 0.81 Friuli V.G. (N) 12.53 0.92 4.9 0.80 13.1\* 0.92 4.05 0.75 4.32 0.77 Liguria (N) 10.83 0.91 NA NA 10.0\* 0.90 3.73 0.73 3.95 0.75 Emilia (N) 12.01 0.92 NA NA 17.5\* 0.94 4.41 0.77 4.69 0.79 Toscana (C) 10.27 0.90 NA NA 9.8\* 0.90 3.60 0.72 3.79 0.74 Umbria (C) 11.01 0.91 NA NA 8.5\* 0.88 3.62 0.72 3.81 0.74 Marche (C) 11.32 0.91 NA NA 10.6\* 0.91 3.83 0.74 4.06 0.75 Lazio (C) 11.83 0.92 NA NA 11.8\* 0.92 4.06 0.75 4.27 0.77 Abruzzo (S) 11.60 0.91 NA NA 14.6\* 0.93 4.64 0.78 5.07 0.80 Molise (S) 12.11 0.92 NA NA 13.2\* 0.92 5.68 0.82 6.62 0.85 Campania (S) 13.30 0.92 7.5 0.87 16.3 0.94 5.91 0.83 6.89 0.85 Puglia (S) 14.43 0.93 7.80 0.87 20.1 0.95 6.01 0.83 7.14 0.86 Basilicata (S) 13.82 0.93 6.7 0.85 19.8 0.95 5.19 0.81 5.86 0.83 Calabria (S) 13.32 0.92 6.1 0.84 18.5 0.95 5.07 0.80 5.62 0.82 Sicilia (S) 13.71 0.93 7.5 0.87 16.6 0.94 5.91 0.83 6.98 0.86 Sardegna (S) 13.17 0.92 6.3 0.84 16.6 0.94 5.29 0.81 5.93 0.83 North 12.32 0.92 5.2 0.81 14.9\* 0.93 4.38 0.77 4.66 0.79 Centre 11.05 0.91 NA NA 10.4\* 0.90 3.76 0.73 3.96 0.75 South 13.27 0.92 6.6 0.85 16.4 0.94 5.45 0.82 6.19 0.84 Italia 12.14 0.92 5.2 0.81 13.2\* 0.92 4.33 0.77 4.64 0.78 Italy-UR 12.93 0.92 6.0 0.83 14.3 0.93 5.04 0.80 5.57 0.82 EURO 17.05 0.94 9.6 0.90 29.3 0.97 7.14 0.86 8.95 0.89 ::: A further point worth of consideration in Fig. [3](#F3){ref-type="fig"} is that, in contrast to the EURO FOI, in the North and Centre the FOI in the highest age group is higher than the FOI in the youngest (a fact shared by most regions in the two clusters, but not occurring in the Southern regions and which occurs regardless of how we define the last age group). This suggests a greater relative importance of contacts between adults (provided one can exclude the effects of poor reporting). Mixing matrices, reproduction ratios and required effort for measles elimination -------------------------------------------------------------------------------- From the values of the FOI we computed mixing-WAIFW matrices. As previously noted, a problem with the computed Italian FOIs is that risk of infection among adults individuals (i.e. age group 18+) is, with the exception of Southern regions, higher than in the \"young\" (0--1 yr.). A verifiable consequence of this fact, is that \"realistic assortative\" mixing matrices (RDIAG) can be non-feasible (they can yield negative coefficients), and this indeed happens in all Central and several Northern regions. In other words, such mixing is not compatible, Southern Regions apart, with observed forces of infection. This may be a problem because \"realistic assortative\" is the mixing pattern that provides under a EURO-type FOI, the upper bound of \"plausible\" values of R~0~, a measure of critical importance from the perspective of disease control. For purposes of comparability with Edmunds et al. \[[@B8]\] we therefore arbitrarily redefined the value of the force of infection in the oldest age group in order to recover, for all Italian regions (and not only the Southern ones) the \"EURO\" shape (i.e. a FOI having its smallest value in the adult age group). In this manner we obtained feasible and \"plausible\" \"realistic assortative\" mixing matrices for all Italian Regions. The results are summarised below (Table [3](#T3){ref-type="table"} gives a synoptic view; we have omitted for sake of brevity the outputs of mixing computations and instead report the more easily interpretable values of reproduction ratios and critical vaccination coverages); the results from \"fully assortative mixing\" are omitted as leading to trivially high values of R~0~: ### 1. Homogeneous mixing Values of R~0~(computed as R~0~≅ L/Awhere Lis the expectation of life, taken as 75 years by assuming type 1 mortality and ignoring regional variability, and Athe average age at infection in the pre-vaccination era, Table [2](#T2){ref-type="table"}) range between a minimum of about 10 (Toscana) up to a maximum around 14.5 in Puglia (17.5 under EURO). ### 2. Realistic assortative mixing (RDIAG) This mixing yields an R~0~around 29 under EURO \[[@B8]\]. Thanks to our correction for the adult age group all Italian Regions yield fully admissible transmission rates; the corresponding R~0~values are in the range 13--20 in the South (critical coverages ranging 95--97%) and 8.5--12 in the Centre (critical coverages 88--92%), with intermediate values in the North. ### 3. Default mixing (DEF) It happens that this mixing pattern is never admissible for all Central Regions and for some Northern ones, as it can yield negative transmission rates when, as is the case in Central Italy, the FOI is rather low in the youngest age groups. Default mixing is, on the contrary, admissible in all Southern regions where R~0~and critical coverages happen to be systematically higher. Critical coverages range from 80% in the North up to 88% in the South (the reference EURO value being 90%). ### 4. Proportionate mixing (PM) PM mixing matrices (always admissible by definition) are the type of mixing \[[@B15],[@B17]\] yielding the lower bound of \"plausible\" values of R~0~. Predicted critical coverages ranged from 72% in the Centre, with intermediate values in the North, up to 83% in the South (EURO = 86%, Table [2](#T2){ref-type="table"}). Nevertheless inspection of the matrices does reveal for all regions (also for EURO) the presence of implausible relationships between groups, e.g. a significant level of disassortativeness. ### 5. Preferred mixing (PREF) We computed preferred matrices by tuning the \"preference for assortativeness\" parameter hstarting from h = 1(proportionate mixing) and then \[[@B15]\] progressively decreasing huntil \"minimally plausible\" contact rates were achieved. Essentially, for all Italian regions (also for EURO), use of a moderate degree (h = 0.9) of assortativeness allowed the \"minimal plausibility\" threshold to be surpassed. This led, compared to the case of proportionate mixing, to increases of 1% to 3% in the corresponding critical coverages (Table [3](#T3){ref-type="table"}). These figures should be considered as providing more reliable lower bounds on R~0~than does Proportionate mixing. An issue is the degree of assortativeness, h, which would lead to the preferred mixing pattern closest to reality (here we only considered the degree guaranteeing a \"minimally plausible\" contact pattern, according to Hethcote \[[@B15]\]). Although one-parameter preferred mixing provides a flexible family of mixing patterns, it is still too inflexible to provide realism, as it assumes the same degree of preference for assortativeness in all age groups. For instance considering schooling as the major source of assortativeness in younger age groups, in pre-vaccination Italy a much smaller proportion, compared to the present, of boys in the youngest age group were attending crèche schools so that a major potential source of their assortativeness was probably absent. This would therefore suggest that at least two \"preference for assortativeness\" parameters (h~1~,h~2~, say) may be needed to describe Italian pre-vaccination infection patterns. This also leads to the nub of the real gain provided by preferred matrices (just because they are \"many\") compared to \"ad hoc\" behavioural matrices. One could ask whether Hethcote\'s \[[@B15]\] preference for an assortativeness criterion is meaningful for mixing matrices which do not conform to the one-parameter \"preferred mixing\" scheme. Using this criterion of \"preference for assortativeness\", in most cases, \"default\" mixing would be discarded, and indeed, under default mixing (quite apart from the over small degree of assortativeness postulated for the youngest age group), most Italian regions do not meet this criterion for age groups 2--4 and 5--10. Nevertheless, from other perspectives, \"default\" is one of the most \"reasonable\" among behavioural mixing matrices. More generally, any answer to such questions is complex, as the problem of how to assess whether one arbitrary given mixing matrix is \"better\", or simply more plausible, than another remains unresolved. This problem is not simply an academic one: the predicted critical coverage for measles at age zero under the EURO FOI ranges between 86% under proportionate mixing (PM) and 97% under realistic assortative (RDIAG) \[[@B8]\]. Using the Hethcote \[[@B15]\] criterion one can marginally increase the lower bound up to 87.5% by replacing proportionate mixing (unacceptable under Hethcote criterion) with the corresponding \"minimally acceptable\" preferred matrix (PREF), but the extent of the uncertainty that remains is hardly less significant. A modelling result ------------------ Our analysis of the pre-vaccination force of infection of measles in Italy formed a step toward developing a mathematical model for the transmission dynamics of measles in Italy. The model has the following features: i\) it provides bounds for the uncertainty surrounding the estimate of the FOI (Table [4](#T4){ref-type="table"}), by taking the \"EURO\" FOI as upper bound, and as a lower bound the FOI (denoted as Italy-UR in Table [2](#T2){ref-type="table"} & Figure [3](#F3){ref-type="fig"}) estimated from Italian case reports by correcting regional figures using the estimates of under-reporting in Williams et al. \[[@B12]\]; ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Force of infection estimates. Estimates of EURO force of infection (FOI) and that from Italian case notifications; computations were based on standard techniques described by Anderson & May \[9\]. ::: Force of infection/FOI (% / year) ----------------------- --------------------------------------- ------ ------- -------- ----- Age group (years) 0--1 2--4 5--10 11--17 18+ Italy-UR 7 15 31 19 6 EURO 12 28 40 20 10 ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Pre-vaccination forces of infections in the Italian regions.Age-related pre-vaccination forces of infection (FOI) estimated from case notifications for North, Centre and South divisions of Italy compared with the \"EURO\" FOI estimated by Edmunds et al\[8\] ::: ![](1478-7954-3-1-3) ::: ii\) it incorporates \"realistic\" demography in order to mirror the broad process of population ageing observed in Italy in the past 20 years (and which would be observed in the future if the present state should continue) as a consequence of the onset of sustained low fertility; iii\) it considers two distinct options as regards interaction between the demography and the epidemiology: the first (D1) assumes no effects on transmission arise as a consequence of population ageing; the second (D2) assumes low fertility and population ageing could lead to a significant decline in transmission (e.g. a decline in the FOI), via proxy mechanisms such as a substantial decline in intra-family transmission which might occur as a consequence of contraction in average family size. By crossing the two FOI assumptions, EURO and UR (i.e. Italy-UR), with the two assumptions D1, D2 we obtain four assumptions: EURO/D1, EURO/D2, UR/D1, UR/D2. The model simulations were carried out by adopting for the post-vaccination era what we considered the \"best\" approximation for the profile of routine vaccination coverages (Fig. [4](#F4){ref-type="fig"}), obtained by combining data collated from the few available regional profiles with the few available national data, such as the 1998 vaccination survey \[[@B2]\]. Fig. [5](#F5){ref-type="fig"} reports the immunity profile derived from the seroprevalence data obtained by the national survey conducted in 1996--97 (3,182 samples collected from residual sera from routine laboratory testing, in 18/20 Italian regions \[[@B13]\]) compared with the immunity profiles predicted by the model for the same years under the four assumptions EURO/D1, EURO/D2, UR/D1, UR/D2. Overall, the UR/D1 and the EURO/D2 assumptions match the observed profile rather well, whereas EURO/D1 and UR/D2 seem to fulfil well their expected roles of upper and lower bounds. Thus, if one disregards younger ages, where the proportion of children from 2 to 5 year of age immune to measles in 1996--97 is higher than predicted by the model (perhaps a result of increased routine coverage observed in several Italian areas since mid-1990\'s), the EURO FOI, as embedded in assumption EURO/D1, does not seem to perform well in the reproducing the seroprevalence profile, in contrast with UR/D1 (e.g. under EURO/D1 the same level of immunity is reached by ages 9--10 years as is reached by the data and UR/D1 by ages 17--19 years). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### The reconstructed time profile of measles routine vaccination coverage. This plausible profile of vaccination coverage was reconstructed from the very limited available national and regional data. ::: ![](1478-7954-3-1-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Observed (ESEN) vs predicted measles immunity profiles. The serological profile from the ESEN survey is compared with model projections assuming the EURO force of infection of Edmunds et al\[8\] or the FOI estimated from Italian pre-vaccination case notifications but corrected for under-reporting (UR). In each case one of two assumptions is made: i) that the process of population ageing predicted for Italy has no effect upon transmission (D1) or ii) that low fertility and population ageing could lead to significant decline in transmission (D2). ::: ![](1478-7954-3-1-5) ::: Discussion ========== An attempt has been made to characterise the measles pre-vaccination epidemiological \"landscape\" in Italy, and also with reference to what we have referred to as the \"EURO conjecture\" (i.e. that age-selective under-reporting disguises underlying homology with FOIs elsewhere in Europe). There are points open to criticism in this analysis: first, the fact that by looking at \"local\" forces of infection, e.g. treating spatial sub-areas as autonomous epidemiological units, we disregard spatial correlations between the various local dynamics; this makes the present analysis, at best, a first step. Second, the present work relies only on case reports, the only available pre-vaccination data. Third, the increasing temporal distance between the present time and the pre-vaccination era (ending in 1976) seems to make more and more heroic the assumption that mixing patterns remain unchanged since then. Nevertheless several interesting facts emerge. Degree of effort required for eradication at the regional level --------------------------------------------------------------- Clearly, good data on immunisation coverage and reports of outbreaks of infection would provide some broad picture of patterns of susceptibility. However, as the level of vaccine-based immunity increases, the mean interval between outbreaks also increases so that case reports, even if unbiased, become much less useful as a source of timely information on patterns of susceptiblity. Also in Italy, historically, data on immunisation coverage has been very poor or in many instances completely absent; in many regions case reporting has also been very poor, forming an insensitive tool for detecting small pockets of infection, and large outbreaks, almost by definition, cannot be timely indicators of the presence of susceptibles. Hence the first motivation for the paper was to construct a map of the efforts needed for elimination of measles in Italy, which could then constitute a useful tool for prioritising regional intervention priorities in the context of the agreed WHO target for measles elimination and with the aim of avoiding such outbreaks. During the second half of the 1990\'s several attempts have been made in Italy to increase routine coverages, which the available data suggested had remained until then disappointingly low (in some regions there have also been campaigns targeted at older age groups, and here it should perhaps be noted that the rationale for such campaigns is re-inforced by the tendency for a lower FOI to increase the accumulation of susceptibles in older age groups which results from sub-optimal vaccination coverage). Even though surveillance data suggests incidence has reached a historic minimum during 1999--2001, national coverage was still well below 70% in 2000, with much regional heterogeneity. It is unsurprising therefore that there have been substantial epidemics causing serious concern, especially in Campania (the region with the lowest coverages) during the spring of 2002, with an estimated 20,000 cases between January and May 2002 \[[@B4]\]. From this standpoint the results here (i.e. basic reproductive ratios etc) suggest that the regions which seem to be the most demanding in terms of effort needed to eradicate the infection are systematically the Southern ones. This is rather problematic, as the Southern regions are those presently characterised by the lowest vaccination coverages \[[@B2],[@B3]\]. The absolute size of the required eradication effort, as summarised by the critical coverages for routine birth vaccination, is somewhat variable and depends on the chosen mixing pattern. For Southern regions critical coverages range from 82%, under \"minimally plausible\" preferred mixing, to values around 95--97% under realistic assortative mixing, which represent lower and upper bounds for realistic contact patterns. Intermediate assumptions lead to intermediate figures: under \"Default\" mixing critical coverages for the major Southern Regions (Campania, Puglia, Sicilia) are in the range 86--88%, only two points less than the \"EURO\" value of 90% \[[@B8]\]. Keeping in mind a) that vaccination at birth is simply a \"theoretical standard\" (indeed critical coverages quickly increase as the age at vaccination is delayed), b) that we are assuming a 100% effective vaccine, and that, last but not least, c) the strong and persistent presence of anti-vaccination pressure groups in Italy, all these facts indicate that a substantial effort is still needed, especially in the Southern part of the country in order to approach, even in a minimal fashion, the WHO target. A major target of the Italian Measles Elimination Plan currently being initiated will be to strongly reduce regional heterogeneity in vaccine compliance. The EURO conjecture ------------------- The \"EURO conjecture\" suggests that the measles FOI in Italy is, in fact, homologous to that observed in other European countries but that, in the case notifications data, this fact is camouflaged by a strong under reporting bias with age. Under-reporting of childhood diseases has been a major problem in Italy. It is known \[[@B7],[@B12]\] that in the pre-vaccination era the overall reported measles incidence was an order of magnitude less than true incidence, and that the degree of under-reporting varied widely between regions. The existence of large and heterogeneous overall rates of under-reporting at the regional level does not necessarily imply, however, a bias in the (national) estimate of the force of infection: this additionally requires that i) reporting rates exhibit a significant variation with age, or that ii) spatial heterogeneity in overall reporting rates coexist with a marked spatial heterogeneity in infection patterns by age. We therefore made an extensive investigation of forces of infection at the spatial level (Regions, but also large conurbations and cities). Our feeling was that by going more deeply spatially it should have been possible to find some \"footprint\" of the presence of the presumed underlying EURO pattern (e.g. areas with a \"high\" FOI, or with significant heterogeneity in FOIs). As documented in the Results, we have not been successful in detecting such a \"footprint\". All \"local\" forces of infection are systematically substantially smaller compared to the EURO FOI, and surprisingly similar to each other in levels and shape: significant heterogeneity exists only in the youngest age groups. This raises the point, if the EURO conjecture were to be true, of what might be the social processes, if any, underlying the poor age reporting of measles cases, which are capable of so effectively camouflaging the true underlying force of infection. Indeed, in order to appear so stable over space and time, such processes should be profoundly rooted in social responses of families toward diseases of their children and/or medical practitioners\' behaviour. In the absence of the relevant data any discussion of the nature of such processes must remain in the realm of speculation, but clearly there is room for bias at many points in the chain leading to reporting of a case \[[@B20]-[@B22]\](Fig. [6](#F6){ref-type="fig"}): perhaps in the historically larger families of Italy because of their size there was more direct family experience of measles and less inclination to seek medical attention when there was an uncomplicated case in the family; perhaps, with measles in young children being a normal stage of life, medical practitioners could have been less inclined to conform to the \"bureaucratic\" requirements of case reporting; or perhaps there could have been failures in the bureaucratic process itself. Such speculations rely on caricatures of contrasts between Italy and more northern European countries and, clearly, to provide soundly based insights a detailed study would be necessary. Nevertheless, by use of a sentinel survey selective under-reporting by age has indeed been documented in Italy by Ciofi et al\[[@B23]\] in recent years for varicella (still in its pre-vaccination period). However their work shows that reporting rates are somewhat higher at young ages (0--10 yr.) and subsequently tend to decrease. Therefore, assuming that current under reporting rates for varicella are in some way similar to those for measles during the pre-vaccination period, their result would not provide evidence in favour of a bias leading to underestimation of the force of infection. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Potential sources of error in case reporting. An illustration of potential sources of failure or systematic bias in processes of case notification. ::: ![](1478-7954-3-1-6) ::: We then investigated the measles incidence time series, encouraged by the fact that \[[@B12]\] reporting rates of measles at the regional level seemed to have remained fairly constant over time, so that (though unreliable in absolute terms) we can reasonably trust that the available incidence data should be broadly representative of the true qualitative patterns. Even in this case, however, we could not find significant footprints of an underlying \"higher\" force of infection. Rather the results appear to be more or less consistent with the Italian age patterns observed from case reports. Indeed the observed long-term oscillation of measles is around three years or more for most Italian regions. Such figures, though substantially different from the biennial oscillations observed for England & Wales, are by no means uncommon \[[@B24]\]; additionally, compared to England & Wales, they are consistent with a much higher value of the average age at infection, and more generally, with the force of infection. Though one must be cautious about these facts, it is also clear, however, that we cannot quickly dismiss them as the consequence of the presumed existence of age biases in reporting rates: it would mean that patterns of under-reporting are capable of also hiding true time patterns. Though this latter possibility can not be excluded a priori(one possibility could be that there is an association between practitioners\' reporting rates and the phase of the epidemic cycle, e.g. an epidemic year vsa non-epidemic year), it appears rather unlikely in most circumstances. Indeed, if the \"true\" percentage age distribution of cases is broadly stationary over time, as we would expect in the pre-vaccination era (a fact that Italian data strongly suggest), then significant (and periodic) time changes in age-specific reporting rates would be needed to effectively mask true incidence patterns. Finally, our modelling work on measles in Italy, shows that the EURO assumption matches rather poorly the serological profile observed for measles in Italy in 1996/7 in the European Sero-epidemiology Network (ESEN) survey \[[@B8],[@B25]\]. Which then was the true force of infection acting in Italy during the pre-vaccination era: the higher EURO FOI, or the lower FOI emerging from Italian case notifications data, or indeed perhaps something in between? Rather than being essentially homologous, as implicit in the EURO conjecture, the possibility that patterns of infection by measles throughout Europe could be and have been largely different (as by the way suggested for rubella by Edmunds et al. \[[@B26]\]) is a stimulating one. Our results provide many indications that infection patterns in Italy could indeed deviate from the \"EURO\" standard. However the uncertainty still present in many factors suggests that more work is urgently needed in order to better understand measles infection patterns in Italy. Be this as it may, we believe that, from a public health perspective, a quite prudent attitude is necessary: knowing that the eradication coverages computed here could just represent lower bounds of the true values, which could be substantially higher, seems to be a sufficient argument for not deviating from the target coverages suggested by WHO. Indeed, the higher R~0~values estimated for the South suggest that, even with achievement uniformly of WHO targets across the regions, elimination of measles will take longer than in the North. Conclusions =========== The results suggest that critical vaccination coverages for elimination are likely to be higher in the south of Italy, precisely where the existing record of coverage is lowest. Substantial efforts are still required if there is to be a realistic hope of achieving WHO targets for measles elimination in Italy, particularly in the south. Notwithstanding this, the evidence does appear to suggest that the force of infection for measles in Italy is indeed somewhat lower than that applicable to other regions participating in the European Sero-Epidemiology Network. More particularly, the evidence suggests that it is probably unlikely that age biases in reporting (suggested elsewhere) could have led to an underestimate of the measles force of infection in Italy. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= PM conceived the work, provided the main theoretical analysis and drafted the manuscript, EMC conducted the time series analysis, JRW undertook the modelling work and SS and MCdA contributed epidemiological insight into the Italian public health context of the work. All authors read and approved the final manuscript. Acknowledgements ================ This paper was partly funded by \'Ministero della Sanità, progetto 1%\'. We thank Gianni Gallo and Donatella Mandolini for useful discussions.	PubMed Central	2024-06-05T03:55:53.991803	2005-2-17	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554971/", "journal": "Popul Health Metr. 2005 Feb 17; 3:1", "authors": [ { "first": "Piero", "last": "Manfredi" }, { "first": "Eugene M", "last": "Cleur" }, { "first": "John R", "last": "Williams" }, { "first": "Stefania", "last": "Salmaso" }, { "first": "Marta Ciofi degli", "last": "Atti" } ] }
PMC554972	Background ========== Ribosomes are vital organelles, which catalyze protein synthesis in all living organisms. Eukaryotic ribosomes consist of four RNA molecules (rRNAs) and 79 different proteins. The mammalian genes encoding the rRNAs are multicopy and clustered at a few loci, whereas those encoding the ribosomal proteins (rp genes) are single copy and scattered throughout the genome \[[@B1]\]. In addition to the functional rp genes, all of which contain introns, mammalian genomes contain many nonfunctional intronless rp pseudogenes \[[@B2]\]. The earliest determinations of mouse rp gene sequences and of transcriptional start points (tsp\'s) revealed a salient feature of rp genes, namely that transcription is initiated at a C residue within a polypyrimidine tract \[[@B3]-[@B5]\]. A recent study by Kenmochi and coworkers \[[@B6]\] has demonstrated that this is a general property of virtually all human rp genes. Because of this novel feature, the rp mRNAs contain a 5\' terminal oligopyrimidine sequence (TOP), which is essential for their translational control \[[@B7]\]. Coordinated expression of the rp genes at transcriptional and post-transcriptional levels is required to ensure a roughly equimolar accumulation of ribosomal proteins. Transcriptional run-on measurements with nuclei of rapidly proliferating cells indicated equivalent loading of RNA polymerases on three unlinked mouse rp genes \[[@B8]\], consistent with the equal abundance of the corresponding rp mRNAs \[[@B9]\]. Moreover, the promoters of these genes were of comparable strength in driving the expression of a common reporter gene \[[@B8]\]. These results suggested that similar promoter strength and mRNA processing efficiency might provide a basis for the coordinated expression of rp genes. However, whether this concept applies to all rp genes or to distinctive subsets of genes is presently unclear. Despite the obvious importance of rp gene expression for cell viability, there have been very few experimental studies of rp promoter architecture and transcriptional regulation in higher eukaryotes. To date, efforts to identify functionally relevant cis-acting regulatory elements and transcription factor binding sites have been made for only 9 mammalian \[[@B8],[@B10]-[@B23]\] and 2 amphibian \[[@B24],[@B25]\] rp genes, less than 15 % of the total rp gene complement. These studies identified regulatory elements in the promoter-proximal regions, both upstream and downstream of the tsp. Some of these elements contained binding sites for known transcription factors, notably GABP, Sp1 and YY1. When the binding of any individual factor was eliminated by a site-specific mutation, transcriptional activity was reduced, but not abolished, indicating that the overall transcription efficiency is determined by a combinatorial effect of multiple factors. No regulatory element common to all rp genes was found, although certain elements were present in several of the genes that were studied. None of these rp genes contained a canonical TATA box in the -25 to -30 region, although some had a \"TATA-like\" A/T-rich sequence, which might bind the general transcription factor TBP under certain circumstances. Because the previous experimental studies were limited to a small subset of rp genes, it is important to know whether the results of those studies can be used to predict special features of promoter architecture that characterize the entire set of rp genes or that could be used to sort the rp genes into classes with similar features. This applies not only to transcription factor-binding sites, but also to the polypyrimidine tract that spans the tsp. As an initiator element, this tract is atypical because the tsp of initiators is normally an adenine residue, flanked on both sides by pyrimidines \[[@B26]\]. To address these issues, I have applied the principle of \"phylogenetic footprinting\", which holds that important regulatory sequences will have a tendency to be evolutionarily conserved and thus revealed by a sequence comparison of corresponding regions of orthologous genes \[[@B27]\]. Some regulatory promoter elements may escape detection by this approach, but a substantial majority will probably be recognized with confidence \[[@B28]\]. Although previously done on a small scale for a few selected rp genes \[[@B19],[@B21],[@B29]-[@B32]\], such an analysis has not heretofore been made for the entire rp gene population. I have therefore compared the promoter regions of all 79 orthologous human and mouse rp genes and have extended the comparisons to include chicken, amphibian and fish counterparts when these sequences were available. I have compared the sequence organization of the rp promoters with that of the promoters of non-rp TOP genes and other genes in the housekeeping category. This analysis has provided some useful insights into the general architecture of mammalian rp promoters and has identified parameters that might coordinately regulate the transcriptional activity of certain subsets of rp genes. Results ======= Compilation of the promoter sequences of orthologous rp genes ------------------------------------------------------------- To make a study of the evolutionarily conserved features of ribosomal protein gene promoters, I first had to assemble a set of orthologous human and mouse rp gene sequences, in which the transcription start sites are reasonably well defined. For all of the human rp genes, annotated sequences are now available owing to the studies of Kato, Kenmochi and coworkers \[[@B6],[@B33]\], who systematically determined the 5\' termini of human rp mRNAs by the oligo-capping method. While these studies indicated some small variations in the exact 5\' ends of individual rp mRNAs, the tsp\'s could usually be specified to within a few nucleotides. Independent determinations of the human tsp\'s by the oligo-capping procedure are also available on the database of transcription start sites (DBTSS) compiled by Sugano and colleagues \[[@B34]\]. The agreement between the two sets of oligo-capping data and the results of primer extension/nuclease protection (PE/S1) experiments is generally very good \[see [Additional file 1](#S1){ref-type="supplementary-material"}\]. In all cases, the human tsp\'s selected in the present study correspond either to the most abundant oligo-capped cDNA or to an observed variant. In contrast to the situation with the human rp genes, accurate determinations of the tsp\'s of mouse rp genes have been made in only a few cases, and therefore the annotations of rp genes in the mouse genome databases are generally incomplete. To overcome this problem, I used the mVISTA alignment program \[[@B35]\] to compare mouse sequences extending several kb upstream of the coding regions with the sequences of the corresponding human orthologues. Fortunately, the strong conservation of sequences in the proximal promoter regions and in the first exons enabled me to identify the probable tsp\'s of most mouse rp genes with confidence. For the most part, the tsp\'s that were predicted by this strategy corresponded exactly to those previously determined by primer extension and/or nuclease protection assays of murine rp genes \[see [Additional file 1](#S1){ref-type="supplementary-material"}\]. Some information on putative tsp\'s of mouse rp genes is available on the DBTSS database. However, the most prominent of these tsp\'s did not agree with the PE/S1 data. Moreover, they were not coincident with the aligned C tsp\'s of the human orthologues, but were frequently at adjacent or nearby T residues. Conceivably, these discrepancies are caused by a technical bias, as is sometimes encountered in the generation of genome-wide full-length cDNA libraries \[[@B36]\]. Therefore, relying on maximum sequence conservation and the best agreement with previous experimental data, I compiled aligned sequences of all 79 pairs of orthologous human and mouse rp genes, and used these sequences for further analysis of the promoter regions. In a preliminary analysis, I used the mVISTA sequence alignment program to examine six to ten kb of 5\' flanking sequence of three pairs of orthologus rp genes (rpS16, rpL30and rpL32). These genes were selected because the mouse orthologues have been previously studied extensively in my laboratory. Scans with a 50 bp window indicated that the conserved (\> 75 % identity) flanking sequences are largely confined to regions within a few hundred base pairs of the tsp. There was very little alignment of the upstream region sequences and many gaps caused by the presence of numerous insertion elements. With this analysis, I detected only one short block of conserved sequence at -2 kb in rpL30(77 % identity over 84 bp), one short block at -1.5 kb in rpL32(82 % identity over 60 bp), and none in rpS16. Neither of the conserved blocks contained any recognizable transcription factor binding sites or other remarkable characteristics. The lack of any credible long-range regulatory elements is consistent with earlier conclusions based on transient transfection experiments with these genes \[[@B8],[@B17],[@B37]\]. Given these results, I restricted the analysis of the full set of rp genes to the transcribed portions of the genes and about one kilobase of 5\' flanking sequence \[see [Additional file 2](#S2){ref-type="supplementary-material"}\]. An initial analysis of the large data set revealed the presence of repetitive sequence insertion elements (sines for human and B1/B2 for mouse) in the 5\' flanks of a very high proportion of the rp genes \[see [Additional file 2](#S2){ref-type="supplementary-material"}\]. Indeed, as seen in Figure [1](#F1){ref-type="fig"}, half of the rp genes contain such an element within 500 bp of the tsp and over 80 % contain elements within 900 bp. The distribution is very similar in the human and mouse rp genes. Both ancestral elements, which are moderately conserved between mouse and human, and lineage-specific elements could be identified. As in the case of the three examples discussed above, very little sequence conservation was evident more than 200 bp upstream of the tsp. Accordingly, for the refined analysis of rp promoter architecture, I analyzed segments extending from 200 bp upstream to 100 bp downstream of the tsp. These segments were scrutinized for the quality of TATA box motifs in the -25 region, for conserved sequences in the initiator region, for transcription factor binding sites, and for the location of the AUG translation start codons. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The location of insertion elements in the 5\'-flanking regions of rp genes. Insertion (ins) elements were identified by the RepeatMasker program in scans of up to one kilobase of sequence 5\' of the tsp, and the location of the element nearest to the tsp recorded for 79 human and mouse rp genes \[see [Additional file 2](#S2){ref-type="supplementary-material"}\]. The distance from the tsp was divided into 200 bp intervals, the percentage of rp genes with an element within each interval determined, and the values plotted cumulatively against the distance from the tsp. ::: ![](1471-2148-5-15-1) ::: Criteria for the annotation of rp promoter sequences ---------------------------------------------------- To evaluate the quality of TATA box motifs, I established criteria based on rules derived from a crystallographic analysis of TBP-DNA complexes \[[@B38]\]. With these rules one can classify each nucleotide in the motif as being \"preferred\" or \"acceptable\" or \"incompatible\" with TBP binding (Figure [2a](#F2){ref-type="fig"}). I considered motifs with a string of 6 or more compatible nucleotides, of which at least 5 are \"preferred\", as being capable of binding TBP with high affinity (+ motifs). Those with a string of 6 compatible nucleotides, 3 or 4 of which are \"preferred\", were considered as possible low affinity sites (+/- motifs). Sequences that do not satisfy these criteria were judged to be incapable of unaided binding to DNA (- motifs). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Criteria for rp promoter annotation. (a). Quality of the TATA box for TBP binding based on a structural analysis of TBP-DNA complexes. Rules adopted for + and +/- ranking and some examples in rp promoters are shown at the right. (b). Criteria used for identification of potential transcription factor-binding sites by the rVISTA program for motifs aligned in human-mouse promoter sequence comparisons and with the FindPatterns program for unaligned motifs. ::: ![](1471-2148-5-15-2) ::: Employing stringent criteria (Figure [2b](#F2){ref-type="fig"}), I used the rVISTA program \[[@B39]\] to search systematically for conserved (aligned) sites that would be predicted to bind the three transcription factors (GABP, Sp1, and YY1) that were identified in earlier experimental studies of rp promoters and also for highly conserved sites that might bind other ubiquitously expressed factors. In addition, I scanned both the human and mouse rp promoter sequences for unaligned optimal sites for the above-mentioned factors and for a motif termed Box A, which was previously implicated in rp gene expression \[[@B19]\]. I used the results of these analyses to complete the annotation of all 79 pairs of aligned human and mouse rp promoter sequences, which can be viewed individually on pages 1 through 80 of a supplementary file \[see [Additional file 3](#S3){ref-type="supplementary-material"}\]. Annotated comparisons of human/mouse promoter sequences ------------------------------------------------------- Illustrative examples of four annotated promoter sequence comparisons are shown in Figure [3](#F3){ref-type="fig"}. All four of these examples illustrate the strong sequence conservation of the non coding portions of exon 1, which is a general feature of the entire rp gene family \[see [Additional file 3](#S3){ref-type="supplementary-material"}\]. The AUG translation initiation codon is located within exon 1 of rpL13a(panel d) and within exon 2 of rpL30(panel b), whereas in rpS18(panel a) and rpS4(panel c), it is at the extreme 3\' end of exon 1. Many rp genes have this latter feature, which may be relevant to the evolution of vertebrate rp promoters (see below). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Examples of comparisons of human and mouse rp promoter sequences from -200 to +100. Exon I is in uppercase letters, highlighted in yellow with the ATG translation initiation codon, if present, highlighted in gray. The sequences evaluated for TATA box quality are highlighted in green. Putative transcription factor-binding sites in aligned sequences are shown above the sequences, highlighted in fuchsia; sites in unaligned sequences are shown above the human or below the mouse sequences and enclosed in carets. An upstream sequence identified as an insertion element is in red letters. Gaps inserted by the alignment program to maximize overall sequence identity are indicated by dashes. (a) rpS18, (b) rpL30, (c) rpS4, (d) rpL13a. In rpL30the elements with known functional relevance are underlined with asterisks. ::: ![](1471-2148-5-15-3) ::: The rpS18promoter contains a conserved TATA (+) motif, while the TATA motifs of rpS4and rpL13awere scored as (+/-) and that of rpL30as (-). Well-conserved (aligned) consensus binding sites for GABP and/or Sp1 are located upstream of the tsp in all four examples; downstream YY1 sites are evident in rpL30and rpL13a. The rpS18and rpL13apromoters also contain conserved consensus binding sites for the ubiquitous AP1/ATF factors. The promoter elements that were found to have functional significance in experimental studies of the mouse rpL30gene \[[@B11]\] are all conserved in the human orthologue (panel b). This includes sites for RFX1 and the Gamma Factor as well as the GABP and YY1 sites. In addition to the conserved sites, a few non conserved (unaligned) GABP and Sp1 sites of uncertain relevance are evident in rpL30and rpL13a. The results of this type of analysis for the full set of rp promoters are tabulated in Table [1](#T1){ref-type="table"}. The identity between human and mouse sequences in the 300 bp segment (-200 to +100) that was analyzed ranged from 50 to 75 % with an average of 61 %. The location of the translation initiation codon was conserved in all except one rp gene (rpL29). In 29 % of the rp genes the AUG codon is at the extreme 3\' end of exon 1 and in 47 % it is in exon 2. Thus, in 3/4 of the rp genes, the genetic elements involved in transcriptional/translational regulation are spatially separated from those with protein encoding function. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Ribosomal protein promoters Quality of the TATA Box Motifs, the Number of Transcription Factor-Binding Sites and the Location of AUG Codons. ::: RP GENE TATA Quality % H/M identity in 300 bp ‡ GABP (5\' of tsp) Sp1 (5\' of tsp) YY1 (3\' of tsp) OTHER (5\' of tsp) AUG † ------------- ------------------ -------------------------------- ----------------------- ---------------------- ---------------------- ------------------------ ----------- ---------- ------ --------------------------- -------- SA \+ \+ 58 1 2 1 ATF, CREB E2 S2 \+ \+ 54 1 1 E2 S3 \+ \+ 60 1 1 1 E1 S3a -- -- 75 2 1 AP1 E1 S4 +/-- +/-- 70 1 1 1 E1e S5 +/-- \+ 62 1 E2 S6 -- -- 56 3 \* 1 1 Box A \* E1 S7 +/-- \+ 56 4 2 Box A E2 S8 +/-- +/-- 60 1 E1e S9 \+ \+ 50 1 1 ATF, CREB, AP1 E2 S10 -- -- 64 1 1 1 1 Nrf1 E2 S11 +/-- +/-- 53 1 1 1 1 Box A, ATF, CREB, AP1 E1 S12 \+ \+ 62 1 Nrf1 E2 S13 -- +/-- 61 1 1 AP1 E1 S14 -- -- 62 3 \* 1 AP1 E2 S15 +/-- +/-- 52 1 1 1 Nrf1 E1e S15a +/-- +/-- 59 1 2 1 Box A E2 S16 -- +/-- 74 2 \* 2 Nrf1 E1 S17 +/-- \+ 60 1 1 2 1 E1e S18 \+ \+ 64 3 1 ATF, CREB E1e S19 +/-- +/-- 72 1 1 Box A (2) E2 S20 \+ \+ 63 1 1 E1e S21 \+ +/-- 52 3 1 E2 S23 -- -- 65 1 1 1 E1e S24 \+ \+ 59 1 1 2 E1e S25 -- -- 56 1 1 E1e S26 \+ \+ 53 1 E1e S27 \+ \+ 71 2 1 1 E1 S27a -- -- 57 1 1 1 E2 S28 \+ \+ 67 1 1 1 1 AP1 E1 S29 -- -- inc E1 S30 -- -- 68 4 1 ATF, CREB E2 L3 \+ \+ 56 2 1 1 1 E1e L4 \+ \+ 68 2 E1e L5 \+ \+ 62 1 1 E1e L6 +/-- +/-- 62 1 1 1 E2 L7 +/-- +/-- 69 1 1 Nrf1 E1 L7a \+ \+ 51 2 1 1 1 *Box A\, B E1e L8 +/-- +/-- 50 1 1 1 1 1 AP1 E2 L9 -- -- 57 1 ATF, CREB (2) E2 L10 \+ \+ 60 1 E2 L10a \+ \+ 55 Box A, Nrf1 E1 L11 +/-- +/-- 64 1 1 1 E2 L12 \+ \+ 73 1 Box A E1 L13 -- +/-- 55 1 1 E2 L13a +/-- +/-- 67 2 2 1 ATF, CREB, AP1 E1 L14 +/-- +/-- 58 2 1 Nrf1 E1e L15 -- -- 66 1 1 1 2 E2 L17 -- -- 69 1 1 1 Box A E2 L18 -- -- 51 1 2 ATF, CREB, AP1 E1e L18a -- -- 67 1 1 E1 L19 +/-- +/-- 65 1 1 E1 L21 -- -- 62 1 1 E2 L22 -- -- 61 E1 L23 \+ \+ 64 1 1 1 E1 L23a +/-- +/-- 63 1 Box A E1 L24 +/-- +/-- 56 1 1 E1 L26 +/-- +/-- 64 1 1 2 Box A E2 L27 -- -- 66 3 1 1 E2 L27a -- -- 56 1 1 1 Box A E1e L28 \+ +/-- 56 2 1 1 1 E2 L29 +/-- +/-- 68 2 1 1 E1e//2 L30 -- -- 60 2 \* 1 1 \* RFX1\* Gamma\* E2 L31 -- -- 62 1 1 E2 L32 -- -- 68 1 \* 1 \*** 1 \* Gamma \* E2 L34 -- -- 55 1 1 2 1 E2 L35 -- -- 59 2 4 1 E1e L35a -- -- 64 1 1 1 E2 L36 +/-- \+ 51 1 1 E2 L36a \+ \+ 62 1 2 2 1 E1e L37 \+ -- 60 2 2 1 1 2 E1e L37a +/-- +/-- 52 1 Nrf1 E1e L38 +/-- -- 62 2 E2 L39 -- +/-- 61 1 1 1 1 E1e L40 -- -- 62 2 3 ATF, CREB E2 L41 \+ \+ 54 1 1 1 ATF, CREB, AP1 E2 LP0 \+ \+ 59 1 E2 LP1 +/-- +/-- 62 1 E1 LP2 +/-- -- 55 1 E2 Bolded numbers indicate that the sites are aligned in orthologous human and mouse genes. An asterisk indicates that there is experimental evidence for the functionality of one or more of the sites. ‡ From --200 to +100 relative to the tsp. † E1, within Exon 1; E1e, at the extreme 3\' end of Exon 1; E2, within Exon 2. The locations are conserved in all rp genes except rpL29. ::: Contrary to previous impressions based on an incomplete set of rp genes, the rp promoters cannot generally be classified as \"TATA-less\". Thirty-five percent of the promoters contain, in the -25 region, a TATA box that should theoretically bind TBP with high affinity. An additional 25 % have an A/T-rich tract in this region, which might bind TBP with lower affinity, and the remainder would not be predicted to bind TBP without help from other proteins. For the most part, the TATA box quality is well conserved between the two mammalian species: the assessed quality of human and mouse TATA motifs was the same in 82 % of the rp promoters. The prevalence in rp promoters of evolutionarily conserved GABP, Sp1 and YY1 binding sites is readily apparent from these results. Conserved upstream GABP and Sp1 sites are present in 54 % and 48 % of the rp promoters, respectively. Conserved downstream YY1 sites are present in 52 % of the rp promoters. The ratio of aligned to unaligned sites in the human rp promoters is approximately 2:1 for GABP, 1:1 for Sp1 and 4:1 for YY1. The occurrence of unaligned sites in the mouse rp promoters is similar to that in the human rp promoters \[see [Additional file 3](#S3){ref-type="supplementary-material"}\]. It is noteworthy that 76 % of the rp promoters contain at least one conserved upstream GABP and/or Sp1 site within 200 bp of the tsp. If unaligned sites are included, the proportion of human rp genes with an upstream GABP and/or Sp1 site is 92 %. Conserved consensus motifs for other ubiquitous transcription factors are present in a much lower proportion of the rp promoters, namely 12 % for the Box A-binding factor, 11 % for AP1, 10 % for ATF/CREB and 8 % for Nrf1. The rp gene initiator --------------------- The existence of a novel polypyrimidine initiator sequence in which the conventional A residue at the tsp is replaced by a C residue is well known. Moreover, the roles of this sequence in rp gene transcription and rp mRNA translation have been previously demonstrated experimentally for a few rp genes \[[@B7],[@B40]-[@B42]\]. What is not known is whether there is a consensus initiator sequence that characterizes the entire rp gene set. To address this issue, I used the pairwise alignments of human and mouse orthologues to produce an occupancy matrix for positions -8 to +10 of the initiator \[see [Additional file 4](#S4){ref-type="supplementary-material"}\]. With this matrix and the standard consensus rules, I determined that the rp consensus initiator sequence is (Y)~2~C^+1^TY(T)~2~(Y)~3~. A striking result is that T\'s are strongly preferred over C\'s at positions +2, +4 and +5. This preference, which is graphically illustrated in Figure [4](#F4){ref-type="fig"}, might be related to a transcriptional function of the initiator or a translational function of the TOP or to both functions. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### The consensus initiator sequence of mammalian rp genes. Seventy-nine pairs of orthologous human and mouse rp gene sequences were compared at positions -8 to +10 and the occurrence of each nucleotide or pair of nucleotides depicted by the height of the letters: A, G, C. T, Y = C/T, R = A/G, W = A/T, K = G/T, S = C/G, M = C/A. The tsp is the C at position +1. ::: ![](1471-2148-5-15-4) ::: The extent of conservation of rp promoter features in non-mammalian vertebrates ------------------------------------------------------------------------------- To determine the features of rp promoter architecture that are conserved over large evolutionary periods, I compared the promoter sequences of five chicken, six amphibian and five fish rp genes to their mammalian orthologues using the Clustal W multiple sequence alignment program. An example of such an alignment for rpS3(Figure [5](#F5){ref-type="fig"}) shows strong conservation of both the coding and non-coding portions of the first exon, the TATA box motif, the initiator sequence, and a downstream YY1 site that spans the translation initiation codon. In contrast, the overlapping GABP and Sp1 sites, which are aligned in the human and mouse promoters, are not conserved in the amphibian and fish promoters. However, each of these promoters contains an unaligned consensus site for GABP or Sp1. A summary of the multi-sequence analysis for 11 rp genes (Table [2](#T2){ref-type="table"}) indicates that the extent of conservation observed for rpS3is fairly typical, although some small variations are evident. The conformance to the consensus initiator sequence is the same (90 %) for mammals and lower vertebrates. Yet, the particular C residue within the initiator that is used as the major tsp may differ slightly. Also the adjudged quality of the TATA box for TBP binding is not always the same. Nevertheless, except for the location of upstream transcription factor-binding sites, the general features of rp promoter architecture are usually well conserved over large evolutionary distances. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### *Comparison of rpS3promoter sequences in human (Hs), mouse (Mm), Xenopus laevis (Xl) and Fugu rubripes (Fr). Alignment by the ClustalW program with 100% and 75% identities enclosed in boxes. The first exon is highlighted in yellow with the ATG translation initiation codon in gray. Putative binding sites for TBP, GABP, Sp1 and YY1 are highlighted in green, red, blue and pink, respectively. ::: ![](1471-2148-5-15-5) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Comparison of promoter features in mammals and lower vertebrates ::: Gene* Species Initiator TATA GABP Sp1 YY1 Box A AUG Acc. No. ---------- ------------- --------------- ------------ ---------- --------- --------- ----------- --------- ------------------------------- rpS3 Hs/Mm `TTCCTTTCCT` + 1 1 1 E1 AB061838, NM\_012052 Xl `GC-C------` + † 1 E1 Z34530 Fr `C---------` + † 1 E1 X97794 rpS6 Hs/Mm `SCCTCTTTTY` -- 3 1 E1 X67309, D28348, Z54209 Xl `C---C---C-` -- E1 AF020551 rpS7 Hs/Mm `SYCTCTTYCT` +/-- + 1 E2 Z25749, AB055774, NM\_011300 Xl `-CC-------` -- 1 E2 X71081 Fr `----------` + 1 E2 X94942 rpS15 Hs/Mm `WYCTYTTCYG` +/-- 1 1 1 E1e M32405, AB055776, NM\_001018 Gg `C--C------` +/-- 1 † 1 E1e D10167 rpS24 Hs/Mm `TYCTCTTTTC` + 1 E1e AB062069, U12202, NM\_011297 Xl `----TCC--T` + 1 E1e M33517 Fr `-----C---T` +/-- 1 E1e AJ001398 rpL5 Hs/Mm `GYCYTTTTCC` + 1 E1e AB055762, NM\_00969, mCG13589 Gg `-G-C------` -- 1 E1e D10737 rpL7a Hs/Mm `TTCTYTCTCC` + 2 1 1 E1e X61923, X54067 Gg `GCCC--T-TA` +/-- 1 1 E1e X62641 Fr `-C------GC` +/-- E1e Y15171 rpL14 Hs/Mm `YYCTTCTCGC` +/-- 2 E1e AB061822, mCG22708 Xl `--C--T--T-` -- E1e X06552 rpL18 Hs/Mm `STCTTTCCGG` -- 1 2 E1e AB061825, mCG132477 Xl `TCC-----TC` +/-- † 1 E1e X05025 Om `TCC---T-CC` -- † E1e AF240376 rpL30 Hs/Mm `TTCCTTTCTC` -- 2 1 E2 AB070559, K02928 Gg `C--C-----G` -- 1 E2 D14521 rpL37a Hs/Mm `TCCTYTYYGG` +/-- 1 E1e NM\_000998, NM\_009084 Gg `--C-----CT` +/-- 1 E1e D14167 Hs/Mm: Homo sapiens/Mus musculus; Gg: Gallus gallus; Xl: Xenopus laevis; Fr: Fugu rubripes; Om: Oreochromis mossambicus (tilapia). For the initiator sequences, [C]{.underline} represents an alleged tsp based on various types of experimental evidence. For the transcription factor binding sites, only those sites that are conserved (aligned) between human and mouse are listed. A † symbol indicates that the promoter contains an unaligned site for the factor. Designation of AUG locations as in Table 1. ::: Comparison of rp promoter features with those of non-rp TOP genes and other housekeeping genes ---------------------------------------------------------------------------------------------- It is of interest to know which features of rp promoter architecture are specific and which might be common to other ubiquitously expressed genes. To this end, I examined the promoters of two additional sets of genes. One set consisted of non-rp genes that also produce translationally controlled mRNAs with 5\'-terminal oligopyrimidine tracts (non-rp TOP genes). At the present, there are nine known genes in this category for which the tsp of at least one orthologue has been experimentally determined \[see [Additional file 5](#S5){ref-type="supplementary-material"}\]. The non-rp TOP set contains genes encoding translation elongation factors (eEF1A1, eEF1B and eEF2), RNA-binding proteins (PABPc1 and hnRNPA1), a major nucleolar protein (nucleoplasmin/B25), a protein with tubulin-binding properties (TCTP/p23), and two genes that do not encode proteins, but rather have small nucleolar RNA-encoding sequences embedded within their introns (gas5and U17HG). Annotated aligned sequences of the nine human and mouse non-rp TOP genes are presented on pages 1 through 9 of a supplementary file \[see [Additional file 6](#S6){ref-type="supplementary-material"}\] and the results are summarized in Table [3](#T3){ref-type="table"}. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Non-rp top genes ::: GENE TATA Quality % H/M identity in 400 bp \* GABP SP1 YY1 Other Initiator -2 to +7 † AUG ----------- ------------------ --------------------------------- ---------- --------- --------- ----------- -------------------------- --------- ---------- ------------- ----- eEF1A1 \+ \+ 44 1, 2 `TTCTTTTTC` E2 eEF1B \+ \+ 68 2 1 `TCCTTTTTY` E1 eEF2 \+ \+ 55 4 2 `KTCTCYYCC` E1e PABP cl \+ \+ 66 2, 1 `TTCCCCTTC` E1 hnRNPA1 -- -- 71 2 `YTCCTTTCT` E1 B23 \+ \+ 73 1, 1 BoxA `TTCCYTGGC` E1 Tpt1 \+ \+ 67 1 1 1 `GCCTTTTCC` E1 gas5 \+ \+ 55 1, 2 `RKCYTTTCG` \-- U17HG \+ \+ 43 1 `STCYYTYTW` \-- \* From -300 to +100 relative to the tsp. † The bolded Cis the tsp. Other symbols are as in Table 1. ::: There are some notable differences in the promoter architecture of rp genes and non-rp TOP genes. First, GABP- and YY1-binding motifs, which are prevalent in the rp promoters, are rarely found in the non-rp TOP promoters (Table [4a](#T4){ref-type="table"}). Second, eight of the nine non-rp TOP promoters have conserved TATA boxes that would be expected to bind TBP with high affinity, whereas only a third of the rp promoters have such TATA boxes (Table [4b](#T4){ref-type="table"}). These differences were considered to be statistically significant when analyzed by Fisher\'s exact test (Table [4](#T4){ref-type="table"}). The initiator sequences of the non-rp TOP genes resemble the rp consensus (89 % identical from -2 to +7) except for the roughly equal occurrence of C\'s and T\'s at +2. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Comparison ofthe promoters ofrp genes, non-rp top genes, and other housekeeping genes ::: (a) Occurrence of transcription factor binding sites: proportion of genes with at least one binding site. --------------------------------------------------------------------------------------------------------------- -------------- ---------------------- ------------------------ ----------- ----------- Aligned Total Aligned Total Total GABP (5\' of tsp) 54 % 68 % 22 % \* 33 % 35% SP1 (5\' of tsp) 48 % 70 % 89 % 89 % 90 % GABP or SP1 76 % 92 % 100 % 100 % 95 % YY1 (3\' of tsp) 52 % 59 % 11 % † 11 % † 10 % † (b) Potential for TBP binding to TATA boxes in the -25 region: proportion of genes in each category. TATA BOX QUALITY RP GENES NON-RP TOP GENES HOUSEKEEPING GENES \+ 35 % 89 % ‡ 30 % +/-- 25 % -- 20 % -- 39 % 11 % ‡ 50 % Based on analyses of 79 pairs of orthologous human and mouse rp genes, 9 pairs of orthologous non-rp TOP genes and 20 randomly selected human or mouse housekeeping genes. The symbols \, †, and ‡ indicate the percentages considered to be significantly different from the corresponding values for rp genes according to a Fisher\'s two sided exact test of the three sets of data. \: p = 0.086; †: p = 0.031 and 0.00075; ‡: p = 0.0029. For the rp genes, the TATA box quality was scored as: \+ when both orthologues are + or when one is + and the other +/-, +/-- when both orthologues are +/-- or when one is + and the other is --, and -- when both orthologues are -- or when one is - and the other is +/--. For the non-rp TOP genes, both orthologues were either + or --. ::: The second set of ubiquitously expressed genes consisted of 20 housekeeping genes randomly selected from the eukaryotic promoter database \[see [Additional file 7](#S7){ref-type="supplementary-material"}\]. The promoters of these genes also have an under-representation of YY1 sites compared to the rp promoters, and like both the rp and non-rp TOP genes, contain abundant motifs for Sp1 (Table [4a](#T4){ref-type="table"}). The proportion of these genes that have TATA boxes with TBP-binding capability is similar to that observed for the rp genes (Table [4b](#T4){ref-type="table"}). Discussion ========== The foregoing analysis of 79 mammalian ribosomal protein genes has revealed several features of rp promoter architecture, some of which are largely conserved over long periods of vertebrate evolution (about 450 million years) and others that are strongly conserved only in mammals. (about 90 million years). One highly conserved feature, present in over 3/4 of the rp genes, is the separation by an intron of the sequences involved in transcriptional and/or translational regulation from the sequences with protein-encoding function. In 47 % of the rp genes, the AUG translation initiation codon is in exon 2, and in 29 % of the genes, it is at the extreme 3\' end of exon 1. It would seem that at an early stage of vertebrate evolution, these regulatory sequences were appended as discrete units to the loci containing the protein-encoding sequences. The polypyrimidine tract that spans the tsp is present in all vertebrate rp genes. This tract can function as a transcriptional initiator \[[@B40],[@B41]\] and also embraces the TOP sequence, which is essential for the translational control of rp mRNAs \[[@B7],[@B42]\]. Based on the assignments of human and mouse tsp\'s used in the present study, the average lengths of the polypyrimidine tracts and TOP sequences are 12.2 bp and 8.2 bp, respectively. A compilation of conserved sequences in the -8 to +10 regions of orthologous human and mouse rp genes revealed the consensus sequence (Y)~2~C^+1^TY(T)~2~(Y)~3~. Thus, in addition to the C at the tsp, there is a clear preference for T over C at positions +2, +4, and +5. This preference may reflect a structural bias for transcription, e.g., ease of strand separation, or for translation, e.g., affinity of rp mRNAs for a putative repressor. The presence of transposable element residues in the 5\' flanks of the rp genes is noteworthy. Half of the rp genes contain an element (sines for humans, B1/B2 for mouse) within 500 bp of the tsp, and over 80 % of the genes contain an element within 900 bp. Some elements are moderately conserved between mouse and human, but most appear to be lineage-specific. These elements are unlikely to have any specific role in rp promoter function. They may be passively tolerated because the vast majority of conserved 5\' sequence is confined to within 200 bp of the tsp. Within a segment from -200 to +100, the sequence identity between human and mouse rp orthologues ranges from 50 to 75 % with an average of 61 %, whereas the sequence match beyond -200 is of borderline significance. The observation that 35 % of the rp promoters contain a TATA box motif at -25, which would be predicted to bind TBP with high affinity, and that an additional 25 % possess A/T-rich motifs, which might bind TBP with lower affinity, was unexpected. The assessed quality of these motifs for TBP binding, made according to rules established by a detailed structural study of TBP-DNA complexes \[[@B38]\], was largely conserved between human and mouse rp orthologues. Some of the promoters classified as poor (-) binders, e.g., rpL32, might bind TBP weakly without help from an additional protein \[[@B43],[@B44]\], so that the true proportion of rp promoters with TBP-binding capability might actually be greater than 60 %. Thus, contrary to earlier views based on an analysis of a small subset of rp genes, many of the rp promoters should not be classified in the \"TATA-less\" category. When human and mouse rp promoter regions from -200 to +100 were scanned for conserved (aligned) transcription factor-binding sites with the rVISTA program \[[@B39]\], which is based on consensus sequences and matrix tables in the TRANSFAC database, three ubiquitously expressed factors that had previously been implicated in rp promoter activity predominated. Using high stringency criteria, I detected aligned GABP- and Sp1- binding sites upstream of the tsp and aligned YY1-binding sites downstream of the tsp in approximately half of the rp promoters. The occurrence of aligned motifs for other ubiquitously expressed factors is considerably less, i.e., only about 10 % for any single factor. Whereas Sp1 sites are also commonly found in the promoters of many housekeeping genes, including the non-rp TOP genes, the prevalence of GABP and YY1 sites appears to be a more prominent feature of the rp promoters. GABP is a heteromeric protein consisting of an ETS family member, which has DNA-binding capability, and an ankyrin repeat-containing subunit, which greatly improves the stability of GABP-DNA interactions \[[@B45],[@B46]\]. Two-thirds of the human rp promoters contain one or more potential GABP-binding sites upstream of the tsp, 79 % of which are perfectly aligned in the orthologous mouse rp promoters. Previous experimental studies have implicated GABP as a positive transcriptional regulator of the mouse rpL30and rpL32genes \[[@B10],[@B14]\], the human rpS14and rpS6genes \[[@B20],[@B22]\], and the Xenopus rpL18gene \[[@B24]\]. The GABP sites that positively contribute to transcriptional activity are generally located upstream of the tsp, although in some cases, sites overlapping the initiator or downstream of the tsp may also be relevant. In the mouse rpS16gene, GABP binding at the initiator decreases transcription activity both in vivo and in vitro \[[@B12]\]. For simplicity, only upstream, presumably positively acting, GABP sites were included in the tabulation. YY1 is a zinc finger-containing protein with a variety of gene-specific functions, including transcriptional activation and repression, positioning of RNA polymerase II, and chromatin modification \[[@B47],[@B48]\]. Fifty-nine percent of the human rp genes contain at least one YY1 site downstream of the tsp, 88 % of which are conserved in the mouse orthologues. Two functionally relevant binding sites for YY1 (originally termed \"delta\") were detected downstream of the tsp in mouse rpL32\[[@B15]\]. In mouse rpL30, a downstream YY1 interaction also contributed positively to transcriptional activity, but only when a strong upstream interaction with GABP was eliminated \[[@B11]\]. Interestingly, the vast majority of YY1 sites are located downstream of the tsp, and therefore, for simplicity, only downstream YY1 sites were included in the tabulation. The alignment of mammalian YY1 sites is frequently preserved in chickens, frogs and fish, in contrast to the GABP and Sp1 sites, which rarely have aligned counterparts in those species (Table [2](#T2){ref-type="table"}). The aligned downstream site in Xenopus rpL18was shown to interact with the frog homologue of YY1, but the functional significance of this interaction was not demonstrable with the reporter constructs that were used in these experiments \[[@B25]\]. The diverse activities of YY1 in different promoter contexts and its propensity for interactions with a wide variety of proteins \[[@B49]\], have led to the idea that it may have multiple mechanistic roles in transcriptional regulation. Which of these roles apply to the rp promoters remains to be established. Based on the results of the analysis of rp promoter sequences, I have sorted the different rp promoters into eight classes according to whether they possess conserved binding sites for the three prevalent transcription factors, GABP, Sp1 and YY1 (Figure [6](#F6){ref-type="fig"}). Only 10 of the rp promoters do not appear to contain conserved binding sites for any of the three factors, and considering the high stringency used for the analysis, the true number may even be lower. Moreover, 8 of these 10 promoters contain non-conserved GABP, Sp1 or YY1 sites and/or conserved sites for other ubiquitously expressed factors. TATA boxes with predicted affinity for TBP are distributed among the promoters of all eight classes. This classification might prove useful for interpreting the results of experiments in which cell-specific or physiologically induced variations in the expression of different subsets of rp genes are observed. In addition, when more extensive data on the relative transcription rates of rp genes or the relative abundance of rp mRNAs become available, this classification might help account for any as yet undetected variability. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Classification of rp gene promoters. The data in Table 1 were used to classify the rp promoters into eight groups according to whether they contain aligned sites for GABP, Sp1, YY1, or various combinations of these factors. The promoters highlighted in green are those in which the TATA box quality of both orthologues was ranked as + or in which one ranked as + and the other as +/-. The promoters highlighted in yellow are those in which both orthologues ranked as +/- or in which one ranked as + and the other as -. Non highlighted promoters are those in which both orthologues ranked as -- or in which one ranked as +/- and the other as -. ::: ![](1471-2148-5-15-6) ::: It is worth noting that some ribosomal proteins can also have extraribosomal functions \[[@B50],[@B51]\]. Among the mammalian proteins that have been demonstrated or presumed to have additional functions are rpS3, rpS4, rpL5, rpL7, rpL10, rpL13a, rpLP0 and rpLP2. While, collectively, the promoters of the genes encoding these proteins do not fall into any particular class, most contain conserved binding sites for one or more of the three prevalent transcription factors and have TATA boxes with TBP-binding capability. Recently, the protein RACK1/Asc1p, which had previously been implicated in various signal transduction processes, was shown to have the properties of an authentic 40S ribosomal protein \[[@B52]\]. When I analyzed the promoter structures of the orthologous human (NM\_006098) and mouse (NM\_088143) genes that encode this protein, I observed several features in common with the rp promoters. The tsp of the mammalian RACK1gene is embedded in a polypyrimidine tract that conforms perfectly to the rp initiator consensus sequence. Moreover, the promoter contains a TATA box of +/- quality, an aligned upstream binding motif for Sp1 and an aligned downstream motif for YY1, but no readily detectable motifs for other ubiquitous transcription factors. Interestingly, the features of this promoter resemble those of the rpL13agene, which also encodes a protein with apparent pleiotropic function. This analysis has highlighted features of rp promoter architecture that are shared by a high proportion of the rp genes. The evolutionary conservation of these features lends strong support to their functional relevance. Yet, superimposed on this general design are variations that confer certain idiosyncratic characteristics on each promoter. There does not seem to be a single master switch that co-regulates all rp genes at the transcriptional level. Rather, the rp promoters are tuned to respond to a combination of factors, including components of the general transcription machinery, a relatively small group of sequence-specific transcription factors, and modifiers of chromatin structure. The inherent functional redundancy and lack of dependence on any single factor are useful design features for genes that must be expressed in a broad spectrum of cell types and environmental situations. Conclusions =========== A sequence comparison of the promoter regions of all 79 orthologous human and mouse ribosomal protein genes has revealed several evolutionarily conserved features that are characteristic of a high proportion of the rp gene set. One such feature, which is also evident in the rp genes of lower vertebrates, is the separation by an intron of the sequences involved in transcriptional and translational regulation from the sequences with protein encoding function. Another conserved feature is the polypyrimidine initiator, which in mammals conforms to the consensus (Y)~2~C^+1^TY(T)~2~(Y)~3~. Contrary to previous impressions based on studies of a small subset of rp genes, the majority of rp promoters contain a TATA box or an A/T-rich motif at -25 that should theoretically have TBP-binding capability. Similarly, approximately half of the rp promoters contain conserved binding motifs for transcription factors previously implicated in rp gene expression, namely upstream GABP and Sp1 sites and downstream YY1 sites. Conserved motifs for other ubiquitous factors occurred much less frequently. Transposable element residues within 900 bp of 5\'-flanking sequence were present in over 80 % of the rp genes; very little sequence conservation was evident more than 200 bp upstream of the tsp. Some of these architectural features were seen to be specific for rp promoters. From the results of this analysis, it was possible to sort the rp promoters into eight classes according to their possession of putative binding sites for GABP, Sp1 and YY1, and also to specify which promoters should have intrinsic affinity for TBP. This classification might prove useful for interpreting the results of experiments in which cell-specific or physiologically induced variations in the expression of different subsets of rp genes are observed. Methods ======= The rp gene sequences were extracted from three database sources \[see [Additional file 2](#S2){ref-type="supplementary-material"}\]. The vast majority of sequences were obtained from the UCSC database \[[@B53]\], which conveniently uses uppercase and lowercase letters to distinguish exon and flanking/intron sequences, respectively. The remaining sequences were obtained from the ncbi \[[@B54]\], Celera \[[@B55]\] and the recently available RPG \[[@B56]\] databases. For alignment of human and mouse rp promoter sequences I used mVISTA \[[@B57]\], which is based on the AVID global alignment program \[[@B58]\]. The locations of repetitive insertion sequence elements were determined by the RepeatMasker program supplied with mVISTA and, in many cases, corroborated by ncbi annotations. Transcription factor-binding sites were detected with the rVISTA program and the FindPatterns tool of the GCG program. The analysis of non-rp TOP genes was made similarly to that of the rp genes. A graphic representation of the rp initiator consensus sequence was obtained with the Weblogo program \[[@B59]\]. For alignment and viewing of three or more orthologus rp genes, I used the ClustalW program \[[@B60]\] and SeqVu shareware l.0.1 (Garvan Institute, Sydney Australia). List of abbreviations ===================== rRNA, ribosomal RNA; rp, ribosomal protein; tsp, transcriptional start point; TOP, terminal oligopyrimidine sequence; TBP, TATA-binding protein. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 ST1: Identification of the transcriptional start points (tsp\'s).Comparison of experimental determinations of the tsp\'s of human and mouse rp genes. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 ST2: Promoter region sequences of rp genes.Sources of rp sequences, amount of analyzed sequence 5\' of tsp, and locations of insertion elements nearest the tsp. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 Aligned promoter sequences of rp genes.Annotated sequence alignments of 79 orthologous human and mouse rp promoters and a key to the annotation. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 4 ST3: Characterization of the rp initiator.Occupancy matrix for determination of the consensus sequence of the mammalian rp initiator. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 5 ST4: Promoter region sequences of non-rp TOP genes.Sources of non-rp TOP gene sequences, the amount of analyzed sequence 5\' of the tsp, and location of insertion elements nearest the tsp. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 6 Aligned promoter sequences of non-rp TOP genes. Annotated sequence alignments of 9 orthologous human and mouse non-rp TOP promoters. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 7 ST5: Housekeeping gene promoters.List of 20 housekeeping gene sequences extracted from the Eukaryotic Promoter Database, which were analyzed for GABP, Sp1 and YY1 binding sites and for TATA box quality. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ The author would like to thank Dr. Tom Moloshok of the FCCC Bioinformatics Facility for his valuable help with various bioinformatics tools and Dr. Sam Litwin for help with statistical analyses. He is also indebted to Drs. Oded Meyuhas, Francisco Amaldi, Ken Zaret and John Burch for their critical reviews and comments concerning this paper. This research was supported by a grant from The National Institutes of Health (CA006927) and an appropriation from the Commonwealth of Pennsylvania. Support to R.P. Perry from the Stanley P. Reimann Endowed Chair in Research is also acknowledged.	PubMed Central	2024-06-05T03:55:53.998570	2005-2-13	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554972/", "journal": "BMC Evol Biol. 2005 Feb 13; 5:15", "authors": [ { "first": "Robert P", "last": "Perry" } ] }
PMC554973	Background ========== Favorable dietary habits promote health, whereas unfavorable habits are linked to development of various chronic diseases, such as cardiovascular diseases, type-2 diabetes and dental caries. An excess intake of energy, excess refined sugars and saturated fatty acids, and too little fibers and antioxidants can contribute to the development of chronic diseases \[[@B1]-[@B4]\]. In general a favorable dietary pattern is characterized by a rich content of fruits, vegetables and fiber-rich cereal products and a low content of fat and refined sugar. A diet rich in saturated fat and sugar, but low in fiber, fruit and vegetables is considered unfavorable \[[@B5]\]. The public health message during the latest decades, i.e. to reduce total fat intake, especially saturated fat, and to eat more vegetables, fruits and whole meal cereals, has been constant \[[@B6],[@B7]\]. An individual\'s dietary pattern is largely set by cultural traditions and availability, but both physiological and psychological influences have been described \[[@B8]\]. However, understanding what determines an individual\'s dietary preferences and how to change his/her behavior is limited. The medical sociologist, Aaron Antonovsky \[[@B9]\] was of the opinion that approaches to health and disease can be either salutogenic (origins of health) or pathogenic (origins of disease). He showed health to be connected to an individual\'s \"Sense of coherence\"(SOC), and \"generalized resistance resources\" (GRRs), such as income, education, ego strength, knowledge, which would provide energy to combat various stressors, and thus influential factors in the salutogenic model. Central to sense of coherence are comprehensibility (the cognitive component), manageability (the instrumental component), and meaningfulness (the emotional component). Individuals with low SOC scores are reported to have a higher frequency of various diseases \[[@B10]-[@B13]\], some of which are also linked to dietary habits \[[@B14]-[@B17]\]. An association between SOC and food selection/eating pattern is indicated by the reported (i) higher sucrose intake in adolescents with low SOC scores \[[@B14]\], (ii) lower ability to change dietary habits and lose weight in over-weight individuals with moderate to low SOC scores \[[@B15]\], and (iii) better blood sugar control in type-2 diabetics with high SOC scores, whereas those with low scores have poorer sugar balance \[[@B16]\]. The aim of the present study was to evaluate the association between dietary intake and SOC in adults. The hypothesis was that low SOC scores were associated with less favorable habits and vice versa. Methods ======= Study population ---------------- The Northern Sweden MONICA Project was performed in Västerbotten and Norrbotten, the two most northerly counties in Sweden, with a total population of around half a million inhabitants and a high prevalence of cardiovascular disease. Surveys were performed in 1986, 1990, 1994 and 1999 \[[@B18]\]. In each of the age groups 25--34, 35--44, 45--54 and 55--64 years 250 men and 250 women were randomly selected and invited to participate. In 1994 and 1999, the age group 65--74 years was added. Every person selected was invited to an examination at the nearest health care centre. In the 1999 survey, all participants from the three previous cohorts (n = 5,129), as well as a new cross-sectional sample of 2,500 randomly selected people, were invited. In total 6,000 individuals (71.8 %) participated. For the present paper all individuals aged 75 years or older (n = 206) were excluded. In the MONICA project various cardiovascular risk factors, including weight, height and education level, were monitored. Education was grouped into three levels: primary school (≤ 9 years), secondary school (10--12 years) and university education (≥ 13 years). Body mass index (BMI) was calculated as the ratio between weight (kg) and height^2^(m). BMI was grouped as \<20 underweight, 20--24.9 normal weight, 25 -- 26.9 moderate overweight, 27 -- 29.9 overweight, and ≥ 30 obese. Special forms for questions about food intake and sense of coherence were added. Recording of sense of coherence (SOC) ------------------------------------- Sense of coherence was monitored using the 13-item questionnaire by Antonovsky \[[@B9],[@B19]\]. Participants who had not answered all 13 questions were excluded. The SOC scores, with a theoretical range from 13 to 91, were calculated as described previously \[[@B9],[@B10],[@B19]\]. High scores denote strong SOC. Recording of dietary intake --------------------------- The subjects were requested to complete a self-administered, semi-quantitative and optically readable food frequency questionnaire \[[@B20]\]. Frequencies of consumption of 84 food items were reported on an increasing, nine-level scale, including never, maximum once a month, 1--3 times per month, once a week, 2--3 times a week, 4--6 times a week, once a day, 2--3 times a day, and 4 or more times a day. The questionnaire included eight questions on various types of fats, nine on milk and other dairy products, eight on bread and cereals, ten on fruit, greens and root vegetables, and nine on soft drinks and sugar-containing snacks, and five questions on spirits, wine and beer. Twenty nine of the remaining 35 questions recorded intake of potatoes, rice, pasta, meat and fish, and six varied items, such as salty snacks, coffee, tea and juice. The respondents indicated their average portion of a) potatoes/pasta/rice, b)vegetables and c) meat/ground meat/sausages/fish by comparison of four color photos illustrating four plates with increasing portion sizes of potato, vegetables and meat. For the other food items, we assumed gender- and age-standard portion sizes \[[@B20]\]. The reported frequencies of consumption were converted to number of intakes per day, and energy and nutrient intakes were calculated by multiplying these frequencies by portion size and energy or nutrient content from a food composition database from the Swedish National Food Administration. The energy and nutrient contents were calculated using the software MAT\'s (Rudans Lättdata, Sweden). Participants who had more than 10% missing answers were excluded. Single missing answers in sections where normally only one of several options is consumed frequently, such as type of milk, were not grounds for exclusion. Nutrient intake could not be estimated if portion size indication was missing. Statistical methods ------------------- Data were analyzed separately for men and women using the SAS System for Windows (Release 8.02, SAS Institute, Cary, NC). Dietary variables were logarithmically transformed to improve normality. Univariate Pearson correlation coefficients were calculated between ln-transformed diet measures and SOC score. Differences between means for men and women were tested with Student\'s t-tests, and differences between more groups, i.e., age, BMI and education level groups, by ANOVA. P-values ≤ 0.05 were considered significant. Dietary intake and reporting thereof are highly influenced by gender, age, BMI, and educational level \[[@B21]\]. Therefore, mean intakes were standardized for age, BMI and educational level using general linear model (proc GLM). Significance of food item/nutrient predictors was based on (i) type I and (ii) type-III sum of square estimates, where (i) corresponds to a univariate regression, i.e., only the SOC score had entered the model, and (ii) corresponds to a multiple regression, i.e., all independent variables (SOC, gender, age, BMI, and educational level) were included in the model. To evaluate food selection pattern in individuals in relation to SOC score, PLS multivariate projection to latent structures was applied \[[@B22]\]. PLS is a method to relate two data matrices X and Y to each other by linear multivariate modeling. In contrast to traditional linear modeling, co-varying variables may be included. The PLS parameters carrying information about the x- and y- variables, e.g. R^2^, Q^2^and VIP, were generated as previously described \[[@B22]\]. The R^2^- and Q^2^-values give the capacity of the X matrix to explain (R^2^) and predict (Q^2^; equals cross-validated R^2^) the variance of the Y matrix. The relative importance of each x-variable for the correlation structure among X and Y is given as a VIP-value ([V]{.underline}ariables of [I]{.underline}mportance in the [P]{.underline}rojection); VIP-values \>1.0 are influential and VIP-values ≥ 1.5 highly influential. Results ======= In total 2,446 men and 2,545 women answered the SOC- and food-frequency-questionnaires in such a way that the inclusion criteria were met. This corresponded to 86.6% of the 25--74 year old participants in the northern Sweden MONICA 1999 study. In the present cohort SOC scores varied from 23 to 91 points (Fig. [1](#F1){ref-type="fig"}). Means in quartile groups based on SOC distribution increased by approximately 9 points per quartile among both men and women (see [Additional file 1](#S1){ref-type="supplementary-material"}). Mean scores neither differed significantly between men and women, nor between normal weight, overweight or obese individuals, nor between groups with different length of education. However, mean scores increased significantly with increasing age in both sexes (see [Additional file 1](#S1){ref-type="supplementary-material"}). Univariate correlation analyses between SOC scores and daily intake of nutrients or servings per week in food groups revealed significant associations among women for intakes of energy (kCal/day), total and saturated fat (g/week), ascorbic acid (mg/week), sucrose (g/week), and servings/week of fruits, vegetables, cereals, and sweets (data not shown). Among men significant correlations were seen for daily intake of total and saturated fat (g/day), ascorbic acid (mg/day), fiber (g/day), and alcohol (g/day), and servings of fruits, vegetables, bread, bread and cereals, fish, and potatoes (data not shown). Except for ascorbic acid and cereal intake in women, and bread, bread and cereals, and potato intake in men, variations in intakes of these foods/nutrients were significantly explained by SOC quartile scores in linear GLM models (Type I SS; Tables [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}). After standardization for age, BMI and education level SOC quartile scores still contributed in explaining intake of saturated fat, vegetables, sucrose and sweets in women and vegetables and alcohol in men (Type III SS; Tables [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}). In accordance, mean (age, BMI and education standardized) intakes of vegetables were significantly higher in women and men (1.1 and 0.8 servings more per week, respectively) in the highest, compared to the lowest, quartiles (Tables [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}). Women in the highest quartile also had higher intakes of fruits (+1.0 serving/week), but lower intake of energy (-365 kCal/week), total and saturated fat (-15 and -8 g/week, respectively), sucrose (-14 g/week), and sweets (-1.3 servings/week) (Table [1](#T1){ref-type="table"}). PLS multivariate modeling of intakes of the 84 single food items and food aggregates simultaneously as an X-matrix on SOC-scores (Y-matrix) rendered models which supported low SOC to coincide with a less health promoting dietary preference and vice versa. Thus, in both men and women low SOC scores coincided with high intake of pizza, soft drinks, candies, sausages for main course, hamburgers, mashed potato, chips and other snacks, potato salad, French fries, and traditional broth soaked bread and potato dumplings (Table [3](#T3){ref-type="table"}, items in bold). In contrast, both men and women with high SOC scores were characterized by a high intake of rye crisp bread (whole meal), boiled potato, and vegetables (Table [3](#T3){ref-type="table"}, items in bold). In addition, some more gender specific characteristic food selections are seen in Table [3](#T3){ref-type="table"}. Discussion ========== The present study shows that a high sense of coherence (SOC) is associated with health-promoting food choices, whereas low scores are associated with a less favorable food pattern. SOC score independently contributed in explaining variations in intakes of vegetables in both men and women, and in intakes of saturated fat, sucrose, sweets, and fruits among women, and alcohol intake among men. Correlations for other nutrient/food intakes seen in the initial univariate evaluations, disappeared when tested together with age, BMI and educational level in multiple linear GLM modeling. This indicates that some dietary preferences and food selections are linked to socioeconomic status, whereas others are more bound to the individual\'s sense of coherence, i.e. his/her personal way of grasping and handling life situations. Although the present results support earlier findings that associate high SOC scores with a more healthy diet \[[@B14]-[@B17]\], the interpretations should be considered in relation to methodological strengths and limitations of the study. The Northern Sweden MONICA study cohort of 1999 consisted of 6,000 participants, corresponding to 71.8 % of the invited subjects. Of these 86% could be included in the present evaluation. All subjects were primarily invited to the study by random from a continuously updated population ledger. Taken together, this allows for generalization of the results to a population level, at least to a population similar to that in northern Sweden. Within the MONICA project, extensive quality assessments of various methods and measures have been performed \[[@B23]\], and both the SOC and food frequency questionnaires were printed in an optically readable format to minimize errors due to faulty entry of data. Furthermore, the food frequency questionnaire has been found valid in a random sub-sample of representative adults \[[@B20]\], and the Cronbach alpha value \[[@B24]\] among SOC answers, was 0.81, which is in accordance with other studies \[[@B10],[@B13],[@B15]\], indicates reliability and internal consistency among the answers \[[@B19]\]. However, the cross-sectional design of the study, where the SOC scores were measured at the same time as the food preferences, precludes the possibility of interpreting the identified associations as causally related. The sense of coherence instrument has been constructed to measure an individual\'s capacity to cope in a salutogenic way \[[@B9]\]. In accordance with this the present study shows that the SOC level is associated with a \"healthy\" eating pattern. It has previously been shown that physically active individuals have higher SOC scores than inactive persons \[[@B25],[@B26]\]. The hypothesis that SOC links to life style in a wider sense is therefore supported, even though we are aware that it cannot be ruled out that a healthy life style per semay influence SOC. The four cross-sectional MONICA screenings in 1986, 1990, 1994 and 1999 have demonstrated a distinct time trend in dietary intake in northern Sweden \[[@B27]\]. The present results therefore raise some questions. Are individuals with higher SOC more prone to change eating pattern? Are those changes more likely to follow dietary recommendations? Is a low SOC level a marker for a less healthy life style in general? Since all previous participants were re-called in 1999 answers may be searched for within the frame work of the MONICA project. Notably, both men and women in the highest, as compared with the lowest SOC score quartile, reported more food choices from the contemporary public message of a healthy life style \[[@B6],[@B7]\]. In contrast, food selection in those in the lowest SOC-score quartile may render these individuals at a higher risk for various chronic diseases. Further knowledge of the influence of an individual\'s sense of coherence on his/her willingness and ability to change dietary habits may be of advantage when designing preventive programs. Conclusion ========== Both men and women in the highest, as compared with the lowest, SOC score quartile reported more \"healthy\" food choices. Dietary habits for individuals in the lowest SOC quartile therefore may render a higher risk for various endemic diseases. Competing interests =================== The author(s) declare that they have no competing interests Authors\' contributions ======================= All participants have contributed to study design and manuscript preparation; IJ has been main author, UL has done data processing, BN has been responsible for SOC, BL for general behavior aspects, and BS is PI for the Northern Sweden MONICA study. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 SOC scores in gender, age, BMI, education level and SOC-quartile groups. Differences between means for men and women are tested with t-test, and among more groups, i.e. age groups, BMI education level, with ANOVA. ns for p \> 0.05 ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This study was supported by The Swedish Research Council (BS), The Norrbotten and Västerbotten counties, the Joint Committee of Northern Sweden Health Care Region, the Heart and Chest Foundation, the Social Science Research Council and other funds supporting the Northern Sweden MONICA Project. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Cumulative frequencies of SOC scores in men and women. The vertical lines denote the upper and lower cut-off values for the lowest and highest quartiles, respectively. ::: ![](1475-2891-4-9-1) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Diet intake in women. Food items with significant univariate correlations between diet intake and SOC score are listed. ns for \>0.05. ::: GLM PROCEDURE^1^ MEAN INTAKES OF NUTRIENTS AND FOODS^4^ ---------------------------- ---------------------- -------------------------------------------- ------- ------- ------- ------- Energy (kCal/day) 0.066 ns^a,b,e^ 1 531 1 492 1 478 0.037 Fat total (g/week) 0.012 ns^a,b^ 373 362 358 0.027 Fat saturated (g/week) 0.017 ns^a,b^ 154 148 146 0.023 Ascorbic acid (mg/week) ns ns^a,e^ 520 528 533 ns Fruits (servings/week) 0.028 0.088^a,e^ 8.0 8.5 8.9 0.018 Vegetables (servings/week) 0.007 0.017^a,e^ 8.6 9.2 9.7 0.002 Cereals (servings/week ns ns^a,b^ 3.1 3.1 3.5 ns Sucrose (g/week) 0.037 0.067^a,b^ 188 181 174 0.011 Sweets (servings/week) 0.014 0.019^a,b^ 8.8 8.1 7.5 0.002 1\) GLM modeling of SOC-quartile classification scores, including age, BMI, and level of education group in the model, ln-transformed diet intake values. ns for p \> 0.09. 2\) Type I sum of square estimates are when only SOC quartile scores have entered the model, and 3\) Type III sum of square estimates when all variables are kept in the model. Independent contribution by (a) age group, (b) BMI, and (e) education level in explaining nutrient/food intake variations are indicated by the respective superscript. 4\) Geometric mean intakes standardized for age, BMI, and education in SOC-quartile (Q1, Q2+Q3, Q4) groups. Mean (95% CI) SOC score in the Q2+Q3 group (n~women~= 1303, 49.6% of all women) is 69.2 (69.0--69.4). 5\) The difference between mean intakes (standardized for age, BMI and education) in the lowest (Q1) versus highest (Q4) SOC quartile groups. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Diet intake in men. Food items with significant univariate correlations between diet intake and SOC score are listed. ns for \>0.05. ::: GLM PROCEDURE^1^ MEAN INTAKES OF NUTRIENTS AND FOODS^4^ ----------------------------------- ---------------------- -------------------------------------------- ------- ------- ------- ------- Energy (kCal/day) ns ns^a,b^ 1 964 1 980 1 936 ns Fat total (g/week) 0.001 0.020^a,b,e^ 545 545 525 0.064 Fat saturated (g/week) 0.006 0.057^a,b,e^ 227 227 219 ns Ascorbic acid (mg/week) 0.021 ns^a,e^ 428 456 458 0.063 Fiber (g/week) 0.006 ns^a,b^ 123 127 127 ns Fruits (servings/week) 0.058 ns^a,e^ 5.2 5.6 5.5 ns Vegetables (servings/week) 0.009 0.037^a,e^ 5.6 6.2 6.4 0.009 Bread (servings/week) ns ns^a,b,e^ 17.0 17.6 17.4 ns Bread and cereals (servings/week) 0.039 ns^a,b,e^ 21.5 22.3 22.2 ns Fish (servings/week) 0.010 ns^a,e^ 1.0 1.1 1.1 ns Potato (servings/week) ns ns^a^ 4.9 5.0 4.9 ns Alcohol (g/week) 0.007 0.011^a,b,e^ 16.1 23.1 20.5 0.064 1\) GLM modeling of SOC-quartile classification scores, including age, BMI, and level of education group in the model, ln-transformed diet intake values. ns for p \> 0.09. 2\) Type I sum of square estimates are when only SOC quartile scores have entered the model, and 3\) Type III sum of square estimates when all variables are kept in the model. Independent contribution by (a) age group, (b) BMI, and (e) education level in explaining nutrient/food intake variations are indicated by the respective superscript. 4\) Geometric mean intakes standardized for age, BMI, and education in SOC-quartile (Q1, Q2+Q3, Q4) groups. Mean (95% CI) SOC score in the Q2+Q3 group (n~men~= 1323; 50.4% of all men) is 69.3 (69.0--69.5). 5\) The difference between mean intakes (standardized for age, BMI and education) in the lowest (Q1) versus highest (Q4) SOC quartile groups. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Food selection pattern by SOC score. VIP-values denotes the influence in explaining variation in SOC-scores. VIP values ≥ 1.2 are listed, and + denotes associations with high SOC and -- association with low SOC scores. ::: MEN WOMEN ------------------------------------- ----------- ------ ------------------------------------- ---- ------ *Influential for high SOC* *Influential for high SOC* Bread \+ 2.18 Fruits \+ 1.83 Bread and cereals \+ 1.96 Vegetables \+ 1.83 Rye crisp bread (whole meal) \+ 1.89 Liver paste \+ 1.64 Fish \+ 1.76 Boiled potato \+ 1.47 Boiled potato \+ 1.71 Rye crisp bread (whole meal) \+ 1.43 Berries \+ 1.51 Blood foods \+ 1.32 Wine \+ 1.49 Cereals \+ 1.29 Light beer \+ 1.32 Oat flake porridge \+ 1.24 Vegetables \+ 1.23 *Influential for low SOC* *Influential for low SOC* Pizza \- 2.41 Mashed potato \- 2.98 Soft drinks \- 2.25 Sausage as main course \- 2.11 Pasta \- 2.14 Potato salad \- 1.98 Candies \- 2.02 Soft drinks \- 1.94 Sausage as main course \- 1.97 French fries \- 1.89 Hamburgers \- 1.94 Hamburgers \- 1.82 Mashed potato \- 1.87 Beer (high alcohol) \- 1.71 Fried potato \- 1.65 Pizza \- 1.68 Butter for cooking \- 1.63 Sweets \- 1.67 Chips, cheese doodles, nuts \- 1.57 Candies \- 1.60 Potato salad \- 1.48 Rice \- 1.50 Banana \- 1.37 Pancakes \- 1.43 French fries \- 1.22 Syrups \- 1.42 Traditional broth soaked bread \- 1.21 Cookies \- 1.39 Fruit soup \- 1.21 Traditional broth soaked bread \- 1.29 Traditional potato dumpling \- 1.20 Chips, cheese doodles, nuts \- 1.29 :::	PubMed Central	2024-06-05T03:55:54.005546	2005-2-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554973/", "journal": "Nutr J. 2005 Feb 28; 4:9", "authors": [ { "first": "Ulrika", "last": "Lindmark" }, { "first": "Birgitta", "last": "Stegmayr" }, { "first": "Berit", "last": "Nilsson" }, { "first": "Bernt", "last": "Lindahl" }, { "first": "Ingegerd", "last": "Johansson" } ] }
PMC554974	Background ========== On average, the worldwide incidence of low birthweight (LBW) is 17% per year, making LBW an important infant health problem in many populations \[[@B1]\]. The incidence of LBW varies among countries, ranging from 4% to 6% in Western countries like Sweden, France, United States and Canada (UNICEF 2003) and much higher in developing countries. In Latin America, the overall LBW rate varies according to geographical region. The Pan American Health Organization has estimated that the overall LBW rate is 8.27% in South America and Mexico and ranges from 6% in Peru to 10% in Bolivia and Venezuela. In Central America the overall LBW rate is 11.2%, ranging from 6% in Belize and El Salvador to 15% in Nicaragua \[[@B2]\]. The LBW rate can also vary within countries. For example, in Mexico, the incidence ranges from 8.2% to 12%, depending on geographic al region \[[@B3]\]. In addition to its impact on infant mortality, LBW has been associated with higher probabilities of infection, malnutrition and handicapping conditions during childhood, including cerebral palsy, mental deficiencies and problems related to behavior and learning during childhood \[[@B4]-[@B6]\]. Children who survive LBW have a higher incidence of diseases, retardation in cognitive development, and undernourishment. There is also evidence that LBW or its determinant factors are associated with a predisposition to higher rates of diabetes, cardiac diseases and other future chronic health problems \[[@B7]-[@B9]\]. The biological processes that affect the fetus in uteroare related to the mother\'s physiology, including her nutrition (mother\'s weight before pregnancy and history of having newborns with LBW), exercise, infections, and consumption of tobacco, alcohol and other drugs \[[@B10],[@B11]\]. During the fetal phase, growth depends on the nutritional condition of the mother, indicating that pregnant women should not only increase their weight but also consume essential nutrients. For many women in the developing world, however, economic, social and cultural factors make it difficult for them to obtain the necessary food and healthcare, which are closely interrelated \[[@B12]\]. Associations between poorer child health and poverty, inequity and social exclusion have been documented worldwide and have been shown to be independent of research methods, local culture, and available health care services \[[@B13]\]. While the relationship between socio-economic conditions and health have been of interest and concern for centuries, recent studies have sought to identify the social factors most relevant to health \[[@B14]\]. Some authors consider that health therefore may be an important determinant of opportunities in life and this process, termed \"selection by health\", and suggests that health \"selects\" people in different social strata \[[@B15],[@B16]\]. Among the socio-economic factors are income, education, occupation, household leadership and gender differences related to roles within the family \[[@B17],[@B18]\]. In Mexico, there are also differences in socio-economic conditions resulting from geographic area and political organization. These may affect various health parameters, including mortality, morbidity and reproductive behavior. Several studies have shown different results on whether socioeconomic factors affect pregnancy outcomes and newborn conditions \[[@B19]-[@B21]\]. The inconsistency of these findings may be due to poor clarification of the mechanisms by which socioeconomic status affects LBW. This is especially true in relationship to the mother\'s nutritional conditions, although low maternal weight before pregnancy and small weight gain during pregnancy have been shown to be associated with higher risks of preterm infants and LBW \[[@B22]-[@B25]\]. Other studies have questioned whether maternal occupation or educational level is associated with LBW, or whether the latter is related to a group of socioeconomic factors. In this study, we have analyzed the socio-economic factors related to birthweight adjusted by others known factors in the urban population of Mexico in three hospitals located in three different geographic areas of Mexico City. Methods ======= A case-control study was carried out in three hospitals with gynaecological and obstetrics services in Mexico City during the first semester of 1996. We recruited 154 LBW newborns, defined as newborns weighing \<2500 grams, from these three hospitals, as well as 474 controls chosen from births on the same day to control for time of birth. All newborns included in this study provided a statistical power of 80% when we assumed a = 0.05 (one-sided test), a case/control ratio 1:3, and a 3% difference among both groups in the low socioeconomic level \[[@B26]\]. Upon arrival at the hospital for delivery, women were interviewed to determine if they met our inclusion criteria. Women with a history of previous chronic conditions or those with twin or multiple pregnancies were not included. Following delivery, and after obtaining informed consent, each woman was administered a questionnaire by a trained interviewer to obtain information about socio-economic, reproductive, and nutritional factors. Socioeconomic factors included age, level of education, civil status, occupation, income and owning certain goods; reproductive factors included parity, history of preterm delivery and LBW, which were classified as positive or negative for all previous pregnancies; and nutritional factors included calcium and iron supplementation, pre-gestational weight, prenatal care, morbidity during pregnancy and tobacco exposure. Clinical records were also reviewed to verify information about each newborn. To create a socioeconomic level index, we used two variables that have been considered as proxies \[[@B27]\]. Ownership of goods was defined as to whether a woman owned her house or flat and if she had a car and whether the woman and her partner were employed. Using these two variables, we constructed a three category socio-economic level index, in which High indicated that the woman and her partner had jobs and goods; Medium indicated that the woman or her partner had jobs or goods; and Low indicated that the woman and/or her partner did not have jobs or goods. Statistical analysis was performed by describing sociodemographic, reproductive and prenatal care characteristics of the mother and newborn. Univariate analysis was used to evaluate the association between the independent variable (socioeconomic level) and covariates with LBW (outcome variable). Stratified Mantel-Haenszel analysis was performed to evaluate confounding and/or interaction (i.e. parity and age). To obtain the association magnitude of the socioeconomic level adjusted by the covariates, multivariate analysis was performed (logistic regression). Variables with clinical and statistical significance were included in multivariate modeling. We used three models to classify factors relevant to LBW into three groups as known, controversial and unknown risk factors for LBW. Model 1 included socioeconomic level index and maternal age, maternal education, marital status and accessibility to public services. In model 2 we included all variables of the model 1 besides, prenatal care, tobacco exposure, and morbidity during pregnancy were considered as adjusted variables. Finally, model 3 included all variables previously mentioned (model 1 and model 2 besides reproductive variables. Analyses were performed using StataCorp. 2002. Stata Statistical Software: Release 7.0 College Station, TX:Stata Corporation. Results ======= The sociodemographic characteristics of the LBW and normal birthweight groups are shown in Table [1](#T1){ref-type="table"}. There was a high proportion of married women in both groups, and 90% of women in both groups had access to public services, including electricity, water and a sewage system. The effect of maternal education had the expected direction, although it was not statistically significant. Women in the lower socio-economic level had a higher risk for LBW (OR, 2.19; 95% CI, 1.18--4.07) than those in the medium and high socioeconomic levels. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Sociodemographic characteristics of mothers of low and normal birthweight infants ::: Variable Birthweight OR 95% CI ---------------------------------- ---------------------------- ---------------------------- -------- --------------- *\<2500 grs (n = 158)* *≥ 2500 grs (n = 474)* Socioeconomic level High 81 267 1.0 Medium 57 177 1.06 \[0.71,1.56\] Low 20 30 2.19\* \[1.18,4.07\] Maternal age (years) \<19 22 83 0.77 \[0.46,1.29\] 20--30 110 321 1.0 \>30 26 70 1.08 \[0.65,1.80\] Maternal education (years) \>12 13 50 1.0 10--12 56 143 1.50 \[0.76,2.98\] 7--9 52 165 1.21 \[0.61,2.40\] \<7 37 116 1.22 \[0.60,2.50\] Marital Status Married 138 429 1.0 Unmarried 20 45 1.38 \[0.78,2.42\] Accessibility to public services No 12 51 0.68 \[0.35,1.31\] Yes 146 423 1.0 \* p \< 0.05 ::: We observed no significant between group differences in tobacco exposure prior to or during pregnancy, or in the frequency of urinary tract infections (Table [2](#T2){ref-type="table"}). However hypertension (OR, 1.53; 95% CI, 0.93--2.53) and calcium supplementation (OR, 1.86; 95% CI, 0.97--3.56) during pregnancy were marginally significant. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Tobacco exposure, morbidity during pregnancy and prenatal care characteristics of mothers of low and normal birthweight infants ::: Variable Birthweight OR 95% CI ------------------------------------------ ---------------------------- ---------------------------- -------- --------------- *\<2500 grs (n = 158)* *≥ 2500 grs (n = 474)* Smoking before pregnancy No 126 384 1.0 Yes 32 90 1.09 \[0.70,1.71\] Smoking during pregnancy No 149 451 1.0 Yes 9 23 1.19 \[0.53,2.66\] Hypertension during pregnancy No 131 416 1.0 Yes 27 58 1.53 \[0.93,2.53\] Urinary tract infection during pregnancy No 100 318 1.0 Yes 58 156 1.22 \[0.83,1.77\] Calcium supplementation Yes 146 409 1.0 No 12 65 1.86 \[0.97,3.56\] Iron supplementation Yes 64 216 1.0 No 94 258 1.20 \[0.83,1.74\] Prenatal care Yes 150 454 1.0 No 8 20 1.07 \[0.45,2.52\] ::: Reproductive characteristics, including maternal weight, previous preterm-birth, and previous LBW infants, were significantly different between mothers of LBW and normal birthweight infants (Table [3](#T3){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Reproductive characteristicsof mothers of low and normal birthweight infants ::: Variable Birthweight OR 95% CI ------------------------------ ---------------------------- ---------------------------- -------- --------------- *\<2500 grs (n = 158)* *≥ 2500 grs (n = 474)* Parity Multiparous 92 284 1.0 Primiparous 66 190 1.04 \[0.73,1.51\] Maternal weight (kgs) \<48 54 107 1.61\* \[1.09,3.84\] 49--54 37 120 0.99 \[0.58,1.67\] 55--60 37 119 1.0 \>60 30 128 0.75 \[0.43,1.29\] Previous^a^preterm birth No 58 213 1.0 Yes 34 71 1.75\* \[1.06,2.90\] Previous low birth weight^a^ No 66 246 1.0 Yes 26 38 2.55\* \[1.44,4.50\] Previous abortion^a^ No 67 222 1.0 Yes 25 62 1.33 \[0.77,2.28\] \p \<0.05 ^a^Only applicable for women with more than one pregnancy ::: Multivariate analysis showed that low socioeconomic level was the most important risk factor for LBW (Table [4](#T4){ref-type="table"}). Using model 1, the OR was 2.57 (95% CI, 1.19--5.58) after adjustments for maternal age, education, marital status, and accessibility to public services. When adjustments for prenatal care, tobacco exposure, and morbidity during pregnancy were also included (model 2), we obtained an OR of 2.45 (95% CI, 1.13--5.36) for low socioeconomic level as a risk factor for LBW (Table [4](#T4){ref-type="table"}). When we also included adjustments for reproductive variables (model 3), we obtained an OR of 2.68 (95% CI, 1.19--6.03) for low socioeconomic level as a risk factor for LBW (Table [4](#T4){ref-type="table"}). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Adjusted odds ratios for LBW infants by socioeconomic level among women of Mexico City ::: Variable* Model 1 Model 2 Model 3 ----------------------------------------- ------------- --------------- ------------- --------------- ------ ------------------- OR 95% CI OR 95% CI OR 95% CI Socioeconomic level Medium 1.14 \[0.75,1.74\] 1.16 \[0.76,1.77\] 1.12 \[0.72,1.74\] Low 2.57 \[1.19,5.58\] 2.45 \[1.13,5.36\] 2.68 \[1.19,6.03\] Maternal age (years) \<19 0.74 \[0.43,1.25\] 0.74 \[0.43,1.26\] 0.57 \[0.32,1.03\] \>30 1.17 \[0.70,1.98\] 1.09 \[0.64,1.84\] 1.34 \[0.76,2.38\] Maternal education (years) \< 7 1.21 \[0.58,2.56\] 1.11 \[0.52,2.40\] 1.05 \[0.47,2.34\] 7--9 1.29 \[0.63,2.62\] 1.23 \[0.59,2.55\] 1.39 \[0.66,2.94\] 10--12 1.56 \[0.78,3.13\] 1.61 \[0.79,3.27\] 1.82 \[0.87,3.77\] Unmarried 0.99 \[0.49,2.01\] 1.08 \[0.52,2.28\] 0.96 \[0.44,2.08\] No accessibility to public services 0.68 \[0.34,1.33\] 0.58 \[0.29,1.17\] 0.56 \[0.27,1.14\] Smoking before pregnancy 0.99 \[0.57,1.72\] 1.07 \[0.60,1.90\] Smoking during pregnancy 1.09 \[0.43,2.78\] 1.14 \[0.43,2.99\] Hypertension 1.61 \[0.95,2.73\] 1.55 \[0.90,2.68\] Urinary tract infection 1.23 \[0.83,1.83\] 1.14 \[0.76,1.71\] No calcium supplementation 1.98 \[1.01,3.87\] 2.30 \[1.14,4.63\] No iron supplementation 1.23 \[0.84,1.80\] 1.32 \[0.89,1.96\] No prenatal care 1.15 \[0.47,2.87\] 1.27 \[0.52,3.19\] Primiparous 1.73 \[1.05,2.83\] Pre-gestational weight(kgs) \<48 2.33 \[1.33,4.08\] 49--54 1.28 \[0.71,2.31\] \>60 1.32 \[0.74,2.36\] Previous preterm birth^a^ 2.95 \[1.04,8.38\]\\ Previous low birth weight^a^ 2.61 \[1.36,5.04\] Previous abortion^a^ 0.52 \[0.17,1.63\] ^a^only applicable for women with more than one pregnancy. ::: Discussion ========== LBW is a public health problem linked to lack of equity in populations. Despite efforts to decrease the proportion of newborns with LBW, success has been quite limited, and the problem persists in both developing and developed countries \[[@B28]\]. In recent years, studies focused on explaining how social factors influence this problem have shown that populations with greater inequities have a greater proportion of newborns with LBW \[[@B25]\]. These inequities are caused by both social conditions of populations and gender differences. Although these differences are not explicit and conclusive, they are revealed by social indicators such as access to health care services, occupation, income, education, and social exclusion or isolation. Differences found in the results about the effect of socioeconomic factors on LBW are probably due to the use of different socioeconomic indicators. It should be pointed out, however, that obtaining information that accurately reflects social and economic characteristics can be difficult, leading to the generation of proxy variables. Thus, education has been used as a proxy variable of social class, and occupation has been used as a proxy of socioeconomic status \[[@B29]\]. In addition, studies performed in European countries have used education as a proxy for socioeconomic level \[[@B29],[@B30]\]. A recent Mexican study, however, found no association between LBW and household infrastructure (including lack of indoor sanitation or water facilities and lack of electricity) when used as a socioeconomic indicator, although this study found that LBW was related mainly to access or utilization of prenatal services and disadvantageous maternal lifestyle behaviors \[[@B31]\]. In our study, we used ownership of goods and having a job as indicators of socioeconomic level in a Mexican urban population with social security. Using these indicators, we found that low socioeconomic level is the most important risk factor for LBW, independent of other factors such as reproductive and nutritional characteristics, smoking, morbidity during pregnancy, and accessibility to health services and prenatal care. These results are similar to those of other studies describing a positive relationship between socioeconomic condition and effects on health \[[@B32]-[@B34]\] It is important to mention that to diminish the possible error of misclassifying we used at least two indicators to construct the socioeconomic level variable (ownership of goods and occupation). However we thought that it will be a non differential error. Although we analyzed other proxies of socioeconomic level, we found that each of them was irrelevant. For example, although education has been used as a proxy of social status, we found that this indicator was not important in our population, perhaps because the women in our study were incorporated into the work force, making exposure to intermediate factors, such as occupational stress and load work, an influence on pregnancy outcomes. Family circumstances and biological processes may also be affected by a wider social context, including cultural and historic issues such as educational opportunities, parent\'s divorce, unemployment, risk of poverty risk, and risks factors for smoking and obesity. Although many socioeconomic factors related to LBW have been identified, the specific role of each of them is not known, limiting the ability to use preventive actions in exposed populations. Interventions aimed at reducing the number of LBW infants have had limited success on conditions of the newborn, although some showed benefits in pregnant women \[[@B35]\]. To decrease the incidence of LBW, it is important to consider health services interventions to get better quality of care for pregnant women. Investments in the health and development of the most vulnerable populations, such as pregnant women and children, are important in themselves, because they prepare the context and environment for a more productive and healthy life, with the full development and use of mental and physical human potential \[[@B36]\]. Conclusion ========== Heterogeneity among different populations makes findings related to interventions in one population not applicable to others. Thus, it is necessary to design studies that account for the geographic, racial, cultural, social and economic context of each country and specific group. In the case of Mexico, we believe that our results provide information representing a closer approach to the effect of socioeconomic status on LBW. Our results provide a starting point in the search for better indicators for evaluating socioeconomic status in Mexican populations with other social conditions, including suburban, rural, and indigenous populations. Another aspect to consider within the context of each country is the availability of information. In Mexico, there is no way to obtain uniform information about the socioeconomic characteristics of the population. Information about conditions of different populations is important in designing programs aimed at solving existing inequities. It is a challenge to create a socioeconomic index that will reflect the real living conditions of pregnant women in Mexico. This effort will require including other variables, as well as refining proxy variables. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= LPTA, PCC, SFH contributed in the conception and design of the study and statistical analysis. JPVB, ERM reviewed for important intellectual content. All authors participated in the interpretation of data and read and approved the final version to be published. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/5/20/prepub> Acknowledgements ================ Authors would like to thank medical personnel of the participating hospitals.	PubMed Central	2024-06-05T03:55:54.008697	2005-3-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554974/", "journal": "BMC Public Health. 2005 Mar 3; 5:20", "authors": [ { "first": "Laura P", "last": "Torres-Arreola" }, { "first": "Patricia", "last": "Constantino-Casas" }, { "first": "Sergio", "last": "Flores-Hernández" }, { "first": "Juan Pablo", "last": "Villa-Barragán" }, { "first": "Enrique", "last": "Rendón-Macías" } ] }
PMC554975	Introduction ============ The viral protein R (Vpr) of HIV-1 is a small basic protein (14 kDa) of 96 amino acids, and is well conserved in HIV-1, HIV-2 and SIV \[[@B1]\]. The role of Vpr in the pathogenesis of AIDS is undeniable, but its real functions during the natural course of infection are still subject to debate. The Vpr role in the pathophysiology of AIDS has been investigated in rhesus monkeys experimentally infected with SIVmac, and it was initially shown that monkeys infected with a vprnull SIV mutant decreased virus replication and delayed disease progression \[[@B2],[@B3]\]. Moreover, monkeys infected with a SIV that did not express the vprand vpxgenes displayed a very low virus burden and did not develop immunodeficiency disease \[[@B4],[@B5]\]. Regarding these in vivophenotypic effects, numerous laboratories have dissected the role of Vpr in various in vitro, in vivoand ex vivosystems to explore the contribution of this protein in the different steps of the virus life cycle. Despite its small size, Vpr has been shown to play multiple functions during virus replication, including an effect on the accuracy of the reverse-transcription process, the nuclear import of the viral DNA as a component of the pre-integration complex (PIC), cell cycle progression, regulation of apoptosis, and the transactivation of the HIV-LTR as well as host cell genes (Fig. [1](#F1){ref-type="fig"}). Furthermore, Vpr is found in virions, in cells, and exists as free molecules found in the sera and the cerebrospinal fluid of AIDS patients, indicating that it may exert its biological functions through different manners. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Schematic view of the early steps of the HIV-1 infection of a target cell. The functional events in which the Vpr protein is involved are highlighted. Vpr has been shown to play multiple functions during the virus life cycle, including an effect on the accuracy of the reverse-transcription process, the nuclear import of the viral DNA as a component of the pre-integration complex, cell cycle progression, regulation of apoptosis, and the transactivation of the HIV-LTR as well as host cell genes. ::: ![](1742-4690-2-11-1) ::: Structure of the HIV-1 Vpr protein ---------------------------------- Because the full length protein aggregated in aqueous solution, the overall structure of Vpr has been difficult to access \[[@B6]\], and preliminary strategies used two distinct synthetic peptides corresponding to Vpr (1--51) and (52--96) fragments for NMR and circular dichroism studies \[[@B6]-[@B9]\]. As previously predicted \[[@B10]\], the structure of the Vpr(1--51) fragment has a long motif of α helix turn-α helix type encompassing the Asp17-Ile46 region, and ends with a γ turn \[[@B8]\]. The Vpr(52--96) fragment contains an α-helix encompassing the 53--78 region that is rich in leucine residues \[[@B7]\]. One side of the helix offers a stretch of hydrophobic residues that can form a leucine-zipper like motif \[[@B11]\]. This structure may account for the formation of Vpr dimers \[[@B7],[@B12],[@B13]\] and/or for the interaction with cellular partners \[[@B14]\]. Finally, NMR analysis of a soluble full length Vpr (1--96) polypeptide was recently performed, and gave access to the tertiary structure of the protein (Fig. [2](#F2){ref-type="fig"}), confirming the amphipathic nature of the three α-helices of HIV-1 Vpr. The helices are connected by loops and are folded around a hydrophobic core \[[@B15]\] surrounded by a flexible N-terminal domain and a C-terminal arginine-rich region that are negatively and positively charged, respectively. Four conserved prolines (positions 5, 10, 14 and 35) which present cis/transisomerization are found in the N-terminal domain \[[@B16]\]. It was reported that the cellular peptidyl-propyl isomerase cyclophilin A was able to interact with Vpr via prolines in position 14 and 35, which insured the correct folding of the viral protein \[[@B17]\]. The carboxy-terminus of Vpr contains six arginines between residues 73 and 96. This domain shows similarity with those of arginine-rich protein transduction domains (PTD), and may explain the transducing properties of Vpr, including its ability to cross the cell membrane lipid bilayer \[[@B6],[@B18]-[@B20]\]. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Three-dimensional structure of the HIV-1 Vpr protein (from \[15\]). The three α-helices (17--33, 38--50, 55--77) are colored in pink, blue and orange, respectively; the loops and flexible domains are in green. We can the Trp54 residue localized between the second and the third a-helix, and that is likely accessible for protein-protein interaction with UNG2 \[54\]. ::: ![](1742-4690-2-11-2) ::: Vpr is packaged into virus particles ------------------------------------ Vpr is expressed at a late stage of the virus life cycle, but it is present during the early steps of infection of target cells since it is packaged into virions released from the producing cells. The incorporation of Vpr occurs through a direct interaction with the carboxy-terminal p6^Gag^region of the gag-encoded Pr55^Gag^precursor \[[@B21]-[@B24]\]. While the integrity of the α-helices of Vpr is required for efficient packaging into virions \[[@B25]\], a leucine-rich motif found in the p6^Gag^region of the Pr55^Gag^precursor is directly involved in the interaction with Vpr \[[@B23],[@B26]\]. After assembly and proteolytic cleavage of Pr55^Gag^in matrix, capsid, nucleocapsid (NCp7), and p6 mature proteins, Vpr is recruited into the conical core of the virus particle \[[@B27],[@B28]\] where it is tightly associated with the viral RNA \[[@B29],[@B30]\]. Interestingly, Vpr displays a higher avidity for NCp7 than for the mature p6 protein \[[@B23],[@B24],[@B31]\]. Since p6 is excluded from the virion core \[[@B27],[@B28]\], Vpr could switch from the p6^Gag^region of the precursor to the mature NCp7 protein to gain access to the core of the infectious virus particle budding at the cell surface. It seems that Vpr is less avid for the fully processed p6 protein than for the p6^Gag^region in the context of the p55^Gag^precursor. Because of this differential avidity, Vpr is recruited into to the core of the particle where it could interact with nucleic acids, NCp7 \[[@B24],[@B31]\] and/or the matrix protein \[[@B32]\]. Since it was estimated that Vpr is efficiently incorporated with a Vpr/Gag ratio of \~1:7 \[[@B33]\], that may represent 275 molecules of Vpr per virion. The incorporation of Vpr has been also used as a unique tool to target cargoes (i.e., cellular and viral proteins, drugs) into viral particles \[[@B34],[@B35]\]. This property was extensively used to study the respective functions of integrase (IN) and reverse transcriptase (RT) during virus replication by expressing Vpr-IN and Vpr-RT fusions in transin virus-producing cells \[[@B36]-[@B38]\]. This strategy of trans-complementation also allowed the analysis of mutant of IN without altering assembly, maturation and other subsequent viral events \[[@B37],[@B39]\]. Furthermore, Vpr fused to the green fluorescence protein (GFP) has been recently used to tag HIV particles in order to follow intracellular virus behavior during the early steps of infection of target cells \[[@B40],[@B41]\]. Vpr influences the fidelity of the reverse transcription process ---------------------------------------------------------------- Following virus entry, the viral core is released into the cytoplasm of the target cell and the reverse transcription of the viral RNA takes place in the cytoplasm within a large nucleoprotein complex termed the reverse transcription complex (RTC) containing the two copies of viral RNA and the viral proteins: RT, IN, NCp7, Vpr and a few molecules of the matrix protein \[[@B42]-[@B46]\]. It is generally believed that the reverse transcription process is initiated in virus particles and is then completed, after virus entry, in the cystosol of the target cell. This process is likely concomitant of both virus uncoating and trafficking through the cytosol (for reviews, see \[[@B47],[@B48]\]). Recent studies confirmed that Vpr co-localizes with viral nucleic acids and IN within purified HIV-1 RTCs \[[@B41],[@B45],[@B49]\], and remains associated with the viral DNA within 4 to 16 h after acute infection \[[@B43]\]. In addition to a potential role in the initiation step of the reverse transcription process \[[@B50]\], it has been shown that Vpr modulates the in vivomutation rate of HIV-1 by influencing the accuracy of the reverse transcription. The HIV-1 RT is an error-prone RNA dependant DNA polymerase, and quantification of the in vivorate of forward virus mutation per replication cycle revealed that the mutation rate was as much as fourfold higher in the absence of Vpr expression when measured in actively dividing cells using a genetically engineered system \[[@B51],[@B52]\]. Furthermore, recent analysis in non-dividing cells shows that this phenotype is exacerbated in primary monocyte-derived macrophages (MDM) leading to a 18-fold increase of the HIV-1 mutation frequency \[[@B53]\]. This activity strikingly correlates with the interaction of Vpr with the nuclear form of uracil DNA glycosylase (UNG2) \[[@B54]\], an enzyme involved in the base excision repair pathway that specifically removes the RNA base uracil from DNA. Uracil can occur in DNA either by misincorporation of dUTP or by cytosine deamination. Initially identified from a yeast two-hybrid screening using Vpr as a bait, the interaction with UNG was confirmed both in vitroand ex vivoin Vpr-expressing cells. While the Trp residue in position 54 located in the exposed loop connecting the second and the third α-helix of HIV-1 Vpr has been shown critical to maintain the interaction with UNG, the Vpr-binding site was mapped within the C-terminal part of UNG2 and occurs through a TrpXXPhe motif. Currently, three distinct cellular partners of Vpr contain a WXXF motif including the TFIIB transcription factor, the adenosine-nucleotide translocator (ANT) and UNG2 \[[@B55],[@B56]\]. The association of Vpr with UNG2 in virus-producing cells allows the incorporation of a catalytically active enzyme into HIV-1 particles where UNG2 may directly influence the reverse transcription accuracy \[[@B54]\], and this plays a specific role in the modulation of the virus mutation rate. The model supporting the direct contribution of incorporated UNG2 in the reverse transcription process was recently demonstrated by using an experimental system in which UNG2 was recruited into virions independently of Vpr. UNG2 was expressed as a chimeric protein fused to the C-terminal extremity of the VprW54R mutant, a Vpr variant that fails to recruit UNG2 into virions and to influence the virus mutation rate, even though it is incorporated as efficiently as the wild type (wt) Vpr protein. The VprW54R-UNG fusion is also efficiently packaged into HIV-1 virions and restores a mutation rate equivalent to that observed with the wt Vpr, both in actively dividing cells and in MDMs. In agreement with this phenotype on the virus mutation frequency, it was finally documented that the Vpr-mediated incorporation of UNG2 into virus particles contributes to the ability of HIV-1 to replicate in primary macrophages. When the VprW54R variant was introduced into an infectious HIV-1 molecular clone, virus replication in MDMs was both reduced and delayed whereas replication in PBMC was not altered by the lack of UNG2 incorporation into virus particles. Although it was proposed that the viral integrase was also able to mediate interaction with UNG2, Vpr seems the main viral determinant that allows for the incorporation of cellular UNG2 into virus particles. However, preliminary results obtained from in vitrobinding assays suggest that both Vpr and IN associate with UNG to form a trimeric complex (ELR and SB, unpublished results), but further analyses are required to document the nature of the interactions between UNG2, Vpr, IN as well as RT both in virus-producing cells and then in target cells. HIV-1 and other lentiviruses are unusual among retroviruses in their ability to infect resting or terminally differentiated cells. While Vpr has been shown to facilitate the nuclear import of viral DNA in non-dividing cells, the virion incorporation of UNG2 via Vpr also contributes to the ability of HIV-1 to replicate in primary macrophages. This implies that UNG2 is a cellular factor that plays an important role in the early steps of the HIV-1 replication cycle (i. e. viral DNA synthesis). This observation is in good agreement with a recent report showing that the misincorporation of uracil into minus strand viral DNA affects the initiation of the plus strand DNA synthesis in vitro\[[@B57]\]. This observation suggests that UNG is likely recruited into HIV-1 particles to subsequently minimize the detrimental accumulation of uracil into the newly synthesized proviral DNA. While further work is needed to explain the precise mechanism for how UNG catalytic activity may specifically influence HIV-1 replication in macrophages, it is worth noting that nondividing cells express low levels of UNG and contain relatively high levels of dUTP \[[@B58]\]. Similarly, most non-primate lentiviruses, such as feline immunodeficiency virus (FIV), caprine-arthritis-encephalitis virus (CAEV) and equine infectious anemia (EIAV), have also developed an efficient strategy to reduce accumulation of uracil into viral DNA. These lentiviruses encode and package a dUTP pyropshophatase (dUTPase) into virus particles, an enzyme that hydrolyzed dUTP to dUMP, and thus maintains a low level of dUTP. Interestingly, replication of FIV, CAEV or EIAV that lack functional dUTPase activity is severely affected in nondividing host cells (e.g., primary macrophages). Taken together, these results indicate that uracil misincorporation in viral DNA strands during reverse transcription is deleterious for the ongoing steps of the virus life cycle. The presence of a viral dUTPase or a cellular UNG will prevent these detrimental effects for replication of non-primate and primate lentiviruses in macrophages, respectively. In addition, it is intriguing to note that two viral auxiliary proteins from HIV-1, Vpr and Vif, can both influence the fidelity of viral DNA synthesis. The Vif protein forms a complex with the cellular deaminase APOBEC-3G (CEM15) preventing its encapsidation into virions \[[@B59]-[@B63]\], while Vpr binds the DNA repair enzyme, UNG, to recruit it into the particles. It is tempting to speculate that the action of both viral proteins may influence the mutation rate during the course of HIV-1 infection, and their balance may play a key role during disease progression in infected individuals. Vpr and the nuclear import of the viral pre-integration complex --------------------------------------------------------------- Nondividing cells, such as resting T cells and terminally-differentiated macrophages, are important targets for viral replication during the initial stages of infection, since primary infection of these cell populations contributes to the establishment of virus reservoirs, crucial for subsequent virus spread to lymphoid organs and T-helper lymphocytes \[[@B64]\]. Infection of lymphoid histoculture using human tonsil or splenic tissue showed that Vpr greatly enhances HIV replication in macrophages but did not influence productive infection of proliferating or resting T cells \[[@B65]\]. After virus entry into the cell, the viral capsid is rapidly uncoated and the reverse transcription of the genomic HIV-1 RNA leading to the full length double-strand DNA is completed. This viral DNA associates with viral and host cell proteins into the so-called pre-integration complex (PIC). In contrast to oncoretroviruses which require nuclear envelope disintegration during mitosis to integrate their viral genome into host chromosomes, lentiviruses, such HIV and SIV, have evolved a strategy to import their own genome through the envelope of the interphasic nucleus via an active mechanism 4--6 h after infection (for review, see \[[@B66]\]). Vpr has been reported to enhance the transport of the viral DNA into the nucleus of nondividing cells \[[@B67]-[@B69]\], by promoting direct or indirect interactions with the cellular machinery regulating the nucleo-cytoplasmic shuttling \[[@B70]-[@B74]\]. ### PIC en route to the NE The exact composition of the PIC is still an area of debate but it contains the viral DNA at least associated with integrase, and many recent studies have confirmed that Vpr is also an integral component of this complex (for reviews, see \[[@B75]-[@B77]\]). Of course, the PIC likely contains cellular factors that participate in both intra-cytoplasmic routing and nuclear translocation of the viral DNA. While actin microfilaments seem to play a role in the early events of infection by acting as a scaffold for the appropriate localization and activation of the RTC \[[@B78]\], the PIC is tightly associated with microtubular structures in the cytoplasm. An elegant system using Vpr fused to GFP as a probe was developed to follow the movement of the PIC soon after virus entry in living cells \[[@B40]\]. It has been shown that the GFP-Vpr labeled-PIC progresses throughout the cytoplasm along cytoskeletal filaments and then accumulates in the perinuclear region close to centrosomes. More precisely, it was observed that the viral complex uses the cytoplasmic dynein motor to travel along the microtubule network to migrate towards the nucleus. It is not yet known whether Vpr plays an active role during this movement of the PIC along microtubules or whether it is only associated with the complex and then actively participates in the subsequent steps, including the anchoring of the PIC to the nuclear envelope (NE) and the nuclear translocation of the viral DNA. ### Vpr docks at the NE Indeed, Vpr displays evident karyophilic properties and localizes in the nucleus, but a significant fraction is anchored at the NE and can be visualized as a nuclear rim staining in fluorescence microscopy experiments \[[@B73],[@B79]-[@B81]\]. The NE consists of two concentric inner and outer membranes studded with nuclear pore complexes (NPC) that form a conduit with a central aqueous channel which allows selective trafficking between the nucleus and cytoplasm and creates a permeability barrier to free diffusion of macromolecules or complexes. NPC corresponds to a 125-MDa structure consisting of 30 distinct nuclear pore proteins, named nucleoporins (Nups) \[[@B82]\]. A specific subset of Nups contain FG- or FxFG peptide repeats that constitute most of the filamentous structures emanating from both sides of the NPC and that provide docking sites for various transport factors \[[@B83]\]. Initial studies revealed that HIV-1 Vpr bound to the FG-rich region of several nucleoporins including the human p54 and p58 Nups, the rodent POM121, and the yeast NUP1P \[[@B71],[@B73],[@B74]\], but a direct interaction with the human CG1 nucleoporin was more recently reported \[[@B70]\]. This interaction is not mediated by the FG-repeat region of this Nup but rather via a region without consensus motif located in the N-terminus of the protein. Using an in vitronuclear import assay, it has been demonstrated that the association with the N-terminal region of hCG1 is required for the docking of Vpr to the NE, whereas the FG-repeat region does not participate in this process \[[@B70]\]. The role of Vpr at the NE is not clear but two explanations can be proposed. First, this localization may account for the targeting of the PIC to the NPC before its translocation into the nuclear compartment. In this model, the virion-associated Vpr would be primarily involved, after virus entry and uncoating, in the initial docking step of the viral DNA to the NPC, while other karyophilic determinants of the PIC, such as IN, would then allow for the second step of nuclear translocation to proceed \[[@B81],[@B84]-[@B86]\]. Alternatively, another explanation may come from the observation that Vpr was able to provoke herniations and transient ruptures of the NE \[[@B87]\]. The molecular mechanism supporting the local bursting induced by Vpr is not known but the interaction of Vpr with nucleoporins may cause initial misassembly of the NPC leading to alterations of the NE architecture. Consequently, these transient ruptures may provide an unconventional route for nuclear entry of the viral PIC \[[@B87],[@B88]\]. ### Translocation of Vpr into the nucleus Despite the lack of any identifiable canonical nuclear localization signal (NLS), Vpr displays evident karyophilic properties and is rapidly targeted to the host cell nucleus after infection \[[@B89]\]. Even though the small size of Vpr does not strictly require an NLS-dependent process, experiments performed both in vitroor in transfected cells have shown that Vpr is able to actively promote nuclear import of a reporter protein, such as BSA, β-galastosidase or GFP \[[@B10],[@B13],[@B90]-[@B94]\]. Like proteins containing a basic-type NLS, it was initially proposed that Vpr uses an importin α-dependant pathway to access the nuclear compartment \[[@B72],[@B73]\]. In addition, Vpr may enhance the inherently low affinity of the viral MA for importin α to allow nuclear import of MA \[[@B95],[@B96]\], but conflicting data exists on the nuclear localization of this viral protein \[[@B81],[@B85]\]. Finally, it was reported that Vpr nuclear import was mediated by an unidentified pathway, distinct from the classical NLS- and M9-dependant pathways \[[@B92]\]. Two independent nuclear targeting signals have been characterized within the HIV-1 Vpr sequence, one spanning the α-helical domains in the N-terminal part of the protein and the other within the arginine-rich C-terminal region \[[@B92],[@B94]\]. These results are consistent with data showing that the structure of the α-helical domains of Vpr must be maintained both for its nuclear localization and for Vpr binding with nucleoporins \[[@B25],[@B70],[@B80]\]. In conclusion, the nucleophilic property of Vpr and its high affinity for the NPC, associated with its presence in the viral PIC, at least support a role during the docking step of the PIC at the NE, a prerequisite before the translocation of viral DNA into the nucleus. Even though there is no evidence that Vpr directly participates in the translocation process, it is worth noting that purified PICs also dock at the NE before nuclear translocation using a pathway also distinct from the NLS and M9 nuclear import pathways \[[@B49]\]. One can suggest that among the redundancy of nuclear localization signals characterized within the PIC, both in associated viral proteins (i.e. IN, MA, Vpr) and also in the viral DNA \[[@B97]\], Vpr primarily serves to dock the PIC at the NE, while IN and MA act in cooperation with the central DNA flap to target the viral DNA to the nucleus (for review, see \[[@B98]\]). ### Vpr, a nucleocytoplasmic protein In addition to its nonconventional NLS for targeting into the nucleus, Vpr is a dynamic mobile protein able to shuttle between the nucleus and cytoplasmic compartments \[[@B23],[@B99],[@B100]\]. Photobleaching experiments on living cells expressing a Vpr-GFP fusion confirmed that Vpr displays nucleocytoplasmic shuttling properties \[[@B70]\]. This shuttling activity has been related to the distal leucine-rich helix which could form a classical CRM1-dependant nuclear export signal (NES) \[[@B99]\]. The exact role of this NES in the function of Vpr is not known but since Vpr is rapidly imported into the nucleus after biosynthesis, the NES could redirect it into the cytoplasm for subsequent incorporation into virions through direct binding to the viral p55^Gag^precursor during the late budding step of the virus life cycle \[[@B23],[@B100]\]. Vpr and the cell cycle ---------------------- A further important biological activity of SIV and HIV Vpr proteins is related to their ability to induce an arrest in the G2 phase of the cell cycle of infected proliferating human and simian T cells \[[@B91],[@B101]-[@B105]\]. Cell cycle arrest does not require de novo synthesis of Vpr, but is induced by Vpr molecules packaged into infecting virions \[[@B87],[@B106]\]. This indicates that induction of the G2 cell cycle arrest might happen before the integration step of the viral DNA genome. It is noteworthy that the S. pombefission yeast as well as S. cerevisiaeoverexpressing HIV-1 Vpr are also blocked in the G2 phase of the cell cycle \[[@B107]-[@B109]\], supporting the idea that the cellular pathway altered by Vpr is well conserved in all eukaryotic cells. Moreover, infection of caprine cells with a caprine arthritis encephalitis virus (CAEV) expressing the vprgene from SIV similarly provoked a G2 arrest \[[@B110]\]. The biological significance of this arrest during the natural infection is not well understood, but the HIV-1 LTR seems to be more active in the G2 phase, implying that the G2 arrest may confer a favorable cellular environment for efficient transcription of HIV-1 \[[@B111]\]. In agreement, the Vpr-induced G2 arrest correlates with high level of viral replication in primary human T cells. The determinants of the G2 arrest activity are mainly located in the C-terminal unstructured basic region of HIV-1 Vpr and phosphorylation of the protein is required \[[@B112],[@B113]\]. Regulators of the cell cycle, such as cyclin-dependant kinases (CDKs), control progression through the cell cycle by reversible phosphorylation \[[@B114]\]. The p34/cdc2 CDK associates with cyclin B1 in the G2 phase (for review, see \[[@B115]\]) to regulate the G2 to M transition. Accumulation of the cells expressing Vpr in the G2 phase has been correlated to the inactivation of the p34/cdc2-cyclinB kinase \[[@B102],[@B103]\]. The activity of cdc2 is controlled by opposite effects of the Wee-1 and Myt1 kinases and the cdc25 phosphatase. Wee1 inhibits cdc2 activity through tyrosine phosphorylation, while dephosphorylation of cdc2 by the phosphatase cdc25 promotes cdc2-cyclinB activation that drives cells into mitosis. The activities of both cdc25 and Wee-1 are also regulated by phosphorylation/dephosphorylation. It was initially described that Vpr-expressing cells contained both hyperphosphorylated cdc2 and hypophosphorylated cdc25, their inactive status \[[@B101]-[@B103]\]. Consequently, these two regulators of the G2/M switch are blocked preventing any cell cycle progression. The molecular mechanism leading to this inhibition is not yet clear, but different cellular partners interacting with Vpr which could play a role in cell cycle regulation have been proposed as potential mediators of the Vpr-induced G2 arrest. hVIP/MOV34, a member of the eIF3 complex, was identified as a Vpr-partner in a yeast two-hybrid assay \[[@B116]\], and was associated with the cell cycle arrest activity of Vpr \[[@B117]\]. eIF3 is a large multimeric complex that regulates transcriptional events and is essential for both G1/S and G2/M progression. Intracellular localization studies revealed that expression of Vpr induces a relocalization of MOV34 that shifts from a cytoplasmic to a nuclear localization pattern \[[@B116],[@B117]\]. Two other cellular partners of Vpr, UNG and HHR23A (i.e., the human homologue of the yeast rad23 protein), are implicated cellular DNA repair processes. Since a clear relationship exists between the DNA damage response pathway and the progression of the cell cycle, it was initially suggested that Vpr binding to these DNA repair proteins could account for the observed G2 arrest \[[@B118]-[@B120]\], but subsequent analyses indicated that there was no correlation between the association of Vpr with HHR23A and/or UNG and the block in G2 \[[@B121],[@B122]\]. These analyses are in agreement with a previous report showing that the Vpr-mediated arrest is distinct from the cell cycle arrest in G2 related to DNA damage. However, it has also been reported that Vpr induces cell cycle arrest via a DNA damage-sensitive pathway \[[@B123]\]. The G2 DNA damage checkpoint is under the control of the phosphatidylinositol 3-kinase-like proteins, ATR and ATM \[[@B124]\], which lead to the inactivation of the cdc2-cyclinB complex. The ATR protein has been recently linked to the G2-arrest induced by Vpr \[[@B125]\]. Inhibition of ATR either by drugs, a dominant-negative form of ATR or by siRNA reverts the Vpr-induced cell cycle arrest while activation of ATR by Vpr results in Chk1 phosphorylation, the kinase regulating cdc25c activity. These authors suggested that the G2 arrest induced by Vpr parallels the ATR-DNA damage pathway, but additional work is needed to demonstrate that Vpr causes DNA damage or mimics a signal activating one of the DNA damage sensors. The protein phosphatase 2A (PP2A) has been shown to be directly associated with Vpr via its B55α subunit \[[@B126]\]. PP2A is a serine/threonine phosphatase involved in a broad range of cellular processes, including cell cycle progression. PP2A inactivates cdc2 indirectly both by the inactivation of the Wee1 kinase and by activation of cdc25 (for review, see \[[@B127]\]). Genetic studies performed in S. pombesuggest the involvement of PP2A and Wee1 in the Vpr-induced cell cycle arrest \[[@B128]\]. Intriguingly, expression of Vpr and B55α results in the nuclear localization of B55α subunit while it remains cytoplasmic in normal condition. Together, these studies emphasized the fact that Vpr might play a role in the subcellular redistribution of several regulatory protein complexes involved in the progression of the cell cycle. Indeed, the mitotic function of cdc2-cyclinB complex is triggered not only by the turn of phosphorylation/desphorylation of both subunits on specific residues, but also by spatio-temporal control of their intracellular distribution. For example, cyclinB is predominantly cytoplasmic throughout the G2 phase until it translocates rapidly into the nucleus 10 min before nuclear envelope breakdown \[[@B129]\]. As mentioned earlier, Vpr induces herniations and local bursting of the nuclear envelope leading to redistribution of key cell cycle regulators, including Wee1, cdc25, and cyclin B into the cytoplasm of the host cell \[[@B87]\]. It seems evident that alterations of the subcellular localization of segregated cell cycle regulators could explain the G2 arrest induced by Vpr; this may also explain the overall variety of cellular factors that have been involved in this process. Alternatively, nuclear herniations induced by Vpr could also affect chromatin structure leading to the activation of ATR. However, it not known if the Vpr-induced alteration of the NE architecture could cause DNA damage such as double-strand breaks, but disruption of the nuclear lamin structure is sufficient to block DNA replication, another abnormality recognized by the ATR protein (for reviews, see \[[@B130],[@B131]\]). Vpr and apoptosis ----------------- HIV infection causes a depletion of CD4^+^T cells in AIDS patients, which results in a weakened immune system, impairing its ability to fight infections. The major mechanism for CD4^+^T cell depletion is programmed cell death, or apoptosis, that can be induced by HIV through multiple pathways of both infected cells and non-infected \"bystander\" cells (for review, see \[[@B132]\]). Even though the exact contribution of Vpr as a pro-apoptotic factor responsible for the T cell depletion observed in the natural course of HIV infection is still unknown, it was repeatedly evidenced that Vpr has cytotoxic potential and is able to induce apoptosis in many in vitrosystems. In addition, transgenic mice expressing Vpr under the control of the CD4 promoter show both CD4 and CD8 T cell depletion associated with thymic atrophy \[[@B133]\]. However, controversial results indicating that Vpr can also act as negative regulator of T cell apoptosis have been reported \[[@B134],[@B135]\]. Initially proposed as a consequence of the prolonged cell cycle arrest \[[@B136]-[@B140]\], other investigations have then revealed that the Vpr-mediated G2 arrest was not a prerequisite for induction of apoptosis, suggesting that both functions are separated \[[@B79],[@B87],[@B141],[@B142]\]. However, the recent observation that the activity of the cell cycle regulatory Wee-1 kinase is decreased in Vpr-induced apoptotic cells led to the hypothesis of a direct correlation between the G2 arrest and apoptotic properties of Vpr \[[@B143]\]. Hence, reduction of Wee-1 activity, probably related to its delocalization provoked by Vpr \[[@B87]\], results in an inappropriate activation of cdc2 leading to cell death with phenotypical aberrant mitotic features, a process known as mitotic catastrophe \[[@B144],[@B145]\]. Using an established cell line expressing Vpr, it was observed that after the long G2 phase, cell rounded up with aberrant M-phase spindle with multiple poles resulting from abnormal centrosome duplication \[[@B138],[@B146]\]. The cells stopped prematurely in pro-metaphase and died by subsequent apoptosis. However, works from the G. Kroemer\'s group have then well established that synthetic Vpr, as well as truncated polypeptides, are able to induce apoptosis by directly acting on mitochondria leading to the permeabilization of the mitochondrial membrane and subsequent dissipation of the mitochondrial transmembrane potential (ΔΨm) \[[@B56]\]. This direct effect of Vpr was related to its ability to interact physically with the adenine nucleotide translocator (ANT), a component of the permeability transition pore of mitochondria localized in the inner mitochondrial membrane. Since ANT is a transmembrane protein and presents a WxxF motif on the inner membrane face which is recognized by Vpr \[[@B56],[@B147]\], this interaction implies that Vpr must first cross the outer mitochondria membrane to access ANT. The interaction between Vpr and ANT triggers permeabilization of the inner membrane followed by permeabilization of the outer mitochondrial membrane with consequent release of soluble intermembrane proteins, such as cytochrome cand apoptosis inducing factors, in the cytosol. Cytochrome cthen associates with Apaf-1 in a complex with caspase-9 to create the apoptosome, allowing activation of effector caspases, such as caspase-3, and subsequently the final execution of the apoptotic process (for review, see \[[@B148]\]). While numerous reports have shown that Vpr mediated-apoptosis was associated with activation of caspase-9 and capase-3 \[[@B56],[@B79],[@B137],[@B140],[@B147],[@B149]\], it is intriguing that Vpr was still able to induce cell death in embryonic stem cells lacking Apaf-1, caspase-9 and IAF \[[@B150]\]. These results suggest a model in which the direct action of Vpr on mitochondria may be sufficient to cause cell death in HIV-1 infected cells \[[@B149]\]. Although the causal role of Vpr in the induction of apoptosis is evident both in vitroand ex vivo, its real contribution with other viral determinants, such as gp120 envelope, Tat, Nef and the viral protease, in the physiopathology of AIDS needs to be further documented during the course of HIV infection \[[@B151]\]. However, it was recently revealed that long term non-progressor HIV-1 infected patients show a highest frequency of mutation at the position Arg77 of the Vpr protein than patients with progressive AIDS disease. Interestingly, this residue seems crucial for the capacity of the protein to induce apoptosis through permeabilization of the mitochondrial membrane \[[@B152]\]. Conversely, it was reported that mutation of the Leu64 residue enhanced the pro-apoptopic activity of Vpr \[[@B153]\], indicating that mutations affecting the C-terminal region of the protein may generate Vpr molecules with different pro-apoptotic potentials during the course of natural HIV-1 infection. In addition, soluble Vpr protein is found in the sera as well as in the cerebrospinal fluid of HIV-infected patients, and was proposed to play a role related to its pro-apoptotic activity in AIDS-associated dementia \[[@B154],[@B155]\]. The involvement of Vpr in these neurological disorders has been suggested, since recombinant Vpr has neurocytopathic effects on both rat and human neuronal cells \[[@B156]-[@B158]\]. Neurons killed by extracellular Vpr display typical features of apoptosis evidenced by direct activation of the initiator caspase-8 that will lead to subsequent activation of effector caspases. These effects have been linked to the property of the first amphipathic α-helix of Vpr to form cation-selective ion channels in planar lipid bilayers, causing a depolarization of the plasma membrane \[[@B6],[@B157],[@B159],[@B160]\]. These observations indicate that Vpr can trigger apoptotic processes by different alternative pathways depending of the target cells. Nuclear role(s) of Vpr ---------------------- The first reported function of Vpr was a modest transcriptional activity on the viral LTR promotor as well as on heterologous cellular promotors \[[@B161],[@B162]\]. While the connection between cell cycle arrest and LTR-transactivation by Vpr is not well understood, it was concluded that activation of the Vpr-induced viral transcription is secondary to its G2/M arrest function \[[@B111],[@B163]\]. An increase transcriptional activity is indeed observed from the viral LTR in arrested cells expressing Vpr \[[@B164]-[@B166]\]. The transactivation of HIV-1 induced by Vpr is mediated through cis-acting elements, including NF-κB, Sp1, C/EBP and the GRE enhancer sequences found in the LTR promotor \[[@B167]-[@B170]\]. Also related to this activity, Vpr regulates the expression of host cell genes such as NF-κB, NF-IL-6, p21^Waf1^and survivin\[[@B171]-[@B173]\]. Finally, Vpr seems also able to interact directly with the ubiquitous cellular transcription factor Sp1 \[[@B168]\], the glucocorticoid receptor \[[@B174],[@B175]\], the p300 coactivator \[[@B163],[@B176]\], and with the transcription factor TFIIB, a component of the basal transcriptional machinery \[[@B177]\]. This latter interaction is also mediated by a WxxF motif found within the TFIIB primary sequence \[[@B55]\]. Vpr displays high affinity for nucleic acids but no specific DNA sequence targeted by Vpr has been yet identified \[[@B19],[@B29]\]. Interestingly, Vpr does not bind to the Sp1 factor or cis-acting elements alone but it associates with Sp1 in the context of the G/C box array \[[@B168]\], as well as in a ternary complex with p53 \[[@B178]\], indicating that Vpr might bind specific DNA sequence once associated with cellular partners to subsequently drive expression of both host cell and viral genes. Consistently, it has been reported that Vpr can directly bind to p300 via a LXXLL motif present in the C-terminal α-helix of the protein \[[@B179]\], suggesting that Vpr may act by recruiting the p300/CBP co-activators to the HIV-1 LTR promotor and thus enhance viral expression. Since p300 is a co-activator of NF-κB, Vpr can also mediate up-regulation of promotors containing NF-κB and NF-IL-6 enhancer sequences in primary T cells and macrophages. In addition, Vpr markedly potentiates glucocorticoid receptor (GR) action on its responsive promotors \[[@B174],[@B175]\]. The Vpr-mediated LTR transcription was inhibited by the addition of the GR antagonist, RU486, in cultured macrophages \[[@B175]\]. That Vpr-mediated co-activation of the GR is distinct from the G2 arrest and required both LLEEL^26^and LQQLL^68^motifs contained within the first and third α-helical domains of HIV-1 Vpr \[[@B174],[@B180]\]. Vpr may also function as an adaptor molecule for an efficient recruitment of transcriptional co-activators (GRE, p300/CBP\...) to the HIV-1 LTR promotor and thus enhances viral replication. Additionally, it may be involved in the activation of host cell genes inducing cellular pathways in relation with the AIDS pathogenesis. Indeed, cDNA microarray analysis using isogenic HIV-1 either with or without vprexpression revealed that Vpr induces up and down regulation of various cell genes \[[@B181]\]. Conclusion ========== By interfering with many distinct cellular pathways all along the virus life cycle, it is now evident that Vpr\'s contribution to the overall pathogenesis of HIV-1 infection in vivois likely crucial. While major efforts have been made during the last years to define the molecular mechanisms and cellular targets of Vpr, additional work is needed for the complete understanding of its wide range of activities. An important issue now is to define the precise contribution of each activity to the viral replication and pathogenesis during the natural course of HIV infection. The involvement of Vpr in key processes of the early steps the viral life cycle (i.e., reverse transcription and nuclear import of the viral DNA) represents a good target for developing novel therapeutic strategies for AIDS therapy. In addition, this viral factor represents a valuable tool to elucidate many fundamental cellular processes. List of abbreviations ===================== HIV, human immunodeficiency virus; SIV, simian immunodeficiency virus; CypA, cyclophilin A; nup, nucleoporin; PIC, pre-integration complex; RTC, reverse transcription complex. Acknowledgements ================ We thank Louis Mansky for critical review of the manuscript, Guillaume Jacquot, Serge Bouaziz and Nelly Morellet for the kind gift of the figures. E.L.R. is supported by \"Ensemble contre le SIDA/SIDACTION\"and the French Agency for AIDS Research (\"ANRS\").	PubMed Central	2024-06-05T03:55:54.012278	2005-2-22	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554975/", "journal": "Retrovirology. 2005 Feb 22; 2:11", "authors": [ { "first": "Erwann", "last": "Le Rouzic" }, { "first": "Serge", "last": "Benichou" } ] }
PMC554976	Introduction ============ Clinicians, healthcare administrators, researchers, regulators and policymakers are concerned with optimizing mortality ascertainment using administrative data. In addition to its clinical importance, mortality informs program planning, quality assessment and improvement, and public reporting \[[@B1]-[@B8]\]. Veterans are an important, vulnerable population in which mortality has been examined as a function of race / ethnicity, service characteristics, access, and quality of care. Valid, complete reporting is critical to the success of such endeavors, and limitations in using death certificates have been acknowledged \[[@B9],[@B10]\], although VA mortality data is generally regarded as accurate \[[@B11]-[@B15]\]. To understand the limitations of single source ascertainment, we described decedents who would not have been identified by a strategy using either VA decedent files alone or death certificates alone. We compared cases that would have been missed using either single source with the rest of the cohort based on their demographic and clinical attributes and the settings in which they received care. Methods ======= In order to evaluate the implications for improving veterans\' end-of-life care, we constructed a population-based decedent cohort \[[@B16]\]. For such purposes, it is particularly important to understand whether death was recorded elsewhere for veterans who were under VA care since the VA system may be responsible for much of their end-of-life care even if they do not die while receiving health care in a VA facility. Data Sources ------------ The VA Beneficiary Identification and Records Locator System (BIRLS) contains records of all beneficiaries including veterans whose survivors applied for burial benefits. It includes records of discharged military veterans post-1973 and recipients of Medals of Honor and VA education benefits. After submission to the Veterans Benefits Administration (VBA), deaths are recorded in the BIRLS Death File. A submission to the VBA is typically triggered by a family claim for death benefits (e.g. burial assistance, pension) \[[@B17]-[@B19]\]. The VA maintains a National Patient Care Database (NPCD) that contains a record of Social Security Number (SSN) linked VA and contracted health services provided to all veterans \[[@B17]-[@B19]\]. Death certificates are required for burial in California and are available for public use \[[@B20]\]. We first identified 345,380 decedent veterans who died during FY2000 (30 September 1999 -- 1 October 2000) from the BIRLS Death File. We used SSNs to link cases to VA NPCD outpatient, inpatient, or long term care records restricted to recipients of any VA services in California within 12 months of death. We extracted records including any inpatient or long term care admission, or outpatient encounters. Veterans who entered the cohort on the basis of using outpatient services were required to have at least one clinical encounter (e.g., other than laboratory, radiology, or administrative). In addition, we used California death certificates as second source to identify decedent veterans by linking SSNs from death certificates directly to VA utilization files. California death certificates contained 462,561 records for calendar years 1999 and 2000, and we primarily matched decedents identified through death certificates to BIRLS by SSN. We manually inspected matches on SSN only and we also examined matches on criteria other than SSN (e.g. last name, first name, date of birth, date of death). Additional cases we accepted after manual inspection involved transpositions of one and rarely more than one SSN digit but agreement in other fields. Thus, the cohort included recipients of VA clinical services verified as deceased based on either BIRLS or death certificates, and all cases were linked to VA utilization files by SSN. In the final decedent cohort, we excluded cases of non-veterans receiving care at VA facilities by examining indicators of veteran status associated with visits. The VA assigns specific codes to non-veterans rendered care for various reasons (e.g., emergency or charitable care). We also considered the possibility of erroneous decedent status by looking for evidence of healthcare utilization during the 12 months after death. We excluded cases with evidence of utilization more than one month after the date of death. Variables and Analysis ---------------------- We used VA encounters and ICD-9-CM codes to demographically (e.g., age, gender, marital status, state of residence, and race / ethnicity) and clinically characterize decedents \[[@B21]-[@B26]\]. We identified veterans with any visit or admission for congestive heart failure (CHF), ICD-9-CM 398.91, 402.x1, 404.x1, 404.x3 428.x excluding procedures, chronic obstructive lung disease (COPD), ICD-9-CM 491--492.x, 494.x, 496, end-stage liver disease (ESLD), ICD-9-CM 571.2--571.9,572.2--572.8, dementia, ICD-9-CM 046.1, 290.0--290.43, 331.0--331.7, 333.4, 438.0, and malignant neoplasia, ICD-9-CM 140.0--208.9 \[[@B25]\]. To identify end-stage renal disease (ESRD), we used procedure and clinical stop codes that identify the type of care received (e.g., dialysis) \[[@B26]\]. We developed a complexity index of co-morbidity based on a simple count of advanced illnesses. To understand the limitations of single source mortality ascertainment, we described decedents who would not have been identified by a strategy using either death certificates alone or VA decedent files alone. We compared these cases with the rest of the cohort based on their demographic and clinical attributes and the settings in which they received care. Based on distributions, we used Wilcoxon tests for continuous and chi-square tests for categorical variables. Results ======= From 345,380 deaths during the period 30 September 1999 to 1 October 2000 identified in BIRLS, we distinguished 6,071 decedents who were users of VA inpatient, outpatient, or long term care services in California. California death certificates included 227,308 deaths during the same period, including 3,580 additional users of VA inpatient, outpatient, or long term care services in California. Using SSN and other identifiers to match decedent cases to VA utilization data, we excluded non-veterans (n = 365), users of only non-clinical care such as laboratory tests (n = 251), those possibly alive based on subsequent VA encounter data (n = 229), and 3 cases for other reasons. Of the final cohort of 8,813 veteran decedents, 5,698 (65%) cases were identified in both source files, while 689 (8%) were only identified in VA decedent files, and 2,426 (28%) additional cases were only identified through death certificates (Figure [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Cohort Development ::: ![](1478-7954-3-2-1) ::: We examined potential biases associated with veteran decedents missed by either single source of mortality ascertainment (e.g., VA BIRLS or California death certificates). Ninety-nine percent of decedents missed by using VA data alone were California residents (vs. 92% of the remainder cohort, p \< 0.001); whereas, 62% of those missed by using death certificates alone were out-of-state residents (vs. 1% of the remainder cohort, p \< 0.001). Relatively fewer veterans of white or black ethnicity and relatively more veterans of missing ethnicity were represented among decedents missed by either single source strategy. The proportion of married or previously married veterans was higher and single or missing marital status lower among those decedents missed using only BIRLS, and relative proportions were reversed for a strategy using only death certificates. Decedents missed by either single source approach were less likely to have been diagnosed with an advanced chronic illness than the identified cohort. Veteran decedents missed by using only BIRLS were less likely to be diagnosed with any condition except HIV and dementia, and those missed by using death certificates alone were less likely to be diagnosed with any condition except HIV. With a BIRLS only approach, 37% of missing cases vs. 35% of the remainder cohort (p \< 0.001) had no diagnosed chronic illness (death certificate only approach; 69% vs. 32%, p \< 0.001). Veteran decedents missed by either single source approach were equally or more likely to have been users of the outpatient setting, but missed cases were less likely to have been users of inpatient healthcare settings (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Potential Bias Associated with Alternative Strategies For Veterans\' Mortality Ascertainment \* ::: BIRLS Only Strategy Death Certificate Only Strategy --------------------- ------------------------- ------------------------------------- ---------- ------- ------- --------- Number of cases 6,387 2,426 8,124 689 Age (years) 70.86 71.15 0.8253 70.95 70.79 0.6891 Gender Male 98 97 0.2733 98 98 0.8662 Race / Ethnicity White 57 54 58 31 \<0.001 Black 12 8 11 6 Hispanic 5 5 5 1 Other 2 2 2 1 Missing 24 31 \<0.001 23 61 Marital Status Married 46 49 47 45 0.0028 Single 16 13 15 16 Divorced 23 24 23 23 Widowed 11 12 11 11 Missing 4 2 0.0002 3 6 State of Residence California 92 99 99 38 Non-California 8 1 \<0.001 1 62 \<0.001 Diagnosis Cancer 35 32 0.0426 35 17 \<0.001 CHF 22 19 0.0175 22 7 \<0.001 COPD 28 24 0.0002 28 11 \<0.001 ESLD 6 4 0.0327 6 3 0.0010 ESRD 3 1 \<0.0001 3 0 0.001 Dementia 11 11 0.9656 11 3 \<0.001 HIV 1 1 0.1586 1 0 0.0621 Complexity Index 0 35 37 32 69 1 35 39 38 22 2 22 18 22 6 3 7 5 7 2 4 1 1 \<0.0001 1 0 \<0.001 Site of Utilization Any inpatient Any long term 45 29 \<0.0001 42 21 \<0.001 care 20 12 \<0.0001 19 7 \<0.001 Any outpatient 95 96 0.0225 95 96 0.1218 \*Findings are expressed as proportions unless otherwise identified. P-values reflect Wilcoxon two-sided probabilities for continuous variables and chi-square for categorical variables. Categorical tests reflect tests for differences including missing. ::: Discussion ========== Veterans\' mortality ascertainment was significantly improved by using both VA and death certificates as source files. Our findings indicate that either single source approach for mortality ascertainment may misrepresent veteran mortality based on comparisons of race / ethnicity, marital status, severity of illness, and settings of care. Diagnoses associated with serious medical co-morbidity and the likelihood of receiving any inpatient services (e.g. hospital or long term care) were both significantly lower among veterans missed by either single source approach. Our findings are consistent with Washington State where the deaths of 25% of 533 veterans who only used outpatient services were only identified with death certificates, and 5% were only identified in BIRLS. \[[@B9]\] Using BIRLS only for mortality determination, it is unclear why generally healthier, primarily outpatient users are less likely to be noted. Death notification is typically triggered by benefit claims (e.g., burial assistance, pension and related benefits). Affluent veterans whose families might be less likely to file benefit claims were drawn to the VA recently \[[@B27]\]. However, poverty or low social support might also make it harder to file claims. On the other hand, a death certificate only approach to ascertainment misses relatively fewer non-resident veterans. Such veterans may be homeless or mobile, retired veterans, and they may seek care transiently in California, or their deaths may be recorded elsewhere. One limitation of our study is that we did not identify cases that were only decedents by virtue of VA utilization files alone rather than BIRLS, although Dominitz, et. al., identified only 2.7% of deaths this way \[[@B11]\]. We did not compare VA files or death certificates to the National Death Index (NDI), as have previous studies that have used the NDI as a gold standard. The NDI is a central data repository of state vital statistics that is often used as a gold standard in US mortality studies \[[@B28]\]. We report findings for only one state, but given similar findings in Washington State, it would be helpful to determine if this is a national issue or there are particular state issues related to BIRLS death file agreement, or concerns related to veteran morality ascertainment with California death certificates. Conclusion ========== Researchers, managers, and policy makers should understand the limitations of sources of mortality ascertainment. The relationship of missing data to bias is related somewhat to how \"missingness\" is distributed by the outcome of interest. Our findings suggest these concerns may be relatively more important for studies involving veterans and racial-ethnic disparities, co-morbidity, certain disease comparisons, or settings of care. Additional study is needed to compare BIRLS, death certificates, and the NDI for mortality ascertainment in veterans. If our findings are confirmed, the VA may need to consider improving its system for mortality ascertainment through routine linkages to national mortality data. Studies of end-of-life care using decedent cohorts need to pay particular attention to the incompleteness of VA data as the sole source of mortality information. List of Abbreviations Used ========================== VA, Veterans Administration; FY, fiscal year; BIRLS, Beneficiary Identification and Records Locator System; NPCD, National Patient Care Database; SSN, Social Security Number; CHF, congestive heart failure; COPD, chronic obstructive lung disease; ESLD, end-stage liver disease; ESRD, end-stage renal disease. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= KL originated and oversaw all aspects of the conception, design, analysis, and publication of the study. SA, LR, and EY contributed to conception, design, and analysis. MW contributed to analysis and is responsible for programming. All authors reviewed and approved of the manuscript. Acknowledgements ================ Dr. Lorenz is a recipient of a VA HSR&D Career Development Award These analyses were also supported by a Locally Initiated Project award (LIP \# 2001-001) from the VA Greater Los Angeles HSR&D Center of Excellence. The views expressed in this article are those of the authors and do not necessarily represent the views of the Department of Veterans Affairs.	PubMed Central	2024-06-05T03:55:54.015743	2005-2-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554976/", "journal": "Popul Health Metr. 2005 Feb 24; 3:2", "authors": [ { "first": "Karl A", "last": "Lorenz" }, { "first": "Steven M", "last": "Asch" }, { "first": "Elizabeth M", "last": "Yano" }, { "first": "Mingming", "last": "Wang" }, { "first": "Lisa V", "last": "Rubenstein" } ] }
PMC554977	Background ========== Median laparotomy is the most common technique of abdominal incisions because it is simple, provides adequate exposure to all four quadrants, is rapid to open and usually bloodsparing \[[@B1]\]. A major problem after median laparotomy remains the adequate technique of abdominal fascia closure. In prospective studies the incidence of incisional hernias varies from 9% to 20% \[[@B2],[@B3]\]. Wound infection, obesity and suture closure technique are addressed as major risk factors for the development of an incisonal hernia \[[@B4],[@B5]\]. Whereas patient related factors such as age, gender, body mass index (BMI), underlying disease, co-morbidities, prior surgical procedures and life-style factors (e. g. smoking) cannot be controlled or standardised, the decisive chance to lower the incidence of incisional hernias is to optimise the surgical technique. Therefore, a great variety of suture materials and needles has been developed to provide an adequate closure of the fascia and thus the abdominal wall. Thousands of patients have been included in trials in order to answer the question which is the optimal method in abdominal fascia closure and today a number of reviews and a meta-analysis are available. However, the reliability of the existing evidence is compromised by the low number of relevant randomised controlled trials (RCT\'s) \[[@B6]-[@B8]\]. Therefore the discussion regarding the optimal technique of abdominal fascia closure continues and most surgeons practice according to their own experience rather than acting evidence-based. This attitude resulted in an unchanged frequency of incisional hernias over the last decades \[[@B5]\]. None of the prior studies comparing rapidly absorbable braided materials with interrupted sutures versus slowly absorbable monofilament suture materials in a continuous technique were able to determine a definite superiority for one technique. Possible reasons may be small numbers of patients in each group or short follow-up \[[@B9]-[@B12]\]. We therefore conclude that there is a lack of data from a truly well-designed long-term trial performed in the daily practice of surgery. This has led us to develop a large randomised controlled trial comparing different surgical techniques of abdominal closure after median laparotomy. INSECT is a multi-centre, intraoperatively randomised controlled trial comparing three different standardised surgical techniques with certain needle/suture combinations on the occurrence of incisional hernia in patients with elective primary midline laparotomy. Two groups will use running sutures with different longitudinal elasticity (one group PDS™ and the other MonoPlus™) combined with an atraumatic needle and one group interrupted sutures (Vicryl™) with a traumatic needle. A three-group parallel equivalence design was selected because due to the results of the latest published meta-analysis \[[@B11]\] the superiority for one closure method has not been definitely proven. The randomisation procedure will be done stratified for participating centers. The planned sample size is at least 600 patients with a follow up period of three years. INSECT is the first large-scale trial that started after a detailed theoretical and practical training of the participating surgical centers in March 2004 in order to reduce surgical bias in the study. INSECT will provide internal valid data for an adequate surgical technique of abdominal closure. Although we are well aware that this study cannot answer all open questions, the results should help to further improve evidence based surgery. Design ====== Trial organization ------------------ INSECT has been designed and carried out by the Study Centre of the German Surgical Society (SDGC). The SDGC is an independent research group of the German Surgical Society and the Medical School of the University of Heidelberg that has to design, conduct and analyse large randomised surgical trials in order to improve daily surgical practice. The role of the sponsor (BBD Aesculap) is limited to material supply and local first-level-support. The sponsor is not involved in the database management and has no access to the randomisation code. Coordination ------------ The trial is coordinated by the SDGC, which is responsible for overall trial management, trial registration (International Standard Randomised Controlled Trial Number (ISRCTN 24023541), <http://www.controlled-trials.com>), database management, quality assurance including monitoring, reporting and for the scientific program of all trial related meetings. Investigators ------------- Patients will be recruited by over 20 surgical centres in Germany. All investigators are hospital-based surgeons with a focus on general surgery. In order to obtain a representative trial result hospitals of all levels of care and education (county/community, private and university centres) are participating in this trial. Adverse events committee ------------------------ This committee consists of 3 surgeons and decides on the final diagnostic classification of critical clinical events. For all serious adverse events the documentation and relevant patient data are verified by co-ordinating personnel of each centre before submitting the data to the Adverse Events Committee for diagnostic classification. Burst abdomen, pulmonary infection and wound infection are secondary endpoints, but are also defined as Adverse Events (definitions see table [1](#T1){ref-type="table"}). Burst abdomen and postoperative pulmonary infection will even be always a Serious Adverse Event. The term Adverse Eventcovers any sign, symptom, syndrome or illness that appears or worsens in a patient during the period of observation in the clinical trial and that may impair the well-being of the patient. The term also covers laboratory findings or results of other diagnostic procedures that are considered to be clinically relevant. A Serious Adverse Eventis any adverse event that occurs at any time during the period of observation, that results in death, is immediately life-threatening, requires or prolongs hospitalisation, results in persistent or significant disability or incapacity. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Definition of early onset and late complications ::: Complication Definition ------------------------------------------ ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Burst abdomen Postoperatively missing continuity of the abdominal fascia in combination with a wound dehiscence with consecutive relapse operation. Wound infection Redness, wound dehiscence with secretion either of putrid or caliginous, smelly fluid or requiring antibiotic treatment or surgical intervention. Postoperative pulmonary complication Infection of the lung with either evidence of increased infection parameters (CRP \> 2 mg/dl and/or leukocytes\> 10 0000/ml) which are not caused by a different pathologic process or evidence of pulmonary infiltration in the chest x-ray, requiring antibiotic therapy. Incisional hernia Postoperative evidence of a fascia dehiscence after completed superficial wound healing with or without prolapse of abdominal organs, confirmed by abdominal ultrasound. ::: Analysis of safety related data is performed with respect to frequency of: • Serious Adverse Events and Adverse Events stratified by body-system • Adverse Events stratified by severity • Adverse Events stratified by causality. Study material supply --------------------- Study materials for all centres will be acquired by the BBD Aesculap company. Each type of suture material derives from a single batch to eliminate material inconsistencies. All materials are delivered to the participating centres by local representatives of the sponsor who also guarantee local first-level-support. On-site monitoring ------------------ During recruitment of patients each centre is monitored on site according to good clinical practice (GCP) guidelines. The data monitoring for this trial will be performed by an independent study nurse who is not involved in the trial or in completion of the case report form (CRF). The surgical monitoring will be done by independent surgeons being not involved in conducting this trial. Ethics, Informed Consent and Safety ----------------------------------- The final protocol was approved by the ethics committee of the University of Heidelberg, Medical School. Secondary approval is gathered from all local ethics committees responsible for the participating centres. Informed consent will be obtained from each patient in oral and written form before inclusion in the trial. Patient selection ----------------- INSECT focuses on hospitalised patients over 18 years of age who are planned for an elective primary abdominal operation and are eligible for a vertical abdominal incision in order to perform the planned surgical procedure. A detailed overview of all eligibility criteria is given in Table [2](#T2){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Eligibility Criteria ::: --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Inclusion criteria Exclusion criteria ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------- • Age equal or greater than 18 years\ • Peritonitis\ • Expected survival time more than 12 months\ • Emergency surgery\ • Patients undergoing primary and elective median laparotomy (patients with prior laparoscopy or abdominal operation via paramedian incision (e.g. appendectomy) may be included in the trial)\ • Participation in another intervention-trial with interference of intervention and outcome of this study\ • BMI \< 35\ • Coagulopathy\ • Expected length of incision \> 15 cm\ A group of disorders of the blood clotting (coagulation) system in which bleeding is prolonged and excessive with abnormal values in the blood laboratory.\ • Patient must be able to give informed consent\ • Severe psychiatric or neurologic diseases\ • Patient has given informed consent • Lack of compliance\ • Drug- and/or alcohol-abuse according to local standards\ • Current immunosuppressive therapy (more than 40 mg of a corticoid per day or azathioprin)\ • Chemotherapy within 2 weeks before operation\ • Radiotherapy of the abdomen completed longer than 8 weeks before operation\ • Inability to follow the instructions given by the investigator or the telephone interviewer (insufficient command of language, dementia, lack of time)\ • Lack of informed consent --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: Study objectives ---------------- The primary objective of this study is to compare the frequency of incisional hernias between three different abdominal fascia closure methods after one year postoperatively: two continuous slowly absorbable monofilament suture materials with and without longitudinal elasticity respectively (MonoPlus™ USP 1, 150 cm loop, with a HRT-48 needle, BBD Aesculap Tuttlingen, Germany and PDS II™ USP 1, 150 cm loop, Ethicon Norderstedt, Germany) and a interrupted suture with an absorbable braided suture material (Vicryl™ USP 2, 6 × 45 cm, non-needled plus a traumatic needle, Ethicon Norderstedt, Germany). Secondary objectives are the frequencies of early and late onset complications such as burst abdomen, postoperative pulmonary complications, wound infections and incisional hernias after three years postoperatively. Additionally a set of surgical and non-surgical parameters related to the operation will be analysed as secondary objectives such as the frequencies of various complications, the lung function and the postoperative length of hospital stay. A qualitative analysis is included in the study to assess the relevance of the primary endpoint from the patient\'s and the surgeon\'s perspective. The following aspects are ranked in a descending order from 1 (= most important) to 9 (= least important): postoperative complication, intraoperative complication, length of hospital stay, onset of enteral nutrition, death, postoperative pain, postoperative fatigue, convalescence of the complete physical maximum resilience and cosmetic result \[[@B13]\]. The ranking by the surgeon is done once for each surgeon before the operation. Patients are completing the ranking twice: first at inclusion and second at their discharge, to investigate if the patient\'s initial perspective changes within the hospital stay. Those various outcome parameters will be evaluated as part of an additional scientific project to build up a basis for further relevant questions in abdominal wall closure. Randomisation and surgical technique ------------------------------------ A block-randomisation-list is generated via computer system (SAS Version 8.2, SAS Institute Inc., Cary, USA) and stratified for the individual centre. Each centre will contribute 30 patients (10 to each group). The sealed randomisation list is stored in the investigator file. Patients are randomised using sealed opaque envelopes in the operation theatre after the surgical procedure has been started and before the abdominal wall will be closed. Major challenge in this surgical trial compared to pharmaceutical trials is the standardisation of the surgical technique. All patients undergo a skin incision using electric cautery. Upon completion of the surgical procedure, closure of the abdominal wall is performed in all three groups in a standardised manner: four sharp Mikulicz-Clamps are placed at the corners of the incision and in the middle of the edges of the abdominal fascia and then the closure technique will be performed according to randomisation. Three groups are available: a continuous, all-layer closure technique with either two monofilament loops or an interrupted technique using a braided material (material as described above). In all three groups suturing is initiated at both ends of the incision towards the middle, whereas the continuous suture line is overlapping at the centre for at least 2 cm to secure each other (for details see figure [1](#F1){ref-type="fig"}). Neither a subcutaneous closure nor a subcutaneous drainage is to be inserted. Skin closure is done with skin clips. Measurement of the length of scar in centimeters (cm) is performed. A detailed description of the required surgical technique is given in the INSECT-study protocol enhanced by images and sketches as well as video material used during the investigator meeting and provided to all centres and investigators. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Principles of continuous abdominal wall closure.A Anchorage of the suture cranially/caudally outside the incision B Intersection of the loops in the middle of the incision C Knotting of each loop Images reprinted with courtesy of Mrs. B. Wiehn, BBD Aesculap, Tuttlingen Germany ::: ![](1471-2482-5-3-1) ::: In an investigator meeting before trial initiation (March 5^th^-- 6^th^, 2004, AESCULAPIUM, Tuttlingen, Germany) all participating centres have been trained in the required techniques using abdominal wall models of mini-pigs. An evaluation of the investigator meeting was performed by all participants and will be published shortly. Investigators being unable to attend were trained on-site using the same training materials as in the investigator meeting. Training materials were supplemented by videos demonstrating the abdominal wall closure techniques (both continuous and interrupted) in the in vivo situation and in the animal model giving all sub-investigators good insight into the required techniques. Furthermore, a trial manager of the SDGC is available and responsible for all trial related issues and questions. Blinding -------- The patient is blinded for the technique of abdominal wall closure as the randomisation is performed intra-operatively. The patient will remain blinded until the assessment of the primary end-point at one year post-operatively. If feasible within the infrastructure of the participating centre, observers should be independent and not involved in completing the CRF and would thus also be blinded for the used technique. Follow up --------- Patients are observed for 30 days postoperatively for early onset complications defined as secondary endpoints such re-admittance for burst abdomen. One year after the operation the primary end-point will be assessed by documenting the incidence of incisional hernia with a physical examination and ultrasound of the abdominal wall. Follow up is completed 3 years after the primary operation to register any long-term complications of the used method for abdominal wall closure and to document any late-occurring hernias (see table [3](#T3){ref-type="table"} for detailed follow up). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Flow Chart INSECT-Trial ::: Visit 1(=Screening) 2(OP) 3(day 2 post OP) 4(day of discharge) 5(6 months +/- 1 month post OP) 6(12 months +/- 1 month post OP) 7(three years after operation) Extra-visit(secondary endpoint, AE or SAE) -------------------------------------------------------------------------- --------------------------------------------- ---------------------------- ---------------------- -------------------------------------------- ------------------------------------- -------------------------------------------- ------------------------------------ ------------------------------------------------ Past medical history X Informed consent X Physical examination including the personal data X X X Basic study-related examination I (for each secondary endpoint, AE, SAE) X (ranking of the patient before operation) X (ranking of the surgeon) X X (ranking of the patient after operation) X X (ranking of the patient after operation) X Basic study-related examination II X Ultrasound of the abdominal wall X X X Lung function test X X Medication X X X X Past medical history: past medical history, past surgical history, indication for operation, diabetes, renal insufficiency, smoking, lung disease Personal data: gender, date of birth, height in cm, weight in kg Basic study related examination I: physical examination for evaluation of all secondary endpoints including adverse events/serious adverse events where appropriate Basic study related examination II: telephone visit of patient and/or practitioner and physical re-examination if necessary in case of unclear abdominal wall status Physical examination: vital signs (blood pressure systolic/diastolic in mmHg, heartrate in /min), prior abdominal incisions, rectus diastasis Ultrasound: standard abdominal wall investigation (if hernia present: length and width in cm) Lung function test: FEV, vital capacity % ::: Data management and quality assurance ------------------------------------- Investigators enter data directly in paper-based case report form (CRF). These are arranged for each visit time-point and contain instructions and relevant definitions. All treatments are recorded in treatment logs. Standard adverse events forms are used to document (serious) adverse events and relevant clinical procedures that have been carried out. After verification of the data entered according to \"Good Clinical Practice\" (GCP), one copy of each completed CRF is sent by mail to the SDGC. A concurrent database is maintained there. All incoming CRF are scanned to be electronically archived. Data are entered in a specially developed relational data base management system. The data entry module contains on-line range and logical checks. For data that are found missing, illegible or inconsistent, data clarification forms are generated which are sent to the on-site monitor for resolution. Certain events must be reported immediately by the investigator by fax on preprinted forms directly to the coordinating centre. Examples are: informed consent and randomisation form, serious adverse events and premature withdrawal form the trial. The reporting of serious adverse events complies with national regulatory requirements. Statistical considerations and sample size estimation ----------------------------------------------------- Statistical methods are used to assess the quality of the data, homogeneity of treatment groups, endpoints and safety of the three different techniques. The analysis is performed on the basis of an intention to treat (ITT) population and with respect to ITT principles. A patient belongs to the ITT population after the randomisation. The primary endpoint will also be analysed on the basis of a \"per protocol\" population. To enable multiple comparisons in this three-armed study the closed testing procedure will be used\[[@B14]\]. All three treatment groups are considered separately without assuming any pre-specified monotonic trend among groups. All testing is done two-sided. The three elementary hypotheses address pairwise comparisons of the incisional hernia rates R~1~, R~2~, and R~3~. These hypotheses H~12~: R~1~= R~2~, H~13~: R~1~= R~3~, and H~23~: R~2~= R~3~, are all contained in the global null hypothesis H~123~: R~1~= R~2~= R~3~. Sample size estimation, however, will be based on the elementary hypotheses, thus leading to sufficient power in the global test. Bauer had recommended that \"sample sizes should be chosen large enough to give a high chance of jumping over the initial hurdle.\" \[[@B15]\]. The calculation of sample size is based on literature data, as summarised most recently by van \'t Riet et al \[[@B11]\]. With interrupted suturing with an absorbable material, such as Vicryl^®^, incisional hernias were seen in about 13% of patients. This number is primarily based on the four-armed trial by Wissing et al. \[[@B16]\]. With the use of slowly absorbable materials, such as PDS^®^, MonoPlus^®^or Maxon^®^, lower herniation rates were found, but this holds true only for continuous suturing. So far, only one trial has compared fast and slowly absorbable materials for interrupted sutures \[[@B17]\]. The comparison of interrupted rapidly absorbable versus continuous slowly absorbable sutures has been the aim of four previous studies. The results of these studies showed a non-significant tendency towards a lower rate of incisional hernia. Taking this data and the improved suture properties into consideration, a hernia incidence of 4% could be reasonably expected in one or both of the non-Vicryl^®^groups over the first postoperative year. Smaller differences are also unlikely to be of clinical relevance. Assuming annual hernia incidences of 13% and 4% in at least two of the groups, a raw sample size of 172 patients per group can be calculated with a two-sided alpha of 0.05 and a beta of 0.20 (employing Fisher\'s exact test). We used the PS power and sample size program of Dupont and Plummer (Version 1.0.17) for sample size estimation (freely available at: <http://www.mc.vanderbilt.edu/prevmed/ps.htm>). To account for an estimated 10% loss to follow-up and 2% surgical non-compliance with treatment allocation, sample size should be increased to 200 patients per group. In summary, the trial should recruit a total of 600 patients, with equal randomisation into the three groups. Thus a total of 720 patients have to be screened according to the CONSORT statement (Figure [2](#F2){ref-type="fig"}) \[[@B18]\]. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### INSECT-Trial according to CONSORT (Moher et al. Lancet 2001) ::: ![](1471-2482-5-3-2) ::: In the primary intention-to-treat analysis, testing will start with the global null hypothesis H~123~: R~1~= R~2~= R~3~. Only if this global test is significant at the 0.05 level, the three elementary hypotheses H~12~: R~1~= R~2~, H~13~: R~1~= R~3~, and H~23~: R~2~= R~3~will be tested next. These pairwise comparisons (Vicryl^®^versus PDS^®^, Vicryl^®^versus MonoPlus^®^, and PDS^®^versus MonoPlus^®^) use the same alpha level of 0.05, because closed test procedures in three-armed designs do not require alpha level adjustment. If, however, the first global hypothesis cannot be rejected at alpha level, the family of elementary hypotheses will not be tested at all, except for exploratory reasons. To control for possible differences with regard to surgical procedures (colorectal vs. gastric or pancreatic operations), centre, and surgical expertise (board-certified surgeon vs. assistant surgeon), logistic regression will be used. We expect that the use of logistic regression will not essentially comprise power assumptions when compared to univariate testing. The inclusion of time-to-event data in the primary statistical analysis (e.g. by applying Kaplan-Meier-curves or Cox-regression) does not confer specific advantages and seems unwarranted, also because an incisional hernia causes similar consequences for the patient irregardless of whether the hernia occurred after 3, 6 or 9 months. Secondary endpoints, demographic and other variables ---------------------------------------------------- The analysis of the secondary endpoints and demographic variables will be descriptive. The description of continuous variables includes at least: numbers of observations, mean, standard deviation, median, minimum and maximum. The description of categorical variables (ordinal or nominal) includes at least the number and percentage of patients belonging to the relevant categories in the trial population as well as in each treatment group. The description of the ranking of parameters of interest (patient\'s view before, after operation (day of discharge and 12 months later) and surgeon\'s view) includes the mean and median for each category in the trial population. There will be a listing for all measurements taken for the patients. In order to improve the presentation of the observed data, graphical methods will be applied. Closing of the clinical database and follow up database ------------------------------------------------------- The clinical database including all information until 12 months after the operation will be closed six months after the last visit, complete documentation of all cases and resolution of all queries. At this time the primary endpoint of the trial will be ascertained. The information of the final follow-up three years after the operation will be added later. This database will be closed three months after the last telephone three years after the last operation. Current status and planning --------------------------- The initial idea and hypothesis for the study was developed in March 2003. After a systematic review of the literature according to Cochrane standards has been performed the study protocol was completed in October 2003. The study protocol was approved by the local ethics committee of the University of Heidelberg in December 2003. Preparation of all study and instructional materials (including video clips) was completed in February 2004 and the first investigator meeting was held in Tuttlingen, Germany on March 5^th^and 6^th^, 2004. In June 2004, following completion of contracts and approval of local ethics committees, the first centres were initiated and the first patient was recruited in July 2004. Currently 23 centres are in an active status recruiting the required 30 patients per centre. Further 6 centres are processed and will be activated within the next months. Assuming an enrolment of 5 patients per month and centre the end of recruitment is assumed to be in October 2005. Discussion ========== The strategy to publish study protocols in surgery and thus to enhance transparency in a clinical trial might be relatively new in surgery, but has been practised in other medical fields for several years \[[@B19],[@B20]\]. This mentioned transparency increases reliability and validity of the results when a detailed description of the experiment is published prior to the conduction. The assumption how many patients should be screened to include the sufficient number of patients based on the sample size calculation needs to be transparent in order to evaluate whether results from this trial are transferable to daily practice. Therefore a flow chart according to the CONSORT statement should be included in study protocols (Figure [1](#F1){ref-type="fig"}). However, many important details of a randomised surgical trial are interesting to the reader and can not be published together with the final results. It also seems out of the question that medical journals that surgical trials should be internationally registered \[[@B21]\]. INSECT is designed to help answering the question which is the optimal method for abdominal wall closure. Although there are a substantial number of randomised studies and several meta-analyses examining different techniques of abdominal fascia closure the optimal and definite method for closing the abdomen has not yet been found \[[@B6],[@B7],[@B11]\]. Therefore, the technique and materials for abdominal wall closure are still determined by local material supply and surgical tradition. These preconditions and a detailed literature research gave us reason to design a trial comparing three standardised common techniques of abdominal fascia closure in abdominal surgery. Several design features of INSECT were discussed before the trial started. A two armed or a three armed study design was debated before writing the protocol. As we wanted to compare the clinically most relevant and most evidence-based suture materials and suture techniques for closure of midline abdominal incisions the most recent and most relevant meta-analysis as a data-base for choosing the different groups in our trial was used \[[@B11]\]. Two different suture techniques and materials became clear to show the best results in abdominal fascia closure and revealed the lowest incidence for incisional hernias: slowly absorbable continuous (or running) sutures and absorbable interrupted sutures. Aside from those two groups of suture techniques (continuous versus interrupted sutures) we included a third group using the same technique as one of the other groups (continuous suture) but using a different suture material (monofilament suture with longitudinal elasticity) which is supplied by a different company. INSECT depicts the surgical reality and variety of abdominal wall closure as it compares not only different suture techniques but also several suture materials offered by different companies. Therefore, the results of INSECT will be relevant for the majority of surgeons as the materials and techniques used in this trial are widely accepted in daily use. An important issue for a good trial is to define adequate and well designed end-points. Regarding the optimal method for abdominal closure a great variety of objectives has to be considered: wound infection, wound dehiscence, incisional hernia, suture sinus, wound pain, etc. The socio-economic and surgical most relevant objective is the incidence of incisional hernias \[[@B4]\]. Because 50--70% of incisional hernias will occur within one year after operation we selected the frequency of incisional hernias at one year postoperatively as the primary end-point of our study. All other relevant objectives such as wound infection, pain, pulmonary impairment, etc. were included as secondary endpoints into the trial design. Additionally a qualitative analysis for surgeons and patients was planned to evaluate the importance of different outcome variables between surgeons and patients. A major challenge in this INSECT trial compared to other studies, e. g. pharmaceutical trials, is the standardisation of the surgical technique. Lack of standardisation in surgical trials is often an argument of opponents who state that the achieved trial results would not be transferable to surgical routine. Therefore all patients in the INSECT trial undergo a standardised skin incision and opening of the abdominal cavity as well as the closure of the abdominal wall is performed in all three groups in a standardised manner. To provide an overall highly standardised surgical procedure in every study site all participating centres have been trained in the required techniques using abdominal wall models of mini-pigs during an investigator meeting before trial initiation. For investigators who were unable to attend the meeting training materials and videos demonstrating the different abdominal wall closure techniques (both continuous and interrupted) used in the study were provided. For further quality assurance there will be a regular surgical monitoring during the trial. Additionally, randomisation is stratified for each individual centre and the trial results will be therefore comparable among the different participating centres. Conclusion ========== Over the last years, surgeons\' attitude towards randomised surgical trials has changed. It becomes more and more evident that surgical procedures could also be tested in randomised studies. Surgical expertise is largely a personal conviction and an apprenticeship with surgical techniques passed from one surgical generation to the next \[[@B6],[@B22]\]. Most surgeons rather use particular techniques because they are trained in than using the most evidence-based technique. The Study Centre of the German Surgical Society (SDGC) was founded 2003 in order to ensure that surgical techniques are more evidence based and not simply the result of a surgical dogma. This is the first multi-centre trial designed and conducted by the SDGC after a special Study Group was founded. We are aware of the difficulties and problems of performing large surgical multi-centre trials and, therefore, we decided to start with a trial examining a fundamental, \"simple\" and daily performed surgical technique. As the abdominal fascia closure is largely based on tradition rather than evidence, the results of the INSECT trial will help to create more evidence and to guide surgeons to a critical review of their surgical routine. Abbreviations ============= AWC Abdominal wall closure CRF Case Report Form GCP Good Clinical Practice ICH International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use IEC Independent Ethics Committee ITT Intention-to-treat-analysis PDS^®^Polydioxanone (absorbable suture material) RCT Randomised Controlled Trial SDGC Study Centre of the German Surgical Society Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= Hanns-Peter Knaebel and Moritz Koch designed and wrote the manuscript, Stefan Sauerland prepared bio-statistics of the trial and is in charge of the data management, Markus K Diener searched and assessed the literature, Markus W Büchler sponsors the trial and provides necessary infrastructure, Christoph M Seiler searched and assessed the literature and overlooked the completion of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2482/5/3/prepub> Acknowledgements ================ The trial is designed, managed and conducted by the Study Centre of the German Surgical Society (SDGC) and supported by the BBD Aesculap Company, Tuttlingen, Germany. Finally recruitment is performed by the participating surgical centres of the INSECT study group (Klinikum Augsburg, St. Josefs-Hospital Bochum, Klinikum links der Weser Bremen, Fürst-Stirum-Klinik Bruchsal, St. Josefs-Hospital Dortmund, Klinik Eichstätt, Westküstenklinikum Heide, Chirurgische Universitätsklinik Erlangen, Krankenhaus Mittleres Erzgebirge, Evangelische Kliniken Gelsenkirchen, Waldklinikum Gera, Chirurgische Universitätsklinik Heidelberg, Klinikum Herford, St. Bonifatius-Krankenhaus Lingen, Klinikum Lüdenscheid, Städtisches Klinikum Magdeburg, Kreiskrankenhaus Mosbach, Chirurgische Universitätsklinik München-Grosshadern, Chirurgische Universitätsklinik Regensburg, Klinikum Südstadt Rostock, Marienhospital Stuttgart, Robert-Bosch-Krankenhaus Stuttgart, Kreiskliniken Traunstein, Krankenhaus Gerresheim Düsseldorf, Dreifaltigkeits-Krankenhaus Wesseling, Krankenhaus Wermelskirchen, Westpfalz-Klinikum Kaiserslautern).	PubMed Central	2024-06-05T03:55:54.018421	2005-3-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554977/", "journal": "BMC Surg. 2005 Mar 8; 5:3", "authors": [ { "first": "Hanns-Peter", "last": "Knaebel" }, { "first": "Moritz", "last": "Koch" }, { "first": "Stefan", "last": "Sauerland" }, { "first": "Markus K", "last": "Diener" }, { "first": "Markus W", "last": "Büchler" }, { "first": "Christoph M", "last": "Seiler" } ] }
PMC554978	Background ========== In disorders resulting in poor fetal growth, and in pre-eclampsia, thrombotic lesions are frequently observed in the maternal and fetal vascular components of the placenta, \[[@B1]-[@B3]\] and hence have been implicated in the pathophysiology of these conditions. In addition, it has been reported that in vivo generation of thrombin, in maternal plasma, is higher in patients with small for gestation age fetuses and with pre-eclampsia, than in normal pregnancy \[[@B4]\]. It is well established, for many years, that thrombin plays a role in blood coagulation, but its effects in many other cell and tissue types (smooth muscle cells, endothelial cells, lymphocytes) \[[@B5],[@B6]\] have been the subject of more recent attention. It is now apparent that thrombin can regulate target cells by cleaving and activating a family of G-protein-coupled protease-activated receptors (PARs)\[[@B5]-[@B7]\]. This proteolytic cleavage of PARs is mediated by a family of enzymes that require serine within the active site i.e. serine proteases \[[@B5]\]. There are 4 major PAR subtypes (PAR1-4) with diverse reported functions in various tissues \[[@B6]\]. PAR activation has been closely linked to inflammation \[[@B6],[@B8]\], contraction of vascular \[[@B5]\] and non-vascular \[[@B9],[@B10]\] smooth muscle, and platelet activation \[[@B6]\]. Although PAR-1, PAR-3 and PAR-4 \[[@B11]\] are all known to be thrombin receptors, the mechanism of activation by thrombin at these different PARs varies \[[@B12],[@B13]\]. There is growing evidence, from vascular tissue studies in several animal models, that non-thrombotic thrombin-mediated signalling events are central to the response to the disease process typical of vascular lesion formation in atherosclerosis \[[@B14]\]. The direct effects of thrombin on vascular cells, via the PAR receptors, and particularly PAR-1, have been the main focus of investigation for this hypothesis. PARs 1, 3 and 4 are activated by thrombin \[[@B11]\], but PAR-1 is activated at low thrombin concentrations and most of what is known about thrombin signalling downstream of the receptors, has been derived from studies of PAR-1 \[[@B14]\]. While other PAR subtypes are present in human arterial vessels, it appears that PAR-1 is primarily involved in endothelium-dependent relaxation to thrombin and trypsin \[[@B15]\]. To our knowledge, there are no data outlining the potential effects of thrombin, or specific PAR-1 receptor modulation, on the feto-placental circulation, despite the critical role of thrombin in disorders of this vasculature. The aims of this study were to evaluate the direct effects of thrombin, the specific PAR-1 activating peptide (PAR1-AP), TFLLR-NH~2~(Thr-Phe-Leu-Leu-Arg-NH~2~), and the PAR-1 specific antagonist (N-trans cinnamoyl -p-fluoroPhe-p-quanidinoPhe-Leu-Arg-Orn-NH~2~) on human umbilical artery tone in vitro. Methods ======= The study was carried out in the Department of Obstetrics and Gynaecology, University College Hospital Galway, Ireland between May 2002 and April 2003. Sections of human umbilical cord approximately 10 cm in length were excised from the proximal segment of the cord (i.e. closest to the placental attachment) immediately after elective cesarean delivery. Samples were obtained from 17 women after elective cesarean section at term and from one patient after normal vaginal delivery. All pregnancies were uncomplicated and there was no evidence of hypertensive disease or intrauterine fetal growth restriction. The mean maternal age was 32.47 years (range 25--40 years). The median period of gestation was 38 weeks (range 37--42 weeks). The reasons for cesarean section included previous cesarean section (n = 9), breech presentation (n = 6), previous myomectomy (n = 1) and unstable fetal lie (n = 1). At the time of recruitment 4 women were nulliparous and 13 women were parous. Samples were immediately placed in cold buffered Krebs Henseleit physiological salt solution (pH 7.4) of the following composition: potassium chloride 4.7 mmol/L, sodium chloride 118 mmol/L, magnesium sulphate 1.2 mmol/L, calcium chloride 1.2 mmol/L, potassium phosphate 1.2 mmol/L, sodium bicarbonate 25 mmol/L and glucose 11 mmol/L. Maternal written informed consent was obtained prior to tissue collection, and the tissue collection procedure was approved by the Research Ethics Committee at University College Hospital Galway. Umbilical arteries were carefully dissected free of Wharton\'s Jelly and cut in rings 4--5 mm in axial length. Rings were suspended individually on stainless steel hooks inserted into their lumens and mounted under 2 g (30 mN) of isometric tension, in glass-jacketed tissue baths, as previously described \[[@B16],[@B17]\]. Each bath-contained 10 mL of oxygenated (95% O~2~/ 5% CO~2~) Krebs Henseleit physiological salt solution (PSS) at 37°C and pH 7.4. Rings were allowed to equilibrate for 90 minutes with regular washouts of PSS. During this interval, spontaneous tone developed. After the equilibration period, the vessel rings were challenged with 60 mM potassium chloride (KCl). Three washouts with PSS were carried out once the maximum response had reached a plateau, and a 20 minute recovery period was allowed in order that baseline be attained again. The KCl challenge was performed three times. After the last KCl challenge, 40 minutes recovery was allowed, and contraction was then stimulated by bath exposure of the vessel rings to 5-hydroxytriptamine (5-HT) (10^-7^M). Once maximum contractile response to 5-HT was attained, the rings were allowed to remain at plateau for 20 minutes. Concentration- effect experiments were performed by cumulative additions of thrombin, the PAR1-AP, or the PAR-1 antagonist to the tissue bath. Thrombin was added to the tissue bath at an initial concentration of 0.5 units/mL, and this was increased at 20-minute intervals to 1 unit/mL, 2 units/mL and 3 units/mL respectively. The bath concentrations ranges investigated for PAR1-AP were 1 nanomol/L, 10 nanomol/L, 100 nanomol/L and 1 micromol/L (i.e. 10^-9^- 10^-6^M), and for PAR-1 antagonist were 1 nanomol/L, 10 nanomol/L, 100 nanomol/L, 1 micromol/L and 10 micromol/L (i.e. 10^-9^-10^-5^M), all at 20-minute intervals. The effects of thrombin, PAR1-AP and PAR-1 antagonist were demonstrated by expressing the mean amplitude calculated during the 20-minute period following addition of each drug concentration, as a percentage of the mean amplitude obtained in the 20 minutes prior to any drug addition. This measurement represents percentage contractility or tone, and subtracted from 100%, provides the percentage relaxation value for each bath concentration of vehicle and study compounds. The net relaxant effect of each compound was calculated by subtracting the percentage contractility value calculated for its respective vehicle (for thrombin, PAR1-AP or PAR-1 antagonist), control experiment, at each similar bath concentration. All of the umbilical artery samples used for experimentation for each compound were obtained from different women (i.e. n = 6 for PAR1-AP), for example, was achieved by using umbilical artery samples from 6 different women). The allocation of umbilical artery samples for the different experiments was entirely random. Fresh Krebs Henseleit physiological salt solution was made and buffered daily. KCl solutions were prepared on the day of experimentation. A stock solution of 5-HT (Sigma-Aldrich, Dublin, Ireland) was made up in de-ionised water and diluted with Krebs solution. Thrombin was purchased from Sigma-Aldrich (Dublin, Ireland) and a stock solution of 1100 U/ml prepared in de-ionised water and stored at -20°C. PAR1-AP was purchased from Tocris Cookson Ltd (Bristol, UK) and a stock solution of 1 millimol/L was prepared using deionized water, with subsequent dilutions in PSS. The PAR-1 antagonist was prepared and assayed by methods previously described \[[@B18]\]. The IC~50~value for antagonist inhibition of platelet aggregation stimulated with 1 micromol/L SFLLRN agonist was determined to be 0.1 micromol/L. It was prepared as a 10 millimol/L stock solution in dimethysulphoxide (DMSO) and stored in room temperature protected from direct light. Final bath concentrations of DMSO, at the highest concentration of PAR-1 antagonist, did not exceed 1%, for both study and vehicle control strips. Serial dilutions of thrombin, PAR1-AP and the PAR-1 antagonist were made using Krebs Henseleit physiological salt solutions. Comparisons of measurements of amplitude for each bath concentration of thrombin, PAR1-AP and the PAR-1 antagonist, or respective control vehicle, were made using a one-way ANOVA. Post-hoc comparisons were made using the Tukey HSD test. A P value \<0.05 was accepted as statistically significant. The statistical package SPSS version 10 was used for statistical calculations. Results ======= A recording from control experiments (i.e. without addition of vehicle, thrombin, PAR1-AP or PAR-1 antagonist) demonstrating umbilical artery tone due to bath exposure of the ring to serotonin, for the entire duration of an experiment, is shown in Figure [1A](#F1){ref-type="fig"}. The mean net spontaneous relaxation of tone observed was 14.39% (SEM = 2.76). Thrombin exerted a potent and cumulative vasodilatory effect on umbilical artery tone in comparison to simultaneous vehicle (PSS) only experiments. A representative recording of the effects of thrombin is shown in Figure [1B](#F1){ref-type="fig"}. At bath concentrations at or greater than 0.5 units/mL thrombin exerted a significant vasodilatory effect. The net vasodilatory effects of thrombin are provided in Table [1](#T1){ref-type="table"}. The mean net inhibition of tone observed, at maximum thrombin concentration investigated (i.e. 3 units/mL) was 53.5% (SEM = 4.62; n = 6; P \< 0.001). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Representative recording of A)Serotonin induced contraction of umbilical artery, B)Serotonin induced contraction followed by cumulative additions of Thrombin and C)Serotonin induced contraction followed by cumulative additions of PAR1-AP. ::: ![](1477-7827-3-8-1) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Net inhibitory effect of thrombin, PAR-1 and PAR1-AP on human umbilical artery tone. ::: Thrombin (n = 6) (% ± SEM) PAR1-AP (n = 6) (% ± SEM) PAR-1 Antagonist (n = 6) (% ± SEM) (P Value) ---------------------- ---------------- --------------------- ---------------- ------------------------------ ------------------ --------------- *0.5 U/ml* \#28.90 ± 2.60 *10^-9^M* \23.91 ± 5.64 10^-9^M* 2.83 ± 3.05 (NS) (0.630) *1 U/ml* \35.39 ± 3.91 10^-8^M* \37.32 ± 2.29 10^-8^M* 3.80 ± 2.33 (NS) (0.471) *2 U/ml* \44.72 ± 2.31 10^-7^M* \52.39 ± 1.28 10^-7^M* 5.60 ± 3.74 (NS) (0.228) *3 U/ml* \53.51 ± 4.62 10^-6^M* \61.50 ± 1.43 10^-6^M* 7.03 ± 5.74 (NS) (0.148) *10^-5^M* 6.87 ± 4.48 (NS) (0.815) Values presented represent the mean inhibitory effects on umbilical artery tone i.e. after adjusting for control / vehicle experiments. The values provided represent % inhibition ± standard error of the mean (SEM). The PAR1-AP used was Threonine-Phenylalanine-Leucine-leucine-Arginine-NH~2~. The PAR-1 antagonist used was Ser- pFPhe-pGPe-Leu-Arg-Orn-NH~2~. The values were compared with amplitude measurements observed prior to drug addition (NS not significant, \# P \< 0.01, \* P \< 0.001). ::: TFLLR-NH~2~, the PAR1-AP, similarly exerted a cumulative vasodilatory effect on umbilical artery tone. A representative recording of the cumulative effects of PAR1-AP are shown in Figure [1C](#F1){ref-type="fig"}, with the mean difference in amplitude measurements (i.e. in comparison to simultaneous control experiments) provided in Table [1](#T1){ref-type="table"}. The mean net inhibition of tone observed, at maximum PAR1-AP concentration investigated (i.e. 10^-6^M / 1 micromol/L) was 61.5% (SEM = 1.43; n= 6; P \< 0.001). For the PAR-1 antagonist, N-trans cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH~2~, no relaxation of umbilical artery tone in vitro was observed, in comparison to vehicle only control experiments. Figure [2A](#F2){ref-type="fig"} demonstrates a representative recording of umbilical artery tone, after exposure to vehicle only (i.e. DMSO added cumulatively). The mean net relaxation of tone observed with vehicle was 70.28% (SEM = 2.98). In Figure [2B](#F2){ref-type="fig"}, a representative recording of the effects of cumulatively increasing bath exposure of arterial rings to the PAR-1 antagonist is shown. The mean net maximal inhibition exerted was 6.87% (SEM = 1.57; n = 6; P = 0.280). Finally, there was no observed difference in the vasodilatory effects of thrombin, PAR1-AP or the PAR-1 antagonist in relation to parity. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Representative recordings of A)umbilical artery tone after serotonin induced contraction with exposure to vehicle only (i.e. DMSO added cumulatively), and B)the effects of cumulatively increasing bath exposure of arterial rings to Ser- pFPhe-pGPe-Leu-Arg-Orn-NH~2~after serotonin induced contraction. ::: ![](1477-7827-3-8-2) ::: Discussion ========== This study demonstrates that thrombin exerts a potent vasodilatory effect on human umbilical artery vasculature in vitro. While it is known that thrombin has an inhibitory effect on vessel tone in other animal and human vascular tissue types \[[@B19]-[@B21]\] this is the first report, to our knowledge, of the direct effects of thrombin on human umbilical artery vasculature. Receptors for thrombin, the PAR family of receptors, are present on vascular smooth muscle cells, and on endothelial cells \[[@B14],[@B22]\], with the effects of thrombin mediated mainly via the PAR 1, 3 and 4 receptor subtypes \[[@B11]\]. The potential physiological function of thrombin in mediating vascular tone in the umbilical circulation in normal pregnancy is unknown. Our findings also raise questions in relation to the role of the non-thrombotic effects of thrombin in the feto-placental vasculature in disorders of pregnancy such as pre-eclampsia and intrauterine growth restriction, which are classically associated with thrombotic lesions or a relative excess of thrombin \[[@B1]-[@B3]\]. The possibility that the vasodilatory effect of thrombin may serve to counteract the diminished perfusion associated with the pathophysiology of these conditions, at least in the early stages of disease, is one hypothesis. A further theory is that the much enhanced uterine contractility elicited by thrombin and PAR1-AP \[[@B13]\] may be concomitantly associated with a feto-placental vasodilatory effect to maintain good utero-placental blood flow. We have not elucidated the exact mechanism, or mechanisms, of the thrombin mediated alteration in vascular tone in human umbilical artery. We have however demonstrated that a similar effect is elicited by bath exposure of the arterial rings to the specific PAR1-AP, TFLLR-NH~2~. PAR1-APs are recently designed small synthetic peptide ligands which mimic the effects of proteases by binding directly to the activation site of the PAR-1 receptor, bypassing the need for proteolytic cleavage of the receptor. Soluble peptide ligands, as PAR-APs are, can vary greatly in potency as agonists in comparison with proteases \[[@B10]\]. TFLLR-NH~2~is reportedly one of the more selective PAR1-APs and is deemed most preferable for purposes of studying the physiologic role of PAR-1 \[[@B10]\]. In view of the fact that PAR-1 function is central to a thrombin-mediated effect in other tissues \[[@B11],[@B14],[@B15]\] and that a PAR-1 mediated effect in human umbilical artery vasculature is similar to that elicited by thrombin, the findings from this study are suggestive of a major role for PAR-1 in mediating the vasodilatory effect of thrombin in umbilical artery vasculature. Other possible mechanisms, and the potential role of PARs 3 and 4, have not been evaluated in this study. The PAR-1 antagonist, N-trans cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH~2~, did not alter umbilical artery tone. This peptide is a competitive antagonist i.e., it competes for the agonist binding site and does not appear to have any other activity \[[@B23]\]. The direct effects of PAR-antagonism had not previously been evaluated on umbilical artery preparations in vitro, and hence their inclusion in this study. We have also previously demonstrated that this specific PAR-1 antagonist exerted a relaxant effect on human myometrium in vitro, the mechanism of which is unknown \[[@B24]\]. The results observed here serve to confirm that N-trans cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH~2~is inactive towards the PAR-1 receptor. The true role of PAR antagonists, in such experiments, is for the purpose of investigation of the potential effects of an agonist at a PAR subtype. Pre-incubation of the arterial rings with PAR-1 antagonist, for example, should alter the response elicited by PAR1-AP. Such experiments, with selective blockade of the PAR subtypes, in normal and diseased pregnancies, are a subject of further studies. There are some limitations to this study. All umbilical cord artery samples were obtained at elective cesarean section. This was included in the design of the study in order to maintain uniformity in terms of mode of delivery, and, to avoid using cord samples that may have undergone excessive traction in the third stage of labor at vaginal delivery. It is unknown whether this latter point is valid, or not, in studies using in vitro umbilical preparations. We are currently performing comparative studies from samples obtained at vaginal delivery to further evaluate this matter. Secondly, the effects of thrombin and PAR-1 modulation, on smaller vessels in the feto-placenta circulation (i.e. placental arteries), and in association with disorders of pregnancy, would compliment our knowledge of the importance of this pathway. Finally, there are limitations in extrapolating from in vitro experiments to the in vivo situation, but the experiments conducted here represent a reliable and valid in vitro model for these vascular preparations. Conclusion ========== In conclusion, thrombin exerts a potent vasodilatory effect on umbilical artery preparations in vitro. A similar effect is also observed using a specific PAR1-AP. The potential non-thrombotic role of thrombin, and PAR subtype modulation, in regulation of the feto-placental circulation in normal pregnancy, and in pregnancies complicated by hypertensive disease or intrauterine growth restriction, is highlighted by these findings. Authors\' contributions ======================= AJO\'L performed the experiments and wrote the manuscript. CJO\'S and NR performed the experiments. AMF analysed the data and wrote the manuscript. JTE provided the PAR-1 antagonist (N-trans cinnamoyl- p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH~2~) and wrote the manuscript. JJM designed, supervised the study and wrote the manuscript. All authors read and approved the final manuscript. Acknowledgements ================ Presented at the Fiftieth Meeting of the Society for Gynecologic Investigation, Washington, DC, March 26--30, 2003.	PubMed Central	2024-06-05T03:55:54.021963	2005-2-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554978/", "journal": "Reprod Biol Endocrinol. 2005 Feb 24; 3:8", "authors": [ { "first": "Aonghus J", "last": "O'Loughlin" }, { "first": "Crochan J", "last": "O'Sullivan" }, { "first": "Nandini", "last": "Ravikumar" }, { "first": "Anne M", "last": "Friel" }, { "first": "John T", "last": "Elliott" }, { "first": "John J", "last": "Morrison" } ] }
PMC554979	Background ========== Hypersensitivity pneumonitis (HP) is an interstitial lung disease (ILD) caused by the inhalation of and sensitization to a variety of environmental organic antigens. The immune mediated nature of the disorder is testified to by the characteristic sequel of events taking place in the lung after antigenic inhalation: an acute pulmonary neutrophilia occurs early followed by an interstitial T-cell infiltration of CD8 T-cell showing a limited expression of the T-cell receptor \[[@B1]\]. A number of data point to chemokines as orchestrators of inflammatory disorders which are characterized by a massive accumulation of immunocompetent cells within affected organs, including the lung \[[@B2]\]. Chemokines, which can be divided into four groups based on the positioning of the cysteine residues in the mature protein \[[@B3]-[@B6]\], induce directional migration of immune cells through their interactions with G-protein coupled receptors. Three chemokines induced by IFN-γ, IFN-γ-inducible protein-10 (IP-10, CXCL10), monokine induced by IFN-![](1465-9921-6-20-i1.gif) (Mig/CXCL10), interferon-inducible T-cell α-chemoattractant (I-TAC/CXCL11) bind to the CXCR3 receptor molecule which is expressed by activated T lymphocytes and natural killer cells \[[@B7],[@B8]\]. We have recently found that CXCR3 is expressed in vivo by CD4+ Th1 infiltrating the lung of patients with sarcoidosis and by T cells accumulating in the pulmonary parenchyma of lung-transplant recipients with rejection episodes \[[@B9],[@B10]\], providing evidence that CXCR3 expression constitutes an important mechanism in the regulation of T-cell migration to the lung. Furthermore, recent data in the animal model suggest that CXCR3/CXCL9, CXCL10, CXCL11 interactions are central in the pathogenesis of hypersensitivity reactions to Saccharopolyspora rectivirgula(SR) and successive granuloma formation \[[@B11]\]. Using immunohistochemical studies of tissue sections and a flow cytometry evaluation of cells recovered from the bronchoalveolar lavage (BAL), we studied the role of CXCR3/CXCL10 interactions in the regulation of T-cell migration into the lung of patients with hypersensitivity pneumonitis. We have shown that CXCR3 is expressed by T cells accumulating in the lower respiratory tract of patients with this hypersensitivity disorder. In addition, we found that signalling of CXCR3 with CXCL10 induces the in vitromigration of CXCR3(+)T cells. The ligand CXCL10 can be detected in pulmonary macrophages and is released by these cells. Materials and Methods ===================== Study population ---------------- 12 HP patients were included in the study (9 males and 3 females; mean age 38.3 ± 6.4 yr). The majority of the patients had farmer\'s lung disease (10 patients); 1 patient had bird fancier\'s lung, 1 patient had mushroom worker\'s lung. The following criteria for HP diagnosis were used: a) history of exposure to HP antigens, b) a symptomatic acute episode with chills, fever, cough, breathlessness 4 to 8 hours after exposure to specific antigens, c) radiological features (mainly diffuse reticular pattern) and/or a functional pattern of interstitial lung disease, and d) evidence of antibodies against S. rectivirgula in all except one case (bird fancier\'s lung). Each patient underwent bronchoscopy for transbronchial biopsy (TBB) and BAL analysis. BAL was performed according to the technical recommendations and guidelines for the standardization of BAL procedures \[[@B12]\]. Briefly, a total of 200 ml of saline solution was injected in 25-ml aliquots via fiberoptic bronchoscopy, with immediate vacuum aspiration after each aliquot. Immediately after the BAL, the fluid was filtered through gauze and the volume measured. A volume of 100-200 ml of BAL recovery and a sample of 50% of the instilled volume with a minimum of 50 ml was considered acceptable. The percentage of BAL recovery was 54.9% ± 4.2. Cells recovered from the BAL were washed 3 times with PBS, resuspended in endotoxin tested RPMI 1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 20 mM HEPES and L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FCS (ICN Flow, Costa Mesa, CA) and then counted. A standard morphological and immunologic analysis of BAL cellular components was performed and included cell recovery, differential count of macrophages, lymphocytes, neutrophils, and eosinophils, and flow cytometry analysis of the CD4/CD8 BAL T-cell ratio. Five healthy controls were selected (3 men and 2 women; average age 37.3 ± 4.3 yr; 2 non-smoking healthy adults and 3 non-smoking subjects evaluated for complaints of cough without lung disease). They showed normal physical examinations, chest X-rays, lung function tests and BAL cell numbers. Purification of alveolar macrophages and T cells ------------------------------------------------ Alveolar macrophages (AMs) and T cells were enriched from the BAL cell suspensions by rosetting with neuraminidase-treated SRBC followed by F/H gradient separations and residual CD3^+^lymphocytes were removed using high-gradient magnetic separation columns (Mini MACS, Miltenyi Biotec, Germany) \[[@B13]\]. Following this multistep selection procedure more than 95% of the above cells were viable, as judged by the trypan blue exclusion test. Staining with mAb showed that more than 99% of purified lymphocytes were CD3+ T cells. Monoclonal antibodies and cytokines ----------------------------------- The commercially available conjugated or unconjugated mAbs used belonged to the Becton Dickinson and PharMingen series and included: CD3, CD4, CD8, isotype matched controls. Anti-IL-4 and anti-IFNγ mAbs were purchased from PharMingen (San Diego, CA). Purified rabbit anti-human CXCL10 polyclonal antibody (R&D Systems Inc, Minneapolis, MN) and anti-hCXCR3 mAb (R&D Systems Inc) were also used. Immunohistochemical analysis of CXCR3+ cells and CXCL10 producing cells ----------------------------------------------------------------------- Open lung biopsies from 8 patients with clinical and histological diagnosis of hypersensitivity pneumonitis were studied by immunohistochemistry for the immunophenotype characterization of inflammatory cells and for CXCR3 and CXCL10 expression. Immunohistochemistry for the characterization of inflammatory infiltrate was carried out using the following antibodies (Dako Glostrup, Denmark): CD45 (1:20), CD43 (1:40), CD45RO (1:100), CD20 (1:100), CD3 (1:50), CD68 (1:50), CD4 (1:100), and CD8 (1:100). The immunoreaction products were developed using the avidin-biotin-peroxidase complex method. Immunostaining for CXCR3 was performed as previously described. Briefly, after the microwave antigen retrieval procedure and neutralization of endogenous peroxidase activity, the slides were incubated with primary antibody for 1 hr in a humidified chamber at 37°C (anti-hCXCR3 mAb 1:100). Immunoreactivity was detected using biotinylated secondary antibodies incubated for 45 min followed by a 30 min incubation with avidin-peroxidase and visualized by a 7 min incubation with the use of 0.1% 3,3\'-diaminobenzidene tetrahydrochloride as the chromogen. Parallel control slides were prepared either lacking primary antibody or lacking primary and secondary antibodies, or stained with normal sera to control for background reactivity. The intensity of antibody staining was classified in three groups: strong, weak, negative. Confocal microscopy for the identification of CXCR3+ cells ---------------------------------------------------------- Paraffin sections were prepared for immunofluorescent labelling. Briefly, primary antibodies against CD3 and CXCR3 (1:100 diluted and 1:100 diluted in phosphate-buffered saline with 5 g/L bovine serum albumin and 1 g/L gelatine, respectively) and secondary antibodies (goat anti-mouse IgG and donkey anti-goat IgG) conjugated with TEXAS red or ALEXA 488 (Sigma) were used. Double labelling using both antibodies on the same section was performed. Primary antibodies and secondary antibodies were incubated for 1 h at room temperature. Nuclear staining was carried out with DAPI (Sigma) in PBS. Slides were stored at 4°C and analysed within 24 h. As a control, the primary antibody was omitted. Immunofluorescence was evaluated with a confocal microscopy (Biorad 2100 Multiphoton; Hercules, CA), We used an argon laser at 488 nm in combination with a helium neon laser at 543 nm to excite the green (CD3) and red (CXCR3) fluorochromes simultaneously. Emitted fluorescence was detected with a 505--530 nm band pass filter for the green signal and a 560 nm long pass filter for the red signal. Images were analyzed using the Adobe Photoshop 7.0 program. Phenotypic evaluation of BAL cells ---------------------------------- The frequency of BAL cells positive for the above reagents was determined by overlaying the flow cytometry histograms of the samples stained with the different reagents as previously reported \[[@B12]\]. Cells were scored using a FACScan^®^analyzer (Becton Dickinson), and data were processed using the Macintosh CELLQuest software program (Becton Dickinson). The expression of cytoplasmic cytokine was evaluated following permeabilization of cell membranes using 1:2 diluted PermeaFix (Ortho, Raritan, NJ) for 40 min. After permeabilization procedures anti-IL-4, anti-IFN-γ and anti-CXCL10 antibodies were added. Since pulmonary cells bear cytoplasmic cytokines in a unimodal expression pattern, indicating that the entire cell population exhibits relatively homogeneous fluorescence, the percentage of positive cells does not represent the most accurate way of enumerating positive cells. Mean fluorescence intensity (MFI) was used to compare the positivity of these specific antigens on different cell populations. To evaluate whether the shift of the positive cell peak was statistically significant, the Kolmogorov-Smirnov test for analysis of histograms was used according to the Macintosh CELLQuest software user\'s guide (Becton Dickinson). For immunofluorescence analysis, control IgG1 and IgG2a and IgG2b were obtained from Becton-Dickinson; control rat antiserum consisted of ascites containing an irrelevant rat IgG2b; control rabbit antiserum consisted of rabbit IgG (purified protein) purchased from Serotec (Serotec, U.K.); goat-anti-rabbit IgG and goat F(ab\')2 anti-rat IgG were obtained from Immunotech (Marseille, France). Determination of IP-10/CXCL10 and Mig/CXCL9 mRNA levels ------------------------------------------------------- Each PCR product was analysed and quantitated by Bio-Rad\'s Image Analysis System Gel Doc using Quantity One software (Bio-Rad, Hercules, CA). Briefly, the images of the gels were acquired from the Gel Doc system densitometer and saved in digitised forms to perform volume analysis. The intensity of each band was differentiated by the intensity of the background, whose value was subtracted from each individual band and the resulting PCR product value was expressed in mm\mm\intensity of the pixels of the specific band in the gel. Generation of macrophage supernatants ------------------------------------- To verify the ability of AMs to release CXCL10, AMs (1 × 10^6^/ml) were isolated from the BAL of HP patients, resuspended in RPMI medium and cultured for 24 hr in 24-well plates at 37°C in 5% CO~2~. In separate experiments AMs were stimulated with IFN-γ (100 U/ml), PMA (10 ng/ml) and LPS (10 μg/ml; Difco Lab., Detroit, MI). Following the incubation period, supernatants were harvested, filtered through a 0.45 μm Millipore filter and immediately stored at -80°C. At the end of the culture time AM viability was always greater than 95%. Chemotactic activity of supernatants was determined as reported below. Migration activity of pulmonary T cells in response to CXCLIO ------------------------------------------------------------- T-cell migration was measured in a 48-well modified Boyden chamber (AC48 Neuro Probe Inc., USA). The chamber is made of two sections: different chemotactic stimuli were loaded in the bottom section while cells were added in the top compartment. Polyvinylpyrrolidone-free polycarbonate membranes with 3 to 5 μm pores (for lung T cells obtained from HP patients and the CXCR3+ and CXCR3- T-cell lines, respectively) (Osmonics, Livermore, CA) and coated with fibronectin were placed between the two chamber parts. Only the bottom face of filters was pretreated with fibronectin; the fibronectin pretreatment maximizes attachment of migrating cells to filters, avoiding the possibility that they may not adhere. Using this procedure in preliminary experiments we demonstrated that only a trivial number of cells may be recovered in the bottoms of the wells. To avoid the shedding of fibronectin, fibronectin-treated filters were extensively washed. In preliminary experiments, fibronectin-treated filters did not induce spontaneous chemotaxis in absence of chemokines. To evaluate the migratory properties of pulmonary T lymphocytes rhIP-10/CXCL10 (200 ng/ml) were used. The CXCR3- and CXCR3+ cell lines (300-19, kindly provided by Dr. B. Moser, Theodor-Kocher Institute, University of Bern, Switzerland) were used as negative and positive controls. 30 μl of chemokines or control medium were added to the bottom wells, and 50 μl of 5.0 × 10^6^cells/ml T cells or CXCR3-/+ cells resuspended in RPMI 1640 were added to the top wells. The chamber was incubated at 37°C with 5% CO~2~for 2 hrs. The membranes were then removed, washed with PBS on the upper side, fixed and stained with DiffQuik (Dade AG, Düdingen, Switzerland). Cells were counted in three fields per well at 800× magnification. All assays were performed in triplicate. In blocking experiments, cell suspensions were preincubated before chemotaxis assay for 30 min at 4°C with anti-human CXCR3 mAb at a concentration of 20 μg/ml. Chemotactic activity of the fluid component of BAL and macrophage supernatants ------------------------------------------------------------------------------ The CXCR3(-) and CXCR3(+) cell lines were also used to evaluate both the chemotactic activities of macrophage supernatants and the fluid component of BAL samples. Supernatants from cell cultures and the fluid components of BAL were obtained as reported above and used undiluted; different concentrations of CXCL10 were utilized as a positive control. Chemotactic assays were performed as reported above. In blocking experiments, anti-CXCL10 was added to the cell supernatants before chemotaxis assay at a concentration of 20 μg/ml. Statistical analysis -------------------- Data were analysed with the assistance of the Statistical Analysis System. Data are expressed as mean ± SD. Mean values were compared using the ANOVA test. A P value \<0.05 was considered as significant. Results ======= Immunohistochemical analysis of CXCR3 expression in lung biopsies ----------------------------------------------------------------- In all cases typical pathological examination showed features with poorly formed non-necrotizing granulomas and widespread thickening of the alveolar walls by a diffuse lymphocytic infiltrate. Pleural lymphoid aggregates were seen in a few cases and pleural lymphoid aggregates were seen in a few samples. Diffuse interstitial lymphocytic infiltrates were characterized by an accumulation of T cells and a few B-lymphocytes. Sub-pleural and peri-bronchiolar nodules consisted mostly of T lymphocytes mainly represented by CD8 cytotoxic T lymphocytes which strongly stained for CXCR3 in all cases (Figure [1A](#F1){ref-type="fig"} and [1B](#F1){ref-type="fig"}). Marked CXCR3 immunostaining was also seen in peribronchial lymphocytic infiltrate and in the interstitial non-necrotising granuloma (Figure [2A](#F2){ref-type="fig"} and [2B](#F2){ref-type="fig"}) Both interstitial and intra-alveolar macrophages (CD 68 positive) showed weak or negative CXCR3 staining and multinucleated giant cells always stained negatively (Fig. [2C](#F2){ref-type="fig"} inset). Endothelial and epithelial cells close to more intense lymphocytic infiltrate were sometimes positively marked. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Immunohistochemistry for CD8 and CXCR3 in lung biopsy from HP patient. Most lymphocytes positive for CD8 (panel a) and CXCR3 (panel b) were clearly visible in a subpleural focus. Original Magnification × 25. ::: ![](1465-9921-6-20-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Immunohistochemistry for CXCR3 in lung biopsy from HP patient: positive marked lymphocytes while negative or weak staining macrophages were also seen in peribronchial space (panel A) and in the setting of non-necrotizing interstitial granuloma (panel B) (original magnification × 50). Note negative or weak staining of macrophages and giant cells forming the central core of the granuloma (inset panel C, original magnification × 100). ::: ![](1465-9921-6-20-2) ::: Confocal microscopy analysis of lung biopsies confirmed that lymphocyte infiltrates were formed by T cells coexpressing CXCR3 (Figure [3](#F3){ref-type="fig"} panels A, B and C). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Immunofluorescence confocal laser scanning microscopy analysis shows the presence of CXCR3 (panel B, red) on CD3+ T cells (panel A, green). Panel C shows the overlay image of A and B in yellow (original magnification × 1000). ::: ![](1465-9921-6-20-3) ::: Morphological and phenotypical features of cells obtained from the BAL ---------------------------------------------------------------------- Morphological and phenotypical features of cells obtained from the BAL of 12 patients with HP and 5 controls are reported in Table [1](#T1){ref-type="table"}. All HP subjects showed a high intensity lymphocytic alveolitis sustained by CD8(+) Tc1 cells (Table [1](#T1){ref-type="table"} and Figure [4A](#F4){ref-type="fig"} and [4B](#F4){ref-type="fig"}). These cells were CXCR3(+) and bore IFN-γ but not IL-4 receptor (Figure [4C](#F4){ref-type="fig"}). Furthermore, pulmonary T cells expressed activatory molecules such as CD103 and IL-12β2 receptor (Figure [4C](#F4){ref-type="fig"}). The percentage and absolute number of BAL CXCR3(+) was significantly higher in HP patients with respect to control subjects (Table [2](#T2){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Broncholaveolar findings in patients with hypersensitivity pneumonitis and controls ::: Study population Cell Recovery Lymphocytes CD4 T cells CD8 T cells ------------------ ---------------- ---------------- ---------------- ------------------ ---------------- ------------------ HP (n. 12) 351.9\* ± 62.3 44.4\\ ± 8.1 25.6\\ ± 5.3 38.6\\\* ± 9.3 53.7\\ ± 6.3 83.4\\\* ± 8.5 Controls (n. 5) 138.6 ± 12.7 8.2 ± 2.2 48.3 ± 3.2 5.4 ± 0.9 23.7 ± 2.2 2.5 ± 0.3 Significance as follows: \p \< 0.05; \\p \< 0.01; \\\p \< 0.001 ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### The flow cytometry profile of BAL T cells recovered from 2 representative patients with hypersensitivity pneumonitis and a control subject. BAL T cells were gated as shown in panel A. In patients with hypersensitivity pneumonitis the majority of lymphocytes were CD8(+) T cells (panel B). Panel C shows that BAL T cells from patients with hypersensitivity pneumonitis are CD45RO(+) T cells which express CXCR3, IFN-γ but not IL-4, or other activation markers including CD103 and IL12Rβ2. ::: ![](1465-9921-6-20-4) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Expression of CXCR3 by CD8+ T cells and expression of IP-10/CXCL10 and Mig/CXCL9 mRNAs by alveolar macrophages from patients with hypersensitivity pneumonitis and controls\* ::: Study population CXCR3+/CD8+ T cells IP-10/CXCL10 Mig/CXCL9 ------------------ --------------------- ------------------ ----------------- ----------------- HP (n. 6) 51.7\\ ± 5.9 80.1\\\* ± 7.7 2.55\\ ± 0.14 2.25\\ ± 0.25 Controls (n. 4) 23.7 ± 2.2 2.5 ± 0.3 0.70 ± 0.05 0.48 ± 0.06 \* Band intensity calculated as follows: mm × mm × pixel Significance as follows: \\p \< 0.01; \\\p \< 0.001 ::: CXCR3 mediates pulmonary T cell chemotaxis ------------------------------------------ To define the biological activities of CXCR3, highly purified T cells obtained from the BALs of patients with HP were assessed for their migratory capabilities in response to different concentrations of CXCL10. The evaluation of the migratory potential of T lymphocytes obtained from the BAL of the controls was prevented by the low number of cells recovered. For this reason, the 300-19 T-cell lines expressing high levels of CXCR3 or not expressing CXCR3 were used as positive and negative controls respectively for the in vitrochemotaxis assay (Figure [5](#F5){ref-type="fig"}, panel B and C respectively). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Chemotactic activity of CXCL10 on BAL CD8(+)/CXCR3(+)T cells highly purified from 2 representative patients with hypersensitivity pneumonitis. The assays were performed using a modified Boyden chamber in triplicate and data are given as mean ± SD. CXL10 shows significant chemotactic activity on BAL T cells (panels A) and the CXCR3(+) T-cell clone (panel B) but not on CXCR3(-) T-cell clone. ::: ![](1465-9921-6-20-5) ::: As shown in panel A of Figure [4](#F4){ref-type="fig"}, the migratory capability of T lymphocytes of patients with HP is regulated by CXCR3. In fact, CXCR3+ lung T cells exhibited a strong, definite migration in response to CXCL10. To further verify the functional role of the CXCL10 receptor, CXCR3+ pulmonary T cells were preincubated with anti-CXCR3 neutralizing antibody: the blocking of the receptor determined a marked inhibition of CXCL10-induced chemotaxis (panel A). These data suggest that pulmonary T lymphocytes that sustain T-cell alveolitis in patients with HP express a functional CXCR3 receptor and actively migrate in response to CXCR3 ligands. Lung macrophages express CXCR3 ligands and release ligands showing chemotactic activity on CXCR3(+) cells --------------------------------------------------------------------------------------------------------- In order to analyse whether CXCL10 is expressed in vivoby lung cells of patients with hypersensitivity pneumonitis, BAL cells were stained with a anti-CXCL10 antibody as described above. Flow cytrometric analysis (Figure [6](#F6){ref-type="fig"}, panels A and B) revealed that AMs of patients with HP express CXCL10; macrophages retrieved from control subjects lacked the CXCR3 ligand (panel C). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Flow cytometric analysis of CXCL10 expression by AMs infiltrating the lung of patients with hypersensitivity pneumonitis. Panels A-C shows the flow cytometry profile of AMs recovered from the BAL of 2 representative patients and a control subject. AMs from patients with hypersensitivity pneumonitis (panels A and B) but not control subject (panel C) express CXCL10. ::: ![](1465-9921-6-20-6) ::: Measurement of mRNA levels of the CXCR3 ligands demonstrated that unstimulated alveolar macrophages isolated from the BAL of HP subjects expressed increased mRNA levels of CXCL9 and CXCL10 with respect to macrophages obtained from control subjects (Table [2](#T2){ref-type="table"} and figure [7](#F7){ref-type="fig"}). Spearman Rank correlation coefficients between BAL T CD8(+)/CXCR3(+) T cell number and levels of CXCR3 ligands were also calculated. Interestingly, a positive correlation was demonstrated between mRNA levels of CXCL10 and CXCL9 and the absolute numbers of lung CD8(+)/CXCR3(+) T cells (r 0.815, p \< 0.001 and r 0.825, p \< 0.001, respectively). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### IP-10/CXCL10 and Mig/CXCL9 mRNA levels expressed by AMs recovered from the BAL of 2 representative patients and 2 control subject. AMs from patients with hypersensitivity pneumonitis express higher amounts of IP-10/CXCL10 and Mig/CXCL9 mRNA levels than control AMs. ::: ![](1465-9921-6-20-7) ::: Cell-free supernatants were obtained from 24-hour cultured AMs in the presence of IFN-γ and tested for their ability to induce T-cell migration. Supernatants obtained from AMs of patients with HP exerted chemotactic activity on the CXCR3(+) cell line; the CXCR3(-) cell line did not migrate in the presence of supernatants (data not shown). The addition of an anti-CXCL10 neutralizing antibody inhibited chemotactic activities of supernatants. The inhibitory activity shown by the neutralizing antibody was not complete, suggesting that other CXCR3 ligands (CXCL9 and CXCL11) are likely to be present in supernatants. CXCR3 ligands may be demonstrated in the fluid component of BAL --------------------------------------------------------------- To assess whether CXCR3 ligands are released in vivoin the lung microenvironment, the fluid component of BAL obtained from 10 HP patients was evaluated for chemotactic activity on CXCR3(+) cell lines (Figure [8](#F8){ref-type="fig"}). Measurable biological activity was demonstrated in 7 out of 10 patients with HP; this migration was partially abrogated by an anti-CXCL10 neutralizing antibody. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### The fluid components of the BAL obtained from 4 patients with hypersensitivity patients exert chemotactic activity on the CXCR3(+) 300-19 T-cell line, indicating the presence of CXCR3 ligand(s). The migration was partially abrogated by an anti-CXCL10 neutralizing antibody (white bars). The assays were performed in triplicate, and data are given as mean ± SD. ::: ![](1465-9921-6-20-8) ::: Discussion ========== We have herein shown that CXCR3 expression represents a crucial mechanism which is involved in the recruitment of activated Tc1 cells in the pulmonary microenvironment of patients with HP. The continuous recruitment of CXCR3(+) T cells might play a role not only in the pathogenesis of T-cell alveolitis but also in favouring granuloma formation since T cells surrounding the macrophagic core of the HP granuloma expressed this chemokine receptor. This mechanism is likely to be shared by various ILDs since we and others have previously demonstrated the presence of a significant infiltrate of CXCR3(+) Th1 cells in other ILDs characterized by T cell alveolitis and granuloma formation, such as sarcoidosis and tuberculosis \[[@B13],[@B14]\]. Our data provide definitive confirmation of the recent findings obtained in an animal model of IFN-γ-knockout (GKO) mice exposed to the particulate antigen Saccharopolyspora rectivirgula(SR) (i.e., the etiologic agent involved in the immunopathogenesis of HP reaction in the majority of our patients) \[[@B11]\]. While WT mice show the production of IP-10/CXCL10, Mig/CXCL9, and I-TAC/CXCL11 during the development of the classic HP reaction, GKO mice have reduced or no levels of IP-10/CXCL10, Mig/CXCL9 and I-TAC/CXCL11 in the lungs and reduced T-cell alveolitis in response to SR exposure. The present study suggests the role of CXCL10/CXCR3 and CXCL9/CXCR3 interactions in driving local CD8 immune responses to SR (Figure [9](#F9){ref-type="fig"}). A logical question is whether our data may have therapeutic implications. Because of the role of CXCR3 expression in the migration of T cells, strategies to block CXCL10 could in theory be proposed to prevent the development of HP reactions, particularly in subjects continuously exposed to inhaled antigens and thus at risk for the development of lung fibrosis. Further data are required to evaluate the in vivorole of IP-10/CXL10 in preventing or favouring pulmonary fibrosis in HP before proposing this strategy. ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Putative scheme of the effect of the CXCR3/CXL10 interactions in the lung of hypersensitivity patients. As a consequence of the antigenic stimulation APC release IFN-γ. This cytokine stimulates AM to release CXCL10, a chemokine which favours the recruitments of activated CD8(+) /CXCR3(+) Tc1 lymphocytes. These cells, which release IFN-γ, in turn contribute to maintain the activation state of APC at sites of hypersensitivity reaction. ::: ![](1465-9921-6-20-9) ::: The relationship between CXCL10 release and the local production of other chemokines involved in HP immune response is another important aspect that deserves further investigation. In a murine model it has recently been shown that monocyte chemotactic protein-1 (MCP-1/CCL2) is increased in the BAL of mice challenged with SR \[[@B15]\]. Furthermore, Pardo et al. \[[@B16]\] have recently examined the expression of dendritic cell (DC)-derived CC chemokine 1 (CK1)/CCL18 in the lungs of patients with HP. CCL18 expression is significantly increased in lungs affected by HP, with higher levels in the subacute rather than in the chronic phase of the disease. Macrophages, dendritic cells, and alveolar epithelial cells are the main sources of CCL18 whose expression is induced by various profibrogenic cytokines including IL-4, IL-10, and IL-13. Interestingly, a direct correlation between the levels of tissue CCL18 and the number of lymphocytes has been demonstrated in the bronchoalveolar lavage fluids. Thus, our findings and the data of Pardo et al. \[[@B16]\] suggest that chemokines ordinarily induced by profibrogenic cytokines (CCL18) and chemokines induced by antifibrotic agents (IFN-γ and CXCL10) can be demonstrated in the lung of HP patients with T cell alveolitis. Whether the presence of the two chemokines is concomitant and there are common molecular mechanisms involved in the CC and CXC chemokine release is unknown. Given the heterogeneous pattern of pulmonary infiltrate during different phases of the disease, a full understanding of the interdependence of the local hyperproduction of chemoattractant molecules may help to clarify the pathogenesis of HP. In this context, it is also mandatory to investigate the production of CC chemokines in relationship with CXCL10 release in individuals with acute, subacute and chronic HP. For instance, it is known that the combination of RANTES/CCL5 and CXCL10 but not other chemokines (MIP-1α/CCL3, MIP-1β/CCL4, Mig/CXCL9, and ITAC/CXCL11) markedly increases T-cell recruitment \[[@B17]\]. Since there are data indicating that circulating, antigen-reactive, memory T cells, generated by previous sensitization to organic antigens, migrate into lung parenchyma in response to chemokines such as RANTES \[18\], it is possible that the interplay of CXCL10 with CCL5 may serve to finely tune inflammatory responses in vivo*in HP lungs. Conclusion ========== Our findings clearly indicate the effects of CXCR3/CXCL10 interactions on hypersensitivity reaction to SR antigens. Considering the importance of CD8 T cells in mediating granuloma formation and lung damage, further studies are needed in animal models to explore the therapeutic potential of CXCR3 antagonists with the ultimate goal of offering new clues for immune intervention in subjects continuously exposed to inhaled antigens and thus at risk of developing HP-related lung fibrosis. List of abbreviations ===================== AM: alveolar macrophage; BAL: bronchoalveolar lavage; CXCR3: Receptor 3 for CXC chemokines; HP: Hypersensitivity pneumonitis; IP10/CXCL10: IFN-γ-inducible protein-10; I-TAC/CXCL11: interferon-inducible T-cell αlfa-chemoattractant; Mig/CXCL10: monokine induced by IFN-γ; TBB: transbronchial biopsy. Authors\' contributions ======================= CA: planned the experimental design, coordinated the research group and drafted the manuscript FC: participated in the study design and in immuohiostochemical analysis VP: participated in the study design and in the bronchoalveolar lavage execution GM: participated in the study design and in the clinical evaluation of the patients MF: participated in the study design and performed migration assays studies MM: participated in the study design and performed cellular studies AC: participated in the study design and performed flow cytometry studies IB: participated in the study design and performed flow cytometry studies RZ: participated in the study design and helped the draft of the manuscript LT: participated in the study design and helped the draft of the manuscript GS: participated in the study design and coordination of the research group Acknowledgements ================ The authors wish to thank Mr. Martin Donach for his help in the preparation of the manuscript and Mrs. Alessandra Dubrovic for the technical assistance in the preparation of lung biopsies for immunohistochemical analysis. This study was supported by a Grant from the M.U.R.S.T (PRIN), Rome	PubMed Central	2024-06-05T03:55:54.023864	2005-2-22	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554979/", "journal": "Respir Res. 2005 Feb 22; 6(1):20", "authors": [ { "first": "Carlo", "last": "Agostini" }, { "first": "Fiorella", "last": "Calabrese" }, { "first": "Venerino", "last": "Poletti" }, { "first": "Guido", "last": "Marcer" }, { "first": "Monica", "last": "Facco" }, { "first": "Marta", "last": "Miorin" }, { "first": "Anna", "last": "Cabrelle" }, { "first": "Ilenia", "last": "Baesso" }, { "first": "Renato", "last": "Zambello" }, { "first": "Livio", "last": "Trentin" }, { "first": "Gianpietro", "last": "Semenzato" } ] }
PMC554980	Background ========== The performance of health care systems is closely related to the numbers, distribution, knowledge, skills and motivation of its workforce, particularly of those individuals delivering the services \[[@B1]\]. Improvements in global health are greatly dependent on how well health systems can meet the demands placed on them by governments, programmes, communities and ultimately individuals. Human resources for health (HRH), all categories of clinical and non-clinical staff who make each individual and public health intervention happen, constitute a sine qua non of health systems. Therefore, developing HRH and fostering appropriate HR management are crucial steps towards achieving and sustaining improved and equitable health. Tuberculosis (TB) constitutes the third most important cause of death and disability \[[@B2]\] among infectious diseases. It is estimated that in 2002, there were 8.8 million new TB cases worldwide (141/100 000) of which 3.9 million were sputum-smear positive (SS+). Despite control efforts, the global incidence of TB continues to grow in some regions, particularly in sub-Saharan Africa \[[@B3]\]. The emergence of AIDS and multidrug-resistant TB (MDR-TB) pose further challenges for global TB control; in 2000 there were 1.82 million TB-related deaths, of which 226 000 (12%) were attributable to HIV \[[@B2],[@B4]\]. The World Health Assembly (WHA), in 1991, pledged countries to achieve detection of at least 70% of estimated infectious TB cases (SS+) and to cure 85% of them by the year 2000 \[[@B5]\]. Slow progress resulted in the deferral of these targets until 2005 \[[@B6]\]. Likewise, the United Nations commitment to sustaining development and eliminating poverty throughout the world led world leaders to formulate the eight Millennium Development Goals (MDGs), among them to halt, and begin to reverse, the global incidence of TB by 2015 \[[@B7]\]. The creation of the Stop TB Partnership in 1998 \[[@B8]\] and the Global Fund to fight AIDS, TB and Malaria (GFATM) in 2001 \[[@B9]\] represented significant developments in the fight against TB, thanks to increased financing and technical assistance made available to endemic countries. However, disease-specific programmes (including TB) are still struggling to meet their targets, and governments and their financial/technical partners have finally recognized this is largely due to shortcomings in the health care workforce \[[@B3],[@B10]\]. Low-income and middle-income countries (LMIC) urgently need a sufficiently large health care staff with appropriate expertise, experience and motivation, working at the right places. Lack of HR strategies, inadequate HR planning and management, poor deployment practices, inflexible contracting arrangements and inability to create new posts or increase salaries resulting from international regulations capping social sector spending have contributed to the global HRH crisis \[[@B10]\]. In addition, poor salaries, low morale and worsening local economic circumstances result in low recruitment/retention, internal and external migration of trained staff and attrition of the health care workforce. The HIV/AIDS epidemic has increased pressure on health systems and causes death and disability of the workforce itself \[[@B11]\]. In 2003, national TB programme (NTP) managers from 18 of the 22 TB high-burden countries (HBCs, countries that together account for approximately 80% of the global TB burden), ranked inadequate HR first within the top five constraints to reaching the WHA global TB control targets \[[@B3]\]. Insufficient numbers, lack of adequately qualified or trained staff at different service levels, inadequate distribution, low motivation and poor staff retention were commonly described. Although some components of disease control programmes still remain vertical (e.g. drug procurement or laboratory QA) the majority of front-line services are now partially or totally integrated to primary health care and therefore a shortage of HR for TB control represents a shortage of HR for health delivery. There is very limited published literature on HRH issues, particularly in LMIC and there are both scanty information on methods to assess HR capacity and lack of evidence on how best to evaluate interventions to strengthen and/or build HR capacity \[[@B12]\]. The dearth of published information probably stems from the persistent neglect of HRH development issues; in addition, research in HRH involves a broad scope of disciplines and often different research methodologies than those recognized in clinical medicine \[[@B13]\]. Currently there is increasing awareness that HRH concerns must be addressed in order to reach the MDGs, to expand access to priority interventions, to promote health systems development and to achieve global health equity \[[@B14],[@B15]\]. However, without reasonably accurate information on numbers, location, qualifications and activities of staff, it is not possible to effectively manage or plan HR for the country\'s health services or for specific programmes \[[@B16]\]. This paper reports the results of a questionnaire sent to NTP staff in the 22 HBCs to assess the workforce available for TB control (staff numbers, cadres and skills), as well as the estimated HR requirements for appropriate TB control in HBCs. It aims to inform the development of more reliable methods of gathering qualitative and quantitative information on HR and to stimulate a long-overdue discussion on HR for TB control issues, so that governments of endemic countries and technical and financial partners can finally begin to address them jointly. Methods ======= Participants ------------ NTP managers and country-based WHO staff in the 22 HBCs: Afghanistan, Bangladesh, Brazil, Cambodia, China, Democratic Republic of the Congo, Ethiopia, India, Indonesia, Kenya, Mozambique, Myanmar, Nigeria, Pakistan, Philippines, the Russian Federation, South Africa, Tanzania, Thailand, Uganda, Viet Nam and Zimbabwe. These countries account for more than half of the world\'s population and approximately 80% of the global TB burden Questionnaire ------------- Discussions with NTP managers and a literature review on HRH informed the development of a questionnaire to ascertain current staff provision; quality and intensity of training; time and type of personnel involved in performing different TB control activities; and estimated staffing needs at different health service levels. The questionnaire was pilot-tested internally within the Stop TB Department (STB), WHO headquarters, Geneva; and externally with staff from the NTP in Indonesia. The first section of the questionnaire assessed staff numbers and skills. It included open questions about absolute numbers of staff involved in delivering TB control activities at each service level (provincial, district and health facility and laboratory personnel). Skills were assessed using a composite of the proportion of staff receiving training in the previous three years (at each level) and the quality of the training provided (length of courses, development of educational materials and/or use of standard -WHO or International Union Against Tuberculosis and Lung Diseases (IUATLD) -- materials) as proxy measures. Section two of the questionnaire addressed estimated HR gap. Since this paper specifically refers to TB control, we estimated the workload involved in adequate TB control in HBCs. Workloads were assessed by means of open questions on the different processes involved in the management of new SS+ patients (diagnosis and administration of a short course chemotherapy regimen under proper case management conditions including directly observed therapy, DOT), the estimated duration of each task and the type of staff involved in delivering them. A series of worksheets containing the different tasks required for TB case management were designed to assist respondents completing the questionnaire and to improve standardization. Capacity of health services at two different CDRs were assessed (current CDR and at the target 70% CDR). Workloads at current CDRs were calculated by multiplying the current numbers of new SS+ patients by the estimated time needed to treat a new SS+ patient; workloads at the 70% CDR were calculated by multiplying the figure corresponding to 70% of the estimated SS+ TB cases for each country by the time needed to treat a new SS+ patient. Eleven hours was used as the time needed to treat a new SS+ patient; this had been estimated previously by a TB experts\' consensus (WHO, unpublished data). Survey ------ In March 2003, the questionnaire, together with detailed instructions in English, was e-mailed to NTP managers and country-based WHO staff in the 22 HBCs. Three e-mail reminders were sent monthly after the return deadline; follow-up with several countries included e-mail and telephone communications to clarify responses. Data were entered into spreadsheet software for the analysis; qualitative answers were discussed by the authors. Results ======= Response rate ------------- Nineteen of the 22 HBCs returned the questionnaire (86% response rate). Two countries provided information on their HR for TB control but did not use the questionnaire format and were thus excluded from the analysis; one country reported that it would take a substantial amount of time to get reliable information from such a large country and provided other readily available HR information; another reported that given the country\'s current staffing deficits and competing priorities they were unable to complete the survey. Further discussions revealed that some HR information was available in this country, although it was scattered in different sources and lengthy to compile. Both countries requested technical support to assess staffing needs at different levels and to assist their government\'s HR development programmes. Despite e-mail and telephone remainders, three countries failed to respond. Numbers of staff ---------------- Information on estimated numbers of staff was available from all 17 countries but complete in nine (53%) of them. Staff numbers within the same service level varied considerably between countries; i.e. numbers of staff at provincial level varied from 8704 in one country to 6 staff in two countries (Table [1](#T1){ref-type="table"}); similar variations were seen at other service levels. More countries provided information on staff numbers at provincial than at district or health facility levels.; only nine countries (53%) provided numbers of staff at laboratory level. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Staff numbers at each level and estimated numbers of trained staff in the previous three years (2000--2002) ::: Country Health service Level ----------------- -------------------------- ----------- ------ ------------ -------- ------------- ------ ------------ Afghanistan 40 22 (55) 360 \- \- \- \- 30 (\--) Bangladesh 460 150 (33) 460 120 (26) 39329 750 (2) 1015 450 (44) Brazil 27 19 (70) \- 20 (\--) \- 6379 (\--) \- \- Cambodia \- 72 (\--) \- 236 (\--) 1120 705 (63) \- \- DR Congo 56 19 (34) 306 \- 4306 \- 1000 \- Ethiopia 12 4 (33) \- \- \- \- \- \- Indonesia\* 70 70 (100) 420 420 (100) 1256 1256 (100) 405 405 (100) Kenya 10^¶^ 7 (70) 94 \- 45900 1148 (2) 2121 350 (16) Myanmar \- 8 (\--) \- 276 (\--) \- 18056 (\--) \- 327 (\--) Nigeria 37 19 (51) 664 149 (22) 27000 295 (1) 3000 160 (5) Pakistan 6 0 (0) 60 60 (100) 21000 500 (2) 600 200 (33) Philippines 156 \- 748 \- 13900 \- 2200 \- Russian Fed.^§^ \- \- \- 4062 (\--) \- 4197 (\--) \- 1167 (\--) South Africa 9 9 (100) \- (70--80) 202265 (70--80) \- \- UR Tanzania 25 2 (8) 156 58 (37) \- 50 (\--) \- \- Uganda 6 3 (50) 55 \- 1050 \- 350 \- Viet Nam 8704 2205 (25) 2705 1248 (46) 10510 10510 (100) 804 624 (77) \* Percentage of training according to 2002 training plan ¶At regional level §Federation ::: Skills ------ Complete information on numbers of staff trained in the previous three years was available in only six countries (35%). There was also great variation in the numbers (or proportions) of trained staff (Table [1](#T1){ref-type="table"}). Indonesia reported it trained 100% of staff at all levels, but this was based on its target for 2002; Viet Nam reported high levels of training at health facility and laboratory levels and South Africa reported high levels of training (70% to 80%) at provincial, district and health facility levels (although absolute numbers were missing). Other countries either reported very low levels of training or had missing information. Only six countries (35%) reported accurate information on training of laboratory staff. Information on length of training courses and development of training materials was also incomplete. Length of courses for staff at the same service level varied greatly between countries; for example, courses for TB coordinators at provincial level (data from 11 countries) varied from 4 to up to 60 days, whereas at district level (information from 10 countries) courses varied from 4 days to 4 months (Table [2](#T2){ref-type="table"}). Similar variations were reported at other levels. Development of training materials was reported by eight (47%); a further four (23%) reported developing lectures and exercises modules for training at all levels, whereas only three (18%) described using participatory educational methods. Nine countries (53%) had not developed specific training modules; five of them used WHO/IUATLD materials and the remaining countries used programme implementation manuals and guidelines as training tools. Altogether eight countries (47%) reported using WHO/IUATLD training materials. The WHO/STB training modules for health facility staff take normally five days to complete if all its tasks and units are included; only one of the countries that reported using it had a five-day course. Training at this level took three days or less in six of the 12 countries with available information. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Length of training courses at different training levels and development of training materials by country ::: Country Length of training in days -------------- -------------------------------- --------- ------------- -------- ----------------------------------------------------------------------------------------------------------- Afghanistan 10 \- \- 7 No materials developed Bangladesh \- 4--6 2--3 6 Developed lectures and exercises, also uses WHO materials for training at provincial and district levels Brazil 5 5 5 5 Developed manuals & guidelines for all levels Cambodia \- 5 3 \- Developed training modules at all levels DR Congo 21 \- \- \- No materials developed, uses WHO materials at all levels Ethiopia \- 7--10 5 5 Ad hoc handouts Indonesia 12 12 6 8 Developed training modules for all levels Kenya 15 \- 2 3 No specific training materials developed, uses national guidelines and WHO & IUATLD training materials Myanmar 5 5 1 5 TB manual for health facility staff and lab. technicians developed in 2002 Nigeria 21 120 3 6 Developed training materials at all levels, did not specify Pakistan \- 10 variable^§^ 10 Developed training modules, translated WHO materials for training lab. staff Philippines \- \- \- \- Use modified WHO materials at all levels Russian Fed. \- \- \- \- WHO materials were developed for Russia in 2002, use manuals and guidelines for staff at different levels South Africa 4 \- 3 \- No specific materials developed, uses WHO & IUATLD materials for different levels UR Tanzania 14 30 5 5 Developed a TB Manual but no specific training materials Uganda 15 \- \- \- Developed training materials but did not specify Viet Nam 10 or 60 5 or 10 3 or 5 15--20 Uses national guidelines for training, for health facility staff it uses WHO & IUATLD training materials \* Development of specific training materials for each country or use of standard WHO / IUATLD modules and courses. §Variable: Medical staff 6 days, paramedical 3 days, health workers 1 day. ::: Performance ----------- The estimated time needed to treat a new SS+ patient is shown in Figure [1](#F1){ref-type="fig"}. Times were positively skewed and ranged from 5 to 36 (mode 10) hours. Eight countries (47%) needed from 9 to 12 hours; Brazil was the only country requiring less than 9 hours; Pakistan, Uganda, Nigeria, Kenya, Russia and Philippines were out-layers requiring between 22 to 36 hours. The Russian Federation (31 hours) and the Philippines (36 hours) took the longest, due to the policy to hospitalize all patients during the intensive phase of treatment in the former and the tendency to perform strict DOT during the continuation phase in the latter. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Average time spent to treat one new sputum-smear positive TB patient ::: ![](1478-4491-3-2-1) ::: Estimated HR gap ---------------- Sixteen countries (Table [3](#T3){ref-type="table"}) reported information on estimated shortages of staff at peripheral level (health facility) at current or target CDRs. Two countries (12%) reported shortages of staff at current CDR, whereas five countries (29%) predicted health facility staff shortages at the 70% CDR. Estimated numbers of staff needed varied from 1009 more nurses in Afghanistan to 8981 health care workers in Myanmar; Uganda reported shortages of staff but did not estimate the numbers needed. Eleven countries (65%) reported no shortages of health facility staff at current or at 70% CDRs; these included countries with low current CDR and no data on staff numbers. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Perceived staff needs at different service levels. NTP managers were asked to report perceived staff needs at different service levels. Staff needs at Health Facility level were evaluated both at current and at the 70% target case detection rates ::: Country CDR\* Perceived staff shortages Comments ----------------- ----------- ------------------------------- -------------- ------------- -------------- ------------ ------------------------------------------------------------------------ Afghanistan 19%^a^ No 1009 No No 17 Poor distribution of staff, staff needed to run new facilities Bangladesh 33% No No No No 1 -- 5 Brazil 84% No No Yes Capacity building of existing staff is a priority Cambodia 52%^a^ No No No No No Poor distribution and training of staff at district and central levels DR Congo 52%^a^ No No No 52 \>5 Capacity building of existing staff a priority Ethiopia 33%^a^ Yes 5 (regional) 2 Lack of data, poor distribution and training Indonesia 30%^a^ No 3670 Yes Yes Yes Kenya 49%^a^ No No No No 8 Poor distribution of existing staff Myanmar 73%^a^ Yes 8981 6 4 4 Nigeria 14% No No No 111 25 Capacity building of existing staff is a priority Pakistan 13% No 2981 Yes Yes \>6 Philippines 58%^a^ No No \>3 Yes \>10 Russian Fed.^§^ 34% No No No No Approx. 48 Not enough data available South Africa 97% No No Yes Yes Yes Lack of funds for recruiting new staff UR Tanzania 43%^a^ No No \>364 \>88 \>11 Staff retention and deployment problems Uganda 47%^a^ Yes Yes Yes (Zonal) 4 Inconsistent data provided Viet Nam 82%^a^ No No No No No Poor training of existing staff at district and provincial levels \* Case Detection Rate for 2002, Ref 3. a No data available for the whole country; case detection rate for DOTS programmes §Federation ::: All countries provided information on their estimated HR gap at district level; nine (53%) countries reported existing staff needs at district level (Table [3](#T3){ref-type="table"}); nine countries reported existing needs at provincial level and all but two countries reported staff needs at central level. Some countries answered qualitatively while others estimated numbers needed, but it is not clear how these estimates were reached. Cambodia and Viet Nam were the only HBCs reporting no staff shortages at any service level. Afghanistan, Cambodia, Ethiopia, Kenya and Uganda reported poor distribution of staff. Brazil, Cambodia, Democratic Republic of the Congo, Ethiopia, Nigeria and Viet Nam reported training and capacity-building shortfalls. Discussion ========== In many HBCs, NTP managers do not have access to accurate information on numbers, types and distribution of staff involved in TB control activities. As previously stated, TB control programmes are partially or fully integrated into health care systems; this lack of HR information thus could at best indicate poor communication and coordination between NTP managers and HR planners or, at worst, a general lack of information on HR in the health care system. In order to adequately manage and plan HR it is important to have up-to-date information on the quantity, distribution and skills of the existing health care workforce \[[@B16]\]. It is therefore imperative to assist HBCs to develop and maintain appropriate HR databases so that the necessary information for planning and managing their health care workforce can be readily accessible. Accreditation is often used as a proxy for competence for professional groups (doctors and nurses); for other health care staff, adequate competence is ensured through regular training and supervision. Apart from Indonesia and Viet Nam, there was no correlation between information on numbers of staff, attendance of training courses, length of courses and development of training materials. In general NTP managers in HBCs had limited information on staff attendance of training courses and on the characteristics, duration and intensity of training activities. HBCs need to develop needs-based comprehensive training policies and training strategies for health care staff at all service levels, incorporating pre-service training, re-training, in-service support and continuous professional/career development. Similarly, it is important to emphasize the role of periodic monitoring and supervisory visits as part of the staff continuous education and support processes. Appropriately designed strategies fostering career paths could increase staff motivation and performance; improve staff recruitment, retention and distribution; and even have a positive effect on enlisting of students into training programmes \[[@B17]\] and expansion of the health care workforce. The quality of training materials was also of concern: many countries used inadequate training tools, few had developed specific modules and even fewer used problem-based learning or participatory methodology to facilitate adequate skill development. The length of courses also varied greatly: while very short courses do not allow the development of skills and competences needed to improve performance, lengthy courses have economic and logistic implications. Differences in the quality and intensity of training could translate into service or performance variations with detrimental effects to programmes and ultimately to patients. There is a need to standardize training in terms of contents, competences, methodologies, course duration and quality of training materials. Improvement and standardization of training curricula and methodologies will facilitate the adoption of a universal standard of care for TB patients in HBCs, leading to a more rational use of the health care workforce, improved staff motivation and productivity and better outcomes for TB patients. Although the majority of countries described deficiencies of staff at central level, few reported shortages in the actual numbers of posts. However, there were concerns about the distribution and/or the skill mix (competences and efficiency) of staff. There was no clear relation between reported data on staff needs and either the TB burden in the country or the actual performance of the NTP. It is of concern that only Uganda and Myanmar reported shortages of staff at current CDRs, whereas countries with very low current CDRs did not. Only five countries projected shortages of staff at the 70% CDR; four of them estimated large numbers of staff required. If the assessment of the HR needs was based on the actual workload, it is possible no substantial gap was determined because most of the current diagnostic and treatment tasks are carried out satisfactorily by existing staff, but an unrecognized HR gap could hamper improvement in case detection rates. The inability of some countries to increase the number of posts because of recruitment ceilings imposed under structural adjustment programmes could lead to countries underreporting their HR gap. Further analysis and recommendations on staff needs at different levels is hindered by the lack of accurate information on numbers, quality and distribution of existing staff. This survey is, to our knowledge, the first attempt to ascertain HR for TB control in HBCs. The response rate was high and responses were analysed in the context of the countries\' estimated TB burden and current CDRs. Limitations of the study are evidenced by the great variation in reported staff numbers and the lack of correlation between information on numbers of trained staff, length of courses, development of materials and performance, suggesting a shortage of valid information, poor standardization and/or data reliability. The fact that the questionnaire was in English could have generated some inaccurate answers. Indonesia, for example, reported 100% training completion based on its 2002 training goals rather than in absolute numbers (the actual proportion of staff trained at health centre level was 35%), but countries were re-contacted (by e-mail or telephone) to clarify inconsistent replies. Responses could have been biased towards greater HR needs if countries perceived this as an opportunity to request increased support; however, this did not seem to happen, since few countries reported staff shortages. The performance assessment component of the questionnaire assumed NTP managers could assess the HR gap by comparing existing staff numbers (at different service levels) with the product of the additional SS+ cases by the time spent in diagnostic/treatment activities. Although all the tasks were listed in the questionnaire, the interpretation of what constituted achieving each task was left to NTP managers; this could have affected their estimation of the time required. We are currently developing a more detailed task analysis tool that includes the breakdown of each TB control activity into specific single tasks and a description of what constitutes each task; this will facilitate future studies. Furthermore, this method accounted only for new SS+ cases: in some countries, health facility staff will spend more time dealing with relapses, treatment failures or other TB patients: On the other hand, in some HBCs some tasks are performed by NGOs or the private sector and this was not discounted. Constraints to HR development such as poor HR data quality, lack of a comprehensive national HR plan/strategy and little attention to continuing education programmes \[[@B18]\] were common to HBCs. This study evidences the huge gap in HR data (more apparent in large countries such as China and India) and the variability in quality and validity of available information. This is not an isolated problem of TB control programmes, given the dearth of published data on HRH in LMICs. Decentralization, ongoing in many HBCs, could have contributed to worsening HR information at central level. Developing HR planning and management capacity at district level and generating HR information systems should accompany decentralization processes \[[@B19]-[@B21]\] so that decentralization does not result in decentralized chaos \[[@B19]\]. A comprehensive HRH database facilitates HR surveillance and the management and planning of HR development in the health system \[[@B20]\]. The 2nd ad hoc committee on the TB epidemic recommended addressing the health-workforce crisis by collaboratively developing policies to reduce barriers to creating and filling posts in HBCs; increasing staff recruitment and retention by improving working conditions in the health sector; promoting task analysis and HR needs assessments, HR planning and training at country level; and working together with other stakeholders to develop strategies to further mobilize HR for TB control \[[@B22]\]. In general there is poor communication between HR planning units in the MoH and other technical programmes. A starting point for many HBCs will be a rigorous appraisal of their current HRH through in-depth assessments. Calculating programme-specific HR requirements, and informing the HR planners in the MoH, are two steps that are not consistently performed, resulting in a health care workforce often unaware of its own capacities and limitations. Conclusion ========== (See Table [4](#T4){ref-type="table"}.) There is urgent need to assist HBCs developing HR information systems so that the up-to-date information required for appropriate planning, managing and supporting their health care workforce can be available. NTP managers in HBCs were generally aware of the need for appropriately trained staff at different service levels and, in some countries, the need to redistribute existing staff. Many HBCs require support in developing HR planning and management capabilities; however, more information is needed from countries to understand what factors most influence HR capacity so that country-specific plans can be developed. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Conclusions ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 1\. A paradigm shift in our approach to HR is needed. The HR impact of health initiatives must be conveyed in an explicit, open and unambiguous way so that governments, planners, and financial and technical partners will have a clearer understanding of the urgency of the HR crisis and will have to take a stand on addressing it. 2\. HR information systems in HBCs must be developed/strengthened. Without some reasonably accurate information on the numbers, location, qualifications and skills of staff it is impossible to administer, manage or plan the health workforce in any effective manner. 3\. There is a dearth of information on HR for disease control programmes in LIMC. There is a need to develop HR assessment tools allowing for the different disciplines involved in HR issues and to conduct in-depth studies using validated methodology. 4\. It is important to improve the communication link between technical programmes and HR planning at central level. There is a need to support some HBCs in developing HR management skills and in capacitating personnel in the area of HR management and planning. 5\. Training is an important component of HRH development; there is a need to identify the minimum requirements of training at different service levels required to obtain a universal standard of care for TB patients and to better standardize training materials, methodologies and courses. 6\. There must be a twin-track approach to addressing the HRH crisis. Current shortages must be addressed with short-term interventions in line with medium/long-term solutions developed within the context of poverty reduction strategies and national medium-term expenditure frameworks. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: Human resources constraints in TB will not be solved by NTPs in isolation; they are and will remain a subset of the general health workforce. The degree of integration of the HR for TB within the health workforce will vary from partial to total, depending on local conditions. The same holds true for the supply side: different health programmes compete for finite HR, posing a strain on local health systems. The health workforce crisis for TB control must be addressed within the broader HR context. Without creative solutions there will not be enough trained health professionals to implement the strategies proposed by the priority disease control programmes. Finally, a paradigm shift in the way we approach HR issues is needed. Up until now, the HR implications of public health interventions have been tacitly understood; the impact of healthcare initiatives on the limited health workforce has always been implicit, unspoken and often underrated. There is now a need to clearly convey the HR impact of health initiatives in an open, unambiguous manner. By endeavouring to make the HR implications of each existing and new public health intervention explicit, we hope governments, planners, and financial and technical partners will have a clearer understanding of the urgency of the HR situation and will have to take a stand on addressing the health care workforce crisis. Conflict of interests ===================== The author(s) declare that they have no competing interests. Acknowledgements ================ This study was supported by the Stop TB Department, World Health Organization. The authors wish to thank NTP managers and regional and country-based WHO and NTP staff at HBCs who kindly provided HR data. The authors are grateful to Tomas Allen for his contribution to the literature review and to Robert Scherpbier, Norbert Dreesch, Carmen Dolea and Gijs Elzinga for their comments and advice.	PubMed Central	2024-06-05T03:55:54.027219	2005-2-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554980/", "journal": "Hum Resour Health. 2005 Feb 24; 3:2", "authors": [ { "first": "José", "last": "Figueroa-Munoz" }, { "first": "Karen", "last": "Palmer" }, { "first": "Mario R", "last": "Dal Poz" }, { "first": "Leopold", "last": "Blanc" }, { "first": "Karin", "last": "Bergström" }, { "first": "Mario", "last": "Raviglione" } ] }
PMC554981	Background ========== Alternative splicing is estimated to occur in 40--60% of all human genes, accounting for some of the discrepancies between the large number of known proteins and the three-fold lower number of human genes in the genome. Alternative splicing generates a multitude of isoforms that have overlapping but distinct functions during embryonic development and that also contribute to maintaining homeostasis in adult differentiated tissues (reviewed in \[[@B1]\]). Alternative splice forms of key proteins in cancer, TP53, MDM2 \[[@B2]\] and c-MYC \[[@B3]\], have been shown to play a role in oncogenesis. Survivin was originally identified by structural homology to IAPs in human B-cell lymphoma \[[@B4]\]. It is composed of a single BIR domain and an extended carboxy-terminal coiled coil domain \[[@B5]\]. Transcription from the Survivinlocus gives rise to alternatively spliced transcripts identified in both human and mice \[[@B6]-[@B8]\]. To date, three alternatively spliced isoforms have been described in humans \[[@B6]-[@B8]\]. Survivin-2B is generated by the insertion of an alternative exon, exon 2B; Survivin-Δ Ex3 arises from the removal of exon 3 resulting in a frameshift and translation of part of the 3\'UTR generating a unique carboxy-terminus; Survivin-3B results from the introduction of a novel exon 3B resulting in a frameshift and premature termination of the protein (Figure [1A](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Structural analysis of survivin 2α compared with the other human survivin isoforms. A: The survivin pre-mRNA generates at least five mature mRNA transcripts. Boxes represent exons, with the sizes indicated below. The size of the additional nucleotide sequences for survivin 3B and survivin 2α are shown. In survivin 2α, 197nt of intron 2 are added, of which 195nt are non-coding. Protein domains and motifs are indicated above the diagrams. The black arrow indicates the stop codon; the coiled-coil domain is shown by the dotted line; and the double lines represent the BIR domain. B: Survivin 2α transcript and predicted protein sequence. The sequence for the survivin 2α transcript was obtained by sequencing the insert contained within the IMAGE clone. C: Predicted 3D structural modeling of survivin 2α and survivin. The amino acid sequence of survivin 2α was used with SWISS-MODEL to predict a 3D structure by homology modeling. The resulting PDB file was visualized and manipulated using Swiss PBD-Viewer for the view presented. The 3D structure for survivin was obtained from Swiss-PROT database (PDB entry 1F3H). The yellow arrows indicate regions of differences between survivin 2α and survivin. The red arrows represent the first 2 alpha helices of the BIR domain, and the pink arrow represents the 3^rd^helix, that is absent in survivin 2α. The green arrow points to the C-terminal coiled-coil domain. D: Protein Analysis of survivin 2α. Total HeLa cell lysate was loaded on a 18% SDS-PAGE and transferred into nitrocellulose membrane. The proteins were detected by immunoprobing with a polyclonal survivin antibody. A protein of approximate molecular weight 8.5 kDa, corresponding to the predicted size of survivin 2α is detected in HeLa cell lysates. ::: ![](1476-4598-4-11-1) ::: Survivin has 2 main functions; one as a chromosomal passenger protein \[[@B9]\] and the other as an inhibitor of apoptosis \[[@B10]\]. Survivin 2B has been shown to be a pro-apoptotic protein that sensitizes resistant leukemia cells to chemotherapy in a p53-dependent fashion \[[@B11]\]. Survivin-Δ Ex3 functions as an anti-apoptotic protein and is upregulated in malignancies (Mahotka et al., 1999). No function has yet been described for survivin-3B. In this report we identify and characterize a novel isoform of survivin, survivin 2α. We show that survivin 2α is expressed at high levels in malignant cells, co-localizes with survivin and has the potential to attenuate the anti-apoptotic effect of survivin. Results and Discussion ====================== Structural Characteristics of Survivin 2α ----------------------------------------- In this work, we characterized a novel isoform of the survivin locus. We surveyed the aligned survivin EST sequences available in the UCSC Human Genome Browser and identified an EST from a human breast tumor cDNA library (I.M.A.G.E. clone 1631662). We sequenced the entire cDNA and designated it Survivin 2α. The complete cDNA sequence is shown in Figure [1B](#F1){ref-type="fig"}. The protein contains the coding sequences from exon 1 and exon 2, and one additional amino acid before termination (Figure [1B](#F1){ref-type="fig"}). This 74 amino acid protein, with a predicted molecular weight of 8.5 kDa, contains a truncated BIR domain and lacks the carboxy-terminal coiled-coil domain in its entirety (Figure [1A](#F1){ref-type="fig"}). There are no defined localization signals in the protein, and PSORTII predicts localization within the nucleus and the cytoplasm (Table [1](#T1){ref-type="table"}). Alignment with the known human survivin isoforms shows that the sequence of Survivin 2α is identical to exons 1 and 2 of the other survivin splice variants, with the exception of the last amino acid. Alignment of Survivin 2α with the three mouse survivin isoforms also reveals some similarity with survivin40, a 40-amino acid mouse splice variant (not shown). The 3D predicted structure of Survivin 2α shows the absence of the alpha-helical coiled-coil domain, present in survivin (Figure [1C](#F1){ref-type="fig"}). It also shows minor predicted rearrangements in the structure that may occur to stabilize the protein (Figure [1C](#F1){ref-type="fig"}, yellow arrows). These re-arrangements occur within the BIR domain, and could have functional implications for the role of Survivin 2α in apoptosis. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Table of the predicted localization and structural features of survivin and the novel isoform survivin 2α. ::: Localization Survivin Survivin 2α ---------------------------- -------------- ----------------- Cytoplasm 56.5% 39.1% Nucleus 17.4% 34.8% Cytoskeleton 0% 4.3% Golgi Apparatus 0% 0% Plasma membrane 4.3% 0% ER 4.3% 4.3% Peroxysome 0% 0% Mitochondria 13.0% 13.0% Lysosomes 4.3% 4.3% Features BIR 1 Partial Coiled-Coil 1 0 Protein Size 142 aa 74 aa Predicted Molecular Weight 16.4 kDa 8.5 kDa ::: The BIR domain has been shown to be important for homodimerization and coordination of the zinc atom co-factor \[[@B12]\]. In the survivin protein, Histidine 80 (H80) is required for zinc atom coordination and homodimerization. Expression constructs containing mutations at this residue within the Survivin protein have previously been shown to accelerate PCD (Programmed Cell Death) in vitro. Similarly, mutations in Cytosine 84 (C84) enhance PCD, as a result of displacement of the wild type Survivin protein \[[@B13]\]. The Survivin 2α protein, truncated at amino acid 74, lacks both of these amino acid residues. Additionally, Survivin 2α lacks the third alpha helix in the BIR domain. As the anti-apoptotic function of Survivin is mediated both by the BIR domain and by the interaction of its C-terminal coiled coil domain with microtubules of the mitotic spindle \[[@B10],[@B14],[@B15]\], it would be predicted that Survivin 2α might not have anti-apoptotic properties. Survivin 2α is highly expressed in tumor cells ---------------------------------------------- Survivin is critical for global normal embryonic development, as demonstrated by the early embryonic lethality of mice with homozygous deletions in the survivin gene locus \[[@B16]\]. Survivin proteins are virtually absent from most normal differentiated tissues, however these proteins are expressed in certain highly proliferative areas within normal tissues \[[@B17]-[@B19]\]. In contrast, survivin is highly expressed in the majority of human malignancies, derived from different cell origins. We evaluated the expression of survivin 2α in 7 different cancer cell lines, 2 non-transformed tissues and 7 primary medulloblastoma tumors by quantitative PCR. We designed primers that will specifically amplify Survivin 2α after selection of polyadenylated RNA. Survivin 2α expression in tumor cells and primary medulloblastoma tumors varied from 2--100 fold above non transformed cells (Table [2](#T2){ref-type="table"}). The levels of Survivin 2α transcripts are comparable to those of Survivin ΔExon3 (Table [3](#T3){ref-type="table"}). Like Survivin, Survivin 2α is expressed at increased levels in transformed cells compared to non-transformed cells, and therefore it suggests that it could have a role in tumorigenesis. Additionally, we detected expression of endogenous Survivin 2α protein in HeLa cells, suggesting that the transcript is translated (Figure [1D](#F1){ref-type="fig"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Survivin 2α expression (relative to normal tissue). ::: Cell Type Relative Increase ---------------------------- ----------------------- Normal Cerebellum 1.00 Normal Breast (MCF10A) 0.97 Breast Carcinoma (MCF7) 8.17 Osteosarcoma (U2OS) 39.06 Lung (A549) 3.03 ALL (Jurkat) 1.84 Soft Tissue Sarcoma (RH28) 94.90 Cervical Carcinoma (HeLa) 58.22 Medulloblastoma (Daoy) 34.23 Primary Tumors Medulloblastoma \#1 4.68 Medulloblastoma \#2 154.55 Medulloblastoma \#3 93.24 Medulloblastoma \#4 5.69 Medulloblastoma \#5 8.54 Medulloblastoma \#6 9.81 Medulloblastoma\#7 75.10 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Expression of survivin splice variants in medulloblastoma (relative to survivin) ::: Tissue Survivin 2B Survivin ΔEx3 Survivin 2α ------------------------ ----------------- ------------------- ----------------- Medulloblastoma \#3 37.63 0.46 0.36 Medulloblastoma \#4 43.82 0.18 0.18 Medulloblastoma (Daoy) 1290.16 35.10 0.66 ::: Functional Properties of Survivin 2α ------------------------------------ To characterize a function for Survivin 2α, we transfected Daoy cells with Survivin 2α and a combination of Survivin 2α and Survivin. To induce apoptosis in the Daoy cells, we treated them with 2 μM of the chemotherapeutic agent vincristine. Vincristine is a vinca alkaloid that binds to tubulin, inhibiting microtubule polymerization. It kills Daoy cells in culture by PCD. We analyzed early apoptotic events in vincristine-treated transfected cells by Annexin V staining. Survivin 2α antagonized the anti-apoptotic effect of Survivin in co-transfection assays with or without a cell death stimulus (not shown and Figure [2A](#F2){ref-type="fig"}). As inhibition of apoptosis by Survivin involves activation of the caspase pathway \[[@B20]\], we assayed Survivin 2α transfected cells for caspase 3 activation. Caspase-3 was strongly activated in vector control and Survivin 2α transfected cells in the presence of vincristine. Much lower levels of caspase-3 activation were observed in Survivin-transfected cells (Figure [2B](#F2){ref-type="fig"}). In the absence of an apoptotic stimulus we observed a 35% increase of caspase-3 activity in Survivin 2α cells, as well as a 46% increase in early apoptosis, as assessed by Annexin V staining. We also performed electron microscopy analysis of Survivin 2α transfected and non-transfected cells. We sorted transfected cells from non-transfected cells by FACS based on GFP fluorescence, and processed each population for EM analysis (Figure [2C](#F2){ref-type="fig"}). Overall, there was a 43% increase in incidence of apoptosis in Survivin 2α-expressing cells versus non-expressing cells. Our results suggest that Survivin 2α can attenuate survivin\'s anti-apoptotic activity and sensitize tumor cells to chemotherapy. These findings have important therapeutic implications in the treatment of chemoresistant tumors. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Early apoptosis in tumor cells transfected with survivin and survivin 2α. A: Daoy cells were transiently transfected with control vector, survivin, survivin 2α, or a combination of survivin and survivin 2α. Apoptosis was measured using Annexin V/PI staining 24 hours after transfection, in the absence of a cell death stimulus. The results are shown relative to empty vector control. Error bars represent standard deviations from triplicate experiments. Results were adjusted for transfection efficiency based on parallel transfection with a GFP-expressing plasmid. B: Daoy cells were transiently transfected with control, survivin or survivin 2α. Apoptosis was measured by Caspase-3 activity following treatment with vincristine. C: HeLa cells were transiently transfected with control or GFP-tagged survivin 2α. Electron Microscopy analysis of transfected cells shows a representative cell undergoing apoptosis as induced by survivin 2α. Images were taken 12,000× magnification and scale bars are shown. ::: ![](1476-4598-4-11-2) ::: Survivin 2α alters the subcellular localization of survivin ----------------------------------------------------------- To characterize the subcellular localization of survivin 2α we performed direct fluorescence assays in HeLa cells transfected with a GFP- survivin 2α construct. Survivin 2α localized to the nucleus and the cytoplasm in interphase cells (Figure [3A](#F3){ref-type="fig"}). In cells undergoing mitosis, survivin 2α was confined to the cytoplasmic compartment (Figure [3B](#F3){ref-type="fig"}). Interestingly, when co-expressed with survivin, survivin 2α co-localized with survivin to the centromeres of the chromosomes in prometaphase (Figure [3C](#F3){ref-type="fig"}) and metaphase (Figure [3D](#F3){ref-type="fig"}), and at the midbody during late telophase/cytokinesis (Figure [3E](#F3){ref-type="fig"}). Moreover, the normal cytoplasmic localization of survivin shifted to the nucleus in interphase cells. This data suggests a direct interaction between the two proteins, as well as a potential mechanism for the attenuation of survivin\'s anti-apoptotic activity by survivin 2α. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Confocal microscopy analysis of survivin 2α sub-cellular localization. HeLa cells were transfected with GFP-survivin 2α or HcRed survivin, as detailed on the top of the figure. Green pixels correspond to GFP expression, red pixels correspond to HcRed expression and blue pixels represent DNA labeled with Hoechst dye. When co-localization of GFP and HcRed occurs the pixels are yellow. A: Expression of survivin 2α at interphase localizes to nuclear and cytoplasmic structures. B: During M-phase survivin 2α is excluded from the condensed/dividing chromosomes and is localized in the cytoplasm of the dividing cell. C, D, E, F: When co-expressed with survivin, survivin 2α localization does not change at interphase. During M-phase survivin 2α co-localizes with survivin to the centromeres of the dividing chromosomes (D and E), and in the midbody region at cytokinesis (F). Scale bar = 5 μm ::: ![](1476-4598-4-11-3) ::: Survivin 2α physically interacts with survivin ---------------------------------------------- To further investigate the possibility that survivin 2α interacts with survivin we performed co-immunoprecipitation experiments. We co-transfected HeLa cells with constructs encoding a Flag-survivin fusion protein and a myc-survivin 2α fusion protein. We used a Flag antibody to precipitate protein complexes, and a myc antibody to detect myc-tagged survivin 2α. We detected survivin 2α-myc in the complexes precipitated with the Flag antibody, substantiating a physical interaction of survivin with survivin 2α (Figure [4](#F4){ref-type="fig"}). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Co-immunoprecipitation of survivin-survivin 2α. HeLa cells were transfected with constructs encoding tagged forms of survivin (Flag) and survivin 2α (myc). Lysates from transfected cells were subjected to immunoprecipitation with an antibody against the Flag epitope. The resulting immunoprecipitated complexes were resolved by SDS-PAGE and subjected to Western blotting. The membrane was immunoprobed with an antibody against the myc epitope tag. Survivin 2α-myc is clearly visualized in lysates precipitated with a Flag antibody. ::: ![](1476-4598-4-11-4) ::: Conclusion ========== We characterized a novel survivin splice variant that we designated survivin 2α. We hypothesize that survivin 2α can alter the anti-apoptotic functions of survivin in malignant cells. Thus, survivin 2α may be useful as a therapeutic tool in sensitizing chemoresistant tumor cells to chemotherapy. Methods ======= Patient samples --------------- Seven fresh frozen primary medulloblastoma tumor samples were obtained from the Cooperative Human Tumor Network (CHTN), after approval through the Columbus Children\'s Hospital IRB. Sequencing ---------- IMAGE clone 1631662 (Invitrogen) was sequenced using primers that flanked the multiple-cloning-site. Plasmids and Cloning -------------------- The cDNA for survivin 2α was amplified from the EST clone (Invitrogen) and cloned into the KpnI-BamHI sites of pcDNA4/TO/myc-HisB (Invitrogen) generating an in-frame fusion with the C-terminal myc-tag, or into the KpnI-BamHI sites of pEGFP-N3 generating an in-frame fusion with the C-terminal GFP tag. The start codon in both constructs corresponds to the naturally occurring start codon in the cDNA transcript. The resulting clones were confirmed by sequencing. Cell Culture and Transfection ----------------------------- HeLa (cervical adenocarcinoma), Daoy (medulloblastoma), Jurkat (acute lymphoblastic leukemia) and MCF-7 (breast adenocarcinoma) cells (ATCC) were grown in DMEM supplemented with 10% FBS at 37°C, 5% CO~2~; U2OS osteosarcoma cells (kindly donated by Dr. Greg Otterson) were grown in McCoy\'s 5A medium supplemented with 10% FBS at 37°C, 5% CO~2~; RH28 (alveolar rhabdomyosarcoma, kindly donated by Dr. Steve Qualman) and A549 (lung carcinoma) (ATCC) were grown in RPMI1640 supplemented with 10% FBS at 37°C, 5% CO~2~. MCF10-A, a non-transformed breast cell line (ATCC) was grown in MEGM, Mammary Epithelial Growth Medium, Serum-free, (Clonetics) supplemented with 100 ng/ml cholera toxin (Sigma Aldrich) at 37°C, 5% CO~2~. Transient transfections were performed using Effectene transfection reagent (Qiagen) at a DNA: Effectene ratio of 1:10. Drug Treatment -------------- Induction of apoptosis by vincristine was done by treatment of cells with complete growth medium supplemented with vincristine sulfate at a final concentration of 2 μM. RNA isolation and Real Time PCR ------------------------------- RNA was isolated from 10^6^proliferating cells or frozen tumor tissue using TriZol reagent (Invitrogen) as recommended by the supplier. Poly(A) RNA was purified using Oligotex dT kit (Qiagen). 100 ng of poly(A) purified RNA was used as a template in a reverse transcription reaction using random hexamers and Omniscript Reverse transcriptase (Qiagen) and performed according to manufacturer\'s instructions. Quantitative real-time PCR reactions using Taqman probes (FAM/TAMRA) were run in triplicate on an ABI Prism 7700 Real-time PCR machine (Applied Biosystems). Control GAPDH reactions (Applied Biosystems) were run to normalize ΔCt values. Relative change was calculated by the comparative C~T~method, 2^(-ΔΔCt)^. The survivin 2α specific primers consist of: Forward 5\'GCTTTGTTTTGAACTGAGTTGTCAA; Reverse 5\'GCAATGAGGGTGGAAAGCA; and Probe: 6FAM AGATTTGAGTTGCAAAGACACTTAGTATGGGAGGG TAMRA Apoptosis Assays ---------------- Two apoptosis assays were performed: Caspase-3 assay and Annexin-V FLUOS. For caspase assays 2,000 cells from each experimental condition were subjected to the caspase-3 assay, Caspase 3/7 GLO (Promega) and analyzed on a Victor3 plate reader (Applied Biosystems). Experiments were performed in triplicate. Annexin V/propidium iodide staining was carried out using the Roche Annexin-V-Fluos Staining Kit following the manufacturer\'s instructions. Fluorescein and propidium iodide fluorescence measured with a Coulter EPICS XL flow cytometer. Experiments were performed in triplicate. Microscopy ---------- Proliferating HeLa cells, grown on glass coverslips, were transiently transfected with a GFP-tagged survivin 2α expression construct or co-transfected with GFP-tagged survivin 2α and HcRed-tagged survivin. 24 hours post-transfection the cells were fixed in 4% paraformaldehyde and stained with 50 μg/ml Hoechst dye. Cells were analyzed on a Zeiss LSM510 META confocal microscope, using a 63x PlanApochromat objective. For electron microscopy analysis, proliferating HeLa cells were transfected with GFP-tagged survivin 2α construct for 12 hours. The cells were aseptically sorted by FACS based on green fluorescence from GFP-survivin 2α for positive and negative populations. This was done in order to separate an enriched population that consisted of \>90% GFP expressing cells. 10^6^cells for each condition were fixed in 2.5% gluteraldehyde for 24 hours and processed for EM. For cell analysis, 10 to 12 fields containing 8--10 cells per field at a magnification of 3500× were used. At least 100 cells were counted for each experimental condition and assigned to categories of healthy or dying based on their morphological appearance, including nuclear integrity. Image collection was performed on a Hitachi H-600 transmission electron microscope equipped with a GATAN image acquisition system. Co-Immunoprecipitation ---------------------- HeLa cells transfected with Flag-survivin and survivin-2α myc were collected in Cell Lysis Buffer (100 mM Tris-HCl pH8.0, 100 mM NaCl, 0.5% Triton X-100, 0.2 μM PMSF) and incubated at 4°C for 30 min. The cell lysate was clarified by centrifugation and the clarified supernatant dissolved 1:5 in Co-IP buffer (50 mM Tris-HCl pH7.5, 15 mM EGTA, 100 mM NaCl, 0.1% Triton X-100, 1x protease inhibitors cocktail, 1 mM DTT, 1 mM PMSF). The equivalent of 400 μg of lysate total protein was incubated with 2 μg of anti-Flag M2 antibody at 4°C for 1 h with constant rotation. As a control the same amount of lysate protein was incubated in the absence of antibody. Fifty microliters of agarose-conjugated protein A (Invitrogen) were added and the mixture incubated for a further hour in the same conditions. The protein-antibody-protein A complexes were pulled down by centrifugation and subjected to 3 washes with co-IP buffer. The proteins were analyzed through electrophoretic separation in a 20% SDS-PAGE, electroblotted onto nitrocellulose and immunoprobed with an antibody against myc-tag. Detection was performed using the ECL kit (Amersham). Protein standards were used for size determination. Bioinformatics -------------- Subcellular localization predicted by PSORTII program. Coiled-Coil domain predicted by Coils and PairCoil programs Authors\' contributions ======================= HC performed bioinformatic analysis, subcellular localization, functional studies, co-immunoprecipitation and drafted the manuscript. LH performed quantitative real time PCR in cell lines and primary tumors. RA conceived the study and participated in its design and coordination, and was responsible for overseeing the final version of the manuscript. All authors have read and approved the final manuscript. Acknowledgements ================ We would like to thank M. Holloway and J. Fangusaro for general technical assistance, C. McAllister for assistance with FACS and EM and G. Otterson and S. Qualman for kindly donating cell lines. We are grateful to Dr Heithem El-Hodiri for critically reviewing this manuscript. This work was supported by the Elsa U. Pardee Foundation and by the Hope Street Kids Foundation.	PubMed Central	2024-06-05T03:55:54.031174	2005-3-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554981/", "journal": "Mol Cancer. 2005 Mar 2; 4:11", "authors": [ { "first": "Hugo", "last": "Caldas" }, { "first": "Laura E", "last": "Honsey" }, { "first": "Rachel A", "last": "Altura" } ] }
PMC554982	Background ========== Human cytomegalovirus (HCMV) is a ubiquitous beta-herpesvirus that affects 60--80% of the human population \[[@B1]\]. The lytic replication cycle of HCMV is a temporally regulated cascade of events that is initiated when the virus binds to host cell receptors. Upon entry into the cell, the viral DNA translocates to the nucleus, where expression of viral immediate early (IE), early and late genes occurs in a stepwise fashion \[[@B2]\]. While generally asymptomatic in immunocompetent individuals, primary HCMV infection may cause infectious mononucleosis and has been associated with atherosclerosis and coronary restenosis \[[@B3],[@B4]\]. Furthermore, HCMV is the leading contributor of congenital viral infections in the United States and Europe, causing cytomegalic inclusion disease, pneumonia and severe neurological anomalies in infected neonates \[[@B5]-[@B7]\]. Like other herpesviruses, HCMV establishes lifelong latency in its host from which reactivation can occur and cause severe and fatal disease in immunocompromised individuals \[[@B8]\]. Cellular immune responses (MHC class I-restricted T-cells and natural killer (NK) cells) appear to be an important factor in both the control of acute infections and the establishment and maintenance of viral latency in the host \[[@B9]-[@B14]\]; however, the mechanisms by which T-cells affect HCMV replication are currently undefined. While cytotoxic T-cell activity has been shown to correlate with recovery from HCMV infection in patients \[[@B15],[@B16]\], recent studies suggest that immune cytokines such as tumor necrosis factor-α and interferons (IFNs) may have direct inhibitory effects on HCMV replication \[[@B17],[@B18]\]. In particular, the involvement of IFNs as a means of curtailing viral replication without cellular elimination is consistent with the hypothesis that cytokines produced by activated immune cells play a direct role in the control of viral infections \[[@B19]-[@B21]\]. Type I IFNs (IFN-α and IFN-β) and type II IFN (IFN-γ) are important components of the host immune response to viral infections. IFN-α and IFN-β are produced by most cells as a direct response to viral infection \[[@B22]-[@B24]\], while IFN-γ is synthesized almost exclusively by activated NK cells and activated T-cells in response to virus-infected cells \[[@B25]\]. Both types of IFNs achieve their antiviral effects by binding to their respective receptors (IFN-α/β or IFN-γ receptors), resulting in the activation of distinct but related Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathways. The result is the transcriptional activation of IFN target genes and the synthesis of a number of proteins that interfere with viral replication (reviewed in \[[@B26]\]). Although IFNs are effective inhibitors of viruses such as vesicular stomatitis virus and encephalomyocarditis virus \[[@B26]\], almost all RNA and DNA viruses have evolved mechanisms to subvert the host IFN response \[[@B21],[@B26],[@B27]\]. For example, HCMV inhibits IFN-stimulated antiviral and immunoregulatory responses at multiple steps \[[@B24],[@B28]-[@B32]\]. Likewise, the herpes simplex virus (HSV-1) protein ICP34.5 \[[@B33]\], the influenza A virus NS1 protein \[[@B34]\], the simian virus-5 V protein \[[@B35]\], the Sendai virus C protein \[[@B36]\], the hepatitis C virus (HCV) NS5A and E2 proteins \[[@B37]\] and the Ebola virus VP35 protein \[[@B38]\] have all been shown to block IFN-mediated responses in infected cells. However, several studies have shown that viruses normally resistant to the effects of type I or type II IFNs separately, are susceptible to IFNs when used in combination. For example, IFN-α/β and IFN-γ synergistically inhibit the replication of HSV-1 both in vitroand in vivo\[[@B20]\]. In addition, recent reports have indicated that IFNs used in combination have a synergistic antiviral activity against severe acute respiratory syndrome-associated coronavirus (SARS-CoV) \[[@B39]\], HCV \[[@B40]\] and Lassa virus \[[@B41]\]. In the present study, we examined the effects of IFN-α, IFN-β and/or IFN-γ on HCMV replication in human foreskin fibroblasts (HFFs). Treatment of HFFs with IFN-α, IFN-β or IFN-γ separately inhibited HCMV replication by ≤ 40-fold in both plaque reduction and viral growth assays. In contrast, treatment with IFN-α and IFN-γ or IFN-β and IFN-γ inhibited HCMV replication 10--20 times greater than that achieved by each IFN separately. This effect was synergistic in nature and the mechanism of inhibition may involve, at least in part, the regulation of IE gene expression. As with HSV-1 \[[@B20]\], we have found that when used in combination, both type I and type II IFNs potently inhibit the replication of HCMV in vitro. Results ======= IFN-α/β and IFN-γ synergistically inhibit HCMV plaque formation --------------------------------------------------------------- The abilities of human IFN-α, IFN-β or IFN-γ to inhibit the replication of HCMV were initially compared in a plaque reduction assay on HFFs. Viral plaque formation was reduced by 9-, 37- or 29-fold in fibroblasts treated with 100 IU/ml of IFN-α, IFN-β or IFN-γ, respectively (Table [1](#T1){ref-type="table"}). To test the effects of combination IFN-treatments on viral plaque formation, HFFs were pre-treated with 100 IU/ml each of (1) IFN-α and IFN-β, (2) IFN-α and IFN-γ or (3) IFN-β and IFN-γ. As expected, the level of inhibition achieved with both IFN-α and IFN-β was not greater than the level of inhibition achieved by both IFNs separately. In contrast, pre-treatment with both type I IFNs (IFN-α or IFN-β) and type II IFN (IFN-γ) reduced HCMV plaquing efficiency by 164- and 662-fold, respectively (Table [1](#T1){ref-type="table"}). To eliminate the possibility that this effect was merely a result of doubling the total amount of IFNs per culture, we tested the inhibitory effects of 200 IU/ml of each IFN separately. Two-hundred IU/ml of IFN-α, IFN-β or IFN-γ reduced HCMV plaque formation by only 11-, 37- or 30-fold, respectively (Table [1](#T1){ref-type="table"}). The level of inhibition was not significantly greater than the level of inhibition achieved by each IFN at concentrations of 100 IU/ml (P \> 0.05), suggesting that the degree of inhibition observed can be attributed to the presence of two distinct types of IFNs. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effect of IFN-α, IFN-β and/or IFN-γ on HCMV plaque formation ::: Treatment IU/ml^a^ Log (mean no. of plaques) ± sem Fold-inhibition^c^ ------------------------ ---------- --------------------------------- -------------------- Vehicle \-\-- 3.34 ± 0.02^b^ \-\-- IFN-α 100 2.38 ± 0.01\* 9 IFN-α 200 2.30 ± 0.01\* 11 IFN-β 100 1.77 ± 0.05\* 37 IFN-β 200 1.77 ± 0.02\* 37 IFN-γ 100 1.88 ± 0.03\* 29 IFN-γ 200 1.85 ± 0.02\* 30 IFN-α and IFN-β 100 1.95 ± 0.04\* 25 IFN-α and IFN-γ 100 1.13 ± 0.09\* 164 IFN-β and IFN-γ 100 0.52 ± 0.05\* 662 IFN-α, IFN-β and IFN-γ 100 0.66 ± 0.15\* 512 ^a^HFFs were treated with either 100 or 200 IU/ml each of IFN-α, IFN-β or IFN-γ (separately or in combination). ^b^Mean ± sem of viral plaque formation on HFFs observed in 3 replicates per group. Cultures were infected with 2000 PFU/well of Towne-GFP, and plaque numbers were determined 14 d p.i. by fluorescent microscopy. ^c^Fold-inhibition was calculated as: 10^(\[log\ plaques\ /\ PFU\ in\ vehicle-treated\]\ -\ \[log\ plaques\ /\ PFU\ in\ IFN-treated\])^ \* Significant reduction in plaque numbers of IFN-treated groups as compared to vehicle-treated groups is denoted by a single asterisk (P \< 0.001, one-way ANOVA and Tukey\'s post hoc ttest). ::: Figure [1](#F1){ref-type="fig"} shows a representative micrograph of HCMV plaque formation on IFN-treated HFFs. Consistent with the results in Table [1](#T1){ref-type="table"}, HCMV plaque efficiency was reduced and plaque morphology was smaller in cultures treated with a combination of type I and type II IFNs (Figure [1E, F](#F1){ref-type="fig"}). This phenotype was also observed in cultures treated with IFN-γ alone (Figure [1D](#F1){ref-type="fig"}), although the overall inhibitory effect of IFN-γ was similar to that achieved in IFN-β-treated HFFs. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### IFN-α, IFN-β and/or IFN-γ inhibit HCMV plaque formation on HFFs. HFFs were pre-treated with (A) vehicle or 100 IU/ml each of (B) IFN-α, (C) IFN-β, (D) IFN-γ, (E) IFN-α and IFN-γ or (F) IFN-β and IFN-γ. Monolayers were subsequently infected with 1000 PFU of HCMV strain Towne-GFP, and plaque numbers were determined 11 d p.i. by fluorescence microscopy. Plaques were determined by counting a minimum of 10 GFP-positive cells in one foci. ::: ![](1743-422X-2-14-1) ::: The antiviral activity of IFNs on HCMV plaque formation was further assessed by generating dose-response curves (Figure [2A](#F2){ref-type="fig"}). The level of inhibition achieved with individual IFN treatments was ≤ 8-fold for IFN-α or IFN-β and ≤ 18-fold for IFN-γ at all concentrations tested. In contrast, combination IFN treatments achieved levels of inhibition 2--18 times greater than the sum of each individual IFN treatment. To determine if the enhanced inhibition of HCMV observed in HFFs treated with both type I and type II IFNs was synergistic, we employed the synergistic analysis for the determination of the interaction of two drugs \[[@B42],[@B43]\]. Interaction indexes were initially calculated from the data generated in the dose response experiments (Figure [2A](#F2){ref-type="fig"}) to assess the synergistic potential of type I and type II IFN treatment. An interaction index of 0.05 ± 0.03 for IFN-α and IFN-γ combined and 0.04 ± 0.01 for IFN-β and IFN-γ combined indicated a high degree of synergy (Table [2](#T2){ref-type="table"}). Additionally, synergy was confirmed by generating isobolograms in which concave isoboles are indicative of synergy while convex isoboles are indicative of an antagonistic effect (Figure [2B](#F2){ref-type="fig"}). Inhibitory concentrations were determined from dose response experiments, and IC~95~isoboles were generated for HFFs treated with both IFN-α and IFN-γ (Figure [2C](#F2){ref-type="fig"}, concave plot) and HFFs treated with both IFN-β and IFN-γ (Figure [2D](#F2){ref-type="fig"}, concave plot). Consistent with the interaction indexes determined (Table [2](#T2){ref-type="table"}), concave isoboles shown in Figures [1C](#F1){ref-type="fig"} and [1D](#F1){ref-type="fig"} indicate a synergistic relationship between type I IFNs (IFN-α and IFN-β) and type II IFN (IFN-γ), suggesting action via distinct antiviral pathways. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Degree of antiviral interaction between IFN-α/β and IFN-γ ::: IFN Treatment^a^(d~a~+ d~b~) IC~90~D~a~^b^ IC~90~D~b~^b^ interaction index^c^ ------------------------------ --------------- --------------- ---------------------- IFN-α + IFN-γ 300 IU/ml 30 IU/ml 0.05 ± .03 IFN-β + IFN-γ 100 IU/ml 30 IU/ml 0.04 ± .01 ^a^HFFs were treated 12 h prior to infection with various combinations of type 1 IFNs (IFN-α or IFN-β) and type II IFN (IFN-γ). ^b^D~a~and D~b~are the concentrations of each IFN separately that inhibit HCMV plaque formation on HFFs by 90% (IC~90~). ^c^Interaction index is a measure of the divergence between the amounts of IFNs that are observed to produce an inhibitory effect in combination (d~a~+ d~b~) and the amounts that would achieve the same effect separately (D~a~and D~b~). Indexes less than 1 indicate synergy, indexes greater than 1 indicate antagonism and indexes equal to 1 indicate additivity. ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Type I IFNs (IFN-α and IFN-β) and type II IFN (IFN-γ) synergistically inhibit HCMV plaque formation on HFFs. (A) Viral plaque reduction assay. HFFs were treated with vehicle or increasing amounts of IFN-α (■), IFN-β (●), IFN-γ (▲), IFN-α and IFN-γ (□) or IFN-β and IFN-γ (○) prior to infection with 400 PFU of Towne-GFP (n = 3). Fold-inhibition in IFN-treated groups as compared to vehicle-treated groups is plotted as a function of IFN concentration (IU/ml). Significant differences in fold-inhibition for HFFs treated with combination IFNs relative to cells treated with individual IFNs are denoted by a single asterisk (P \< 0.001, one-way ANOVA and Tukey\'s post hoc ttest). (B) Illustration of a representative isobologram for a combination of two drugs. The solid line is the line of additivity. When the isobole lies below the line of additivity, the combinatorial effect of drug A and drug B is synergistic. When the isobole lies above the line of additivity, the combinatorial effect of drug A and drug B is antagonistic. Combination effect of (C) IFN-α and IFN-γ and (D) IFN-β and IFN-γ on HCMV plaque formation on HFFs was plotted in an isobologram. Values used to generate the concave isoboles were derived from a dose response curve and represent a combination dose required to elicit 95% (IC~95~) inhibition of viral plaque formation on HFFs. The dashed line represents the theoretical line of additivity. ::: ![](1743-422X-2-14-2) ::: IFN-α/β and IFN-γ synergistically inhibit HCMV replication ---------------------------------------------------------- To further characterize the inhibitory effect of type I IFNs (IFN-α or IFN-β) and type II IFN (IFN-γ) treatment, four-day viral growth assays were performed. In cultures treated with IFN-α, IFN-β or IFN-γ, viral replication was undetectable or below the lower limit of detection at 1 and 2 days (d) post-infection (p.i.). At 3 d p.i., however, HCMV replicated to average titers of 8350, 1050 or 985 PFU/ml in IFN-α-, IFN-β- or IFN-γ-treated cultures, respectively (Figure [3](#F3){ref-type="fig"}). While vehicle-treated cells replicated to average titers of 3.2 × 10^4^PFU/ml, viral titers recovered from cells treated with IFNs separately were reduced by 6-, 23- or 25-fold, respectively. Moreover, at 4 d p.i., viral titers in cells treated with IFNs separately were equal to viral titers recovered from vehicle-treated cultures. Consistent with our plaque reduction assays, we observed a similar enhanced inhibitory effect when HFFs were treated with a combination of type I and type II IFNs. In cultures treated with 100 IU/ml each of IFN-α and IFN-γ or IFN-β and IFN-γ, HCMV replication was detectable beginning at 3 d p.i. yielding titers at or below the lower limit of detection of the assay. Compared to HCMV titers of 1 × 10^5^PFU/ml at 4 d p.i. in vehicle-treated HFFs, treatment with IFN-α and IFN-γ or IFN-β and IFN-γ inhibited HCMV replication in HFFs by an average of 3125- or 5000-fold, respectively. When compared to ganciclovir (GCV)-treated cells, a known DNA synthesis inhibitor of HCMV, the level of inhibition achieved in GCV-treated cultures was comparable to that in IFN-α and IFN-γ- or IFN-β and IFN-γ-treated cultures at 3 and 4 d p.i. (Figure [3](#F3){ref-type="fig"}). In addition, the potent inhibitory effect observed in the presence of IFN-β and IFN-γ was maintained up to 11 d p.i. (Figure [3](#F3){ref-type="fig"}, inset), indicating that the effect was not merely a delay in viral replication. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### IFN-α, IFN-β and/or IFN-γ inhibit HCMV replication in HFFs. HFFs were treated with vehicle or 100 IU/ml of IFNs 12 h prior to infection with HCMV at a MOI of 2.5: (◆) vehicle, (■) IFN-α, (●) IFN-β, (▲) IFN-γ, (□) IFN-α and IFN-γ, (○) IFN-β and IFN-γ or (◇) GCV (100 μM). On the indicated d p.i., average viral titers (n = 3) were determined by a microtiter plaque assay. HFFs were inoculated for 2 h with serially diluted lysed cultures. Plaque numbers were determined 11 d p.i. by fluorescence microscopy. At 3 d p.i., all IFN treatments significantly reduced viral titers as compared to vehicle-treated cultures (P \< 0.001, one-way ANOVA and Tukey\'s post hoc ttest). At 4 d p.i., only cells treated with GCV or combination IFN treatments inhibited viral titers as compared to vehicle-treated HFFs (P \< 0.001, one-way ANOVA and Tukey\'s post hoc ttest). Significant reduction denoted by a single asterisk. Inset: Represents HCMV titers determined over 11 d for (◆) vehicle-treated and (○) IFN-β and IFN-γ-treated HFFs. The dashed line represents the lower limit of detection of the plaque assay (20 PFU/ml) used to measure viral titers. ::: ![](1743-422X-2-14-3) ::: Treatment with IFN-α/β and IFN-γ does not prevent HCMV entry into HFFs ---------------------------------------------------------------------- The HCMV replication cycle is a multistep process, beginning with viral attachment and entry into the host target cell \[[@B2]\]. To investigate the mechanism(s) by which IFN-α/β and IFN-γ synergistically inhibit HCMV replication, we first examined the effect of IFNs on HCMV entry into HFFs. Cells were treated with vehicle or IFNs for 12 hours (h) prior to infection with HCMV. Two h after viral adsorption, DNA was isolated from the HCMV-infected cells and PCR was used to amplify a 373 bp fragment of the HCMV IE gene (Figure [4](#F4){ref-type="fig"}). For each treatment group, the PCR product yield increased as a function of viral multiplicity of infection (MOI). At all MOIs tested, the amount of PCR product amplified from HFFs treated with IFNs (Figure [4B--F](#F4){ref-type="fig"}) was comparable to that of vehicle-treated HFFs (Figure [4A](#F4){ref-type="fig"}). Co-amplification of a GAPDH 239 bp PCR product served as an internal loading control for normalization of PCR product between treatment groups (data not shown). The amplification of similar levels of PCR products from HFFs suggests that the synergistic inhibitory effect of IFN-α/β and IFN-γ does not occur at the level of viral entry. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Inhibition of HCMV by IFN-α, IFN-β and/or IFN-γ is not a result of decreased viral entry into cells. Ethidium bromide-stained IE exon 4 PCR products amplified from HCMV-infected HFFs pre-treated with either vehicle (A) or 100 IU/ml of IFN-α (B), IFN-β (C), IFN-γ (D), IFN-α and IFN-γ (E) or IFN-β and IFN-γ (F). From left to right, PCR products were amplified from H~2~O control, 100 ng of uninfected (UI) HFF DNA or 100 ng of HCMV-infected HFF DNA harvested from cells inoculated for 2 h at MOIs of 0.3 to 30. GAPDH PCR products were run along side IE exon 4 PCR products and served as internal loading controls (data not shown). ::: ![](1743-422X-2-14-4) ::: IFN-α/β and IFN-γ inhibit HCMV IE mRNA expression ------------------------------------------------- HCMV gene expression is temporally regulated in that the IE genes (IE1 and IE2) are the first class of viral genes expressed after HCMV entry into the cell \[[@B44]\]. Although limited studies have examined the effect of IFN-β or IFN-γ treatment on HCMV IE mRNA expression, the conclusions of these studies are conflicting, most likely due to differences in both IFN and cell type \[[@B45],[@B46]\]. To assess the effect of IFN treatment on IE gene expression, real-time PCR analyses of IE1 and IE2 mRNA levels in IFN-treated cells were performed. Figure [5](#F5){ref-type="fig"} summarizes the fold-repression in IE1 and IE2 mRNA levels in IFN-treated cultures as compared to vehicle-treated controls. At 6 h p.i., IE mRNA levels in HFFs treated individually with either IFN-α or IFN-γ were inhibited by \< 2-fold, whereas in cells treated with both IFN-α and IFN-γ, IE1 or IE2 mRNA expression was inhibited by 6- or 5-fold, respectively. A more enhanced inhibitory effect was observed in HFFs treated with both IFN-β and IFN-γ. In these cultures, IE1 or IE2 mRNA expression was repressed by 11- or 8-fold, respectively. Interestingly, the degree of IE mRNA inhibition observed in HFFs treated with IFN-β alone was greater than that observed in cultures treated with IFN-α alone, suggesting that type I IFN-mediated inhibition of IE mRNA expression is better facilitated by treatment with IFN-β rather than IFN-α. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### IFN-α, IFN-β and/or IFN-γ inhibit HCMV IE mRNA expression. SYBR green real-time PCR analyses of IE1 and IE2 mRNA expression in vehicle- or IFN-treated HFFs 6 h p.i. (n = 3). Presented are fold-inhibition ± standard deviation in IE1 (■) and IE2 (□) mRNA expression in each treatment group. Differences in gene expression were determined as described in Methods. ::: ![](1743-422X-2-14-5) ::: IFN-α/β and IFN-γ inhibit HCMV IE protein expression ---------------------------------------------------- IE protein expression plays a pivotal role in controlling subsequent viral and cellular gene expression during productive HCMV infection \[[@B47]\], such that an inhibitory effect at this level would significantly impair viral replication. To determine whether the inhibitory block in IE mRNA expression correlated with decreased IE protein expression in IFN-treated cultures, western blot analyses were performed (Figure [6A](#F6){ref-type="fig"}). At 12 h p.i., a slight reduction in IE72 and IE86 protein expression was observed in HFFs treated with IFN-β, but not with IFN-α or IFN-γ. Moreover, IE72 and IE86 protein expression was decreased in cells treated with both type I and type II IFNs, with the greatest inhibitory effect observed in HFFs treated with both IFN-β and IFN-γ. This inhibitory block in IE protein expression was consistent throughout a 48 h time period (data not shown). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### IFN-α, IFN-β and/or IFN-γ inhibit HCMV IE protein expression. (A) HFFs were pre-treated with either vehicle (1) or 100 IU/ml of IFN-α (2), IFN-β (3), IFN-γ (4), IFN-α and IFN-γ (5) or IFN-β and IFN-γ (6) 12 h prior to infection with HCMV. At 12 h p.i., cells were harvested and equal amounts of total protein were examined for IE protein (IE72, IE86) expression by western blot analyses. (B-G) Vehicle- or IFN-treated cells were infected with HCMV and the nuclear proteins IE72/86 were detected by indirect immunofluorescence 5 d p.i. Representative images (100X) from cultures treated with (B) vehicle, (C) IFN-α, (D) IFN-β, (E) IFN-γ, (F) IFN-α and IFN-γ or (G) IFN-β and IFN-γ. Immunofluorescent labeling: HCMV IE72/86 -- Alexa Fluor 568 (red), nucleus -- DAPI (blue), overlaid (pink). ::: ![](1743-422X-2-14-6) ::: If IFN-α/β and IFN-γ synergistically inhibit HCMV replication through inhibition of IE gene expression, we hypothesized that this inhibitory effect would be maintained after multiple rounds of viral replication. To address this question, IE protein expression was analyzed by indirect immunofluorescence over a 5-day period. For all treatment groups, IE protein expression was detected as early as 1 h p.i.; however, as viral replication progressed IE protein expression among IFN-treated groups varied (data not shown). Notably, by day 5 p.i., nearly 100% of the cells treated with vehicle, IFN-α or IFN-β alone stained positive for IE72/86, and approximately 87% of the cells treated with IFN-γ alone were expressing the IE proteins (Figure [6B--6E](#F6){ref-type="fig"}). In contrast, the percentage of cells expressing IE proteins was significantly reduced (P \< 0.001) in the treatment groups that received combination IFNs, with only 46% of IFN-α and IFN-γ-treated HFFs and 21% of IFN-β and IFN-γ-treated HFFs positive for IE72/86 (Figure [6F, 6G](#F6){ref-type="fig"}). The observed differences suggest that in cells treated with both type I and type II IFNs, IE expression is (1) differentially regulated and/or (2) viral spread is severely hindered. Discussion ========== The immune response to viral infection is responsible for preventing viral dissemination and uncontrolled replication within the host. Following viral infection, type I IFNs are secreted by infected cells and function to induce an antiviral state in neighboring uninfected cells. Infiltrating immune cells, such as NK cells and macrophages, secrete numerous chemokines and cytokines that contribute to the overall antiviral response. Upon activation of the adaptive immune response, T-cells can further add to the milieu of immune cytokines present at the site of viral infection by secreting additional cytokines, including IFN-γ. Although several studies have examined the effects of proinflammatory cytokines on HCMV replication in vitro, these studies are limited as they only examine the effect of one type of cytokine on viral replication rather than examining cytokines in combination. In support of the latter, recent studies have shown that type I and type II IFNs function, in synergy, to inhibit both RNA and DNA viruses, including HCV \[[@B41]\], SARS-CoV \[[@B39]\], Lassa virus \[[@B40]\] and HSV-1 \[[@B20]\]. These studies may more accurately represent the in vivoinflammatory response that results after viral infection. The results presented herein are consistent with this hypothesis and establish that type I (IFN-α and IFN-β) and type II (IFN-γ) IFNs synergistically inhibit the replication of HCMV. In the present study we have demonstrated that combination treatment with type I and type II IFNs renders cells non-permissive to HCMV replication in vitro. The inhibitory effect by IFN-α/β and IFN-γ was synergistic in nature (Table [2](#T2){ref-type="table"}, Figure [2C, 2D](#F2){ref-type="fig"}) and the degree of inhibition was not matched by increasing the concentrations of each individual IFN (Table [1](#T1){ref-type="table"}, Figure [2A](#F2){ref-type="fig"}). These results indicate that the observed IFN-induced antiviral effects are a direct result of the presence of two distinct types of IFNs. Moreover, inhibition of HCMV replication in cells treated with IFN-α/β and IFN-γ was observed in both HFF and embryonic lung fibroblasts (MRC5) (data not shown) infected with either Towne-GFP (see Methods) or another laboratory strain, AD169 (data not shown). The mechanism(s) by which HCMV replication is inhibited remains unclear. Type I and type II IFNs may synergize by acting on one or more different stages of the HCMV lytic cycle such as (1) viral attachment, (2) viral entry, (3) IE gene expression, (4) early gene expression, (5) DNA replication, (6) late gene expression, (7) virus assembly or (8) viral egress and maturation. To address the question of attachment and entry, PCR was used to amplify viral DNA from IFN-treated and vehicle-treated cultures shortly after infection. As previously observed \[[@B20],[@B46]\], IFN treatment did not prevent viral entry into cells as indicated by equal PCR product yield from all treatment groups (Figure [4](#F4){ref-type="fig"}). These data indicate that IFNs exert their inhibitory effects at a step after viral attachment and entry. Previously, Yamamoto, et al.(1987) demonstrated that treatment of cells with both IFN-α and IFN-γ potently inhibits HCMV replication; however, this study neither determined whether the effect was synergistic nor identified the mechanism of inhibition. However, the authors suggested that IFN-mediated inhibition of HCMV might occur at or prior to early gene expression \[[@B48]\]. Similarly, over the course of our experiments utilizing the Towne-GFP strain, it was noticed that very few cells expressed green fluorescent protein (GFP) when treated with IFN-α/β and IFN-γ together (data not shown). In this recombinant Towne strain, GFP expression is driven by the early promoter UL127. The lack of GFP-positive cells in IFN-α/β and IFN-γ-treated groups suggested to us that the synergistic antiviral activities mediated by type I and type II IFNs occurred at a stage prior to early gene expression. Previous, studies have shown that type I or type II IFN treatment can inhibit HCMV IE mRNA expression \[[@B46]\] and/or HCMV IE protein expression \[[@B45],[@B46]\]. Using real-time PCR, we showed that while IFN-α, IFN-β or IFN-γ treatment inhibited IE mRNA expression by 2--6 fold at 6 h p.i., combination IFN-α and IFN-γ or IFN-β and IFN-γ treatment inhibited IE mRNA expression by 6--11 fold. Of note, this inhibitory effect was abolished by 24 h p.i. (data not shown), suggesting that IE mRNA expression is delayed by IFN treatment. The observed decrease in viral IE mRNA expression was accompanied by a decrease in IE protein expression, as viral IE protein expression was reduced in HFFs treated with both type I and type II IFNs (Figure [6A](#F6){ref-type="fig"}). Furthermore, immunofluorescent microscopy of IE protein expression revealed that nearly 100% of vehicle- and individual IFN-treated cells expressed IE72/86 5 d p.i., as compared to 46% or 21% of cells treated with IFN-α and IFN-γ or IFN-β and IFN-γ, respectively (Figure [6B--6G](#F6){ref-type="fig"}). It appears that although individual IFN treatment results in a marginal inhibition in IE expression early in infection, the effect is not maintained as demonstrated by high viral titers at 4 d p.i. (Figure [3](#F3){ref-type="fig"}) and increased IE protein expression at 5 d p.i. (Figure [6A--6E](#F6){ref-type="fig"}). Additionally, HCMV cytopathic effect, characterized by enlarged cells containing intranuclear and cytoplasmic inclusions, increased over time in vehicle- and individual IFN-treated groups, while morphology was unchanged in cells treated with IFN-α/β and IFN-γ (data not shown). Collectively, these data suggest that the synergistic inhibition of HCMV replication by IFN-α/β and IFN-γ may involve, at least in part, the regulation of IE gene expression. The significance of an inhibitory block at this level is evident when the phenotype of IE1 mutant viruses is considered. Greaves and colleagues have demonstrated that HCMV IE1 mutants exhibit a diminished replication efficiency and a reduced ability to form plaques, as well as defective early gene expression \[[@B47],[@B49],[@B50]\]. Interestingly, in the presence of both type I and type II IFNs, HCMV shows similar replication and gene expression defects. Although our data suggest that IE gene regulation contributes to the synergistic inhibition of HCMV replication by IFN-α/β and IFN-γ, other mechanisms may also affect this dramatic response. Accordingly, the decrease in IE protein levels exceeds that in IE mRNA levels in response to IFN-α/β and IFN-γ, suggesting that additional regulation at the level of translation, post-translational processing and/or protein stability may be involved. Delineating the other putative regulatory mechanisms that contribute to IFN-α/β and IFN-γ synergistic inhibition of HCMV replication is the focus of ongoing studies. Type I IFNs (IFN-α and IFN-β) and type II IFN (IFN-γ) activate distinct but related Jak/STAT signal cascades resulting in the transcription of several hundred IFN-stimulated genes \[[@B26]\]. Although similar genes are activated by all three IFNs, Der, et al.(1998) have identified numerous genes differentially regulated by IFN-α, IFN-β or IFN-γ \[[@B51]\]. In particular, IFN-β stimulation induces twice as many genes as compared to IFN-α. This differential regulation of IFN-induced genes may explain in part the fact that the level of inhibition observed in HFFs treated with both IFN-β and IFN-γ was consistently greater than that observed in cells treated with both IFN-α and IFN-γ, although both IFN-α and IFN-β bind to the same receptor. Similarly, when compared individually, IFN-β consistently inhibited HCMV replication and IE gene expression to levels greater than IFN-α. Therefore, to better understand the cellular factors involved in the synergistic inhibition of HCMV, the profile of IFN-stimulated genes present in cells treated with both type I and type II IFNs should be further examined. Conclusion ========== Guidotti and Chisari have reported a model of noncytolytic control of viral infections by the innate and adaptive immune response, in which cytokines are implicated as having a direct role in viral clearance \[[@B21]\]. Here we demonstrate that IFN-γ, together with the innate IFNs (IFN-α/β) synergistically inhibits the replication of HCMV in vitro. We hypothesize that IFN-γ produced by activated cells of the adaptive immune response may potentially synergize with endogenous type I IFNs to inhibit HCMV dissemination and facilitate the establishment and/or maintenance of latency in the host. Further studies are required to evaluate the role(s) of both type I and type II IFNs in the regulation of HCMV replication. Methods ======= Cells, viruses and interferons ------------------------------ HFFs (Viromed, Minneapolis, MN) were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, penicillin G (100 U/ml), streptomycin (100 mg/ml), 2 mM L-glutamine, 1 mM sodium pyruvate and 100 μM non-essential amino acids at 37°C in 5% CO~2~. HCMV strain RVdlMwt-GFP was propagated in HFFs as previously described \[[@B52]\]. RVdlMwt-GFP, referred to as Towne-GFP throughout this manuscript, is a recombinant of HCMV strain Towne that expresses GFP under the control of the early promoter UL127. This virus was kindly donated by Mark F. Stinski and has been previously described \[[@B53]\]. Recombinant human universal IFN-α, IFN-β and IFN-γ (PBL Biomedical Laboratories, New Brunswick, NJ) were added to cell cultures 12 h prior to HCMV infection and maintained after viral infection. Concentrations of 100 IU/ml of each IFN were used in all experiments unless stated otherwise. Plaque reduction and viral replication assays --------------------------------------------- For plaque reduction assays, vehicle- and IFN-treated HFFs were infected with a fixed inoculum of Towne-GFP. After 2 h adsorption, the inoculum was removed and medium containing 1.0% methylcellulose (Fisher Scientific, Houston, TX) and the respective IFN(s) was added to the cells. Plaque numbers were determined 14 d later by fluorescent microscopy (Nikon TE300 inverted epifluorescent microscope, Nikon USA, Lewisville, TX). For viral replication assays, vehicle- and IFN-treated HFFs were infected with Towne-GFP at a MOI of 2.5. After 2 h adsorption, the inoculum was removed, monolayers were washed twice with 1X PBS, and fresh IFN-containing medium was returned to each well. For GCV-treated groups, 100 μM GCV (Sigma, St. Louis, MO) was added to culture medium immediately following infection. One, 2, 3 or 4 d p.i. cells and medium were harvested and titers of infectious virus were determined by a microtiter plaque assay on HFFs \[[@B20]\]. Synergy assays -------------- To determine the degree of antiviral interaction between type I and type II IFNs, interaction indexes were calculated using the inequalities: d~a~/D~a~+d~b~/D~b~\> 1 and d~a~/D~a~+d~b~/D~b~\<1, where d~a~and d~b~are the IFN concentrations needed to jointly produce the effect under consideration, and D~a~and D~b~are the IFN concentrations capable of producing the effect on their own, termed isoeffective doses \[[@B42]\]. Interaction index values of less than 1 indicate synergism, interaction index values greater than 1 indicate antagonism and interaction index values equal to 1 indicate additivity. Isobolograms were also generated to geometrically assess the degree of antiviral interaction between type I and type II IFNs, as previously described \[[@B43]\]. Using the guidelines described by Berenbaum \[[@B43]\], isoboles were generated for IC~95~values at various concentrations of IFN-α or IFN-β in the presence of various concentrations of IFN-γ. Concave isoboles are indicative of synergy while convex isoboles are indicative of an antagonistic effect (Figure [2B](#F2){ref-type="fig"}). For all synergy experiments, HCMV plaque reduction assays were conducted as described above. Viral entry assay ----------------- Vehicle- and IFN-treated HFFs were inoculated with Towne-GFP at MOIs of 0.3, 1, 3, 10 or 30. After 2 h adsorption, the inoculi were removed, cells were washed twice with 1X PBS, and subsequently treated with 0.05% trypsin for 5 minutes to ensure the release of virus that had adhered but had not entered the cells. Cells were pelleted and washed twice with 1X PBS to remove trypsin and non-adhered virus. DNA was isolated from each sample by a standard phenol:chloroform DNA extraction procedure \[[@B54]\], and HCMV-specific oligonucleotide primers were used to amplify a 373 bp product corresponding to exon 4 of the HCMV IE gene, as described previously \[[@B55]\]. PCR products were resolved in a 2% agarose gel and imaged using an Alpha Innotech gel documentation system (Alpha Innotech, Corp., San Leandro, CA). Real-time PCR ------------- Vehicle- and IFN-treated HFFs were infected with Towne-GFP at a MOI of 2.5. Six h p.i., total RNA was prepared using a RNeasy Mini Prep kit (Qiagen, Inc., Valencia, CA) according to the manufacturer\'s instructions. Samples were treated with DNase I (Ambion, Inc., Austin, TX), RNA concentration and purity were determined spectrophotometrically (A~260~/A~280~) and 250 ng was reverse transcribed in a total volume of 20 μl using the iScript cDNA Synthesis Kit (Biorad, Hercules, CA) according to the manufacturer\'s instructions. For real-time PCR, 1 μl of cDNA was amplified in 1X iQ SYBR Green Supermix containing specific primer pairs using the iCycler iQ Real-Time PCR Detection System (Biorad). The optimal primer concentrations and sequences were as follows: 200 nM IE1, sense 5\' CAAGTGACCGAGGATTGCAA 3\', antisense 5\' CACCATGTCCACTCGAACCTT 3\' ; 200 nM IE2, sense 5\' TGACCGAGGATTGCAACGA 3\', antisense 5\' CGGCATGATTGACAGCCTG 3\' \[[@B56]\]; 100 nM 18S rRNA, sense 5\' GAGGGAGCCTGAGAAACGG 3\', antisense 5\' GTCGGGAGTGGGTAATTTGC 3\'. All samples were run on the same plate where those for the internal control (18S rRNA) and those for the genes of interest were each run in triplicate, for each of 3 independent RNA preparations. PCR parameters were as follows: an initial step to denature at 95°C for 30 seconds followed by 40 cycles at 95°C for 15 seconds and anneal/extend at 60°C for 45 seconds. Following amplification, melt curves were generated to confirm the specificity of each primer pair with 80 cycles of increasing increments of 0.5°C beginning with 55°C for 30 seconds. Relative quantification of the target genes in comparison to the 18S reference gene was determined by calculating the relative expression ratio (R) of each target gene as follows: R = (E~target~^)ΔCT(vehicle-sample)^/(E~18S~^)ΔCT(vehicle-sample)^\[[@B57]\]. Differences in gene expression between the IFN-treated cells and the vehicle-treated control cells were expressed as fold-inhibition. Western blotting ---------------- Vehicle- and IFN-treated HFFs were infected with Towne-GFP at a MOI of 2.5. Twelve h p.i., the cells were harvested in 500 μl of 1X RIPA buffer containing a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) and 1 mM PMSF. Lysates were sheared 3X with a 27G 1/2 needle and cell debris was pelleted by centrifugation at 14,000 r.p.m. at 4°C. Total protein concentrations from cleared supernatants were estimated with a Micro BCA™ Protein Assay Kit (Pierce, Rockford, IL), 50 μg of total protein were resolved on 10% SDS-polyacrylamide gels and transferred by blotting to PVDF membranes (Amersham Biosciences, Piscataway, NJ). Non-specific reactivity was blocked with 5% nonfat dried milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature and blots were incubated for 1 h at room temperature with a polyclonal antibody that recognizes the HCMV IE proteins (IE72/86), kindly provided by Daniel N. Streblow \[[@B58]\]. The blots were then washed in TBST and incubated with donkey anti-rabbit IgG conjugated to horseradish peroxidase (1:5000; Amersham Biosciences) for 1 h at room temperature. Antigen-antibody complexes were detected using an enhanced chemiluminescence system (Amersham Biosciences). Blots were subsequently washed in TBST and tested for immunoreactivity to a rabbit polyclonal antibody to human β-actin (Sigma; loading control). Indirect immunofluorescence --------------------------- Vehicle- and IFN-treated HFFs were infected with Towne-GFP at a MOI of 1.0. Five d p.i., cells were washed 3X with 1X PBS, fixed with 1:1 methanol/acetone for 10 minutes at room temperature, washed again with 1X PBS, and blocked with 4% BSA/PBS for 15 minutes at room temperature. Cells were incubated for 1 h at 37°C with a HCMV IE antibody (IE72/86 kD; Chemicon \#MAB810, Temecula, CA) diluted 1:200 in 0.5% BSA/PBS. Cells were then stained with 1:50 Alexa Fluor 568-conjugated goat anti-mouse IgG F(ab\')~2~(Molecular Probes, Eugene, OR) for 30 minutes at 37°C, followed by a 2 minute incubation with 1 μM 4\',6-diamidino-2-phenylindole, dihydrochloride (DAPI; Molecular Probes) at room temperature. Cells were coverslipped and mounted in Prolong Antifade mounting medium (Molecular Probes), visualized on a Zeiss Axio Plan II microscope (Thornwood, NY) and images were analyzed with deconvolution SlideBook™ 4.0 Intelligent Imaging software (Intelligent Imaging Innovations, Denver, CO). To determine the number of HCMV-infected cells, three fields of view (100X) for each treatment group were considered and the percent of IE-positive cells was calculated as: (average number of IE-stained cells/average number of DAPI-stained cells)×100. Statistics ---------- Data are presented as the means ± standard error of the means (sem). Data from IFN-treated groups were compared to vehicle-treated groups and significant differences were determined by one-way analysis of variance (ANOVA) followed by Tukey\'s post hoc ttest (GraphPad Prism^©^Home, San Diego, CA). Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= BS and HL conceived of the study, participated in the experimental design, performed all experiments and drafted the manuscript. RG and CM participated in the coordination and design of the study. All authors read and approved the final manuscript. Acknowledgements ================ This work was supported by the National Institutes of Health (AI054626, AI054238, RR018229, and CA08921; R.F.G.) and (HD045768; C.A.M.). Bruno Sainz is a recipient of a National Research Service Award from the NIH (AI0543818). The authors would like to thank Dr. Mark F. Stinski (University of Iowa, Iowa City, Iowa) for kindly supplying the recombinant virus Towne-GFP and Dr. Daniel N. Streblow (Oregon Health Sciences University, Portland, OR) for kindly donating the HCMV IE antibody. We also thank Dr. Aline Scandurro for critical review of this manuscript and Dr. Joseph Vaccaro and Joshua Costin for their expertise in statistical analyses. We are also indebted to Dr. David Woodhall for his expertise and assistance with HCMV propagation and plaque assays.	PubMed Central	2024-06-05T03:55:54.033850	2005-2-23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554982/", "journal": "Virol J. 2005 Feb 23; 2:14", "authors": [ { "first": "Bruno", "last": "Sainz" }, { "first": "Heather L", "last": "LaMarca" }, { "first": "Robert F", "last": "Garry" }, { "first": "Cindy A", "last": "Morris" } ] }
PMC554983	\"Dynamics of the Vascular System\" is a new book by one of the world\'s greatest experts on bioengineering aspects of hemodynamics. It provides an excellent elementary introduction to this topic. Following an illustrative historical introduction, the author briefly reviews vascular physiology. This is followed by the basics of fluid mechanics as an introduction to the hemodynamics of large arteries. A dedicated chapter illuminating the dynamic consequences of vascular branching follows this chapter. This is the field to which Professor Li has made his substantial contributions. The following chapters cover the venous system and microcirculation. Finally the book reviews measuring techniques used to study hemodynamic behavior. The author suggests this volume to be \"a companion\" to his own treatise \"The Arterial Circulation\" \[[@B1]\]. For those familiar with the latter book I must compare the two. While the new book adds useful information on the venous system and on microcirculation, a topic that has been neglected in classical treatments of hemodynamics, this new volume is substantially less comprehensive in most topics covered in both. Also its index is regretfully, substantially less detailed. Little new has been published in this field since the first book was published in 2000, with the notable exception of Zamir\'s book \"The Physics of Pulsatile Flow\" \[[@B2]\]. The reader is advised, therefore, to consider this book as a supplement rather than a companion to the former. In brief, this book, which is more affordable than its predecessor, should be regarded as a good introduction to the topic, to be used primarily by bioengineering students, rather than an updated authorative text by its erudite author.	PubMed Central	2024-06-05T03:55:54.037814	2005-2-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554983/", "journal": "Biomed Eng Online. 2005 Feb 28; 4:13", "authors": [ { "first": "Michael", "last": "Anbar" } ] }
PMC554984	From an early age our immune system is introduced to a variety of infectious agents through contact with infected individuals. We also deliberately present our immune responses either to attenuated organisms or to components of infectious pathogens in order to provoke a response. A successful response to these organisms when first encountered provides us with immunological memory to enable our immune systems to make more rapidly responses to the same potentially infectious agents at a later date. In theory the longer we live the better our memory responses and our ability to cope with any potential pathogen seen previously. Since there are no completely sterile environments, individuals who have survived to reach a ripe old age must have combated many possible infectious agents, and have an immune system with a prodigious memory component. Only part of this is true. As we age the memory component of our immune system, as measured by the number of memory cells, does increase. Unfortunately this is not accompanied by improved immunity even to infectious agents that have been overcome earlier in life \[[@B1]-[@B4]\]. The problems associated with the ageing immune system coupled with possible solutions were discussed recently at the British Society for Immunology Annual Congress in Harrogate in December 2004. The session \"Ageing and the Immune System in vivo\" dealt in details with the immune risk phenotype and the potential methods of reversing the problems of an ageing immune system. The concept of the immune risk phenotype was described by Dr Anders Wikby (Jönköping University) who carried out a longitudinal immunological study in order to establish predictive factors for longevity. Dr Anders first worked on a group of normal octogenarians in the OCTA study in Sweden which later became the NONA study as these individuals entered their nineties. Within this group he was able to describe the immune risk phenotype which he characterised by a CD4:CD8 ratio of \<1, poor in vitro T cell proliferation, an increase in the number of CD8^+^CD28^-^cells (or CD8^+^CD28^-^CD27^-^in the very old), low numbers of B cells and the presence of CD8 T cells which were cytomegalovirus (CMV) tetramer positive. Within both the OCTO and NONA groups Dr Anders was able to show that the immune risk phenotype had some predictive input towards morbidity. This was even more apparent when cognitive impairment was included in the calculations. The effect of CMV infection on the immune system in the elderly was also discussed by Professor Paul Moss (University of Birmingham) with particular emphasis on the clonal expansion of T cells. Clonally expanded T cells are usually CD8^+^and show an increased incidence with age, so far it seems that clonal expansion is not due to malignant transformation but may follow antigen stimulation. The control of CMV within the body is mainly attributed to CD8^+^T cells and Professor Moss revealed that in older individuals up to 50% of CD8^+^T cells may be specific for CMV as judged by tetramer staining. It is possible that this increased response to CMV may lead to the impairment of responses to other viruses in the aged. This theme of CMV infection and ageing was then continued by Professor Graham Pawelec (University of Tuebingen) who introduced the use of the KLRG-1 marker as a marker of senescence. T cells which are KLRG-1^+^do not proliferate in vitro when provided with a stimulus which would induced proliferation in T cells which lack this marker. In elderly individuals fewer CMV tetramer positive CD8^+^T cells secreted interferon-γ in Elispot assays when compared with similar cells from younger individuals. Furthermore between 96 and 99%of the CD8+ T cells which stained with the CMV tetramer also expressed the KLRG-1 marker. The theme of CMV infection in the elderly was further continued by Professor Arne Akbar (University College London) whose research group has recently been analyzing telomere lengths in different T cell subsets using a fluorescent staining technique. In this method telomeres are stained with a fluorescently labeled probe and the brighter the fluorescent labeling the longer the telomere. Using this technique Professor Akbar reported that CMV specific CD4+ T cells in older individuals had very short telomeres indicating their limited replicative capacity and that more of these CMV specific CD4+ T cells expressed interferon-γ. This view of a bleak future for displaying the immune risk phenotype was countered by the last two presentations which discussed different approaches to reversing the defects seen in the immune system with age. Both of these methods centered around reversing the atrophy of the thymus seen with age. Dr Jayne Sutherland, currently at the Anthony Nolan Research Institute, detailed her work carried out whilst with Professor Richard Boyd (Monash University) on reversing age associated thymic atrophy by surgical or chemical castration. Studies in mice revealed that reversal of thymic atrophy followed surgical castration and in humans chemical castration produced changes in the TREC levels which indicated improved thymic output. Functional studies showed that this treatment improved immune function in the treated aged subjects. This theme of improved immunity following reversal of thymic atrophy was carried further in work reported by Dr Richard Aspinall (Imperial College, London). He showed how therapeutic intervention with interleukin 7 and derivates could reverse atrophy of the thymus in old animals and also lead to improved immune function compared with age and sex matched controls. In addition he described work done in the Gambia which suggested a link between the development and enlargement of the thymus after birth and the level of interleukin 7 in breast milk. Two speakers from the posters presentations were also chosen to present their results. K. Wang from Professor Janet Lord\'s group (Birmingham) presented work on dehydroepiandrosterone, (DHEA). This steroid hormone declines with age and treatment of NK cells with DHEA increases NK cytotoxicity and further analysis shows that DHEA induces PKC-β translocation and upregulates perforin expression in these cells. D. Silva from Donald Palmers laboratory (Royal Veterinary College) presented work on the expression of neuropeptides in epithelial cells from the thymuses of different species. The meeting revealed that much needs to be done in characterizing the changes in the immune system which are associated with ageing, and members of the audience were asked whether they were interested in receiving more details about the newly formed \"Differentiation and Immunosenesence Affinity Group\" associated with the BSI.	PubMed Central	2024-06-05T03:55:54.038291	2005-2-22	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554984/", "journal": "Immun Ageing. 2005 Feb 22; 2:5", "authors": [ { "first": "Richard", "last": "Aspinall" } ] }
PMC554985	Background ========== Pediatric cataract surgery has always been a challenge for the anterior segment surgeons and short term and long term complications have been reported in the literature \[[@B1]\]. Automated lens aspiration is the preferred technique employed these days for pediatric cataracts and central continuous curvilinear capsulorhexis is the method of anterior capsulotomy in this procedure. Performing a continuous curvilinear capsulorhexis (CCC) is more difficult in children than in adults because the capsular bag is more elastic. It has been reported that mechanized circular capsulectomy with a vitrector is easier to perform and safer than manual CCC in very young eyes \[[@B2]\]. However, most prefer using Utrata capsulorhexis forceps to complete the capsulorhexis after an initial nick in the anterior capsule with a bent 26 gauge needle. We herein, describe a new technique of postage stamp multiple capsulorhexisotomies, a modification in the primary capsulorhexis after performing lens aspiration with intraocular lens implantation in pediatric cataracts. Methods ======= We performed this technique of anterior capsulorhexisotomies in 6 eyes of 4 children; 2 patients with bilateral developmental cataract and 2 with unilateral post-traumatic cataract. Surgery was performed in all the eyes under general anesthesia. A clear corneal 3.2 mm 3-plane tunnel was created superiorly. Two side ports were created at 10 and 2 o\'clock positions using a microvitreoretinal blade (Alcon laboratories, Fort Worth, TX). Trypan blue (Vision blue, DORC, Netherlands) was injected (0.1 ml of 0.1%) into the anterior chamber under air bubble to stain the anterior capsule and was completely washed out after 15 seconds by balanced saline solution. Anterior capsulorhexis was initiated with a bent 26 gauge needle and completed by Utrata capsulorhexis forceps to create a circular, 4.0 -- 4.5 mm, central capsulorhexis. The size of the capsulorhexis was measured on a television monitor according to a previously described method \[[@B3]\]. Hydroprocedure was performed to soften the lens matter and a complete lens aspiration was performed using Universal II (Alcon laboratories, Fort Worth, TX) phaco machine. Vacuum cleaning of the posterior capsule and the undersurface of the anterior capsular rim was performed in all the eyes. After inflating the capsular bag with 1.4% sodium hyaluronate (Healon GV; Pharmacia & Upjohn, Kalamazoo), a single piece foldable Acrysof intraocular lens (IOL) of optic size of 6.0 mm with an overall diameter of 13.0 mm (SA60AT, Alcon laboratories) was implanted in the capsular bag and the viscoelastic substance was aspirated by rock and roll technique. Sodium hyaluronate 1.4% was injected under the anterior capsular rim. A bent 26 gauge needle was introduced through one side port and multiple small cuts were made in one half of the circumference of the anterior capsular rim by making a radial movement of the needle tip centripetally over the margin of the anterior capsular rim taking care not to put any scratch mark on the IOL. The needle was again introduced through the other side port and multiple similar cuts were made in the other half thereby creating nearly 20 -- 30 cuts all around circumferentially at the margin of the anterior capsular rim (Figure [1](#F1){ref-type="fig"}). The viscosurgical device was aspirated by rock and roll technique and 0.1 ml of 1% vancomycin was injected intracamerally. The anterior chamber was reformed with balanced salt solution and the corneal tunnel was hydrated. Postoperatively, patients were prescribed topical betamethasone sodium phosphate 0.1% and ciprofloxacin 0.3% QID each for 4 weeks and tropicamide 1% TID for 1 week. Results ======= The mean age of the patients was 7.87 ± 1.60 (9, 8.5, 8.5 & 5.5 years) years and all patients were males. The mean size of the primary capsulorhexis was 4.33 ± 0.20 mm. The nicks at the margin of the anterior capsular rim could be performed successfully with a bent 26 gauge needle in all the eyes. A uniform enlargement of the capsulorhexis could be performed in all the eyes without peripheral extension in any of the eyes. No eye suffered damage to the posterior capsule. There was no scratch mark of the needle on the optic of any IOL. In one eye, the primary capsulorhexis was slightly eccentric, though it was covering the IOL optic all around. The rhexisotomies in this eye were limited to the capsular rim that was overlapping more on the IOL optic (sectoral anterior capsulorhexisotomies). Discussion ========== Size of anterior capsulorhexis has always been a matter of debate for automated lens aspiration in pediatric cataracts. If the anterior capsulorhexis opening is small, there is risk of anterior capsular opacification, capsular contraction syndrome and phimosis of the anterior capsular opening, decentration of the IOL and capsular bag hyperdistension \[[@B4],[@B5]\]. Despite an intact capsulorhexis, IOL decentration may still occur due to capsular contraction syndrome. If the capsulorhexis is too large, there is risk of development of adhesion between the anterior capsular rim and the posterior capsule. This can have a zipper effect on the IOL, which can result in forward popping up of the IOL and a significant change in the refractive status of the eye. The size of the anterior capsulorhexis is considered adequate when the margin of the anterior capsular rim just covers the IOL optic margin all around. However, it is very difficult to create an exactly similar size of anterior capsulorhexis in all the eyes. Intraoperative enlargement after performing lens aspiration with a previously performed smaller rhexis is possible \[[@B6]-[@B8]\]. But in pediatric eyes, it is very difficult to predict whether the enlarged rhexis opening will be of optimum size. In many of these situations, the size of the capsular opening enlarges more than optimum in one half or one quadrant. In an effort to prevent complications related to both smaller and larger anterior capsulorhexis, we performed multiple anterior capsulorhexisotomies like the configuration of a postage stamp, in which after creating a primary capsulorhexis of slightly smaller than optimum size, multiple nicks were made at the margin of the anterior capsular rim all around after lens aspiration and implantation of IOL. These nicks if made before lens aspiration can extend to the periphery towards the equator of the lens as the capsule is taut due to the presence of positive intralenticular pressure. However, after lens aspiration, the capsule becomes lax and hence these nicks do not extend to the periphery. If performed before IOL implantation, there is risk of damaging the posterior capsule and moreover, during IOL implantation, these nicks can extend to the periphery. We have observed that after curvilinear capsulorhexis, capsular opacification and fibrosis is most marked at the margin of the anterior capsular rim. We performed capsulorhexisotomies to prevent phimosis of the anterior capsular opening and capsular contraction syndrome, as these small nicks act as relaxing incisions. Since there were multiple nicks all around, the direction of the force is well distributed. The vector of the force generated at the opacified margin of curvilinear capsulorhexis is directed inwards, while after performing multiple nicks in this margin (capsulorhexisotomies), the vector is directed in both inward as well as outward directions. Therefore, there is less chance of development of capsular contraction syndrome. More over, there is negligible chance of adhesion between the anterior and posterior capsules as the anterior capsular rim rests well on the IOL optic. Conclusion ========== The postage stamp multiple anterior capsulorhexisotomies is a feasible and safe technique after lens aspiration and IOL implantation in pediatric cataracts. Declaration of competing interest ================================= The author(s) declare that they have no competing interests. Individual contribution of authors ================================== JST designed the study and performed surgeries. RS performed the data collection and wrote the manuscript. NS followed up the patients. RBV performed the surgeries. 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/1471-2415/5/3/prepub> Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Postage stamp multiple anterior capsulorhexisotomies with in-the-bag IOL in a pediatric eye of 8 years. ::: ![](1471-2415-5-3-1) :::	PubMed Central	2024-06-05T03:55:54.039100	2005-3-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554985/", "journal": "BMC Ophthalmol. 2005 Mar 8; 5:3", "authors": [ { "first": "Jeewan S", "last": "Titiyal" }, { "first": "Rajesh", "last": "Sinha" }, { "first": "Namrata", "last": "Sharma" }, { "first": "Rasik B", "last": "Vajpayee" } ] }
PMC554986	Background ========== Pain is a noxious stimulus which can be interpreted in many ways by different individuals but as yet the mechanisms by which the body manages it are not completely understood. Pain has been defined by the International Association for the Study of Pain as \'an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage\' \[[@B3]\]. Though it is frequently related to physical causes (such as inflammatory processes and nociceptive transmission of pain messages), the experience is entirely subjective, making objective measurement of pain difficult. \[[@B4]-[@B6]\], The way in which pain is perceived depends on many factors, including past experiences, memory, understanding of pain, cultural conditioning, and pain threshold \[[@B5],[@B7],[@B8]\]. Children with burn injuries undergo significant physical and emotional trauma, initially from their injury, and subsequently from the dressing changes and related treatment they undergo throughout the healing phase. The latter is referred to, in this article, as \'procedural pain\'. Clinicians involved in the care of children with acute burns use the best available methods to reduce procedural pain. Adequate and appropriate pain management is essential to ensure that symptoms secondary to pain experiences do not become habitual \[[@B9]\]. Moreover, unrelieved pain can produce serious physiological and psychological consequences leading to an increased risk of morbidity and even mortality \[[@B10],[@B1]\]. Therefore pain experiences can significantly impact on immediate and longer-term quality of life and well- being of young people. The burns service at the Women\'s and Children\'s Hospital (Adelaide, South Australia) collaborated with University of South Australia, on this project. Pain management comprises administration of a number of medications, including analgesics, muscle relaxants and hypnotics. These drugs help in reducing procedural pain experienced by children, however they frequently have unwanted side effects such as drowsiness, nausea, reduced postural control and lethargy \[[@B11],[@B8]\]. Pain management of children in hospitals during dressing changes has been reported as inadequate \[[@B1],[@B2]\] and is often described by children to be the most distressing part of the hospitalisation \[[@B12],[@B13]\]. Procedural pain, experienced by children with burns is often distressing for health professionals and parents. Therefore an investigation into nonpharmacological strategies of pain relief for children is warranted from the perspectives of improving pain management, decreasing incidences of side effects and distress in children, parents and health professionals involved with burns dressing changes. Virtual Reality (VR) was initially conceived as a tool for pain modulation by Hoffman et al \[[@B14]-[@B18]\] who found it to be effective in reducing burns pain in adults \[[@B15],[@B16]\] as well as in other situations to manage pain and phobias \[[@B14]-[@B18]\]. In 2003, one of our project teams \[[@B19]\] reported on a single subject, cross over design pilot study at the Women\'s and Children\'s Hospital (WCH), Adelaide for a child during rehabilitation following orthopaedic surgery. This pilot study suggested the possible usefulness of VR to modulate pain in children undergoing burns dressing changes. Why virtual reality? VR can be considered intermediary to reality and computer technology. Owing to its ability to allow the user to immerse and interact with the artificial environment that he/she can visualize, the game-playing experience is very engrossing \[[@B14]-[@B18],[@B20]-[@B22]\]. VR games are different to other games as they give the user a perception of actually being in a different environment. Visual, auditory and touch sensations can be modified based on the stimuli. The game used in this study was developed by the Department of Computer and Information Sciences, University of South Australia. A number of criterions had to be taken into account when designing the game, keeping in mind the different characteristics of prospective players (gender, age groups, intellectual capabilities), the amount of violence portrayed, the complexity of the game and being aware of the amount of control and functions given to the child. The game designers had to keep the structure of the game as simple as possible with minimal controls, to minimise the physical movements required to play the game. Methods ======= This project was an interdisciplinary and inter-sectorial collaboration between the Centre of Allied Health Evidence, and the Department of Computer and Information Sciences, both at the University of South Australia (UniSA), Australia, and Women\'s and Children\'s Hospital(WCH), Adelaide, South Australia. Ethics ------ Ethical approval for this project was obtained from both the institutions (WCH, Adelaide and UniSA). Study sample ------------ All children admitted to one specific ward (Newlands Ward), WCH, aged between 5 and 18 years, having burns to more than three percent of their body surface area, and requiring dressing changes, were eligible for inclusion in the study. Children with burns to their hands, face or head, past history of epilepsy and reduced intellectual capacity were not included, as they would have been unable to appropriately use the VR equipment. Informed consent ---------------- All eligible subjects were identified by ward staff, and were invited to participate in the project by the project team. Written child and parent consent was obtained at every contact. Interventions ------------- The test administrations of routine pharmacological analgesia or routine pharmacological analgesia coupled with virtual reality were randomly assigned to each half of the burns dressing change (removal of existing burns dressings or application of fresh dressings) following a coin toss determining the sequence. The child and parents were given a standard explanation about the VR administration and the VR game. If required, subjects were allowed a short preview to assist them to understand how to play the game. VR equipment ------------ • The VR equipment constituted a laptop (Dell Inspiron 5100, Pentium 4 2.4 Ghz CPU with a Radeon Mobility 7500 Video Card) with the game software, developed by the Department of Computer and Information Sciences, UniSA (Based on the game \'Quake\' by ID Software), a head-mount display (HMD) (IOGlasses Head Mount Display with a SVGA video resolution of 800 × 600 16 million colours), with a tracking system (Intersense IS300 6 degree of freedom Inertia Cube with a USB-Serial converter, required for Inertia Cube), to allow interaction with the virtual environment by moving the head and neck and decoder and a mouse used as a trigger. The game involved a visual simulation giving the children a feel of being on a track, using a pointer to aim and shoot monsters. Figure [1](#F1){ref-type="fig"} illustrates the use of the equipment, and scene from the game. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### a\) Child using the VR equipment, b) Mechanics of the equipment and c) a scene from the game she is playing ::: ![](1471-2431-5-1-1) ::: • The developers considered the applicability of the game through varying age groups, gender, intelligence and intellectual capacities, while designing the game. The game tried to achieve effective distraction via immersion without violence and a simplified game structure requiring minimal control by the player, to allow the smallest possible movement during the dressing change procedure. Administration procedure ------------------------ A within-subject design was used in which the children acted as their own controls. There was no interference with the dosage or type of analgesia which was administered to the children 30 to 45 minutes prior to dressing removal. The burns dressing changes would normally occur every Wednesday morning before the hospital ward, grand rounds. The dressing change involved administration of prescribed medication and application of olive oil on the dressing (if adhesive tape was covering the wound), approximately 30 to 45 minutes prior to the actual procedure. The first treatment half constituted the removal of the adhesive tape/bandages and the under-dressing (acticoat/silver oxide dressing) and the second half comprised of the wound being debrided and a fresh dressing applied, after being assessed by the consultant medical officer/s. Data collection --------------- Following the completion of each half of the dressing change (with or without administration of VR), the researcher obtained scores for average pain using the Face Scale (Figure [2](#F2){ref-type="fig"}), and interviewed the child, mother and the nursing staff regarding their perceptions of the procedure Using standardised questionnaires (Appendix II and Appendix III). One researcher only was involved in data collection, and intra-rater reliability was maintained by using standard protocols for introductions, explanations, VR administrations and data collection procedures. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Pain rating scale used by the children \[23\]. ::: ![](1471-2431-5-1-2) ::: Outcome measures ---------------- The subjects were asked to score their average pain experience at the end of each phase of the dressing change procedure (VR and pharmacological analgesics, and pharmacological analgesics only). Pain was scored using a modified self-report Faces pain scale \[[@B23]\]. The scale depicts increasing levels of pain and is offered in combination with a visual analogue scale of 0 -- 10, associated with each picture representing a level of pain. Parents/carers and nurses were also interviewed by the data collector at these times, using open ended questions to obtain views regarding the child\'s anxiety and perception of pain, and utility of VR in a clinical setting. Data analysis ------------- The data was analysed by a blinded assessor, to reduce any biases and increase the rigour with which a de-identified and coded dataset was probed. Results ======= Subjects -------- There were nine eligible, consenting child subjects (6 boys and 3 girls) in the sample. The average age for both boys and girls was 10.0 years (SD 3.7 and 4.1 respectively), age ranging between 5 to 16 years. The average percent of body surface area burnt was 5.3% (SD 3.4%) and there was no significant gender or age difference in body area burnt. For boys, two had burns from contact with a silencer (muffler) on a four wheeled motor bike, two were burnt from a hot water bag bursting, one was burnt while playing with petrol and fire, and the remaining boy was burnt with hot oil from a BBQ. For the girls, all three were burnt by overturned fluids. All participants were experiencing burns for the first time, and when enrolled into the study, their burns were at either second or third dressing change. Every participants\' pain, prior to enrolling in this study, had been managed either with no pain relief, or by pharmacological means. For every child participant, one parent or guardian with one exception (was not available to observe the dressing change) provided data on the effectiveness of the VR for every post-session interview. One key nurse involved in the burns management was also interviewed following each session. Trials ------ Overall, 13 trials were undertaken from nine children (one subject participating in three trials, two subjects in two trials, and the remainder in one trial each). The results of two subjects were withdrawn for further analysis as the respective participants were too drowsy from the effects of analgesia to participate appropriately in the VR section of the session. Thus the remaining seven child subjects were included for analysis, with a total of 11 useable trials (an average of 1.6 trials per subject). The seven participants in the included trials had an average age of 11.1 years (SD 3.5). ### Time factor There was no significant difference (p\<0.05) in time taken in the two treatment halves (removal and application of fresh dressing). The average difference in administering the two treatment halves was approximately 2 minutes (Figure [3](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Per trial comparison of time taken to complete each treatment half ::: ![](1471-2431-5-1-3) ::: Pain change ----------- With pharmacological analgesia only, the mean pain score (using the Faces Scale), over all included trials was 4.1 (SD 2.9), whilst for VR coupled with pharmacological analgesia, the average pain score was 1.3 (SD 1.8). Because of the small number of child subjects in the study, the data was considered per child, and per trial. Over all included trials, the mean pain score difference between administrations was 3.2 (SD 2.1), which was significant using paired t-tests (p \< 0.01). This indicated the importance of the effect of using VR (coupled with analgesia) in reducing pain experiences during burns dressing changes. The per trial pain responses to VR and analgesia, and analgesia alone, compared with the average trial response per administration is shown in Figure [4](#F4){ref-type="fig"}. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Per trial differences in pain scores compared with average administration scores ::: ![](1471-2431-5-1-4) ::: For each child subject who completed an eligible trial, the average per-child difference in pain scores between administrations of VR & Pharmacological Analgesia, or Pharmacological Analgesia alone, suggested that every child but one obtained an improvement in pain scoring of at least 2 points on the Faces Scale, attributable to VR, as demonstrated in Figure [5](#F5){ref-type="fig"}. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Per child differences in pain response attributable to VR ::: ![](1471-2431-5-1-5) ::: ### Comments made by nurses, parent/s and child subject All nurses or parents agreed that VR helped distract the children and was helpful in reducing pain and there were no negative comments regarding the application of VR. (note - delete \'interestingly\') Nurses\' responses ------------------ The overwhelming response from the nursing staff was that VR administration was helpful to the child. Comments from the interviews are provided below as evidence of this. \"\... probably VR helped to take concentration off \... probably helped take away a lot of the anticipation away from the treatment\". \"\... from my past experience, I can tell that it(changing burns dressing) can be a real problem. It was not a problem today. He did not even flinch while the dressing was being taken off.\" \"\... communication was good -- he understood what I asked him to do. I did not find it invasive or intrusive\...\" \"\... it was great to do the changing (of dressing) without dumping him with medication.\" \"\... no(communication was not effected) \... he responded well to the requests and commands.\" \"yes (pain was significantly less)\... he had lot more pain with cleaning(of the wound) than taking the dressing off\... He seemed to cope better with VR than without.\" \"\... cleaning the dressing in the bathroom made him more anxious, when he saw the wound -- felt more pain.\" \"Yes, he was more anxious when VR was not on.\" \"\... he felt worse when he was looking at it (the wound) compared to when he was not.\" \"\... was more relaxed and concentrated on the game. You could tell that he could feel the pain, but focussed on the game.\" The perception of the assisting nurses were the children were more cooperative and distracted from the administration of VR, which helped reduce the difficulty in changing the burns dressing compared to when routine analgesia was used by itself. There appeared to be no problem in physically using VR within the environmental constraints of the burns dressing area, and in no instance did VR impede communication with the child. Parents\' responses ------------------- All parents agreed with the positive effects of VR in pain management for their child. They all commented that the child\'s anxiety level was perceptibly less when using VR, and the child looked forward to playing the VR game. Comments below from the parent interviews support the positive VR effects: \"\... was a lot calmer and enjoyed the VR.\" \"\...absolutely, she did not remember about the last dressing that was taken off. She had to be prompted, to remind her of the game and change of dressing.\" \"\... much happier than usual. He reckoned he felt it but I think he did not. He did not show any of the same signs.\" \"\...she was not as anxious. Was afraid before hand, but she was all right after the game was switched on. It took her mind, off the pain.\" \"\...was smiling while playing the game.\" \"\... compared to the medication which left him groggy, disoriented, lost track of time and anxious, I think this(VR) allows the continuity of time and reduces anxiety.\" \"Yes(pain was significantly less) \... probably judging it from yesterday -- medication made him worse -- uncooperative and pig-headed, compared to when he was playing the game.\" \"Yesterday he was whinging thinking about the dressing change, this morning, when I told him that you were coming; he had a grin on his face\...\" Comments from child participants on the VR game ----------------------------------------------- However, the current game appeared to have a reasonable level of complexity and engaged the participating children of different age groups. The above comments were randomly selected from the questionnaire deployed at the completion of each trial (to interview the nurses, parents and children participating in the study). Discussion ========== This is the first published randomised clinical trial to our knowledge reporting the use of VR for children with burns. It concurs with the findings of Hoffman et al \[[@B15],[@B16]\] who tested VR on adults with burns, and suggests that VR could provide a significant improvement in the pain management for all children undergoing treatment for this condition. We found that VR coupled with analgesics was significantly more effective in reducing pain responses in children than analgesics only. Although there were 3 occasions where the child, equally scored both treatment halves, the respective carer and nursing staff member, consistently indicated that the child\'s behaviour was less distressed and calmer during the treatment when VR was applied, suggesting that VR made it less distressing for the child. Thus the feedback given by nursing staff and parents provided additional and important information in interpreting the Faces Scale responses given by the children. Distraction by an interactive game was the putative influence in reducing sensitivity to pain. Clinical implications --------------------- Given that the application of VR as a method for pain control in the clinical setting is very simple, the results of this study are encouraging with respect to future use. The present prototype VR game being used appears to be cumbersome due to the number of wires attaching to the laptop and the HMD, but this could be simplified considerably so that the equipment required is simply a console with a trigger and a head mount. Applying this equipment would be as simple as providing medication prior to the dressing change procedure. The equipment is reusable and requires minimal technical knowledge for use. Provided a number of different games were available to cater for different age groups, it could be widely applied, and will allow children to relocate themselves to \'another world\' during dressing changes, decreasing their attention to painful stimuli. It was noted by nurses during several trials that communication was never a problem; they were able to instruct the child to change, or assist change in position without any difficulty in a compliant and relatively pain-free manner. On the other hand, children without VR were often distressed and crying in pain, decreasing their ability to listen and cooperate. Limitations ----------- A number of factors resulted in a small sample size. There were relatively fewer children with burns than anticipated, who fitted the inclusion -- exclusion criteria, and who were admitted to the Women\'s and Children\'s Hospital, Adelaide, during the data collection period. Several potentially eligible children (2 trials) had such severe side effects from the medication (particularly drowsiness) that they were not able to participate, and two refused to participate. The reduced sample size limits ability to generalise the results, and a study with a larger sample size may provide better understanding of the usefulness of VR as a treatment adjunct for pain relief. Another potential limitation is that some children were tested more than once -- in the first instance immediately after their burn and then subsequently after surgery or during another dressing change later in the healing phase. In these cases, there may have been a learning effect which modified the pain scores, or simply an overall decrease in pain due to healing. It was also noted from the feedback that older children found the game too simple and therefore not as absorbing or distracting as the younger children found it. Finally, waterproofing of the VR equipment would allow data collectors to examine pain responses through an entire dressing change including when subjects had their wound debridement carried out in the bathroom. Conclusion ========== There appears to be considerable scope for further research into the potential for using VR in the clinical setting. Larger trials could be conducted, using games appropriate for the varying age groups. The next stage would be to test VR alone against pharmacological pain relief, to investigate whether VR is as effective in isolation, and could decrease use of analgesia, thus avoiding the side effects associated with medication. Another avenue of future research would be to investigate the exact mechanisms by which VR assists pain modulation. It is hypothesized that it works by distracting a child\'s attention from painful stimuli, which in turn reduces the perceived intensity of pain. Competing interests =================== The author(s) declare that they have no competing interests Authors\' contributions ======================= DAD was involved with coordination and recruitment of trial subjects, application of the trial, collection of data and writing up the paper. KAG was involved with statistical analysis of the data and supervising the scientific conduction of trials. ALS was a key medical consultant for the trial subjects and coordinated efforts within the hospital to recruit trial subjects. SEM was the first contact with the prospective trial subjects. She identified and provided them with initial information and got verbal consent from them. She also acted as a coordinator for all the nursing staff members on the ward participating in the trial. BAT provided IT support for building and helped in maintaining the inventory (VR equipment) and the game software. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2431/5/1/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Pain scale scoring ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Caregiver interview ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 Nurse interview ::: ::: {.caption} ###### Click here for file :::	PubMed Central	2024-06-05T03:55:54.040217	2005-3-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554986/", "journal": "BMC Pediatr. 2005 Mar 3; 5:1", "authors": [ { "first": "Debashish A", "last": "Das" }, { "first": "Karen A", "last": "Grimmer" }, { "first": "Anthony L", "last": "Sparnon" }, { "first": "Sarah E", "last": "McRae" }, { "first": "Bruce H", "last": "Thomas" } ] }
PMC554987	Background ========== Gastro-oesophageal reflux (GOR) is the passage of gastric contents into the oesophagus. Once past the upper oesophageal sphincter, the gastric juice may pass into the oral cavity. The continual exposure of the teeth and other oral structures to gastric refluxate may result in dental erosion and other soft tissue symptoms \[[@B1]\]. Any acid with a pH below the critical pH of dental enamel dissolution (5.5) can dissolve the hydroxyapatite crystals in enamel. However the critical pH below which enamel dissolves is not constant but is rather inversely proportional to the concentrations of calcium and phosphate in the saliva and plaque fluid \[[@B2]\]. Gastric refluxate has a pH of less than 2.0 and thus has the potential to cause dental erosion \[[@B3]\]. Acid regurgitation is a common symptom of upper gastro-intestinal tract disorders and dysfunctions such as peptic ulcer (duodenal and gastric ulcers) and reflux oesophagitis \[[@B4]\]. Other symptoms include heartburn, non-cardiac epigastric and retrosternal pain \[[@B5]\]. Prominent among factors precipitating GOR and its complications, gastro-oesophageal reflux disease (GORD) are fatty diets and alcohol. Current understanding of GOR and GORD is that acid reflux into the oesophagus may be caused by three possible mechanisms \[[@B6]\]: \(1) Transient spontaneous or inappropriate relaxations of the sphincter; \(2) Transient increase in intra-abdominal or intragastric pressure; \(3) Functional abnormality of the lower oesophageal sphincter (LOS). Bargen and Austin in 1937 \[[@B7]\] first reported the link between dental erosion and gastro-intestinal disturbances. Since then there has been several other studies confirming the relationship between this loss of tooth structure and GORD in the United Kingdom (UK) \[[@B8]\], United States of America (U.S.A) \[[@B9]\] and Canada \[[@B10]\]. A recent survey among young people in the U.K also revealed an association between dental erosion and symptoms of GOR \[[@B11]\]. Although soft tissue symptoms (non-specific burning sensation in the mouth) have been mentioned in the literature, pathognomonic soft tissue lesions have not been documented \[[@B1]\]. But some researchers have reported a lack of relationship between periodontal lesions and GOR, since the prevalence of periodontal lesions is similar in patients with GORD and in healthy volunteers \[[@B12]\]. In various people of the Western world, GOR has been reported to be a common problem, often related to meals and occurring in about 60% of the population at some point in their lives \[[@B13]\]. Whereas the prevalence of GORD is estimated to range from 6% -- 10% \[[@B14],[@B15]\], Meurman et al\[[@B16]\] examined 117 patients with GORD, of whom 28 (24%) had dental erosion. Also, Schroeder et al\[[@B17]\] identified dental erosion in 11 (55%) of 20 patients with GORD. The authors are not aware of such figures in Nigerians. A search of the literature also revealed a dearth of information on this condition and its complications among Black Africans. It is therefore the aim of the present study to estimate the prevalence of GOR among patients attending the medical out-patient department of the Obafemi Awolowo University Teaching Hospital Complex Ile-Ife, Nigeria; to estimate the prevalence of dental erosion in patients with GORD; to document the oral findings in patients diagnosed with GORD and to compare these findings with previous studies elsewhere. Methods ======= A total of 225 subjects were involved in the study over a period of 6 months, January -- June 2002. The subjects were made up of 100 volunteers attending the medical outpatient department (MOPD) and 125 patients diagnosed with gastro-oesophageal reflux disease among whom the prevalence of GOR and GORD was determined respectively. Their age ranges from 18 -- 72 years, with a mean age ± S.D. of 38 ± 10.87 years. Patients presenting primarily with symptoms of asthma, bronchitis and other respiratory disorders were excluded, since they are also at risk of dental erosion \[[@B18]\]. To assess the prevalence of GOR and GORD, consenting patients attending the medical outpatient department of the Obafemi Awolowo University Teaching Hospital\'s complex Ile-Ife, Nigeria, were questioned with reference to their experience regarding symptoms of GOR such as regurgitation of gastric juice, epigastric and non-cardiac pain (heartburn). The onset, frequency of occurrence and duration of each of the symptoms were ascertained and recorded. Patients presenting with a history of heartburn 2 or more times per week were diagnosed as having GORD^10^. Oral examination was carried out to quantify any loss of tooth structure using the tooth wear index (TWI) designed by Smith and Knight (1984) \[[@B19]\]. To document the oral findings associated with GORD, patients referred to the Gastro-intestinal (GIT) endoscopic unit for investigation of gastro-oesophageal tract disease were also evaluated for dental erosion and other soft tissue symptoms such as non-specific burning sensation in the mouth and sensitivity in the tongue. The dental evaluation included history to determine potential eatiological factors responsible for dental erosion. Patients were also examined clinically to quantify loss of tooth structure using the tooth wear index. The oral examination was performed by one of the authors (AO), blind as to the endoscopic diagnosis of subjects. Patients with positive endoscopic findings plus the occurrence of heartburn two or more times per-week were assessed to meet the criteria for GORD. When a clear-cut endoscopic evidence of oesophageal inflammation was seen (marked redness, fibrinous membrane, and or ulcerations), a diagnosis of oesophagitis was made. Gastroduodenal ulcer was diagnosed through endoscopic evidence of an ulcer with a necrotic base or a clear-cut scar. This was grouped into those with gastric ulcer (ulcer in the pylorus, antrum, corpus or fundus) and those with duodenal ulcer (ulcer in the bulbus). Only one diagnosis was given for each patient. Data collected were entered into a computer and analysed using chi-square. P value \<0.05 were considered significant. Results ======= Thirty-five of the 100 subjects attending the medical outpatient department had acid regurgitation and acidic taste sensation in the mouth. This occurs most of the time following a full stomach meal and is associated with belching. Table [1](#T1){ref-type="table"} shows the prevalence of GOR and GORD. Eleven reported a history of heartburn 2 or more times per week, while 16 and 10 reported weekly and monthly history of heartburn respectively. Comparison of subjects presenting with heartburn 2 or more times per week (M = 5, F = 6) and those with less than 2 times per week (M = 12, F = 14) shows that more female presented with heartburn. However, there was no statistical difference among the genders with regard to the symptoms recorded. None of these subjects presented with any burning sensation in the mouth or sensitivity in the tongue. In five of those that reported 2 or more weekly history of heartburn, there were minimal losses of tooth structure (TWI scores of 1 and 2 in three and in two subjects respectively) limited to the maxillary central incisors (Table [2](#T2){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Prevalence of GOR and symptoms of GORD ::: Symptoms Male (N = 49) Female (N = 51) Total --------------------------------------------- --------------- ----------------- ------- Regurgitation and acidic taste in the mouth 16 19 35 Heartburn ≥ 2 per week 5 6 11 Heartburn per week 7 9 16 Heartburn per month 5 5 10 Chi Square = 0.10; df = 3; p = 0.99. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Prevalence of dental erosion ::: Dental erosion ------------------- ---------------- ------------ Subjects/patients No (%) TWI scores M.O.P. (N = 100) 5 (5) 1 -- 2 GORD (N = 125) 20 (16) 1 -- 3 Mantel Haenszel chi-square = 5.50, p = 0.02. ::: One hundred and twenty five patients were diagnosed with GORD. Twenty presented with dental erosion in the maxillary anterior teeth with TWI scores ranging from 1--3. The prevalence of erosion was found to be statistically significant between GORD patients (16%) and controls (5%) (Mantel Haenszel Chi-square 5.50, p \< 0.05), Table [2](#T2){ref-type="table"}. The result of the gastrointestinal endoscopy reported 41(32.8%) as having reflux oesophagitis, 36 (28.8%) with duodenal ulcer and 48 (38.4%) with gastric ulcer. Eight patients with dental erosion came from the group who had reflux oesophagitis, 7 from the group with duodenal ulcer and 5 from the group with gastric ulcer. Comparison of patients with oesophagitis and those without shows that the mean age of patients with oesophagitis was lower than in those without. The prevalence of erosion was not statistically significant between the endoscopic diagnostic groups (chi-square = 1.33, df = 2, p = 0.51), Table [3](#T3){ref-type="table"}. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Endoscopic diagnostic groups: basic data and number of patients withdental erosion ::: Diagnostic groups Number Mean age (yrs) Gender Dental erosion detected --------------------- ----------- ---------------- -------- ------------------------- --- Reflux oesophagitis 41 (32.8) 36.9 ± 9.7 24 17 8 Duodenal ulcer 36 (28.8) 39.2 ± 11.4 16 20 7 Gastric ulcer 48 (38.4) 38.1 ± 11.0 17 31 5 Prevalence of erosion between diagnostic groups: Chi-square = 1.33, df = 2, p = 0.51. ::: The details of the orodental findings in the twenty patients who had dental erosion are shown in Table [4](#T4){ref-type="table"}. There seems to be a slight association between the duration of gastrointestinal symptoms and the severity of erosion. TWI scores of 3 were seen only in those patients whose abdominal symptoms had lasted 10 years or more. The palatal surfaces of the maxillary anterior teeth were usually involved but the central incisors were the most severely affected. Six patients (4 from reflux oesophagitis and 2 from duodenal ulcer) had dental erosion related symptoms in their teeth: sensitivity to cold and heat. The teeth that presented with dentine sensitivity had TWI scores of 3. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Orodental findings in the twenty patients with GORD who had erosions ::: S/N Diagnostic groups\\\* Age (yrs) Gender Duration (yrs) TWI scores Dentine Sensitivity\\ BMS\* ----- ------------------------- ----------- -------- ---------------- ------------ ------------------------- ------- 1 RO 26 Female 5 1--2 NP NP 2 RO 43 Female 10 1--3 P P 3 RO 44 Male 10 1--3 NP NP 4 RO 38 Male 5--10 1 NP NP 5 RO 48 Female 15 2--3 NP P 6 RO 33 Female 10 1--3 P P 7 RO 52 Male 15 2--3 P P 8 RO 50 Female 15 2--3 P P 9 DU 30 Male 5--10 1--2 NP NP 10 DU 50 Male 20 2--3 P P 11 DU 45 Female 15 1--3 NP NP 12 DU 47 Female 10 1--2 NP NP 13 DU 33 Female 5 1 NP NP 14 DU 36 Male 10 1--3 NP NP 15 DU 43 Male 15 2--3 P P 16 GU 46 Female 10 1 NP NP 17 GU 35 Female 10 1--2 NP NP 18 GU 32 Female 5 1 NP NP 19 GU 54 Male 20 2--3 NP P 20 GU 45 Male 15 1--3 NP P \BMS = Burning mouth sensation and sensitivity in the tongue \\NP = Not present, P = Present \\\RO = Reflux oesophagitis, DU = Duodenal ulcer, GU = Gastric ulcer. ::: Burning mouth sensation and peppery sensation in the tongue where reported by 9 patients who had had gastrointestinal symptoms for 10--20 years. Discussion ========== Various methods have been employed in the investigation of GOR including endoscopy with biopsy and contrast radiography. Although twenty-four hour oesophageal pH monitoring is considered the gold standard investigation of GOR \[[@B20]\], due to non-availability of the ambulatory pH recorder in our center, GOR was diagnosed by endoscopy where visual identification of mucosal inflammation and oesophagitis was used to identify the existence of GOR. Several other authors have used this method \[[@B4],[@B21]\]. The prevalence of GOR and GORD in the present study was 35% and 11% respectively. The prevalence of GOR was much lower than the 60% reported in the U.K. and other Western societies \[[@B13]\], whereas that of GORD was a little above the reported range of 6%--10% \[[@B14],[@B15]\]. A lower percentage (10%) of the subject studied reported a monthly history of heartburn as compared to 59% of the population reported by Lock et al\[[@B22]\]. The present study examined 125 patients with GORD of which 20(16%) had dental erosion. This is also lower than the 24% and 55% reported by Meurman et al\[[@B16]\], and Schroeder et al \[[@B17]\] respectively. Although the authors cannot propose any reason for the reported lower prevalence of GOR and GORD, the low prevalence of dental erosion may be related to difference in diet. More so that the thrust of the European studies support the view that erosion (resulting from acidic and carbonated foods and beverages) is more important than attrition in the aetiology of tooth wear \[[@B23]\], whereas, attrition have been reported to be more important than erosion in the aetiology of tooth wear among Nigerians \[[@B24],[@B25]\]. Also in the study of gastro-oesophageal reflux in children and its relationship to erosion of primary and permanent teeth, diet was considered to be a major contributory factor \[[@B26]\]. The consumption of acidic beverages among the study population was low, hence the silence on its contribution to the overall disease level. Although this is controversial, the authors are of the opinion that intrinsic (gastric) acid results in palatally eroded sites (as shown in Figure [1](#F1){ref-type="fig"}) while extrinsic (dietary) acids lead to labial or vestibular erosions. The authors observed some degree of tooth wear (tooth surface loss) on the molar teeth. It was not documented because we believe they are more likely due to \"attrition\" resulting from rigorous mastication of the more fibrous Nigerian diet. However, acidic refluxate may have been a contributory factor, since tooth surface loss is a multi-factorial disease. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Severe dental erosion affecting the palatal surfaces of the upper anterior teeth in a patient with reflux oesophagitis. ::: ![](1472-6831-5-1-1) ::: In accordance with the result of Gregory-Head et al\[[@B27]\], patients diagnosed with GORD in this study had higher TWI scores compared with control subjects. All the twenty patients that presented with dental erosions in the present study had an underlying gastrointestinal pathosis (Gastric oesophagitis, Duodenal ulcer, and Gastric ulcer) with increased output of acid secretion into the stomach \[[@B28]\]. There was however no statistically significant difference in the prevalence of dental erosions in these diagnostic groupings, Table [3](#T3){ref-type="table"}. This is in agreement with the study of Jarvinen et al\[[@B4]\]. The severity of dental erosions may depend on the frequency of regurgitation and duration of the gastro-oesophageal reflux. In the present study, patients with TWI scores of 3 had their symptoms for more than 10 years as shown in Table [4](#T4){ref-type="table"}. This is supported by the study of Loffeld et al\[[@B21]\], which revealed a significant association between duration of complaints and presence of damage in the upper incisors, but in contrast to the findings of Jarvinen et al\[[@B4]\] who reported no direct association between the frequency of regurgitation symptoms and the severity of the erosive lesions. Six patients presented with erosion related symptoms in their teeth, they had dentine sensitivity to cold and heat. All of these patients had dental erosions with TWI scores of 3 (Loss of enamel exposing dentine for more than one-third of the surfaces). This probably explains the sensitivity. Nine patients reported burning mouth sensation and sensitivity in the tongue. They describe the sensation as peppery/burning feeling in the vestibule of the mouth and mostly on the dorsal surface of the tongue. These may have resulted from the prolonged effect of acidic gastric refluxate on the oral mucosa and on the papillae of the tongue. Conclusion ========== The present study supports the consideration of dental erosion as the extra-oesophageal manifestation of GORD. However the association between GORD and burning mouth sensation needs more investigation. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= AOO conceived of the study, participated in its design, performed the dental examination including the application of the tooth wear index (TWI), and participated in the initial draft and final write-up of the manuscript. EAA and DAN performed the endoscopic examination of patients with gastro-oesophageal reflux disease, participated in the initial draft and final write-up of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6831/5/1/prepub>	PubMed Central	2024-06-05T03:55:54.043003	2005-3-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554987/", "journal": "BMC Oral Health. 2005 Mar 1; 5:1", "authors": [ { "first": "Adeleke O", "last": "Oginni" }, { "first": "Elugwaraonu A", "last": "Agbakwuru" }, { "first": "Dennis A", "last": "Ndububa" } ] }
PMC554988	Background ========== It has been estimated that 6.6% of those attending A&E in any given year have a head injury \[[@B1]\] and over 100,000 people are admitted as a consequence \[[@B2]\]. This incidence of admission to hospital following a head injury is known to vary considerably from locality to locality \[[@B3]\]. Although some technical issues, such as case identification and inaccurate coding may contribute \[[@B3],[@B4]\], there is also evidence to support that demographic- and social factors such as age, gender, environment and ethnicity cause variation in incidence and outcome \[[@B5]-[@B12]\]. For example the adjusted odds ratio for age 15--24 years, and the male sex have been found to be significant independent predictors for medically attended brain injury in the USA \[[@B13]\]. Thus the evidence suggests that there is considerable potential for complex interactions which could elevate or depress incidence rates at the local level to a significant degree. Yet, within the UK, evidence upon which to base local service planning is scarce, as recognised in the recent report of the Health Committee of the UK House of Commons \[[@B14]\]. Consequently the committee recommended that a way is found \'of improving the methods of data collection on incidence, prevalence and severity of head injury and subsequent disability\...\' (vii). This paper makes an initial response to that recommendation and reports on the incidence of head injury leading to a hospital admission at the local level in England, and examines the socio-economic associations of any variation shown to be present. Methods ======= Hospital Episodes Statistics (HES) record all episodes of continuous in-patient care in hospitals in England and can be obtained from the UK Department of Health. For the year 2001--2, statistics are available for the 95 Health Authorities (HA) extant at the time, and for 2002--3 for Primary Care Trusts (PCT\'s). Concerned with incidence of new cases, data in the current study is based upon the postcode of residence of the patient. \'First episode\' was chosen as a filter variable (as approximately 10% of patients have more than one episode, that is, fall under the care of more than one consultant during their stay). A second filter identified the relevant primary cause ICD10 codes for head injury (S00.0 to S09.9). There is a long history of debate about the appropriate codes for inclusion in such studies, often surrounding the debate between \'head injury\' and \'brain injury\' \[[@B15]\]. Much was made of the fact that \'fracture of the facial bones\' (ICD-9 code 802) was not indicative of brain injury, but new research has shown that excluding this group will omit many with brain injury \[[@B16]\]. The current study is inclusive of all codes, and so includes \'superficial injuries to the head\', which nevertheless required admission to hospital. Using census population data for the same areas as the denominator, admission rates per 100,000 are calculated for all ages, and age-specific rates for 0--15 year olds, 16--74 year olds, and those aged 75 years and over. Rates are thus for admission of residents of England and their respective HA or PCT, in an English hospital. Admissions are defined as the first period of in-patient care under one consultant within one healthcare provider. The 16--74 year old band was chosen explicitly to match the census key statistics which report on various indicators for the economically active population, deemed to be 16--74 years of age. Census Key Statistics are available from the UK Office of National Statistics and include a range of demographic, social and economic variables that can be presented as percentage indicators. Those chosen for inclusion in the current study are shown in Table [1](#T1){ref-type="table"}. The indicator for qualification at level 4/5+ represents those with a first degree, a higher degree, NVQ levels 4 and 5, HNC, HND, qualified teacher, medical doctor, dentist, nurse, midwife or health visitor. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Key statistics included in the study and presented as percentage indicators. ::: ---------------------------------------- Indicator \% born outside the UK \% with limiting long-standing illness \% permanently sick of working age \% 16--24 who are unemployed \% age 50+ who are unemployed \% Unemployed \% without any qualifications \% with qualifications at grade 4/5+ \% using private transport to work \% using public transport to work \% of households without a car \% who own (or buying) their home \% who rent home privately \% living in overcrowded homes \% lone parent families ---------------------------------------- ::: In addition, the Townsend Deprivation Index is calculated using the percentage of households with no car, not owner occupied, overcrowded and those economically active who are unemployed \[[@B17]\]. The latter two are transformed logarithmically, and then each variable is transformed to a normal distribution using the means and standard deviations for England as a whole. The variables are summed giving an average for England of zero, with negative values indicating less deprived areas, positive values more deprived areas. The UK Census key statistics are currently made available at the Primary Care Trust (PCT) level, and thus it was necessary to aggregate these data up to the HA level to match the 2001--2 admission data which was provided at the Health Authority level. The majority of PCT\'s fit neatly into the 95 Health Authority areas extant in 2002. However, there are some slight variations such that aggregated populations of the HA derived from their respective PCT\'s match exactly the independent figures (provided separately from the ONS) of 2002 HA populations in 90% of cases, and differ by up to 5% for the rest. For example, a small number of Enumeration Districts (the lowest level census tract) belonging to the Preston PCT, which belongs in North West Lancashire HA, are allocated to East Lancashire. No attempt was made to adjust for these slight variations in boundaries. Key statistic indicators for 2001--2 were thus derived from the aggregated PCT data, based on their aggregated population which, for a few HA\'s, will differ slightly from the population used to estimate the admission rates for head injury. There was a one-to-one relationship between admission data and census data at the PCT level for the 2002--3 data. Patterns of head injury and socio-economic indicators are determined by Two-Step Cluster Analysis. The procedure is an exploratory tool designed to reveal natural groupings (or clusters) within a data set that would otherwise not be apparent. The association between the various socio-economic factors, and the overall rate for head injury is explored through a multiple regression model using the 2001--2 data at the HA level. This model is validated on the 2002--3 data at the PCT level. Funding and ethics ================== The project was funded by the UK Department of Health. The author is independent of the funding body. Ethical approval was not required. Results ======= A total of 112,718 admissions were recorded for 2001--2. The gives an incidence rate for admission to hospital following a head injury in England in 2001--2, for all ages, of 229.4 per 100,000 (Table [2](#T2){ref-type="table"}). Of these, 31.2% were aged 0--15; 56.2% were aged 16--74 and 12.6% aged 75 years and over. The total incidence varies by a factor of 4.6 from 90.7 per 100,000 in Brent and Harrow, to 419.4 in Liverpool. Rates for children and the elderly are much higher than for those aged 16--74 years. The highest incidence rate for children was East Lancashire at 637.7 per 100, 000 aged 0--15; the highest for the elderly was North Cheshire at 799.8 per 100,000 aged 75 years and over (See [Additional File 1](#S1){ref-type="supplementary-material"}). The incidence for 2002--3 was identical at 229.1 per 100,000 all ages (Table [3](#T3){ref-type="table"}). While the magnitude of difference for the overall all-age incidence remained the same at 4.6, at the PCT level the age-specific variability appears greater, with the highest rate for children being 881.7 per 100,000 (North Manchester, see [Additional File 2](#S2){ref-type="supplementary-material"}) and that for those aged 75 years and over, 1116.0 per 100,000 in Central Liverpool. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Hospitalised Incidence rate for head injury in England in 2001--2. Estimated rate per 100,000; for those aged 0--15; 16--74; and 75 years and over, and in total. Estimates for England, and the highest and lowest incidence for health authorities. ::: Health Authority RATE 0--15 RATE 16--74 RATE 75+ Total RATE ----------------------------- ---------------- ----------------- -------------- ---------------- England 355.8 178.1 383.8 229.4 Liverpool 465.9 376.9 741.5 419.4 Tees 621.7 346.9 514.5 416.6 North Cheshire 488.0 356.0 799.8 411.5 East Lancashire 637.7 298.5 638.3 399.9 Sunderland 461.4 344.2 609.9 385.0 South Essex 235.0 116.4 223.2 148.9 Barking and Havering 223.9 117.4 210.0 147.4 North Essex 256.0 103.0 268.1 146.8 Bexley, Bromley & Greenwich 218.1 105.4 181.7 134.4 Brent and Harrow 119.5 81.7 106.7 90.7 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Hospitalised Incidence rate for head injury in England in 2002--3. Estimated rate per 100,000; for those aged 0--15; 16--74; and 75 years and over, and in total. Estimates for England, and the highest and lowest incidence for PCT\'s. ::: PCT RATE 0--15 RATE 16--74 RATE 75+ Total RATE ------------------------- ---------------- ----------------- -------------- ---------------- ENGLAND 339.0 179.5 410.8 229.1 Preston 577.4 478.3 611.9 508.0 Central Liverpool 516.5 429.0 1116.0 488.6 Central Derby 698.0 362.9 895.5 479.0 Middlesbrough 582.4 431.1 550.6 472.5 Birkenhead and Wallasey 473.0 436.3 777.3 471.0 Colchester 175.5 95.4 195.6 118.5 Cherwell Vale 193.7 88.9 174.9 117.1 Brent 134.2 108.7 95.4 113.1 South West Oxfordshire 153.2 98.5 136.2 112.5 Harrow 124.7 97.0 198.5 109.8 ::: Taking the overall incidence rate for 2001--2, together with the key statistics in Table [1](#T1){ref-type="table"}, a Cluster Analysis indicated three clear groupings of Health Authorities in England (Table [4](#T4){ref-type="table"}). The first can be described as \'London\', and includes those authorities located within London. Here, 26.7 % of the population was born outside of the UK and the workforce is better qualified than elsewhere. A lower than average number of people own their own homes, and more people travel to work by public transport than by their own car. It is more overcrowded which contributes to a high deprivation index at 5.5. The admission rate is lower than average at 176 per 100,000 all ages. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Cluster analysis of socio-economic indicators associated with head injury in England. ::: Cluster ------------------------------------ ------------- ----------- ----------------- ------------- Characteristic London Shire Other Urban England Admission Rate -- all ages 176 206 288 229 \% Born out of UK 26.7 5.7 4.8 9.2 \% of working age permanently sick 4.5 4.2 7.6 5.7 \% age 16--24 unemployed 5.5 4.6 7.4 5.9 \% without qualifications 22.4 26.7 35.1 29.7 \% qualified at level 5+ 33.2 19.6 14.8 19.3 \% using private transport to work 35.2 67.0 65.3 62.2 \% using public transport to work 43.8 8.9 14.2 15.6 \% households without a car 38.3 20.1 32.3 27.6 \% owning (or buying) own home 55.8 74.0 66.5 68.6 \% Lone parent household 10.9 8.2 10.9 9.4 \% living in overcrowded homes 18.0 5.1 5.7 7.0 Average Townsend Index 5.5 -2.7 1.2 0.0 ::: The second cluster can be described as the \'Shire\' counties, typical of the more rural areas found within England, including Cambridgeshire and Worcestershire. They are characterised by a large proportion of people owning their own home, travelling to work in their own car, and with few of the population born outside of the UK. Although a quarter of the population are without qualifications, the deprivation Index is low at -2.7. The average incidence admission rate for this cluster is 206 per 100,000 all ages. The third cluster can be described as \'Other Urban\', and typical of all the Midland and Northern cities of England. They are characterised by a higher proportion of people with limited long standing illness; those of working age who are permanently off sick; of those without qualifications and of those aged 16--24 who are unemployed. The deprivation Index is above average at 1.2, and the admission rate is high at 288 per 100,000 all ages. A regression analysis with overall rate as the dependent variable shows how these various indicators come together to predict incidence (Table [5](#T5){ref-type="table"}). For every one percent increase in the 16--24 unemployment rate, the hospital admission rate for head injury increases by 17.4 per 100,000 all ages. For every one percent increase in those permanently sick of working age, the rate increases by 16.0 per 100,000 all ages. For every one percent increase in lone parent families the rate increase by 11.0 per 100,000 all ages. In contrast for every one percent increase in the use of public transport to go to work, the rate decreases by 4.6 per 100,000 all ages. With an adjusted R^2^of 0.698, the model explains a large proportion of the variation in admission rates for a head injury (F 36.77; p \< 0.01; and with an acceptable pattern of residuals \[Figure [1](#F1){ref-type="fig"}\]). The most important variable (highest beta) was use of public transport to work. These significant predictors were then entered into another regression model, along with all possible two-way interactions. The latter were entered in a stepwise fashion, but none gained statistical significance. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Indicators of admission to hospital with a head injury: All ages 2001--2. β is increase (decrease) in rate per 100,000 admissions for each % increase of indicator. ::: Indicator β 95% CI for β ------------------------------------ -------- ------------------- \% Unemployed within age of 16--24 17.431 5.707 to 29.154 \% Permanently sick of working age 16.019 6.226 to 25.773 \% Lone parent households 11.012 2.892 to 19.132 \% Using public transport for work -4.563 -6.204 to -2.923 \% Without qualifications -7.781 -10.741 to -4.821 ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Normal probability plot of regression with Rate per 100,000 as dependent variable. ::: ![](1471-2458-5-21-1) ::: The model was then validated upon the 2002--3 data at the PCT level (Table [6](#T6){ref-type="table"}). A similar model emerged with adjusted R^2^of 0.579, again explaining a large proportion of the variation in admission at the PCT level (F70.078; p \< 0.01). On this occasion the proportion of households without a car was added to the previous set of predictors. Once again, the proportion travelling to work by public transport was the most important variable. ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Indicators of admission to hospital with a head injury: All ages 2002--3. β is increase (decrease) in rate per 100,000 admissions for each % increase of indicator. ::: Indicator β 95% CI for β ------------------------------------ -------- ------------------ \% Unemployed within age of 16--24 7.171 0.630 to 13.711 \% Permanently sick of working age 10.772 4.900 to 16.645 \% Lone parent households 11.034 6.018 to 16.051 \% Using public transport for work -4.412 -5.396 to -3.427 \% Without qualifications -5.214 -7.032 to -3.396 \% No car 4.359 2.982 to 5.735 ::: Discussion ========== Head injury is common; there were 112,718 recorded admissions for English residents during the year April 2001 to March 2002, giving a hospitalised incidence rate of 229.4 per 100,000 all ages. Remaining the same for 2002--3 this incidence is similar to that of stroke although the latter is experienced in a predominately older population \[[@B18],[@B19]\]. However, head injury affects a predominately younger population and carries with it a high potential economic impact. For example, based on these latest incidence figures and published evidence from other studies, we can estimate that about 4700 of those admitted in any given year, and who are considered to be economically active (aged 16--74) and in employment at the time of their injury would be unable to return to their work at 6 weeks \[[@B20],[@B21]\]. Admission rates for England were found to vary by a factor of 4.6 between both health authorities and PCT\'s. The rates may slightly underestimate the true incidence as those residents of England treated elsewhere are not included. Also, under-reporting may depress incidence to an unknown extent \[[@B4],[@B22]\] and, if underreporting varies by locality, will further contribute to variation. Standardised admission policies for those presenting with a head injury were not agreed at the time that these data were collected and this may also contribute to variation in incidence. The publication of the NICE guidelines for the early management of head injury may reduce any variation attributable to differing admission policies \[[@B23]\]. Half of all health authorities in England, and slightly more PCT\'s (55%) had an incidence rate which fell below or above the England average by at least 20%. This has important implications for planning local services in response to head injury. Clearly using the overall England incidence rate is unlikely to be helpful. This variation also causes problems in extrapolating the results from research undertaken in a single locality. Estimates of the number of people disabled by head injury, presented as a rate (e.g. 150 per 100000), may have little relation to reality at the local level. The disability rate is conditional upon the local incidence where the study was undertaken. Consequently it is important that research findings should be presented as proportions of the local base incidence if they are to be of any use for planning elsewhere. The extrapolations given above on return to work are based on this approach. Nevertheless such extrapolation assumes equal case mix (e.g. similar mechanisms of injury) which is also unlikely. Case mix will, to a certain extent, be a reflection of local socio-economic factors, particularly associated with sub-populations displaying chronic conditions, including alcohol misuse, shown to have significant impact on outcome \[[@B20],[@B21],[@B24],[@B25]\]. The extent of deprivation has also previously been associated with higher incidence of head injury as well as general workload for primary care \[[@B26],[@B27]\]. The data presented here tell a slightly different story. Although Camden and East London health authorities are above the English average for incidence, and have the highest levels of deprivation, and likewise Tower Hamlets and City and Hackney PCT\'s, the overall correlation between deprivation and incidence is low (0.21). It is the rate of 16--24 unemployment that contributes to higher incidence, and those of working age who report themselves as permanently unable to work because of ill health. Set against these \'lifestyle factors\', other factors mediate the incidence level. Thus London, of all cities in England, with the highest localities of deprivation, but with its extensive public transport system, displays a lower overall incidence rate. These findings are consistent for both years examined. However, it is unclear if, at all, reductions in the number of Accident and Emergency beds in the capital (generally from a higher base than elsewhere in the country), and the consequent reduced capacity to admit and observe, may also have contributed to this lower incidence. In contrast, the very high incidence of admission for children who are resident in the area of North Manchester PCT may be associated with the accessibility to the children\'s hospital located in that area. Indeed, local service and residential patterns, for example, large residential or nursing homes located with a PCT\'s boundary, may have significant impact on incidence (as expressed by place of residence) at the very local level. Where public transport is not used for journeys to work, incidence is much higher and combined with higher unemployment rates, and other significant indicators associated with \'lifestyle indicators\', high incidence rates are observed in the cities and other urban communities in the Midlands and Northern England, including Teeside and Mersyside. As demographic, social and economic factors appear to account for half the variation observed in hospitalised admission rates, one implication is that incidence can be modified at the macro policy level. For example, ceteris paribus, we might expect to see the London incidence fall as a consequence of congestion charging and associated increase in the use of public transport. Policies targeted at reducing unemployment amongst the 16--24 year old age group may also be expected to reduce incidence. Also, a continuing emphasis on prevention is clearly needed, both for the young and the old, to reduce their very high incidence rates. Some technical limitations are worth mentioning. The likelihood distance measure used in the two-step cluster analysis assumes that variables in the cluster model are independent. Further, each continuous variable is assumed to have a normal (Gaussian) distribution, although in practice the technique is robust to violation of these assumptions. In fact, reassuringly, the results of the linear regression model do suggest independent main effects for several of the key variables and, importantly, the absence of interaction effects. Also it is unknown how, if at all, the slight variation in population denominators used to calculate incidence and the socio-economic indicators for the 2001--2 data could influence these results. However, replication upon the 2002--3 data at the PCT level suggests a robust model. Due to censoring of data of elderly admissions at the PCT level we were not able to confidently include the proportion of females admitted in our validation model, and thus we do not know if variation in male-female ratios at the local level was also a contributing factor to variance in incidence. It is also worth restating that the accuracy of case ascertainment, and the coding of head injuries upon admission to hospital is known to underestimate the true incidence \[[@B4],[@B22]\] but how much this varies across localities, and its consequent potential to bias these results, is unknown. Finally, it is possible that a few patients are double counted in that those transferred will be seen as a new admission into a different hospital, thus leading to an unknown over-estimate of incidence. However, these are likely to be those with the most severe injuries admitted initially to hospitals without neurosurgery, and numbers are likely to be small. Conclusion ========== Incidence of head injury in England is high, similar to stroke if just admissions are considered, and the data show considerable variability at the local level. Given that most of the estimates of the potential impact of head injury rely on studies undertaken in a single locality \[[@B21],[@B28],[@B29]\] those planning for rehabilitation and other services must take care to identify the proportion of those with a head injury that experience the sequelae under scrutiny (e.g disability; job loss), together with the case mix, and not just rely on headline rates which are conditional upon local incidence. To assist in planning for services, data at the local level can now readily be obtained from on-line data sets from the Department of Health. At the national level they can be downloaded on-line at: <http://www.dh.gov.uk/PublicationsAndStatistics/Statistics/HospitalEpisodeStatistics/fs/en> While we have as yet little understanding of how case mix is related to, or interacts with socio-economic factors to mediate medium or longer term-outcomes, the association of such factors with incidence suggests that economic and social policies, for example in the development of prevention programmes, and in encouraging the use of public transport for journeys to work, may have a significant impact on reducing the incidence of head injury. Competing interests =================== The author(s) declare that they have no competing interests. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/5/21/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Hospitalised Incidence rate for head injury in England in 2001--2. Estimated rate per 100,000; for those aged 0--15; 16--74; and 75 years and over, and in total. Estimates for England, and extant Regions and Health Authorities at the time. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Incidence of admission to hospital for head injury for England, and each Primary Care Trust (PCT) in England 2002--3; Estimated rate per 100,000; for those aged 0--15; 16--74; and 75 years and over, and in total. Estimates for England, and PCT\'s. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ The author wishes to thank the members of the Research & Evidence Group of the External Reference Group for the National Service Framework for Long term Neurological Conditions for their invaluable support and comments. Specific thanks must go to members of the local steering group, which includes Professor Lynne Turner Stokes, Ms Maggie Campbell, Dr Vera Neumann, Mr John Sloan, Mr Norman Keen and Dr Jeff Graham. Full incidence figures for Health Authorities and Primary Care Trusts are given in [Additional Files 1](#S1){ref-type="supplementary-material"} &[2](#S2){ref-type="supplementary-material"} respectively.	PubMed Central	2024-06-05T03:55:54.045093	2005-3-4	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554988/", "journal": "BMC Public Health. 2005 Mar 4; 5:21", "authors": [ { "first": "Alan", "last": "Tennant" } ] }
PMC554989	Background ========== The images we deal with are images of endothelial cells. Endothelial cells migrate to sites of injury in the body. They are involved in forming new blood vessels to help repair damaged areas \[[@B1],[@B2]\]. In particular, we are interested in the effects of SHS on endothelial migration. By comparing automated migration analysis with varied exposure to SHS, for cells with and without specific genes, we can examine why exposure to SHS impairs endothelial cell migration and explore possible cures \[[@B3],[@B4]\]. Cell migration is a basic biologic function that can be modified by changes in genetic code and in response to chemical and other stimuli. Upon 24 hours serum starvation, the cells were artificially wounded using P20 pipette tip across the plate, then cultured respectively in regular DMEM or DMEM containing Second Hand Smoke (SHS) (unpublished). The subsequent gaps were imaged at 0 and 6 hours post SHS exposure as previously described to determine the rate of migration of the front lines to close in the gap \[[@B5]-[@B8],[@B23]\]. Figure [1](#F1){ref-type="fig"} gives an example about the cell migration. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Images of a plate of endothelial cells growing in agar. Cells appear as dark spots and contrast is limited because introduction of stains to increase contrast could affect cell function. An early time image (top row) shows a wide trough where a lane of cells was removed by the experimenter. A later time points (bottom row) show the effect of progressive narrowing of this lane as the cells migrate to fill in the gap. Our goal is to measure the width of the \"clear lane\" which corresponds to the amount of cell migration in the time interval. ::: ![](1471-2121-6-9-1) ::: The automated borders are compared blindly by a team of domain experts to manual borders created by a technician to assess accuracy. Results are also evaluated blind to biologic significance to determine concordance and power to demonstrated biological effects. Biologists deal with this by making multiple manual measurements, to report an average. Observers have difficulty deciding where and how many times to measure the width. Besides, there are many pairs of images to be processed. Therefore, automatic measurement is desired. Implementation ============== Generating texture ------------------ The primary difference between cell-populated areas and the clear lane is texture. The cell-populated areas are speckled with cells, the clear lane is not. In order to capture the cellularity characteristic of the source images, we sought to compute a texture index that would emphasize the cellular attribute of the region of interest and also minimize the influence of non-cellular signal variations \[[@B11]-[@B14]\]. Because the image may have non-uniform background where the \"clear lane\" can be \'darker\' than the \'cells\' at other locations, the texture index should be generated from the relative gray value difference. Furthermore, we know the orientation of the experimentally produced clear lane, which we take to be vertical. Then our algorithm generates the texture in this way: For each point in the original image Search for darker point in this line vertically If find Set the distance between start and darker point as the gray value of the corresponding point in the texture map Search for continual darker points and set the distance as the value of them Scale the value to 0--255: ![](1471-2121-6-9-i1.gif) where pv is the new pixel intensity value, cv is the distance value of the corresponding point and max and min are the minimum and maximum distance value. Panel b of figure [2](#F2){ref-type="fig"} shows the texture of Panel a of figure [2](#F2){ref-type="fig"} using this algorithm. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### An example image is shown in panel (a), followed by the derived texture index (b) and the resultant graphic overlay (c & d). Note that the region between the vertical lines of 2D is relatively devoid of cells, and each line represents the front of cell migration, as desired. ::: ![](1471-2121-6-9-2) ::: Migration analysis ------------------ Based on the texture map, the region we are interested in appears as a white vertical band. Thus the second stage of analysis must determine the position and width of this lane. As the information in each vertical column is equivalent to repeated measures, we can combine the data to marginal projection. From the histogram of this we can compute a classifier for lane vs. cells and determine the half-height width. The locations are then mapped to a graphic overlay on the original image to demarcate the front lines of cell migration. The change in distance between the front lines reports the amount of (bi-front) cell migration. 1\. Project the texture values to a marginal profile \"cellularity index profile\" (Figure [3](#F3){ref-type="fig"}) which is an array of P \-\-- P \[1..n\] where n is the width of the image. The value of each P \[i\] is the intensity sum of all the pixels in i column. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### The marginal cellularity index profile. ::: ![](1471-2121-6-9-3) ::: 2\. Compute the discriminant classifier (DC) which is average value of P \[i\]. 3\. Locate the leading and trailing edges based on classifier crossing. If P \[i\] \< DC and P \[i+1\] \> P \[i\], i is the leading edge. Conversely, if P \[i\] \> DC and P \[i+1\] \< DC, i is the trailing edge. Then a few pairs of leading and trailing edges could be obtained. The target pair is identified based on the width, P \[i\] values between leading and trailing edges and \"clear lane\" location in the time neighboring image. 4\. Record locations and generate graphic overlay for original image. Validation ---------- Since the manual assessment is the research gold standard for image processing \[[@B15]-[@B18]\], a technologist specially trained to identify the leading and trailing edges of cell migration was provided a computer tool to mark those edges manually in a manner compatible with the graphics overlay engine. These are called \"manual edges.\" The manual edges and the automated edges were then presented to a team of domain experts in random order pairs (one of each on corresponding image) for preference scoring. The scores ranged from 1--5, where 1 is strong preference for first overlay, 2 mild preference for first overlay, 3 equivalency, 4 mild preference for second overlay, and 5 strong preference for second overlay. Results were analyzed by Kappa statistic as a measure of agreement. Results ======= After analysis, results like the ones shown in the Figure [2](#F2){ref-type="fig"} are obtained. Panel (a) shows a photograph of the cell cultures, while the remaining panels show various aspects of the analysis. Figure [4](#F4){ref-type="fig"} shows the two worst cases of disagreement between automated and manual methods. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Examination of width differences between automated and manual identified two outliers with relatively large differences. These are shown as two pairs. Note the uneven heterogeneous cell distribution in these cases, a result that is technical suboptimal and not desired, likely from an error in the excoriation (creation of clear channel). These are a poor sample pairs for technical reasons, and in retrospect domain expert still had no significant difference in preference of manual result over the automatic result. ::: ![](1471-2121-6-9-4) ::: The results of domain expert preference by quality for automated vs. manual assignation of migration front lines, evaluated blinded to method, randomized, and subsequently decoded. Overall, there is complete equivalence of automated vs. manual with respect to expert preference for quality. The values ranged 2--4. In no cases was manual strongly preferred over automatic. Preference testing of analysis methods showed near equivalence, favoring preference for the automated borders (3.02 ± 0.11). Agreement between observers in preference was examined for two domain experts, revealing good agreement (Kappa = 0.59, p \< 0.003). Agreement in preferences by a technologist without domain expertise was lower (Kappa = 0.23, 0.25, p \> 0.10) but supported the same conclusion: the automated analysis is at least as good as manual selection by domain experts. Application of this method to determine the effect of SHS on endothelial cell migration demonstrates that SHS can reduce the cell migration rate, which is statistically significant (Figure [5](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### The migration distance of control group (NL) is 101.30 ± 10.32 and distance of SHS group 36.25 ± 2.71. Pair t test shows two groups are obviously different with p value = 2.24E-06. ::: ![](1471-2121-6-9-5) ::: Discussion ========== Our migration analysis is based on the texture index of the images. This index should reflect the attribute of the images. Since no global thresholding technique could be used in our images, the segmentation of regions and boundaries (edges) have to consider the local property \[[@B9]\]. Because the target boundaries always show as a vertical band, the line-based segmentation appears to be the most suitable approach for our task. Further analysis of regions and edges is based on a uniform data structure reflecting the texture character in each column. Our results show a robust automatic method with no detected errors. This study is a pilot study demonstrating feasibility and biologic significance in real application. Further collective experience in multi-center applications are needed to establish the full utility of the method. In addition, the program runs on the software platform, ImageJ \[[@B10]\] and the speed is fast. A normal process time for one study of images is less than 3 minutes. Results such as width and percentage can be shown as a table. It offers a convenient way for researcher to process their image data using excel. Conclusion ========== We describe a novel method of cell migration analysis based on texture pre-processing and discriminant analysis. Domain expert preference testing demonstrates that this automated method compares favorably to the much more painstaking manual method. The further study is to apply this to evaluation of the impact of SHS on endothelial cell migration. For that purpose, we have constructed a SHS capture system in which we bubble the SHS through tissue culture medium to assess its impact on cell migration. Our results indicate that this analysis system is very sensitive to biological effects, documenting that SHS impairs cell migration \[[@B19]-[@B22]\]. Availability and requirements ============================= Project name: Cell migration measurement project Project home page: <http://magic.hitchcock.org/jianfeng/index.html> Operating system(s): Platform independent Programming language: Java Other requirements: Java 1.3.1 or higher, ImageJ License: Null Any restrictions to use by non-academics: Licence needed Authors\' contributions ======================= JDP proposed and designed the method to evaluate cell migration objectively and automatically. JQ implemented the method as a plugin of ImageJ and validated the method. TWC performed the cell experiments and captured the images. LG performed manual edge definition and subsequently performed statistic analysis of expert preferences. All authors read and approved the final manuscript. Acknowledgements ================ Funded in part by FAMRI.	PubMed Central	2024-06-05T03:55:54.048788	2005-3-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554989/", "journal": "BMC Cell Biol. 2005 Mar 3; 6:9", "authors": [ { "first": "Jianfeng", "last": "Qin" }, { "first": "Thomas W", "last": "Chittenden" }, { "first": "Ling", "last": "Gao" }, { "first": "Justin D", "last": "Pearlman" } ] }
PMC554990	Background ========== Preferential killing of sick and disabled prey individuals by the predator has been the focus of many ecologists working with different predator -- prey systems. In a variety of prey predator systems, diseased or weaker animals are shown to be consumed in greater proportion by predators \[[@B1]-[@B5]\]. Increased susceptibility of parasitized prey to predation, or predator preference for parasitized prey is possible under a set of conditions \[[@B6]-[@B8]\]. Where the prey species is an intermediate host and the predator is the definitive host for a parasite species, the capture of prey is often an essential part of the life cycle. Therefore any mechanism that makes the prey susceptible to predation would enhance the parasite fitness. In such relationships the susceptibility induced by the parasite can be very specific towards the predator host \[[@B9]\]. A mutualistic relationship can be said to exist between a predator and a parasite \[[@B10]\] if the cost of harboring the parasite is less than the benefit of greater success in catching the prey \[[@B1]\]. Some evidence suggestive of predator-parasite mutualism comes from dhole or Indian wild dog (Cuon alpinus) and a protozoan parasite (Sarcosystis axicuonis) with chital or spotted deer (Axis axis) as the prey-host \[[@B1],[@B11]\]. There can be a potential problem in such a mutualistic relationship. Low virulence of the parasite towards the predator host and parasite tolerance by the predator host are essential factors for the maintenance of a mutualistic relationship. However, it is possible that a virulent parasite can grow faster and invade a mild parasite population. On the other hand a parasite resistant predator can avoid the cost of parasitism but share the benefit of prey capture and therefore invade a tolerant population. Either of the events can destabilize the mutualistic relationship. It is essential therefore to examine the evolutionary stability of the mutualism. In a completely randomized distribution, a mild parasite population can be easily invaded by a virulent one and a tolerant predator can be invaded by a resistant one. Population viscosity, group selection and kin selection can alter the evolution of virulence \[[@B12]\]. The dhole -- Sarcocystissystem makes group selection and viscosity very likely factors in shaping the relationship \[[@B1]\]. The life cycle of the parasite is very short as compared to dhole life span. Dhole groups are stable and retain their territories over a long time. Dhole territories are large and encompass home ranges of several chital packs \[[@B13],[@B14]\]. Therefore the protozoan harboured by a dhole pack is very likely to be recycled to the same pack. The benefit of the parasite is more likely to be gained by the same pack. The distribution of a parasite within a pack is shown to follow a consistent pattern in which only a few individuals carry most of the parasite load. On the other hand, one or two individuals in each pack are found to be parasite free. This suggests that within a pack there can be distribution of labor. \[[@B1]\]. A distribution of labor, in which some individuals do active hunting and some disseminate the parasite, can reduce the effective cost of carrying the parasite. Evolution can take a different route under such conditions. We examine here the effect of parasite recycling on the evolution of a predator-parasite mutualism, using a theoretical model. The model --------- We consider two alternative strategies, namely mild and virulent, for the parasite (Table [1](#T1){ref-type="table"}). The virulent parasite multiplies rapidly in the predator host and therefore enjoys greater success (v) and exerts a higher cost (x) on the predator host. The mild parasite exerts relatively low cost (y) on the predator host and gains a limited success (m). The predator has two alternative strategies, namely tolerant and resistant. A tolerant predator always harbors the parasite population whereas the resistant one attempts to resist or eliminate the parasite. However, since the parasite virulence mechanisms also evolve, there is a probability (p) that the parasite can infect a resistant predator. The predator gets an additional net benefit (z) from capturing a prey infected with the parasite as compared to capturing an uninfected prey. The prey can have only one viable strategy, that of becoming resistant to the parasite. The prey will not get any benefit by tolerating the parasite since it would make it more susceptible for predation. Therefore, we do not consider alternative prey strategies in the model. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Pay -- off matrix for predator and parasite strategies. ::: ---------- ----------- --------------------- --------------------- ------------------------ ----------------------- parasite predator Mild Virulent Tolerant Resistant parasite Mild 1-y fr(1-y)+(1-fr)(1-x) m p\* m Virulent fr(1-x)+(1-fr)(1-y) (1-x) v p\* v predator Tolerant -y -x z (1-fr) p \z + fr \z Resistant \- p \* y \- p\* x (1-fr)\z + fr (p\ z) p\* z ---------- ----------- --------------------- --------------------- ------------------------ ----------------------- The table differs from pay-off matrix tables for classical game theory models. The table accounts for two alternative strategies each for two different types of players namely parasite and predator. The pay-off of the parasite is not only decided by other parasites but also by the predator strategy and vice-versa. Therefore the complete pay-off of a mild parasite invading a virulent population in a tolerant host population is m \* \[fr(1-y)+(1-fr)(1-x)\]. Others to be calculated similarly. ::: If the parasite population consists of the mild type, they enjoy a fitness of \'m\' from the tolerant host and \'p\m\' from a resistant one. Since they exert a cost y on the host, there is erosion of the host resource. The host resource available to them is therefore (1-y). Similarly for a population of virulent parasites the mean fitness gain is v and the host resource available (1-x). A virulent host invading a mild population will gain a fitness of \'v\' such that v \> m. In the absence of recycling the host resource available to it would be (1-y). However with a frequency of recycling \'fr\', the host resource would be, fr (1-x) + (1-fr)(1-y) Similarly, that for a mild parasite invading a virulent population would be, fr (1-y) + (1-fr)(1-x) If the predator population is tolerant the parasite will be harbored in large numbers and disseminated to the prey population. Since the parasitized prey is more susceptible to predation the predator gets a benefit \'z\' of easy catching. A resistant population, on the other hand has a small probability \'p\' of harboring the parasite. Therefore the benefit the predator gets would be \'p\z\'. If a resistant predator invades a tolerant population, with the recycling factor \'fr\', the benefit of prey capture would be, fr \* p \z + z(1-fr). The benefit for a tolerant one invading a resistant population would be, fr \ z + (1-fr)\* p\z. We assume that \'v\', the benefit for a virulent parasite by infecting single host is directly proportional to \'x\' i.e the loss to the host from infection by virulent parasite. V = α \x similarly, m = α \y Results and discussion ====================== A mild parasite will be able to invade a virulent population if the pay-off to the mild invader is greater than that for the virulent population. When the predator population is tolerant, this condition is satisfied when, y \ \[fr (1-y)+(1-fr) (1-x)\] \> x (1-x) The condition under which a virulent invader is unable to invade a mild population is (1-y)\* y \> \[fr \(1-x) + (1-fr)\(1-y)\]\x Since x \> y, for satisfying both these conditions, fr should be large, y should be moderate and x should be large. Thus selection would favour a moderate virulence in the parasite towards the predator host. Unlike our expectation, low virulence is unlikely to be stable. However, a mutualistic relation can remain if the prey capture benefit is sufficiently large. It can be easily seen that the above conditions remain unaffected even if the predator population is resistant. Considering predator strategies, a tolerant predator will be able to invade a resistant population in the presence of a mild predator if, p \z - p\y \< (1-fr) p \z + fr\z - y i\. e. p\fr \* z - p\* y \< fr \z - y This condition will be satisfied if fr \z \> y since p \< 1. A resistant predator will be unable to invade a tolerant population since the necessary condition is z-y \< (1-fr)\z + fr\(p\z) - (p \ y) fr\z - y \< p\fr\z - p\y This invasion is impossible if fr\z \> y. If the parasite is virulent, the necessary condition would be fr\z \> x. Since x is assumed to be large, a resistant predator would be stable if the prevalent parasite strategy is virulent. Thus when fr and z are large and y is small mutualism would be stable. When this condition is not satisfied the predator will evolve resistance to the parasite and the parasite will evolve greater virulence. Conclusion ========== A large recycling frequency (fr) appears to be the only critical factor in the evolution of parasite virulence. However, the parasite is unlikely to evolve towards low virulence. There will be a moderate virulence optimum. For a net benefit to the host the cost associated with this level of virulence should be less than the benefit in terms of ease of prey capture. For the evolution and stability of the tolerant strategy in the predator a large fr as well as large z and small y are necessary. A predator-parasite mutualism therefore critically depends upon these factors, whereas it is independent of p and α. In the case of Dhole-chital -- Sarcocystissystem, these condition are very likely to be satisfied. Field data show that the frequency of sarcocystosis of the heart in dhole kills was approximately double that of chital dyeing of other causes \[[@B1]\]. This suggests a substantially large z. Dhole have large, stable and defended territories upto 80 Km^2^\[[@B13]\]. There is only marginal overlap between neighboring packs unlike the overlap in tigers \[[@B15]\]. The territory of a dhole pack encompasses the home ranges of several chital groups. The home ranges of chital groups are small and stable \[[@B13],[@B16]\]. This can ensure a large \'fr\'. Unlike wolves, which tend to defecate more on the boundaries of the territory \[[@B17]\], the frequently used defecation sites of dhole tend to be towards the center of their home range and close to the major hunting areas \[[@B18]\], further ensuring a large \'fr\'. The intensity of intestinal infection is reflected in the density of sporocyst in Dhole scat. Dhole shading large numbers of sporocysts of S. axicuonisshow no apparent symptoms of disease or abnormality \[[@B19],[@B20]\]. This indicates that the virulence of S. axicuonistowards dhole is low. Further, the division of labor can substantially reduce the effective cost of carrying the parasite. Currently we are unable to quantify these parameters empirically and have no estimate of the actual cost of harboring the parasite. Therefore we are unable to state quantitatively that all the necessary conditions for mutualism are met by the system. The importance of the model is that it helps us identify the gaps in the data and thus orient future empirical work. Although we are far from having an empirical estimate of y, fr and z, the known ecology of chital and dhole suggest that fr and z could be sufficiently large. This makes the system a likely candidate for the evolution of predator parasite mutualism. Any other predator-prey system that satisfies these conditions is also likely to co-evolve with some parasite species towards a predator-parasite mutualism. Parasites of diverse taxa have evolved predator-prey life cycles and any of them could be possible candidates for a mutualism. Predator-prey-parasite systems that satisfy the following three criteria are the most likely candidates for a stable mutualistic relationship: i\) parasitized prey individuals are killed with substantially greater frequency by the predator ii\) pathogenicity of the parasite towards the predator host is low or moderate iii\) there is a high rate of parasite recycling to the predator host. We need to look at a number of systems that could satisfy these criteria. The chital-dhole-Sarcocystissystem may not be unique and many possible examples of predator-parasite mutualism may be present in nature. Authors\' contributions ======================= Both authors have contributed approximately equally to the model development. Acknowledgements ================ We are grateful to the Department of Science & Technology, Govt. of India for financially assisting this work. We thank the forest departments of Tamilnadu and Maharashtra State for their permission for field work. We thank Dr. R. Sukumar, CES, field station in Mudumalai for providing necessary facilities, and Arun Venkatraman and R. Arumugum for their active interaction during field work. Discussions with Dr. S. A. Paranjape, Dr. N. V. Joshi and Arun Venkatraman were useful. We are grateful to Prof. Arun Bhagwat for his help in language and presentation during revision of the manuscript. We also thank Sachin, Krushnamegh, Rahul, Shantanu and Dilip for field support.	PubMed Central	2024-06-05T03:55:54.050074	2005-2-22	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554990/", "journal": "BMC Ecol. 2005 Feb 22; 5:3", "authors": [ { "first": "Maithili M", "last": "Jog" }, { "first": "Milind G", "last": "Watve" } ] }
PMC554991	Background ========== Butein (3,4,2\',4\'-tetrahydroxychalone-Figure [1](#F1){ref-type="fig"}), a plant polyphenol, is one of the major biologically active components of the bark and stems of Rhus vernicifluaStokes. In Far Eastern countries such as Korea, Japan, and China, the compound has been traditionally used for treatment of pain, thrombotic disease, gastritis, stomach cancer, and parasitic infections \[[@B1],[@B2]\]. In Korea, it has also long been used as a food additive \[[@B2]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Chemical structure of butein. ::: ![](1472-6882-5-5-1) ::: Recently, butein has been shown to possess potent activity against fibroblast function \[[@B3]\], possibly related to its ability to suppress differentiation of fibroblasts to myofibroblasts that are characteristically involved in wound healing \[[@B4]\]. Because fibroblasts and myofibroblasts are now believed to play a critical role in promoting the growth of cancer cells \[[@B5],[@B6]\], we performed this study to determine if butein could suppress the growth of human breast cancer cells co-cultured with fibroblasts by interfering with the function of the fibroblasts. Methods ======= Clonogenic assay ---------------- The UACC-812 human breast cancer cell line (ATCC, Manassas, VA) was passaged in Leibovitz\'s medium supplemented with 15% fetal calf serum. Normal fibroblasts (CCD-1068SK, ATCC) obtained from the breast of a 65 year old female were passaged at 37°C in minimal essential medium (Eagle\'s) supplemented with 2 mM L-glutamine, Earle\'s balanced salt solution (1.5 grams/Liter), sodium bicarbonate, 0.1 mM non-essential amino acids, 1 mL sodium pyruvate, and 10% fetal calf serum in a 5% CO~2~atmosphere. All cell culture reagents were obtained from ATCC. Our co-culture experiments used confluent monolayers of fibroblasts that had been passaged no more than 21 days. This precaution assured that the fibroblasts were not senescent or transformed. We seeded 100 UACC-812 breast cancer cells into individual wells of a 96-well cell culture plate containing a confluent monolayer of fibroblasts growing in minimal essential growth medium supplemented as described above. At intervals of 3--4 days, fresh medium was added. After 14 days, the cells were fixed with 70% ethanol for 10 minutes prior to staining for 3 minutes with 0.1% toluidine blue. The wells were then washed with distilled water, and the numbers of colonies of tumor cells containing eight or more confluent cells were counted using inverted microscopy. Each experiment was performed in triplicate, and the means and standard deviations for each treatment and control group were then compared using a two-tailed, unpaired t-test. Co-culture with fibroblast modulators (Experiment 1) ---------------------------------------------------- A monolayer of normal fibroblasts was seeded with 100 UACC-812 tumor cells/well containing complete culture medium (control) or medium supplemented with various modulators of fibroblast function. The fibroblast modulators that we tested included troglitazone (an activator of PPAR-γ in fibroblasts \[[@B7]\]; 1 μg/mL); GW9662 (an inhibitor of PPAR-γ\[[@B8],[@B9]\]; 1 μM); butein (an inhibitor of myofibroblast differentiation; 2 μg/mL); meloxican (a COX-2 inhibitor in fibroblasts \[[@B10],[@B11]\]; 1 μM); or 3,4 dehydroyproline (an inhibitor of collagen synthesis by fibroblasts \[[@B12]\]; 10 μg/mL). Because butein is relatively insoluble in aqueous solution, it was first dissolved in dimethylsulfoxide to produce a stock solution (10 mg/mL) that was then serially diluted into growth medium to produce the final desired concentrations of butein. At intervals of 3--4 days, old medium was removed and replaced with fresh medium containing the drugs. After 14 days, the numbers of colonies of 12 or more tumor cells were counted as described above. As a control to determine the effects of butein on the clonogenic growth of breast cancer cells in the absence of fibroblasts, we seeded 100 tumor cells into wells without fibroblasts but containing butein (2 μg/mL) Co-culture with fibroblasts pre-treated with butein (Experiment 2) ------------------------------------------------------------------ In the next experiment, we sought to determine if pre-treatment of fibroblasts alone with various doses of butein would also inhibit clonogenic growth of breast cancer cells. A monolayer of fibroblasts in individual wells of 96-well plates was incubated for 3 days with growth medium containing serial, 2-fold dilutions of butein ranging from 10 μg/mL to 1.25 μg/mL. The adherent fibroblasts were then washed three times to remove any residual butein, and 100 UACC-812 cells were seeded per well. The co-culture was then incubated in culture medium without butein for 14 days, after which the numbers of colonies of tumor cells were counted as described above. Co-culture of primary breast cancer cells with fibroblasts (Experiment 3) ------------------------------------------------------------------------- This experiment was performed in order to determine if butein could also suppress the clonogenic growth of primary breast cancer cells obtained directly from clinical specimens of human breast cancer. After obtaining appropriate approval from the Institutional Review Board to perform the study in compliance with the Helsinki Declaration, we aseptically dissected small fragments of tumor tissue from five cases of invasive ductal adenocarcinoma of the breast. These specimens were submitted for routine diagnostic evaluation to the Surgical Pathology Department at UCI Medical Center (Orange, CA). The tissues were carefully minced into small pieces and then digested overnight at 37°C in collagenase II (900 U/mL; Sigma-Aldrich, St. Louis, MO) solution in cell culture medium. Epithelial cells and organoids were then isolated by differential centrifugation of the digest \[[@B13]\], washed, and counted. We then seeded 100 tumor cells onto monolayers of fibroblasts that had been pre-treated with butein (2.5 μg/mL) for 3 days as described in the preceding experiment. The co-cultures were then incubated in culture medium without butein for 14 days, after which the numbers of colonies of tumor cells were counted as described above. Results ======= Clonogenic assay ---------------- Co-culture of 100 UACC-812 human breast cancer cells on a monolayer of human fibroblasts without butein treatment typically yielded 50--75 distinct colonies of easily recognizable tumor cells on a background of fibroblasts (Figure [2](#F2){ref-type="fig"}). If a monolayer of fibroblasts was not used, an average of five small colonies of tumor cells was generally observed. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Representative colony of tumor cells in clonogenic assay.Breast cancer cells co-cultured with fibroblasts typically formed clusters of confluent cells and were easily distinguished from the fibroblasts when stained with 0.1% toluidine blue. Original magnification 400×. ::: ![](1472-6882-5-5-2) ::: Co-culture with fibroblast modulators ------------------------------------- The results of Experiment \#1 are presented in Figure [3](#F3){ref-type="fig"}. Only GW9662 and butein significantly (p \< 0.01) reduced clonogenic growth compared to the control (cell culture medium alone with no drug). There was only one small colony of tumor cells visible in one of three wells containing butein. Surviving individual tumor cells were not visible in any of the wells containing butein. Notably, in the absence of fibroblasts, there was an average of 4 colonies of tumor cells regardless of the presence or absence of butein. Thus, butein had no detectable effect on the clonogenic growth of breast cancer cells grown in the absence of fibroblasts. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Co-culture of breast cancer cells and fibroblasts with drugs that modulate fibroblasts.Compared to the Control (no drugs), butein and GW9662 almost completely eliminated clonogenic growth of the breast cancer cells. ::: ![](1472-6882-5-5-3) ::: Co-culture with fibroblasts pre-treated with butein --------------------------------------------------- The results of Experiment \#2 are presented in Figure [4](#F4){ref-type="fig"}. There was no clonogenic growth when 100 UACC-812 breast cancer cells were seeded onto monolayers of fibroblasts that had been pre-treated for 3 days with butein at 10 or 5 μg/mL, and clonogenic growth was significantly reduced at butein concentrations as low as 2.5 μg/mL. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Co-culture of breast cancer cells with fibroblasts pre-treated with butein.Pre-treatment of the fibroblasts with butein at concentrations greater than or equal to 2.5 μg/mL completely eliminated or substantially reduced clonogenic growth of co-cultured breast cancer cells, suggesting an indirect mechanism of action that interferes with the interaction between fibroblasts and breast cancer cells. ::: ![](1472-6882-5-5-4) ::: Co-culture of primary breast cancer cells with fibroblasts pre-treated with butein ---------------------------------------------------------------------------------- The results of Experiment \#3 using primary breast cancer cells are presented in Table [1](#T1){ref-type="table"}. For each of the 5 clinical specimens that we tested, there were significantly fewer colonies of breast cancer cells when the co-culture was performed in the presence of fibroblasts that had been pre-treated with butein. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Clonogenic growth of primary breast cancer cells co-cultured with fibroblasts pre-treated with butein (2.5 μg/mL). ::: Clinical Sample \# Mean number of colonies with butein pre-treatment (1 s.d.) Mean number of colonies without butein pre-treatment (1 s.d.) -------------------- ------------------------------------------------------------ --------------------------------------------------------------- 1 0 (0) 13 (4)\* 2 0 (0) 36 (9)\* 3 0 (0) 6 (4) \* 4 3 (2) 12 (5)\* 5 7 (4) 29 (8)\* \p value \< 0.02 by two-tailed, unpaired t-test comparing means and s.d. of butein pre-treatment versus no pre-treatment ::: Discussion ========== Clonogenic growth of small numbers of breast cancer cells in vivo\[[@B5],[@B6]\] as well as in our in vitroculture system appears to be critically dependent on the presence of fibroblasts. In this study, we have demonstrated that butein can suppress the clonogenic growth of breast cancer cells in vitrothrough an indirect mechanism that involves interfering with the function of co-cultured fibroblasts. Significantly, the concentration of butein that we found to be effective in these experiments (2.5 μg/ml) does not significantly reduce fibroblast viability \[[@B4]\] and is also non-toxic in animal experiments \[[@B1],[@B2]\]. Moreover, this concentration of butein had no significant effect on the clonogenic growth of breast cancer cells cultured in the absence of fibroblasts. Previous studies have shown that higher concentrations of butein extract from Rhus vernicifluaStokes are directly cytotoxic in vitro*to human colon adenocarcinoma cells and lymphoma cells \[[@B2],[@B14]\]. Butein has also been shown to inhibit cell growth and induce apoptosis in murine B16 melanoma cells \[[@B15]\]. The mechanism of induction of apoptosis in tumor cells appears to be related to increased caspase-3 activity, decreased Bcl-2 expression, and increased Bax expression \[[@B16]\]. Butein has also been shown to have multiple other activities, including inhibition of epidermal growth factor receptors \[[@B17]\], tyrosine kinase inhibition \[[@B18]\], suppression of E-selectin expression \[[@B19]\], inhibition of tyrosinase enzymes \[[@B20]\], and inhibition of cyclooxygenase-2 \[[@B21]\]. Our results suggest that butein may also suppress the growth of tumor cells through a second, indirect mechanism that does not involve direct toxicity to the tumor cells themselves. At this time, however, the precise molecular mechanisms by which butein interferes with the interaction between fibroblasts and breast cancer cells remain undefined. Conclusion ========== Clonogenic growth of small numbers of breast cancer cells co-cultured with fibroblasts pre-treated with butein is markedly reduced. These results suggest that butein can inhibit the growth of tumor cells through an indirect mechanism that interferes with the interaction between fibroblasts and breast cancer cells. Our results also suggest that the herbal compound butein should be further investigated as a potentially useful experimental approach for suppressing the growth of small numbers of breast cancer cells in early micrometastases. List of abbreviations ===================== PPAR-peroxisome proliferation activator receptor COX-cyclooxygenase Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= MKS conceived and designed the study, performed the experiments with the primary tumor cells, analyzed the data from all experiments, and wrote this report. JT and GC performed the other experiments and reported the data. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6882/5/5/prepub> Acknowledgements ================ The authors acknowledge the assistance of Richard McPartland and Thuy Vu in obtaining the primary tumor specimens. This work was supported by grants from the Chao Comprehensive Cancer Center (University of California, Irvine) and the California Cancer Research Coordinating Committee.	PubMed Central	2024-06-05T03:55:54.051604	2005-3-9	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554991/", "journal": "BMC Complement Altern Med. 2005 Mar 9; 5:5", "authors": [ { "first": "Michael", "last": "Samoszuk" }, { "first": "Jenny", "last": "Tan" }, { "first": "Guillaume", "last": "Chorn" } ] }
PMC554992	Background ========== Although pain is the presenting symptom of most patients with chronic pancreatitis, its neurobiological basis remains poorly understood \[[@B1]\]. In the past, investigators have focused on the role of anatomical abnormalities such as a strictured pancreatic duct or narrowed intraparenchymal ducts. However, mechanical decompression techniques such as endoscopic stent placement or even surgical pancreatojejunostomy frequently do not provide a permanent solution to the pain \[[@B1]\]. More recently, investigators have begun focusing on the role of neurotransmitters and neurotrophins such as substance P and nerve growth factor with known or suspected roles in nociceptive signaling and/or sensitization and have reported an increased expression of several of them in the pancreas of patients with painful chronic pancreatitis \[[@B2]\]. Mast cells are also increased in both acute and chronic pancreatitis \[[@B3],[@B4]\] but their role in the generation of pain in pancreatitis has not been investigated. We hypothesized that mast cells are involved in the pathogenesis of pain in chronic pancreatitis. This hypothesis is based on the following observations. First, mast cells have been associated with human conditions in which pain is a predominant symptom. Interstitial cystitis and irritable bowel syndrome are both conditions in which pain is out of proportion to the objective pathological findings \[[@B5],[@B6]\]. In both conditions, an increase in the number of mast cells has been described in the bladder and the colon, respectively \[[@B5],[@B6]\]. Further, mast cells are frequently found in close proximity to nerves in the intestinal mucosa and the bladder \[[@B7]-[@B9]\]. This has also been observed in the pancreas -- the total number of mast cells was significantly higher in pancreatic tissue from patients with chronic pancreatitis than in the normal pancreatic controls \[[@B3]\]. One of the preferential locations of mast cells was around and within the perineurium of nerve fibers in tissue samples of patients with chronic pancreatitis, suggesting the potential for interactions between mast cells and the nervous system. Lastly, there is evidence for bi-directional functional communication between mast cells and nerves \[[@B10]-[@B12]\]. Mast cells can not only release mediators that increase excitability of neurons but in turn, neurotransmitters such as substance P can trigger mast cell degranulation \[[@B10]\]. Mast cells may therefore contribute to the pathogenesis of pain in pancreatitis through degranulation products that can sensitize pancreatic afferent neurons in an ongoing vicious circle of neuronally mediated mast cell degranulation. Our first aim was to analyze the presence and distribution of mast cells in autopsy specimens of chronic pancreatitis and study the correlation, if any, with historical documentation of pain. We then explored our hypothesis further using an experimental model of trinitrobenzene sulfonic acid (TNBS)-induced chronic pancreatitis in both wild type and Kit^W^/Kit^W-v^mice, a strain deficient in mast cells (MCDM). Methods ======= Data collection autopsy study ----------------------------- Autopsy records from the University of Texas Medical Branch from the years 1993 to 2000 were searched electronically for the term \"pancreatitis.\" One-hundred sixty-six patients were identified of which 26 patients carried an autopsy diagnosis of chronic pancreatitis and 140 patients carried a diagnosis of acute pancreatitis. The medical charts from patients with an autopsy diagnosis of chronic pancreatitis were reviewed for documentation of a medical history of chronic pancreatitis. If no such documentation was present in the chart, patients were excluded from the study (12/26). Thus, 14/26 patients with both a documented history and an autopsy based diagnosis of chronic pancreatitis, were included in the study. Patients were categorized as painful chronic pancreatitis (8/26) when they fulfilled one of the following criteria: a documented history of chronic abdominal pain clinically attributed to chronic pancreatitis that required the use of narcotics, and/or frequent admissions for recurrent abdominal pain clinically attributed to chronic pancreatitis, and/or a surgical or endoscopic procedure for refractory abdominal pain clinically attributed to chronic pancreatitis. Patients were categorized as non-painful chronic pancreatitis (6/26) if patients did not fit any of the criteria listed under painful chronic pancreatitis. In addition, the following data were collected: demographic factors (age and race), cause of death, comorbidities, clinical history of pancreatitis, etiology of pancreatitis, diagnostic studies supporting a diagnosis of pancreatitis (amylase, lipase, calcifications on abdominal plain film, CT-scan, ultrasound or ERCP). Human pancreatic control tissue was obtained from 8 arbitrarily chosen patients of whom the autopsy records recorded acute myocardial infarction as the cause of death. Their medical records were reviewed to ensure that they did not have a clinical history of pancreatitis. Therefore there were three categories of patients: one with painful chronic pancreatitis, one with non-painful chronic pancreatitis and non-pancreatitis controls. A pathologist, blinded to the group assignment, verified all histological diagnoses and counted mast cells on a Giemsa stained tissue section (average of 10 high-power randomly chosen (40X) fields per specimen). The protocol was approved by the Institutional Review Board of the University of Texas Medical Branch. Mice strains ------------ All mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Male mice were used from the following strain: WBB6F1/j-Kit^W^/Kit^W-v^(MCDM) and the respective littermate control mouse strain, Kit^W-v^-- +/+ (WT). The mice were 3 months of age at the onset of the experiment with body weights of 25--30 gram. Experimental protocols involving mice were approved by our Institutional Animal Care and Use Committee (IACUC) in accordance with the guidelines provided by the National Institutes of Health. Induction of chronic pancreatitis --------------------------------- Mice were anesthetized with sodium Nembutal (50 mg/kg body weigh, i.p.) Following a midabdominal laparotomy, a canula was introduced into the common pancreato-biliary duct; the duct was ligated proximally and distally to ensure perfusion into the pancreas and prevent entry of the injected substance into the liver or duodenum. 0.1 ml of 1% TNBS in phosphate buffered saline (PBS)-10% ethanol, pH 8, was infused into the pancreas (modified after Puig-Divi \[[@B13]\]). The canula was removed and the abdomen closed. Control mice were treated in the exact same fashion but were perfused with saline instead (Figure [1](#F1){ref-type="fig"}). Mice were sacrificed 8 weeks after surgery. Von Frey Filament (VFF) measurements ------------------------------------ VFF hairs consist of a series of filaments of increasing diameter that produce increasing sensations of touch when applied to the skin. When the tip of a fiber of given length and diameter is pressed against the skin, the force of application increases until the fiber bends. After the fiber bends, continued advance creates more bend, but not more force of application. This principle makes it possible to apply a reproducible force to the skin surface. VFF testing is an established behavioral pain assay used to determine mechanical pain thresholds in somatic pain. More recently, VFF testing has been used as a surrogate marker for visceral pain \[[@B14],[@B15]\]. Mice were placed in a cage with a mesh floor and habituated to the environment for 30--60 minutes. Measurements were taken from the abdomen and the plantar surface of both hindpaws over a period of three weeks prior to the surgery and for a total of three weeks after the surgery (Figure [1](#F1){ref-type="fig"}). VFF filaments of various caliber were applied to the mid-abdomen in ascending order 10 times, each for 1--2 seconds with a 10 second interval. A response was defined as: a) sharp retraction of the abdomen; b) immediate licking or scratching of site of application of hair; or c) jumping. The response frequency was defined as the total number of responses out of 10 applications (expressed as a percentage) to the skin per filament. An investigator blinded to the different treatment groups performed the behavioral testing. Pancreatic histology -------------------- Fresh specimens of the mouse pancreas were fixed in 10% formaldehyde in PBS pH 7.4 containing 1 mM MgCl~2~at 4°C overnight. Sections from paraffin-embedded specimens were stained with hematoxyline and eosin and observed under a light microscope. Pathological changes were scored based on a scale described by Tito et al. by a pathologist blinded to the different treatment groups \[[@B16]\]. Data expression and statistical analysis ---------------------------------------- Comparisons of the number of mast cells in autopsy specimen were analyzed using the Mann-Whitney U test. For each behavioral experiment (see figure [1](#F1){ref-type="fig"}), the average response frequency was calculated as the mean of the mean response frequencies for each mouse (across four measures). The \"post-pre response frequency\" was calculated by subtracting the pre-surgical average response frequency from the post-surgical average response frequency. To assess the independent effect of TNBS on VFF response (ie. to control for the effect of the surgery itself), the post-pre response frequency for TNBS infusion was compared with the post-pre response frequency for saline infusion. This comparison was performed using analysis of variance for a two-factor experiment with repeated measures on time at each level of force for each type of mice (WT and MCDM). The two factors were induction of pancreatitis or not (TNBS or saline, respectively) and time (pre-surgical or post-surgical). TNBS infusion was considered to have had an independent effect on the VFF response if the post-pre response frequency was greater for TNBS than for saline infusion. Fisher\'s least significant difference procedure was used for multiple comparisons of least squares means with Bonferroni adjustment for number of comparisons. All effects and interactions were assessed at the 0.05 level of significance. Data analysis was conducted using PROC MIXED with LSMEANS options in SAS^®^, Release 8.2 \[SAS Institute Inc., SAS/STAT^®^User\'s Guide, Version 8, Cary, NC: SAS Institute Inc., 1999\]. Results ======= Autopsy data ------------ Patient demographics are summarized in Table [1](#T1){ref-type="table"}. Alcohol abuse was the most common cause for pancreatitis in both groups. Analysis of our results, using the Mann-Whitney U test, revealed significantly more mast cells in patients with a history of painful chronic pancreatitis (n = 8) when compared to patients with either non-painful chronic pancreatitis (n = 6) (33.8 vs 9.4 average mast cell count/10 high power fields; p \< 0.01) or controls (n = 8), (33.8 vs 6.1 average mast cell count/10 high power fields; p \< 0.01) (Figure [2](#F2){ref-type="fig"}). The increased number of mast cells in patients with painful pancreatitis was noted predominantly in interstitial areas and, to a lesser degree, in the periacinar space. Withdrawal responses to mechanical stimulation of the abdomen and paw after induction of chronic pancreatitis ------------------------------------------------------------------------------------------------------------- Figure [3](#F3){ref-type="fig"} shows the post-pre surgical response frequency for both WT and MCDM. TNBS had a significant independent effect on abdominal VFF response in WT mice at the force levels 4 and 8 mN (p = 0.007 and 0.037, respectively) (Figure [3A](#F3){ref-type="fig"}). There was a trend towards a significant effect at the force level of 16 mN (p = 0.066). In contrast, for MCDM, TNBS had no significant effect on abdominal VFF response at any force level (Figure [3B](#F3){ref-type="fig"}). There was no significant TNBS effect on VFF response in the left hindpaw for either WT mice or MCDM (Figure [4](#F4){ref-type="fig"}). Data not shown for the right hindpaw. Histological analysis of mice pancreas following TNBS injection --------------------------------------------------------------- Pancreatic histology confirmed the presence of chronic pancreatitis in both WT and MCDM with marked fibrosis, inflammatory infiltrates and ductular proliferation mimicking changes seen in human chronic pancreatitis (Figure [5A](#F5){ref-type="fig"}). The pancreas of saline treated controls was normal. There was no significant difference in the overall inflammatory scores between the WT and MCDM (Figure [5B](#F5){ref-type="fig"}). An increased number of mast cells were counted in WT mice with chronic pancreatitis compared to saline treated controls (5.6 vs 1.5; p = 0.05) (Figure [6](#F6){ref-type="fig"}). As to be expected, no mast cells were present in pancreas of MCDM. Discussion ========== Chronic pancreatitis has been defined as a continuing inflammatory disease of the pancreas characterized by irreversible morphologic changes that typically cause pain and/or permanent loss of function \[[@B17]\]. The pathogenesis of pain in this condition remains to be satisfactorily established. We examined the association, if any, of pain with mast cells as quantified in autopsy specimens of patients with a history of painful and non-painful chronic pancreatitis and normal controls. Significantly more mast cells were present in pancreatic tissue from patients with a history of painful chronic pancreatitis, indicating an association with this condition and a potential role for these cells in the pathogenesis of pain in painful chronic pancreatitis. There are clearly limitations to a retrospective, autopsy-based study such as the one we report here. For instance, we do not know whether pain was present at the time of death and there was incomplete information on the different patterns of pain. Also, our findings pertain mainly to patients with a history of alcoholic pancreatitis. Nevertheless, our findings do suggest an association of painful chronic pancreatitis with an increased number of mast cells. This observation provided the rationale for further experimental testing, which we performed in mice. We first developed a model of chronic pancreatitis in mice following a modified protocol first described by Puig- Divi et al. \[[@B13]\]. Histological changes consisted of periductal and lobular fibrosis, duct stenosis, chronic inflammatory cell infiltrates, and gland atrophy, mimicking features of chronic pancreatitis in humans. Significantly more mast cells were present in WT mice with chronic pancreatitis, adding to the validity of this model for use in studies on the role of mast cells in pancreatitis. Both WT and MCDM developed histological changes consistent with chronic pancreatitis, indicating that the elimination of mast cells did not modulate the animals\' ability to mount an inflammatory response. Therefore, any changes observed in pain behavior are unlikely to stem from differences in underlying inflammation. Next we determined whether this mouse model could be used to study behavioral differences associated with chronic pancreatitis. The assessment of spontaneous pain in a visceral organ presents significant difficulties. We have used a behavioral method to assess this, which relies on the association of visceral pain with sensitization of somatic regions of the body that share segmental innervation at the level of the spinal cord (referred pain). This somatic sensitization can be quantified using VFF to stimulate the somatotopically appropriate abdominal region and measuring the abdominal withdrawal response. Thus, VFF testing of the anterior abdominal wall can be used as a surrogate marker for visceral pain. Although this is the first time that this technique has been used for the measurement of referred visceral hyperalgesia in a mouse model of chronic pancreatitis, this method has previously been described and validated to assess the severity of referred visceral pain for models of colonic hypersensitivity \[[@B14]\] as well as rat models of acute necrotizing pancreatitis \[[@B15]\] and chronic pancreatitis \[[@B18]\]. The abdominal VFF response was compared to the hind paw response to assess the specificity of the interventions to the pancreas. TNBS treated mice, but not the saline control, developed increased abdominal wall withdrawal responses to VFF testing when compared to baseline, suggesting the development of force-dependent referred hyperalgesia of the abdominal wall in WT mice. There was no evidence of referred hyperalgesia in the hindpaws, suggesting that the measured effect on abdominal withdrawal is specific for an intra-abdominal origin of the pain. Vera-Portocarrero et al. previously described similar findings, increased withdrawal frequency after VFF stimulation to the abdominal area, in a rat model of chronic pancreatitis \[[@B18]\]. These behavioral changes were abrogated by morphine. Rats that demonstrated behavioral changes also expressed increased substance P expression in the nociceptive layers of the spinal cord, suggestive of central nociceptive changes. Mast cells produce a variety of degranulation products in the setting of inflammation that may activate and/or sensitize primary nociceptive neurons. The neurotrophin growth factor (NGF) is one such product \[[@B19]-[@B22]\]. NGF is released in the setting of inflammation and can not only function as a chemoattractant for other mast cells, but it can also trigger mast cell degranulation \[[@B23]\]. We are speculating that NGF production in the inflamed pancreas is responsible for plastic changes in the sensory neurons by activating proalgesic receptors and channels such as the NGF receptor tyrosine kinase A (TrkA) and Transient Receptor Potential Family V receptor 1 (TRPV1; previously known as VR1) thereby contributing to the generation of pain \[[@B24]-[@B26]\]. Similarly, other mast cell degranulation products such as tryptase and histamine are capable of modulating neuronal function \[[@B27]-[@B32]\]. Tryptase may directly activate the proteinase-activated receptor-2 (PAR-2), a G-protein coupled receptor expressed by pancreatic nerves, important in the pathogenesis of pain in pancreatitis \[[@B33],[@B34]\]. Although the role for mast cells in the mediation of visceral nociceptive signaling needs to be explored further, we speculate that mast cell products released in pancreatitis, contribute to the development of pain by direct effects on nociceptors located on pancreatic afferent neurons (Figure [7](#F7){ref-type="fig"}). However, before concluding a definite role for mast cells from our experimental data, it should be noted that MCDM carry a spontaneous mutation for tyrosine kinase receptor c-kit which not only produces a deficiency of mast cells but may have an independent effect on the function of sensory neurons, which are known to express it \[[@B35]\]. Therefore, it remains to be determined whether the detected differences in nociceptive responses is due to the absence of mast cells per se or a yet unknown change in the responsiveness of sensory neurons due to a congenital lack of the c-kit receptor. Reconstitution of mast cells into the MCDM mice should restore their nociceptive responses close to the wild type phenotype. Conclusion ========== Our data should increase awareness of the importance of mast cells in the pathogenesis of painful inflammatory conditions such as chronic pancreatitis and encourage experimental studies for further testing of this hypothesis. Abbreviations ============= TNBS -- trinitrobenzene sulfonic acid, WT -- wild type, MCDM -- mast cell deficient mice, VFF -- von Frey Filaments, IACUC -- Institutional Animal Care and Use Committee, PBS -- phosphate buffered saline, NGF -- neurotrophin growth factor, TrkA -- tyrosine kinase A, TRPV1 -- Transient Receptor Potential Family V receptor 1 (previously known as VR1), PAR-2 -- proteinase-activated receptor-2 Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= WAH conceived of the study, participated in its design and coordination and wrote the manuscript. KG completed the behavioral studies. SYX completed the histological analyses and mast cell counts. JHW provided technical assistance with the surgical procedures and participated in the data analyses and review of the manuscript. WDW provided support for the behavioral studies as well as their analyses. PJP provided financial support and participated in the review of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-230X/5/8/prepub> Acknowledgements ================ We thank B. Ebert for excellent administrative assistance. We thank Vahakn Shahinian, MD for critical review of the manuscript. We thank J.F. Aronson, MD, Director Autopsy Service, Department of Pathology, for assistance in provision of the pancreatic autopsy blocks. We thank Tatsuo Uchida from the Office of Biostatistics for the statistical analysis. We thank M.A. Micci, Ph.D., for assistance in the composition of the images. Funding for this study was provided by a grant from the John Sealy Memorial Endowment Fund for Biomedical Research and AGA RSA (WAH). Partial funding for this study was provided by a grant from the Galveston Moody Foundation and NIDDK Grant RO1 DK62330-01 (PJP). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Experimental designAll mice underwent pre and post surgical VFF testing. For the VFF testing, 4 measures were taken for each mouse. WT and MCDM were randomized to either saline or TNBS perfusion into the pancreatic duct. ::: ![](1471-230X-5-8-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Mast cell in painful and nonpainful chronic pancreatitisA Histology of autopsy specimen. Giemsa staining of autopsy specimens of controls and of patients with chronic pancreatitis (CP) both with and without pain. Large images represent 40× magnification while the inserts are 100× magnifications of mast cells. Scale bars represent 20 μM. Arrows point at mast cells. B Autopsy specimens of patients with a history of chronic abdominal pain contained a significantly higher number of mast cells as compared to specimens of patients with chronic pancreatitis without a history of abdominal pain (34.1 +/- 7 vs 8.6 +/- 2.9; P \< 0.01) or to control specimens (34.1 +/-7 vs 6.6 +/- 1.4; P \< 0.01). ::: ![](1471-230X-5-8-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Abdominal VFF WT and MCDMPost-pre response frequency was calculated by subtracting the average pre-surgical response frequency from the average post-surgical response frequency for WT mice (A) and MCDM (B). P-values are for the comparison of the post-pre response frequency for TNBS infusion versus post-pre response frequency for saline infusion. ::: ![](1471-230X-5-8-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Left Hindpaw VFF WT and MCDMPost-pre response frequency was calculated by subtracting the average pre-surgical response frequency from the average post-surgical response frequency for WT mice (A) and MCDM (B). P-values are for the comparison of the post-pre response frequency for TNBS infusion versus post-pre response frequency for saline infusion. ::: ![](1471-230X-5-8-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Histology (H&E) of mice with chronic pancreatitisA H&E staining of the pancreas of WT and MCDM 4 weeks after infusion of TNBS into the pancreatic duct. Images represent 40× magnification; scale bars represent 20 μM. Fibrosis and inflammatory infiltrates are present. B Average histological scores for WT and MCDM. There is no significant difference in histological scores between WT and MCDM (P = NS). ::: ![](1471-230X-5-8-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Histology (Giemsa) of mice with chronic pancreatitisA Giemsa staining of the pancreas of WT and MCDM 4 weeks after infusion of TNBS into the pancreatic duct. Large images represent 40× magnification while the inserts are 100× magnifications of mast cells. Scale bars represent 20 μM. B Significantly more mast cells are present in the pancreatic tissue of mice treated with TNBS when compared with saline treated controls (\* P \< 0.05). ::: ![](1471-230X-5-8-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Proposed involvement of mast cells in nociceptive signaling in pancreatitisIn pancreatitis, mast cells may migrate to sites of inflammation, in response to release of mast cell chemoattractants. Mast cell degranulation products may modulate neurotransmission directly by activating proalgesic receptors and channels such as trka (NGF), TRPV1 (NGF) and PAR-2 (tryptase and trypsin). ::: ![](1471-230X-5-8-7) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Patient demographics ::: A Patients with chronic pancreatitis and pain --------------------------------------------------- ------------- ----------- ------------------------ ------------------- ---------------- -------------- --------------- -------------------------- Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Age 54 58 38 63 61 65 38 73 Race/gender B-M C-M LA-M C-M C-F B-M C-M C-M Cause of death arrhythmia AMI Cardio-myopathy pneumonia/ESLD liver-failure CAD/cachexia Gibleed/ARDS PE Comorbidities ESRD/CP/HCV COPD/CP CP/sp Puestow cirrhosis/COPD/CP HTN/CP HTN/CP CP/sp Puestow COPD/CAD/CP/Laryngeal ca CP etiology ETOH ETOH chronic cholelithiasis ETOH ETOH ETOH ETOH ETOH CP imaging CT CT/ERCP CT/ERCP CT CT/ERCP CT/ERCP CT/ERCP CT Enzymes no no yes no no no yes yes Insulin no no yes no no yes yes no Narcotics vicodin vicodin vicodin vicodin vicodin darvocet vicodin vicodin B Patients with chronic pancreatitis without pain Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Age 56 72 56 60 45 40 Race/gender C-M C-F C-M C-M C-F C-F Cause of death AMI sepsis AMI pneumonia sepsis unknown Comorbidities CP CP IDDM/CP CAD/CM/IDDM/CP HIV/asthma/HTN CP CP etiology ETOH ETOH idiopathic idiopathic ETOH ETOH CP imaging CT/ERCP RUQ-US CT CT CT CT Enzymes yes yes no yes no yes Insulin yes no yes yes no yes C Control group Control 1 Control 2 Control 3 Control 4 Control 5 Control 6 Age 56 56 73 51 53 76 Race/gender C-M LA-F C-F LA-M C-M C-F Cause of death AMI AMI arrhythmia AMI PE AMI Comorbidities HTN CAD CAD/HTN/DM DM CAD CAD/HTN/DM C = caucasian; LA = latin-american; B = black; M = male; F = female; ESRD = end stage renal disease; HCV = hepatitis C virus; COPD = chronic obstructive pulmonary disease; AMI = acute myocardial infarction; ESLD = end stage liver disease; HTN = hypertension; CAD = coronary artery disease; ca = cancer; ETOH = alcohol; ERCP = endoscopic retrograde cholangiopancreatogram; IDDM = insulin-dependent diabetes mellitus; CM = cardiomyopathy; HIV = human immunodeficiency virus :::	PubMed Central	2024-06-05T03:55:54.052901	2005-3-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554992/", "journal": "BMC Gastroenterol. 2005 Mar 3; 5:8", "authors": [ { "first": "Willemijntje A", "last": "Hoogerwerf" }, { "first": "Kelly", "last": "Gondesen" }, { "first": "Shu-Yuan", "last": "Xiao" }, { "first": "John H", "last": "Winston" }, { "first": "William D", "last": "Willis" }, { "first": "Pankaj J", "last": "Pasricha" } ] }
PMC554993	Background ========== The postmenopausal loss of bone mass and subsequent increased risk of low-energy (fragility) fractures is an important public health problem, especially in countries with a high proportion of elderly individuals. More than 1 million fragility fractures, primarily in postmenopausal women, occur each year in the US. The annual direct medical costs exceed US\$10 billion \[[@B1]\]. Bone mineral density (BMD) measured with dual energy X-ray absorptiometry (DEXA) has been widely used to estimate the risk of fracture in epidemiological studies and to study treatment effects of antiresorptive agents in clinical trials. There are several well documented environmental and biological factors known to influence bone mineral density and the risk of fragility fractures including female gender, age, previous fragility fracture, low body weight, reduced lifetime exposure to estrogen, low calcium intake, physical inactivity, vitamin D deficiency, smoking, and excessive alcohol consumption \[[@B2]-[@B5]\]. There is also a strong genetic component to interindividual BMD variability, with heritability estimates ranging from 0.46 to 0.84 at different body sites \[[@B6]-[@B8]\]. Numerous candidate genes have been tested for association to BMD and fragility fractures. A polymorphism in a transcription factor-binding site of the collagen 1A1 (COL1A1) gene has shown one of the most consistent associations to osteoporosis, even if the association is generally weak for BMD and varies between populations \[[@B9]-[@B11]\]. Linkage studies have also been performed with the aim of locating genetic loci influencing BMD variation \[[@B12]-[@B19]\]. So far, the genes responsible for the resulting linkage peaks have not been identified. Recently, linkage of a compound osteoporosis phenotype was reported to chromosome 20p12. Subsequent positional cloning efforts implicated BMP2, the gene encoding for bone morphogenetic protein 2, as responsible for the linkage \[[@B20]\]. Nevertheless, the associations reported thus far that have been independently validated account for only a small portion of the genetic contribution to BMD and osteoporosis. Studies that rely on direct association approaches based on linkage disequilibrium within populations are expected to have greater statistical power and be more feasible to implement than traditional linkage studies to identify common variations that influence common, complex traits such as osteoporosis \[[@B21]\]. Recently, there has been increasing interest in the use of whole-genome association methods to identify genes that are involved in complex trait variation. To date, however, few such large-scale studies have been reported. In an effort to identify genes and variants that influence risk of osteoporosis, we conducted a large-scale study using more than 25,000 single nucleotide polymorphisms (SNPs) located within approximately 16,000 genes in DNA pools of unrelated females at the extremes of the lumbar spine bone mineral density distribution. A number of intriguing associations identified in this study are currently being scrutinized in further detail. In this paper we report the most advanced of these, which is the association of a variation in PDE4D, the gene encoding cyclic AMP-dependent phosphodiesterase 4D, with lumbar spine BMD. PDE4D selective inhibitors have been shown to promote osteoblast differentiation in progenitor cells \[[@B22]\] and to increase bone mass by promoting bone formation in normal mice \[[@B23]\] but the gene has not until now been implicated in human bone metabolism. Methods ======= Subjects -------- ### Discovery sample: unrelated females from UK twin collection The population sample from which the discovery samples were chosen consisted of 5,436 female twins collected at the Twin Unit at St Thomas Hospital, London, England. They were selected without regard to health or trait. The volunteers had been recruited through advertisements and had undergone extensive investigation at the Twin Unit at St Thomas Hospital. Investigations included several questionnaires inquiring about present and past diseases, symptoms, family history, socio-economical factors, and medication. Subjects underwent an extensive clinical assessment including DEXA measurements of bone mineral density and anthropometric measurements \[[@B17],[@B24]\]. All individuals with data on lumbar spine BMD were considered for inclusion. To exclude relatives, the individual with the most extreme BMD was kept in each twin pair. Individuals with diseases or medication known to influence BMD were excluded, as were individuals younger than 40 years because of the observed complex relationship between age and BMD. In addition, individuals with fractures were excluded from the high BMD group. BMD values were adjusted for age, weight, BMI, self reported leisure time physical activity, smoking, and menopausal status using an ordinary least squares model including second and third order polynomial terms for age and second order terms for weight and BMI. We included BMI and weight as covariates because both were independently associated with BMD in this sample. Based on the trade-off between group sizes and separation, target sizes of 350 were chosen, resulting in a separation of approximately 1.9 SD between groups. After assessment of DNA availability sufficient for such a large scale study, group sizes were reduced to 319 and 321 individuals in the high and low groups, respectively. Lumbar spine BMD T-scores were calculated with the females between 20 and 35 years of age as the reference population. Based on this, 32% of the women in the low BMD group had osteoporosis and an additional 58% had osteopenia according to WHO criteria. The characteristics of the selected individuals are reported in Table [1](#T1){ref-type="table"}. ### Replication sample: Australian twin collection A twin sample from Royal North Shore Hospital, Sidney, Australia was collected similarly as the UK twin collection. 731 individuals including twin pairs and singletons with lumbar spine BMD assessments were available for genotyping (Table [3](#T3){ref-type="table"}). Groups of unrelated subjects corresponding to the lower and upper quartiles of the age- and BMI-adjusted lumbar spine BMD distribution were defined similarly to the discovery sample. The characteristics of the selected individuals are reported in Table [1](#T1){ref-type="table"}. ### Replication sample: international multi-center sib-pair study The second replication sample was a multi-center (Australia, UK, New Zealand, Belgium) study that collected sib pairs concordant and discordant for bone mineral density \[[@B17]\]. Probands (BMD Z-score \<-1.5 at lumbar spine, femoral neck, or hip) were identified and their siblings were contacted and underwent DEXA measurements at the lumbar spine and hip. Participants had to be between 25--85 years of age. Exclusion criteria included steroid medication, hyperparathyroidism, immobility, amenorrhea, anorexia nervosa, and unstable thyroid disease. Nine hundred and eight individual samples were genotyped from 392 families. In the present analysis we included only females older than 40 years of age. In this sample the distribution of family sizes were 164 singletons, 248 families of 2, 34 families of 3, 7 families of 4, and 3 families of 5 members (Table [3](#T3){ref-type="table"}). Lumbar spine BMD levels were adjusted by age and BMI as described for the Australian sample. Human subjects protection ------------------------- All studies were approved by the appropriate research ethics committees. All participants gave their informed consent to participate in genetic studies before enrollment. Bone mineral density -------------------- Bone mineral density was estimated from the L1-L4 vertebrae, hip, and forearm using DEXA according to the user\'s manual for the Hologic QDR 4500W, (Hologic, Waltham, Massachusetts, United States) at all collection sites. SNP markers and genotyping -------------------------- A set of 25,494 SNP markers was selected from a collection of 125,799 experimentally validated polymorphic variations \[[@B25]\]. This set was limited to SNPs located within gene coding regions, minor allele frequencies greater than 0.02 (95% have frequencies greater than 0.1), and a target inter-marker spacing of 40 kb. SNP annotation is based on NCBI dbSNP database, refSNP, build 118. Genomic annotation is based on NCBI Genome Build 34. Gene annotation is based on LocusLink genes for which NCBI was providing positions on the Mapview FTP site. For pooled DNA assays, 25 ng of case and control DNA pools was used for amplification at each site. All PCR and MassEXTEND™ reactions were conducted using standard conditions \[[@B26]\]. Relative allele frequency estimates were derived from area under the peak calculations of mass spectrometry measurements from four analyte aliquots as described elsewhere \[[@B26]\]. For individual genotyping, the same procedure was applied except only 2.5 ng DNA was used and only one mass spectrometry measurement was taken. Primers used to genotype rs1498608 were ATAACCTCGGGGTCCAGAAA (forward PCR primer), GAATCCCTGTTCATTCCTTG (reverse PCR primer) and CCCTAAAAACTGTTCCAGGTA (extension primer). The primers used to genotype the Ser37Ala polymorphism in BMP2were AGCTGGGCCGCAGGAAGTTCG (forward PCR primer), TCGTCAGAGGGCTGGGATGAG (reverse PCR primer) and TGAGGGGCGGCCCGACG (extension primer). Statistical analysis -------------------- Tests of association between adjusted lumbar spine BMD group and each SNP using pooled DNA were carried out in a similar fashion as explained elsewhere \[[@B27]\]. Briefly, the test statistic is based on the difference in allele frequencies between the two groups divided by the known sources of variation in each allele frequency estimate, including sampling and pool-specific measurement variation. Sources of measurement variation incorporated in the test statistic are pool formation, PCR/mass extension, and chip measurement. When three or more replicate measurements of a SNP were available within a model level, the corresponding variance component was estimated from the data. Otherwise, the following historical laboratory averages were used: pool formation = 5.0 × 10^-5^, PCR/mass extension = 1.7 × 10^-4^, and chip measurement = 1.0 × 10^-4^. Tests of association using individual genotypes were carried out using a chi-square test of heterogeneity to compare allele frequencies, and Fisher\'s exact test to compare genotype frequencies (due to low frequency contingency table cells). Confidence intervals and P-values for odds ratios were derived using Fisher\'s exact test. When one or more cell counts were zero, non-infinite odds ratios were estimated by adding 0.5 to each cell \[[@B28]\]. In the samples that included a combination of singletons, sib pairs, and occasionally additional relatives, we estimated the relationship between genotypes and phenotypes using the generalized estimating equations (GEE) approach with a Gaussian link by clustering on family using an exchangeable correlation matrix \[[@B29]\]. Hypothesis testing was carried out with a Wald test statistic. The geepack implementation of GEE in the R statistical software platform was used \[[@B30]\]. No attempt was made to correct P-values for multiple testing. Rather, P-values are provided to compare the relative strength of association. P-values less than 0.05 are referred to as statistically significant. Results ======= Initial genome scan in UK sample -------------------------------- We carried out a genome-wide association study using 25,494 SNPs located within 10 kb of 15,995 LocusLink annotated genes. An overview of the investigative process is shown in Figure [1](#F1){ref-type="fig"}. The basic design was a two-group study in subjects from the tails of the adjusted lumbar spine BMD distribution. We selected lumbar spine BMD as the phenotype to create the contrasting groups because it had a high estimate of heritability in our twin sample (h^2^= 0.82) \[[@B7]\]. The selected low and high BMD groups consisted of 319 and 321 individuals, respectively. The adjusted BMD range was 0.56--0.87 g/cm^2^in the low BMD group and 1.11--1.60 g/cm^2^in the high BMD group, corresponding to the upper and lower 22^nd^percentiles. Other characteristics of the samples are described in Table [1](#T1){ref-type="table"}. To facilitate the screening of such a large number of SNPs, we employed a high-throughput approach using DNA pools, chip-based mass spectrometry \[[@B26],[@B31]-[@B33]\], and a three-phase SNP selection strategy (Figure [1](#F1){ref-type="fig"}). In the first phase, we performed a single PCR and primer extension reaction for each SNP on two DNA pools consisting of equimolar amounts of DNA from each individual in the low BMD group and high BMD group, respectively. Relative allele frequencies obtained from four mass spectrometry measurements of the extension products were compared between pools. In the second phase, the 1,520 SNPs (\~6%) with the most statistically significant associations (nominal P-values \< 0.05) were re-measured in triplicate on each DNA pool. In the third phase, we genotyped the 140 most significant SNPs (9%) from step two (nominal P-values \< 0.02) on all individuals comprising the pools. Based on the genotype results, 78 SNPs were confirmed to have statistically significant allele frequency differences between cases and controls (P \< 0.05). The liberal criteria for selecting SNPs from each phase represent a practical trade-off between following up false positive versus overlooking false negative associations. We chose to follow up as many SNPs from each phase as seemed reasonable. One of the associations was found with rs1498608, an A/T polymorphism within intron 5 of PDE4Don chromosome 5q12. Allele frequencies for the T allele based on genotyping were 0.91 in the low lumbar spine BMD pool and 0.88 in the high BMD pool (OR = 1.5, P = 0.035). Complete genotype counts and summary statistics are reported in Table [2](#T2){ref-type="table"}. Observed genotype frequencies were consistent with expected frequencies under Hardy-Weinberg equilibrium within each study collection. Menopausal status did not have a significant influence on the effect (P = 0.87). Genome-wide studies using tens of thousands of SNPs and liberal statistical selection criteria are expected to yield a high proportion of false positive associations. To distinguish the true genetic effects from among the false positives, the 78 selected SNPs were genotyped in a second twin sample from Australia. Replication in Australian sample -------------------------------- The Australian replication sample, a combination of female and male twin pairs and singletons, was analyzed in two ways. First, to create a design and carry out an analysis comparable to the discovery sample, unrelated individuals were selected from the lower and upper quartiles of the sex-specific adjusted lumbar spine BMD distribution (Table [1](#T1){ref-type="table"}). A similar effect was observed for the marker SNP rs1498608 in females (OR = 2.14, P = 0.018) and males (OR = 1.55, P = 0.35) as in the original UK collection (Table [2](#T2){ref-type="table"}). The second method of analysis utilized generalized estimating equation (GEE) models to take into account all of the available genotype information by carrying out a regression-type analysis while accounting for familial covariance. The characteristics of this sample are reported in Table [3](#T3){ref-type="table"}. The regression of marker genotypes on adjusted lumbar spine BMD, with sex included as a covariate, found the AA genotype to be associated with significantly higher levels than the AT (β = 7.8 g/cm^2^, P = 0.049) or TT (β = 8.0 g/cm^2^, P = 0.037) genotypes, thus confirming the results observed in the unrelated tails of this sample. Similar GEE analyses carried out for femoral neck and hip BMD were not statistically significant. Replication in international multi-center family study ------------------------------------------------------ Being a sample of mostly affected sib pairs, this sample was unsuitable for formation of groups with contrasting BMD because of the preponderance of individuals with low BMD (Table [3](#T3){ref-type="table"}). Therefore, we restricted the analysis to using a generalized estimating equation, regressing marker genotypes on BMD values. Surprisingly, the estimates in this sample were opposite to that in the Australian sample, as the AA genotype was associated with lower adjusted lumbar spine BMD values than both the AT (β = -5.3 g/cm^2^, P = 0.11) and the TT (β = -5.4 g/cm^2^, P = 0.09) genotypes. Using Z-scores at the femoral neck (P = 0.0007 and 0.0004), total hip (P = 0.003 and 0.007), and lumbar spine (P = 0.03 and 0.02) as dependent variables confirmed this pattern of association. In all cases the AT and TT genotypes had very similar point estimates. Association fine mapping ------------------------ In order to better define the extent of the region of association and possibly identify other SNPs more strongly associated with BMD, we performed DNA pool based association fine mapping in the UK sample using 80 publicly available intronic SNPs in the 100 kb region surrounding the incident SNP (Figure [2](#F2){ref-type="fig"}). This analysis identified a 20 kb region of association encompassing exon 6 of PDE4D. Replication of BMP2association -------------------------------- As described in the discussion below, PDE4D inhibition is known to influence BMP2-induced alkaline phosphatase activity in osteoblast precursor cells. Recently, variation in the gene encoding BMP2 was found to be associated with osteoporosis in a study employing whole genome linkage and subsequent positional cloning \[[@B20]\]. Since we were unaware of any published independent attempts to replicate this finding, we genotyped the Ser37Ala polymorphism in our UK and international samples. In the UK sample, the allele frequency of the rare allele was 2.2% in the low BMD group and 1.4% in the high BMD group, with an odds ratio of 1.6 (P = 0.28). We tested for, but were unable to detect, an interaction between the Ser37Ala polymorphism and rs1498608 on the association with lumbar spine BMD. In the international sample we performed an allele based general estimating equation to estimate the effect of the rare allele on BMD in that sample. The allele frequency of Ala37 in this sample, mainly selected for low BMD, was 1.9%. The effect of the Ala allele was estimated to decrease the adjusted lumbar spine BMD by 0.06 g/cm^2^(P = 0.0029). There were no homozygous Ala individuals in this sample. Discussion ========== In an association study using SNPs in nearly 16,000 genes we obtained evidence that variation in the SNP rs1498608 located within PDE4Dis associated with low bone mineral density at the lumbar spine in females. PDE4Dencodes cyclic AMP-dependent phosphodiesterase 4D. Phosphodiesterases are a superfamily of enzymes involved in degradation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) \[[@B34],[@B35]\]. cAMP and cGMP are important second messengers participating in the response of various cells to hormones. In osteoblasts, cAMP produced in response to parathyroid hormone or prostaglandins regulates osteoblastic differentiation \[[@B36]-[@B39]\], which leads to increases in cancellous bone volume as indicated by experiments in animal models \[[@B40]-[@B45]\]. The intracellular level of cAMP is regulated by G protein-coupled adenylyl cyclase \[[@B46]\], and degradation is mediated by the phosphodiesterases. The phosphodiesterase superfamily consists of seven families, PDE1-7, distinguished by substrate specificity, chromatographic behaviour during purification, and affinity for biochemical activators and inhibitors. Of these, the PDE4 family is specific for cAMP and is selectively inhibited by rolipram. Four PDE4 genes, 4A, 4B, 4C, and 4D, have been cloned from rat and humans, all of which are predicted to have multiple protein products due to alternate spicing of RNAs. PDE4 inhibitors have been shown to increase bone formation in normal mice \[[@B23]\] and to ameliorate loss of bone mass in animal models of osteopenia \[[@B47],[@B48]\]. PDE4A and PDE4D are expressed in two common mouse osteoblastic cell lines, ST2 and MC3T3-E1, that represent different stages in the osteoblast differentiation pathway \[[@B22]\]. PDE4 inhibition with rolipram increased BMP2-induced alkaline phosphatase activity, a marker of early osteoblast differentiation in ST2 cells. Furthermore, rolipram increased the expression of alkalic phosphatase, osteopontin, collagen type I and osteocalcin in the same osteoblast precursor cells \[[@B22]\]. In spite of these experimental data, we are not aware of any published attempts to investigate the role of PDE4genes in human osteoporosis. However, variation in PDE4Dwas recently reported to be associated with the risk of ischemic stroke \[[@B49]\]. Given the central role of PDE4 in second messenger signalling, it is quite conceivable that PDE4Dvariants may have effects on the risk for different common diseases. There are other examples of genes having such pleiotropic effects, the most notable being APOEin hyperlipidemia and Alzheimer\'s disease \[[@B50],[@B51]\]. It should also be noted that Gretarsdottir et al found that the PDE4Dassociation with stroke was strongest for a region in the recently extended 5\' end of the gene, which is close to 1,000 kb upstream of rs1498608 \[[@B49]\]. Assuming a contribution of PDE4Dto the risk of osteoporosis as well as stroke, it is possible that different domains are involved in the different diseases. Given the interaction between BMP2 and PDE4 for the inhibition on osteoblastic differentiation in vitro, it is interesting to note that variants in the gene encoding for BMP2 have also been found to increase risk of osteoporosis in humans \[[@B20]\]. In the current study, we replicated the association between the Ser37Ala variant in BMP2and measures of osteoporosis in an international family-based sample ascertained via low BMD probands. Although not statistically significant, this finding was supported by the results in the discovery sample of unrelated high and low spine BMD subjects. The allele frequency in the low BMD group was 2.2% and in the high group 1.4%, with an odds ratio of 1.6 (P = 0.28). The rare allele was less common in our low BMD group than the low spine BMD group (3%) in the Icelandic sample. However, our allele frequencies in the low and high BMD groups and the resulting OR corresponded well with the figures in the Danish sample (1.8% vs 1.0%, RR = 1.8) reported in the same paper \[[@B20]\]. We found no evidence for statistical interaction between the variations in BMP2and PDE4Din either sample. However, given the low minor allele frequencies of each SNP, there was very little power to test for interaction effects. The starting point of the present study was a large-scale association study of more than 25,000 SNPs located in 16,000 genes. After a stepwise selection process an association between an intronic SNP in PDE4Dand lumbar spine bone mineral density was detected, providing the first evidence that a variant of this gene could contribute to the risk of osteoporosis in humans. The effect was similar in size in premenopausal and postmenopausal women, indicating that the effect would be on the attainment of peak bone mass rather than the rate of decrease in BMD after menopause. The lack of a detectable interaction with female sex hormones is supported by having observed a similar genetic effect in the small sample of males in our study. The genetic contribution to peak bone mass is possibly bigger and definitely better documented than the as yet unproven genetic influence on postmenopausal bone loss \[[@B52]\], and it is possible that PDE4Dcould contribute to this effect, especially in light of the documented anabolic effect on bone by PDE4 inhibitors. An association with an intronic SNP provides little evidence for a change in amount or function of the protein that could explain the association. None of the 80 SNPs investigated as part of the association fine mapping were non-synonymous coding changes, which is consistent with the extraordinary lack of variation that others have reported for all PDE classes \[[@B53]\] and PDE4D in particular \[[@B49]\]. This makes it unlikely (but still possible) that the observed association would be due to a non-synonymous and disruptive single-base coding change in linkage disequilibrium with our marker SNP. Therefore it is more likely that the effect is mediated by a change in RNA splicing or expression. Given the functional similarity between different PDE4 enzymes, we went back and scrutinized our data for associations with SNPs in the other PDE4 genes that may have been overlooked during the first stage of the scan. The only SNP in PDE4Bin our assay set, rs1318475, was taken through to the second stage (Figure [1](#F1){ref-type="fig"}) where it was estimated to have an OR of 0.78 (P = 0.041), but failed the criteria to be taken forward to the genotyping stage. Similarly, a SNP roughly 18 kb downstream of PDE4C, rs874628, was also taken forward to the second stage where it displayed an OR of 1.3 (P = 0.08). These results suggest that further investigation into possible associations between variants in all PDE4 genes and bone mineral density may be justified. The route by which these genetic associations were arrived at and the potential for spurious association must be considered. Recent published work has brought light to the need for proper validation to verify genetic findings for complex traits \[[@B54]-[@B56]\]. In the current study, the initial association found between the PDE4Dmarker and bone mineral density was one result from over 25,000 hypothesis tests. A conservative Bonferroni adjustment to yield an experiment-wide type I error rate of 0.05 would demand a test-wise p-value on the order of 10^-6^. Given the modest sample size, only common variations with relatively large effects (OR \> 2) would reach such significance levels. Instead, we chose to be more mindful of the role of type II error rates and apply a more liberal set of criteria in the initial phases of the study and verify true genetic effects by independent replication. The analysis of 78 selected markers in the Australian replication sample resulted in multiple associations of continuing interest, with rs1498608 displaying one of the strongest associations. A one-sided test of association comparing the results in the discovery and replication samples yields a p-value of 0.0074. This would not be considered significant on an experiment-wide level after Bonferroni adjustment, which would require a p-value on the order of 0.0006 or lower. The analysis in the international replication sample produced contradictory data in that the A allele, which in the first two samples was associated with increased lumbar spine bone mineral density, was associated with decreased BMD at all tested sites. The pattern of association evident from the first two samples, with AT and TT genotypes having very similar point estimates, was preserved in this sample, even in the face of the reverse direction of association. The highly statistically significant association between rs1498608 and femoral neck and hip BMD in this third sample and the consistency in the pattern of association would be unexpected from a spurious result. A possible explanation for the contradictory results could be the fact that the international sample consists mostly of individuals with low BMD since the probands all have a BMD Z-score \< -1.5, and most of the siblings also have low BMD. It is possible that within such a selected sample the relationship between rs1498608 and BMD could be altered due to interactions with other genetic or environmental factors. The well-documented anabolic effect on bone by PDE4 inhibitors lends indirect support for the association reported here, and it would seem that the possible role of PDE4Dvariants in the genetic contribution to BMD in humans merits further investigation. Conclusion ========== The result of the present large scale association study together with data from previously published animal models suggest that genetic variation in the gene encoding PDE4D contributes to the variation in lumbar spine BMD in humans. Competing interests =================== The authors of this manuscript affiliated with Sequenom, Inc. (RR, SM, CH, GM, SK, MN, AB) declare competing financial interests, which may include current or prior receipt of salary and/or stock ownership, as Sequenom, Inc. may be affected financially by the publication of this manuscript. Authors\' contributions ======================= RR drafted the manuscript and participated in study design and data analysis. SM was study project leader and participated in data analysis. SK supervised the operational aspects of the study, and was responsible for the development of the SNP marker assay set. CH and GM participated in the development of the SNP marker assay set. SW was project manager for the international multicenter study. PS was principal investigator for the Australian Twin collection. TS was principal investigator for the UK twin and international collections. MN participated in study design, performed the statistical analyses and helped draft the manuscript. AB had the overall scientific responsibility for the study. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2350/6/9/prepub> Acknowledgements ================ The authors acknowledge R. Prince, N. Gilchrist, J.Y. Reginster, I. Fogelman, I. Smith, and the clinical teams who contributed patients to the study. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Overview of DNA pool-based large-scale association strategy. Phases 1 and 2 are conducted using DNA pools yielding allele frequencies, all subsequent steps involve genotyping of individual samples. hME, homogeneous MassEXTEND; MS, mass spectrometry. See text for more details. ::: ![](1471-2350-6-9-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Association fine mapping of PDE4Dregion. Eighty public domain SNPs in a 100-kb window around the initial marker SNP (indicated as a bold symbol at the center of the plot) were compared between the UK low and high BMD pools. Ten of 80 SNPs were significant at P = 0.05 (horizontal dashed line). The x-axis corresponds to the chromosomal position of each SNP, the y-axis to the test P-values (\--log~10~scale). The continuous dark line presents the results of a goodness-of-fit test for an excess of significant associations (at a 5% significance level) in a 10-kb sliding window assessed at 1-kb increments. The continuous light gray line is the result of a nonlinear smoothing function showing a weighted average of the P-values across the region. The color of each point corresponds to the minor allele frequency of each SNP in the control sample (see legend below graph). Vertical dashed lines are placed every 20 kb. The LocusLink gene annotation for NCBI genome build 34 shows the location of exon six. All investigated SNPs are located within introns. ::: ![](1471-2350-6-9-2) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of subjects in the groups selected for low and high lumbar spine bone mineral density. ::: Discovery (UK) Replication (AUS) Replication (AUS) ------------------------------ ---------------- ------------------- ------------------- ---------------- ---------------- ---------------- Age (years) 48/52/60 48/53/59 44/52/61 45/53/61 48/56/65 48/54/62 Menopausal status Pre 29% (76) 31% (84) 41% (45) 36% (41) N/A N/A Post 71% (184) 69% (190) 59% (66) 64% (73) Smoking Never 56% (177) 53% (168) 78% (93) 67% (93) 41% (14) 62% (21) Former 26% (82) 29% (93) 22% (13) 20% (28) 47% (16) 21% (7) Current 18% (56) 18% (57) 10% (13) 13% (17) 12% (4) 18% (6) Weight (kg) 58/66/74 58/64/72 56/64/74 58/67/76 72/80/87 74/80/90 BMI 23/25/28 22/25/27 22/25/27 23/26/29 24/27/28 24/26/29 BMD spine (g/cm^2^) 0.74/0.80/0.87 1.12/1.19/1.26 0.77/0.87/0.96 0.98/1.10/1.17 0.88/0.92/1.03 1.00/1.14/1.21 Adjusted BMD spine (g/cm^2^) 0.77/0.81/0.84 1.14/1.18/1.23 0.80/0.84/0.87 1.08/1.13/1.19 0.86/0.90/0.93 1.15/1.17/1.25 BMD hip (g/cm^2^) 0.75/0.82/0.89 0.95/1.02/1.10 0.79/0.87/0.95 0.91/0.97/1.06 0.96/1.03/1.11 1.02/1.08/1.20 BMD femoral neck (g/cm^2^) 0.63/0.70/0.77 0.82/0.89/0.98 0.64/0.73/0.81 0.75/0.84/0.91 0.77/0.83/0.92 0.80/0.88/0.99 BMD forearm (g/cm^2^) 0.48/0.52/0.56 0.55/0.59/0.61 0.49/0.53/0.57 0.53/0.56/0.59 0.59/0.62/0.68 0.64/0.67/0.73 Categorical variables are reported as frequency percent (count). Quantitative variables are reported as 1^st^quartile/median/3^rd^quartile. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Allele and genotype frequencies of rs1498608 in high and low lumbar spine BMD groups. ::: Sample Allele/Genotype Low N (Rel. Freq.) High N (Rel. Freq.) OR (95% C.I.) P-value ------------------- ----------------- -------------------- --------------------- ----------------- ---------- Discovery (UK) Female A 52 (0.086) 77 (0.123) 0.035^a^ T 550 (0.914) 547 (0.877) 1.5 (1.0, 2.2) AA 1 (0.003) 9 (0.029) 0.026^b^ AT 50 (0.166) 59 (0.189) 7.6 (0.99, 340) 0.042^c^ TT 250 (0.831) 244 (0.782) 9.2 (1.3, 410) 0.020^c^ Replication (AUS) Female A 15 (0.054) 30 (0.109) 0.018^a^ T 261 (0.946) 244 (0.891) 2.1 (1.1, 4.4) AA 0 (0.000) 3 (0.022) 0.048^b^ AT 15 (0.109) 24 (0.175) 4.4 (0.23, ∞) 0.541^c^ TT 123 (0.891) 110 (0.803) 7.8 (0.45, ∞) 0.108^c^ Male A 9 (0.132) 13 (0.191) 0.352^a^ T 59 (0.868) 55 (0.809) 1.6 (0.56, 4.5) AA 1 (0.029) 3 (0.088) 0.754^b^ AT 7 (0.206) 7 (0.206) 3.0 (0.17, 180) 0.588^c^ TT 26 (0.765) 24 (0.706) 3.3 (0.24, 180) 0.610^c^ Abbreviations: Rel. Freq., relative frequency; OR, odds ratio; C.I., confidence interval. Odds ratios for the AT and TT genotypes are contrasted against the AA genotype. ^a^Chi-square test of heterogeneity comparing allele frequencies between high versus low. ^b^Fisher\'s exact test comparing three genotype frequencies between high versus low. ^c^Fisher\'s exact test comparing genotype frequencies in current table row and AA genotype between high versus low. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Characteristics of complete genotyped replication samples. ::: Australian Twin Replication International Replication ------------------------------ ----------------------------- --------------------------- ------------------- Trait Females (N = 590) Males (N = 141) Females (N = 805) Age (years) 40/52/61 47/54/64 52/59/67 Twin status Singletons 42% (246) 48% (67) N/A Dizygotic pairs 58% (72) 52% (37) N/A Smoking status Never 63% (368) 55% (78) 52% (317) Former 24% (141) 30% (42) 26% (157) Current 13% (77) 15% (21) 22% (130) Weight (kg) 58/64/74 73/81/91 55/60/69 BMI 22/25/28 25/27/29 22/24/27 BMD spine (g/cm^2^) 0.89/0.99/1.09 0.92/1.03/1.13 0.71/0.80/0.94 Adjusted BMD spine (g/cm^2^) 0.89/0.99/1.06 0.93/1.02/1.12 0.74/0.81/0.92 BMD hip (g/cm^2^) 0.85/0.93/1.02 0.96/1.05/1.14 0.68/0.77/0.87 BMD femoral neck (g/cm^2^) 0.71/0.79/0.88 0.78/0.86/0.95 0.57/0.65/0.73 BMD forearm (g/cm^2^) 0.51/0.55/0.59 0.61/0.66/0.69 N/A Categorical variables are reported as frequency percent (count). Quantitative variables are reported as 1^st^quartile / median / 3^rd^quartile. :::	PubMed Central	2024-06-05T03:55:54.056677	2005-3-7	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554993/", "journal": "BMC Med Genet. 2005 Mar 7; 6:9", "authors": [ { "first": "Richard H", "last": "Reneland" }, { "first": "Steven", "last": "Mah" }, { "first": "Stefan", "last": "Kammerer" }, { "first": "Carolyn R", "last": "Hoyal" }, { "first": "George", "last": "Marnellos" }, { "first": "Scott G", "last": "Wilson" }, { "first": "Philip N", "last": "Sambrook" }, { "first": "Tim D", "last": "Spector" }, { "first": "Matthew R", "last": "Nelson" }, { "first": "Andreas", "last": "Braun" } ] }
PMC554994	Background ========== Amplified fragment length polymorphism (AFLP) is a DNA fingerprinting technique using electropherograms \[[@B1]\]. AFLP analysis belongs to the category of selective restriction fragment amplification techniques, which are based on the ligation of adapters to genomic restriction fragments followed by PCR-based amplification with adapter-specific primers \[[@B2]\]. This technique has been widely used for genotyping since it requires no prior knowledge of genomic DNA sequences and offers potentially better discriminatory power and speed than the existing techniques for fingerprinting such as random-amplified polymorphism DNA markers (RAPD) \[[@B3]-[@B8]\]. However, it has only been used to a limited extent for expression analysis \[[@B9]\]. The main problems with the comparison of AFLP patterns are (i) variation in peak height, and (ii) false positive peaks which often overlap with real peaks, probably due to differences in PCR efficiency \[[@B5],[@B10]\]. There is room for tuning selective PCR amplification \[[@B8]\]. Recently, we developed an AFLP-based gene expression profiling method called HiCEP (High Coverage Expression Profiling) \[[@B11]\]. The experimental and analytical procedures are essentially the same as those of AFLP, i.e., the technique is based on the selective PCR amplification of restriction fragments from a total restriction digest of genomic DNA. Refinements of the selective PCR technique improved reproducibility and reduced the rate of false positive peaks as well as the number of peaks. They also enabled the digestion of purified genomic DNA with two four-nucleotide recognition restriction enzymes, having a higher cutting frequency, such as MspI and MseI. Consequently, the HiCEP method can detect a slight expression change of transcript-derived fragments (TDFs) with high coverage. The estimated 30,000 transcripts expressed in a cell are divided into 256 subgroups (16 MspI-NN primers \* 16 NN-MseI primers) containing approximately 120 PCR-amplified TDFs. This number is small enough to be separated by fluorescent capillary electrophoresis using an automated DNA sequencer such as the ABI Prism 310 (Applied Biosystems). We can achieve higher throughput by using several fluorescent dyes at once \[[@B14],[@B15]\]. Normally, digitized electropherograms are imported into image analysis software such as GeneScan (Applied Biosystems), which outputs each fragment (band) together with its length (in bp), area and height (signal intensity), carrying out accurate fragment sizing and background subtraction for most of the operations. GeneScan is capable of separating the signal from each fluorophore to provide higher throughput analysis. However, it should be noted that intense signals from abundant TDFs can breed into each other, potentially confusing the fragment sizing \[[@B7],[@B15]\]. Furthermore, the use of a frequently matching 4-bp cutting endonuclease (MseI) tends to produce many small TDFs (\< 100 bp) and in our experience this range is prone to errors of fragment sizing. Cumulative errors of fragment sizing interfere with normalization across different electropherograms and lead to the mis-assignment of valid TDFs. Hence, more detailed analysis such as observation of gradual expression changes in the time series of a TDF still counts in subjective visual examination \[[@B11]\]. Further preprocessing of the electrophoretic data to be compared, each of which is independently normalized according to molecular weight standards, is needed. The purpose of the present study is to develop a normalization method for the automated analysis of temporal electrophoretic data. We assume the samples to be compared are identical, that TDFs have similar fragment lengths across electropherograms and that expression changes can be detected as variations in peak height using the HiCEP technique. The performance of the method is demonstrated by analyzing a large set of time-course data obtained from mouse embryonic stem (ES) cells, using HiCEP. Results and discussion ====================== We analyzed a total of 2560 HiCEP electropherograms (256 sets of ten), containing time-course data of embryonic stem (ES) cells 0, 12, 24, 48, and 96 h after adding stimulation for differentiation. Reproducibility was confirmed by the duplication. We applied the current method to each of the 256 sets. Delineation of quality profiles for lanes ----------------------------------------- When a set of electrophoretic data is arranged and surveyed, one can often find ranges (called \'dissimilar ranges\') in which peak fragment lengths are incorrectly measured. For example, in Fig. [1a](#F1){ref-type="fig"} three lanes (0 h-1, 12 h-1, and 48 h-2) in the range (35--50 bp) appear to be compressed on the short side. This is probably because another intense peak just under 35 bp is mistaken for the 35 bp marker peak. This reduces the overall similarity between lanes and makes it difficult to recognize identical TDFs such as red filled peaks in Fig. [1a](#F1){ref-type="fig"}. To this end, we first developed a method for displaying dissimilar ranges. The method is based on a moving-fragment approach that continuously determines the average correlation coefficient between particular lane P^target^and the other lanes within a certain range using equation 3. By using the average correlation coefficients, we can make a quality score function Q^k^(t) for all lanes (k= 1, 2, \..., 10) at arbitrary length t(see Methods). An example of the calculation for lane 0 h-1is shown in Table [1](#T1){ref-type="table"}. The \'quality profiles\' delineated from Q(t) take the place of detailed visual evaluation of dissimilar ranges (Fig. [1b](#F1){ref-type="fig"}). Undoubtedly, false peaks must have been used incorrectly at 35 bp in three lanes (0 h-1, 12 h-1, and 48 h-2) and at 75 bp in two lanes (0 h-2and 96 h-1). Detection of dissimilar ranges ------------------------------ Next, we applied a simple method for the automated detection of dissimilar ranges to 256 sets of electrophoretic data (see Method). The method identified a total of 362 dissimilar ranges. Most (289, 79.8%) of the ranges were of 100 bp or less. This is reasonable because the main source of fragment sizing errors is the presence of intense peaks near the marker \[[@B7],[@B11],[@B15]\] and the HiCEP technique tends to produce short fragments. In fact, of a total of 222,108 detected peaks in the range (35--700 bp) analyzed by GeneScan, 58,988 (26.6%) were \< 100 bp. Visual examination revealed many of those ranges to be genuine, but not all. The set of ten electropherograms shown in Fig. [1](#F1){ref-type="fig"} is a good example. Our method identified seven ranges as dissimilar: five lanes (0 h-1, 0 h-2, 12 h-1, 12 h-2, and 48 h-2) in range (35--50 bp) and two lanes (0 h-2and 96 h-1) in range (50--100 bp). Of these, we at first suspected that two lanes (0 h-2and 12 h-2) in range (35--50 bp) were false-positives (mistakenly identified as dissimilar). However, we observed that the range in the two lanes is worthy of being normalized: the fragment lengths on the short side of the range deviate gradually from the mean lengths of lanes 24 h-1, 24 h-2, 96 h-1, and 96 h-2\[see [Additional file 1](#S1){ref-type="supplementary-material"}\]. Visual examination of all the electropherograms did not reveal any false-negative errors (overlooked dissimilar ranges). Recall that the samples to be compared are identical and that the measure of the quality of fragment sizing is based on a calculation of the average correlation between electropherograms. These results suggest that the normalization strategy we present here is useful, especially for temporal expression analysis. The effectiveness of the method depends on the choice of the parameter Tin equation 3 in the Methods section, which is the number of consecutive fragments making up the quality profile examined by the program. Quality profiles using the shortest span (T= 1) are noisier than those using a moderate span, and runs using spans of less than four fragments were found unsatisfactory in our investigation. On the other hand, long spans (T= 10) tended to miss small dissimilar ranges. These trends are essentially the same as those in the delineation of hydropathy plots of proteins using a moving-window approach and in the detection of transmembrane regions \[[@B16]\]. Although we set T= 5 throughout the analysis, further improvement in the choice of parameters as well as the method for the detection of dissimilar ranges remains to be studied. Normalization of dissimilar ranges ---------------------------------- To normalize dissimilar ranges across a set of electropherograms, it is necessary to select one as a reference. In conventional algorithms the reference is selected manually \[[@B17],[@B18]\]. For reproducible automated normalization, it is vital that the choice be objective. Our method selects the lane (electropherogram) having the highest average quality score in a given dissimilar range. In the case of Fig. [1](#F1){ref-type="fig"}, our method selects 96 h-2as the best reference in ranges (35--50 bp) and (50--100 bp). We cannot, of course, reject the possibility that accurate fragment sizing is performed in the minority group (such as lanes 0 h-1, 12 h-1, and 48 h-2in range (35--50 bp) in Fig. [1](#F1){ref-type="fig"}), but it is natural that the best reference should be selected from lanes in the majority group. We prepared two models for accurate normalization of various types of fragment sizing errors. Model 1 is the case of an incorrect fragment sizing at the shortest (or longest) marker peak. Figure [2](#F2){ref-type="fig"} shows an example of normalization using Model 1. The best approximating profile (normalized profile) is determined by considering various combinations of candidates from D× 100% expansion (or - D× 100% compression) to D× 100% compression of the short side of the original profile at intervals of dbp. The best approximating profile is one of the candidate profiles with {x× d- D× (C~e~- C~s~)} / (C~e~- C~s~) × 100% compression of the side in a given range (C~s~- C~e~bp), where x= {0, 1, \..., 2 × (C~e~- C~s~) × D/ d}. There is of course a trade-off between the computation time and the normalization accuracy in the choices of parameters. In Model 1, we set D= 0.4 and d= 0.2. We expected that the normalization would be achieved by a linear expansion of the short side of the dissimilar range (35--50 bp) by anchoring the long-side in the target lane 12 h-1. Indeed, the best approximating profile that achieved the highest correlation coefficient against the reference 96 h-2was the case of x= 9 (28% expansion). Figure [3](#F3){ref-type="fig"} shows an example of normalization using Model 2. Model 2 is the case of an incorrect fragment sizing at the marker length M~j~in a dissimilar range (M~j-1~-M~j+1~bp) (see Methods). Accordingly, the program can easily determine the length of 75 bp because there is only one marker length inside of the range (50--100 bp). We can directly apply the normalization procedure for Model 1 to Model 2 by considering two hypothetical dissimilar ranges, (50--75 bp) and (75--100 bp). The main difference from Model 1 is that the two ranges cannot be normalized independently in Model 2: {x× d- D× (100 - 50)}/(100 - 50) × 100% compression (resp. expansion) of the long-side of the original profile in range (50--75 bp) and {x× d- D× (100 - 50)}/(100 - 50) × 100% expansion (resp. compression) of the short side in range (75--100 bp) affect on each other. In Model 2, we set D= 0.1 and d= 0.2 as a maximal realistic displacement. The best approximating profile is the case of x= 13 and is consistent with the reference profile. Figure [4](#F4){ref-type="fig"} shows the result of normalization for electrophoretic patterns in the primer combination of Fig. [1](#F1){ref-type="fig"}. Seven dissimilar ranges (coloured in red; five in range (35--50 bp) and two in range (50--100 bp); 0 h-2has two normalized ranges) are normalized nearly perfectly (Fig. [4a](#F4){ref-type="fig"}). For example, the electrophoretic pattern of 0 h-2in range (35--50 bp) which is a possible false-positive error are normalized as 2.7% compression of a short side of the range. The correlation coefficients between the target 0 h-2and the reference 96 h-2in the range before and after normalization are 0.674 and 0.798, respectively. A quality profile for lane 48 h-2indicates that an incorrect normalization is performed in range (35--50 bp) of the lane. The low correlation coefficient (0.4) between the normalized profile and the reference 96 h-2in the range, compared to values (\> 0.7) between four other normalized profiles (0 h-1, 0 h-2, 12 h-1, and 12 h-2) and the reference in the corresponding range, strengthens this suspicion \[see [Additional file 2](#S2){ref-type="supplementary-material"}\]. After visual examination it was decided that the dissimilar range (35--50 bp) of lane 48 h-2should be extended on the long side. We searched for the best range to be normalized and chose (35--53.6 bp). The correlation coefficient of the normalized profile, expanded by 26.3% on the short side in the range (35--53.6 bp), was 0.9. Undoubtedly an exhaustive search for edges in dissimilar range might yield better normalization for some cases. However, it also dramatically increases the possible combinations of normalization candidates. It is a balance between the computation time and the number of analyzable TDFs. One way to do objective evaluation of normalized electrophoretic patterns is to re-delineate the quality profiles (Fig. [4b](#F4){ref-type="fig"}). Generally, a higher quality score Q^k^(t) for lane kindicates greater consistency with the other lanes around arbitrary length tif the sample is identical (e.g., time-course data). The quality scores after normalization overall were higher than before (Figs. [1b](#F1){ref-type="fig"} and [4b](#F4){ref-type="fig"}). This means the assignments of the quality scores to time-course electrophoretic data are effective for evaluating reproducibility. Evaluation of the method ------------------------ The normalization method we propose here can be regarded as an image warping method which deforms images by mapping between image domains \[[@B19]\]. There are a number of reports on warping methods especially for dealing with two-dimensional (2-D) images \[[@B19]-[@B21]\]. There are also some methods for 1-D electrophoretic data \[[@B17],[@B18],[@B22]\]. Comparison with these methods might provide an objective evaluation of the current method. However, they are not directly comparable with the current method because of different frameworks such as input data format, the requirement of pre-determined parameters, and so on \[[@B17],[@B22]\]. A critical step in the analysis of 1-D electrophoretic data is the assignment of the correct size to each TDF. In time-course data, one expects that the same TDFs should have quite close fragment lengths across electropherograms and that temporal expression changes are reflected as differences in peak height. We developed the current method aimed at temporal expression analysis by the electrophoretic method and used a scoring system for an objective evaluation of experimental reproducibility using Q^k^(t) which indicates a relative similarity at t(bp) in lane kto the other lanes. We demonstrate two other sets of electrophoretic data and discuss the feasibility of the method. Figure [5](#F5){ref-type="fig"} shows a set of electrophoretic patterns and quality scores which is different from the primer combination used in Figs. [1](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}. This is a representative example of electrophoretic patterns with high quality scores (arbitrary defined as \> 0.7). Visual evaluation confirmed the reproducibility of the set of ten electrophoretic patterns throughout the analyzed range (35--700 bp). There is, of course, no dissimilar range detected by the current method. We should demonstrate the case of normalization to dissimilar range (35--75 bp) where both Models 1 and 2 are applicable. A set of ten electrophoretic patterns and their quality scores shown in Figure [6](#F6){ref-type="fig"} is the good example. There are three lanes with dissimilar range (24 h-2, 48 h-2, and 96 h-1) detected by the method. Of these, 24 h-2and 96 h-1were normalized using Model 1 and 48 h-2was normalized using Model 2. Visual evaluation of the electrophoretic patterns and the quality scores after normalization verified the choices of the models as appropriate (Figure [7](#F7){ref-type="fig"}). The use of normalized electrophoretic patterns facilitates the identification of TDFs (e.g., red filled fragments in Fig. [7](#F7){ref-type="fig"}) having potential temporal expression change. The development of a peak alignment algorithm for multiple lanes and integration with the current method are the next challenge. We also estimated the feasibility of the method with regard to an increasing number of peaks with certain quality score or more. The minimum value of Q(t) necessary for the accurate alignment of valid TDFs across lanes is about 0.7 (Fig. [4b](#F4){ref-type="fig"}). Accordingly, we set the threshold to be 0.7. The number of peaks with Q(t) ≥ 0.7 in the range (35--700 bp) before and after normalization are 202,204 (91.0% of the total number of peaks in the range detected by GeneScan) and 205,829 (92.7%), respectively. Furthermore, 3,334 (92%) of the 3,625 (= 205,829 - 202,204) new high-quality peaks were \< 100 bp, which corresponds to the biased distribution of the detected dissimilar ranges (nearly 80% of which were 100 bp or less). Conclusion ========== When we apply the method to HiCEP time-course data, we assume that the set of electrophoretic data to be compared is identical (i.e., corresponding TDFs across electropherograms should have nearly the same fragment lengths). The monitoring of temporal expression change by the HiCEP technique has great potential for screening of genes related to chemotherapeutic drug resistance, circadian rhythm, and so on \[[@B11],[@B23],[@B24]\]. Although the current method was developed for pre-processing HiCEP data, the algorithm is easily applicable to the processing of other 1-D electrophoretic data such as AFLP and DD if the samples are identical or nearly identical. We strongly recommend the strategy be widely used for data processing for temporal expression analysis by the electrophoretic method. Methods ======= Samples ------- mRNAs were prepared from mouse embryonic stem (ES) cells at 0, 12, 24, 48, and 96 h after removal of Leukemia Inhibitory Factor (LIF) from the culture medium. The samples subjected to HiCEP reaction were duplicated. We designated each sample as 0 h-1, 0 h-2, 12 h-1, 12 h-2, 24 h-1, 24 h-2, 48 h-1, 48 h-2, 96 h-1, and 96 h-2. HiCEP analysis -------------- mRNAs prepared from each sample were digested with two 4-bp-cutting endonucleases (MspI combined with MseI) and ligated with the corresponding adaptors. The resulting HiCEP templates, MspI-MseI-poly(A) mRNAs, were amplified by fluorescently labelled primers; for labelling, FAM, HEX, and NED were used. In total, 256 primer combinations (16 MspI-NN primers combined with 16 NN-MseI primers; N = {A, C, G, T}) were used in the HiCEP analysis. For example, a primer combination of MspI-TA and GC-MseI is capable of amplifying particular transcript-derived fragments (TDFs) corresponding to that combination. The details of the protocol of the HiCEP reaction are described elsewhere \[[@B11]\]. An animation of the principle is provided at the following URL <http://133.63.22.11/english/research/serch03.html>. Electrophoresis and image analysis ---------------------------------- The PCR products were denatured and loaded on an ABI Prism 310 (Applied Biosystems) for capillary gel electrophoresis. The digitized images were analyzed by the GeneScan software (Applied Biotech). The size of the fragments was calculated by the software, according to internal molecular size markers of 35, 50, 75, 100, 139, 150, 160, 200, 300, 340, 350, 400, 490, 500, 600, and 700 bp, on each gel. The fragment sizing and baseline subtraction were performed by the software. The software quantifies each peak by the fragment length L(in bp), peak height H, and area A(in arbitrary units). Accordingly, the subsequent normalization procedure accepts these three-tuples as input for detected TDFs between 35 bp and 700 bp. TDFs smaller than 35 bp or larger than 700 bp were omitted from the analysis because the range was outside the size calibration range. Delineation of quality scores for lanes --------------------------------------- The starting point of normalization is a set of lanes (10 time-course measurements; 0, 12, 24, 48, and 96 h, each experiment duplicated) in each of 256 primer combinations. We explain the procedure using data from the primer combination of \'MspI-CT combined with tt-MseI (designated as CT-tt)\' because the ten electropherograms have some ranges for which fragment sizing is obviously inappropriate (we therefore designated such ranges as \"dissimilar ranges\"). The first step starts from the Gaussian approximation of each lane. The use of the approximating lane is the same as described in Aittokallio et al. \[[@B25]-[@B27]\]. Briefly, a fragment F~i~in lane Pis originally characterized by the three-tuples (L~i~, H~i~, A~i~). If lane Pconsists of nfragments ![](1471-2105-6-43-i1.gif), the approximation of the lane at length tis given by: ![](1471-2105-6-43-i2.gif) where σ~i~is obtained from the following equation: ![](1471-2105-6-43-i3.gif) The approximation is performed independently for each lane. The ten approximate profiles of time-course data in the primer combination of CT-ttare shown in Fig. [1a](#F1){ref-type="fig"}. For the automated identification of \'dissimilar ranges\' from the expression profiles of ten lanes ![](1471-2105-6-43-i4.gif), we next assign quality scores to each of the fragments ![](1471-2105-6-43-i5.gif), where the fragments are originally numbered with respect to their lengths. By using the ten approximate profiles, relative similarity scores ![](1471-2105-6-43-i6.gif) for intervals from fragment ito fragment (i+T-1) (i= 1, 2,\..., n- T+ 1) in lane P^target^(target= {1, \..., 10}) are calculated from the following equation: ![](1471-2105-6-43-i7.gif) where ![](1471-2105-6-43-i8.gif) is the Pearson correlation coefficient between the target lane P^target^and one of the other lanes P^k^in the interval (start-endbp) which always includes Tfragments from fragment ito fragment (i+T-1) (i= 1, 2, \..., n-T+1). The interval is defined as: start= L~i~- 2.5σ~i~and end= L~i+T-1~+ 2.5σ~i+T-1~. In this analysis, the number of fragments Tis held constant at T= 5; other numbers are of course possible. By applying a moving window of Tfragments, most of the fragments (n-T+2 fragments in this case, with the exception of F~1~, F~2~, F~3~, F~4~, F~n-3~, F~n-2~, F~n-1~, and F~n~) have Trelative similarity scores. Finally, the relative quality value Q(L~i~) for fragment F~i~is defined as the average of the similarity scores which satisfy start≤ L~i~≤ end. An example of the calculation is given in Table [1](#T1){ref-type="table"}. Quality scores at arbitrary lengths t, Q(t), are interpolated by the use of cubic splines to ![](1471-2105-6-43-i9.gif). The procedure is applied to each of the ten lanes ![](1471-2105-6-43-i4.gif) and then the quality profiles ![](1471-2105-6-43-i10.gif) corresponding to the expression profiles are created (Fig. [1b](#F1){ref-type="fig"}). The quality profiles delineated from Q(t) have a clear interpretation. The high (or low) score for Q^k^(t) in lane kindicates a high (or low) level of relative similarity between the lane and the others around the length t. Detection of dissimilar ranges ------------------------------ Now we have information (quality profiles) for the automated detection of dissimilar ranges. Here we adopt a simple method for detecting the range. Briefly, 1\) Seek \'seed\' ranges (C~seed\_s~- C~seed\_e~bp) which satisfy two conditions: a) Q(t) ≤ thres~seed~and b) they contain at least two peaks. 2\) Seek C~tmp\_s~which satisfies both ![](1471-2105-6-43-i11.gif) and C~tmp\_s~\<C~seed\_s~; similarly, C~tmp\_e~, ![](1471-2105-6-43-i12.gif) and C~tmp\_e~\<C~seed\_e~ 3\) Substitute the nearest marker length ![](1471-2105-6-43-i13.gif) (in this case, M~1~= 35, M~2~= 50, \..., ![](1471-2105-6-43-i14.gif) = 700) to C~tmp\_s~(resp. C~tmp\_e~) for C~s~(resp. C~e~); accordingly, both C~s~and C~e~= ![](1471-2105-6-43-i13.gif) and C~s~\<C~e~ Aparameter thres~seed~is set to 0.3 empirically. Forexample, P^9^has the following parameters in Fig. [1b](#F1){ref-type="fig"}: C~seed\_s~= 57.04, C~seed\_e~= 89.98, C~tmp\_s~= 52.60, C~tmp\_e~= 104.60, C~s~= M~2~, and C~e~= M~4~. Although fine tuning might be necessary, the procedure enables us to display dissimilar ranges. Selection of the reference lane ------------------------------- When we want to correct a dissimilar range (C~s~- C~e~bp), we have to select the \"reference\" (a kind of mean or typical profile in the corresponding range). One method is to choose lane P^reference^satisfying max {![](1471-2105-6-43-i15.gif)}, where ![](1471-2105-6-43-i15.gif) is the average of Q^k^in the range (C~s~- C~e~bp) in lane ![](1471-2105-6-43-i4.gif). For example, the algorithm selects P^10^(i.e., 96 h-2) as a reference in a particular range (M~1~-M~2~bp) and also in range (M~2~-M~4~bp). Two models for the normalization of dissimilar ranges ----------------------------------------------------- The meaning of the word \"normalization\" here is to correct the fragment lengths (L) and the areas (A) of peaks in a dissimilar range so that the similarity between the normalized electrophoretic pattern and the reference pattern in the corresponding range can be maximized. To normalize a particular lane P^target^against the reference P^reference^, we now consider the following two models. Model 1 is the case of an incorrect fragment sizing at the shortest (or longest) marker peak, i.e, C~s~= M~1~= 35 (or C~e~= ![](1471-2105-6-43-i14.gif) = 700). The peak lengths deviate more and more from the reference length moving from C~e~to C~s~(or from C~s~to C~e~). Model 2 is the case of an incorrect fragment sizing near marker length M~j~(C~s~\<M~j~\<C~e~, j= {2, 3,\..., n~M~- 1}; the inside of dissimilar range (C~s~- C~e~bp)). Roughly, the deviation of peak lengths from the reference length gradually increases starting from C~s~; the maximum deviation is reached at M~j~(C~s~\<M~j~\<C~e~); the deviation decreases gradually; and finally disappears at C~e~bp. Normalization is performed by either expanding or compressing. Consider, for example in Model 1, normalization for the expression profile of P^3^(12 h-1) in range (M~1~-M~2~bp) against the reference P^10^(96 h-2). Undoubtedly, the profile displays a systematic deviation from the reference. The degree of the deviation gradually increases starting from M~2~bp to M~1~bp probably because an intense peak generated near the shortest marker peak for the correction of M~1~bp is used mistakenly. We expect the normalization will be achieved by a linear expansion of the short side (M~1~) of the range (M~1~-M~2~bp) by anchoring the long side. The best approximating profile is found by considering various combinations of normalization candidates starting from D× 100% expansion to D× 100% compression of the short side at intervals of dbp. We set D= 0.4, as a maximal realistic displacement and d= 0.2. Accordingly, in practice, the number of combinations is 2 × (C~e~- C~s~) × D/d+ 1 (for example, there are 61 combinations of normalization candidates in the range (M~1~-M~2~bp)) in Model 1. For each combination x(x= {0, 1, \..., 2 × (C~e~- C~s~) × D/d)}, we make a candidate profile P~x~by changing three parameters (L~i~, A~i~, and σ~i~) accompanied by fragments (F~i~) in the dissimilar range (C~s~- C~e~bp), according to the level of correction (expansion or compression). Those parameters are calculated as follows: ![](1471-2105-6-43-i16.gif) ![](1471-2105-6-43-i17.gif) ![](1471-2105-6-43-i18.gif) Candidates are made by substituting these transformed three-tuples ![](1471-2105-6-43-i19.gif) in a given range (C~s~- C~e~bp) into eq. (1). The best approximate profile is the one that achieves the highest correlation coefficient between P^reference^and P~x~(x= {0, 1, \..., 2 × (C~e~- C~s~) × D/ d}) in the range (C~s~- C~e~bp). In the normalization for the expression profile P^3^in the range (M~1~-M~2~bp) against the reference P^10^, the best normalized profile by our method matches well with the reference (Fig. [2](#F2){ref-type="fig"}). A good example of Model 2 is the expression profile P^2^(0 h-2) in range (M~2~-M~4~bp) with the reference P^10^(96 h-2); there is no possibility of Model 1 (C~s~≠ M~1~) and the number of incorrect marker lengths is only one (M~3~). Model 2 is a mixture of Model 1. The normalization is also done by one of the {2 × (C~e~- C~s~) × D/ d+ 1} combinations starting from D× 100% compression of the long side in (M~2~-M~3~bp) and D× 100% expansion of the short side in (M~3~-M~4~bp) to D× 100% expansion of the long side in (M~2~-M~3~bp) and D× 100% compression of the short side in (M~3~-M~4~bp) at intervals of d(= 0.2) bp. Unlike Model 1, we set D= 0.1 as a maximal realistic displacement. In the normalization for the expression profile P^2^in the range (M~2~-M~4~bp) against the reference, the best normalized profile by our method is matches well with the reference (Fig. [3](#F3){ref-type="fig"}). It should be noted that when a dissimilar range (M~j~-M~j+l~bp) is very wide (j= 1, 2,\..., n~M~- l; l≥ 3), there are two or more possibilities for incorrect marker lengths in Model 2. Of these cases, we only consider cases with j= 1 in Model 1 because such cases are the only realistic ones. For the remaining cases (j= 2,\..., n~M~- l; l≥ 3), the experiment should be redone rather than trying to normalize them by considering numerous possibilities. It should also be noted that there is a case of a dissimilar range (M~1~-M~3~bp) to which both Models 1 and 2 are applicable. In this case, the best approximate profile is decided by comparing the two best possible profiles determined using Models 1 and 2. Authors\' contributions ======================= KK invented the method and wrote the paper. RF made critical comments in light of the HiCEP experimental technique. JJR edited the paper. RA and MA provided critical comments and led the project. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Magnified electrophoretic patterns and the quality profiles in range (35--50 bp) inFig. [1](#F1){ref-type="fig"}. Descriptions are the same as those in Fig. [1](#F1){ref-type="fig"}. Detailed observation of the dissimilar range for two lanes (0 h-2and 12 h-2) confirmed the identification. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Magnified electrophoretic patterns and the quality profiles in range (35--50 bp) inFig. [4](#F4){ref-type="fig"}. Descriptions are the same as those in Fig. [4](#F4){ref-type="fig"}. Visual evaluation confirmed the validity of the normalizations (2.7% compression of the short side of the range) for two lanes (0 h-2and 12 h-2) which are suspected false-positive errors. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This work was supported by a Research Revolution 2002 on Innovative Development Project grant. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### *Electrophoretic patterns and the quality profiles for ten lanes from a primer combination of CT-tt**. Samples are mouse embryonic stem (ES) cells 0, 12, 24, 48, and 96 h after differentiation. There are ten lanes since each sample are duplicated. From bottom to top: 0 h-1, 0 h-2, 12 h-1, 12 h-2, 24 h-1, 24 h-2, 48 h-1, 48 h-2, 96 h-1, and 96 h-2. Data from a primer combination of CT-ttin the interesting range (35--102 bp) are shown. (a) The approximated electrophoretic lane data and, (b) its interpolated quality profile. An example of calculation of quality profiles for lane 0 h-1is shown in Table 1. Note the variation in the lengths of particular TDFs across peaks of lanes (red filled peaks). ::: ![](1471-2105-6-43-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Normalization for lane 12 h-1in dissimilar range (35--50 bp) in Fig. 1 (Model 1). Magnified expression profiles of the target 12 h-1and the reference 96 h-2in the range in Fig. 1 are shown (top). Colours are the same as those in Fig. 1. There are 61 possible combinations in this case: 30 different levels of expansion (x= 0, 1, \..., 29), the original target profile (x= 30), and 30 compressions (x= 31, 32, \..., 60). The highest correlation coefficient between the best approximating profile and the reference in range (3---50 bp) was 0.844 for the case x= 9. The position of xon the X axis corresponds to the new position of the short side (originally, 35 bp) of the original profile after expansion or compression. For example, the new position of the short side after maximum expansion (x= 0) becomes 29 bp, while after maximum compression (x= 60) it becomes 41 bp. Visual evaluation of three representative approximate profiles (x= 0, 9, and 60) in range (35--50 bp) confirmed the validity of the normalization (bottom). ::: ![](1471-2105-6-43-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Normalization for lane 0 h-2in the dissimilar range (50--100 bp) in Fig. 1 (Model 2). Magnified expression profiles of the target 0 h-2and the reference 96 h-2in range (50--100 bp) are shown (top). Colours are the same as those in Fig. 1. There are 51 possible combinations in this case. The highest correlation coefficient between the best approximating profile and the reference in the range (50--100 bp) was 0.911 for the case x= 13. Visual evaluation of three representative approximate profiles (x= 0, 13, and 50) in the range confirmed the validity of the normalization (bottom). ::: ![](1471-2105-6-43-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Electrophoretic patterns and the quality profiles after normalization in Fig. 1. (a) Normalized electrophoretic patterns. Ranges coloured in red were detected as dissimilar and normalized. Note that the 0 h-2consists of two dissimilar ranges: (35--50 bp) and (50--100 bp). After normalization the valid (red filled) peaks are much closer together. (b) Consequently, the more accurate fragment lengths and peak areas in the ranges are accompanied by an increase in the quality scores. ::: ![](1471-2105-6-43-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Reproducible electrophoretic patterns and the quality profiles for ten lanes. Data from a primer combination of AA-gcin the interesting range (35--155 bp) are shown. (a) The electrophoretic data lane and (b) its quality profile. ::: ![](1471-2105-6-43-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Electrophoretic patterns and the quality profiles for ten lanes from a primer combination of GA-gc**. Data from a primer combination of GA-gcin the interesting range (35--80 bp) are shown. (a) The electrophoretic data lane and (b) its quality profile. Three lanes (24 h-2, 48 h-2, and 96 h-1) have a dissimilar range (35--75 bp) suitable for both normalization Models 1 and 2. ::: ![](1471-2105-6-43-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Electrophoretic patterns and the quality profiles after normalization in Fig. 6. (a) Normalized electrophoretic patterns. Ranges coloured in red were detected as dissimilar and normalized. After normalization the valid (red filled) peaks are much closer together. ::: ![](1471-2105-6-43-7) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Calculation of quality scores for lane 0 h-1in Fig. 1. Similarity scores (S) are computed using Equation 3. The quality score at fragment length L~4~is calculated as (0.00 + 0.17 + 0.04 - 0.27)/4 = -0.02. The quality profile for each lane is made by spline interpolation of a set of quality scores of fragment lengths of peaks in the lane. ::: i* L~i~ σ~i~ S^1^in size interval \[L~i-j~- 2.5 σ~i-j~, L~i-j+4~+ 2.5 σ~i-j+4~\] Q(L~i~) ----- ---------- ---------- ----------------------------------------------------------------------------------------------- --------------- ------- ------- ------- ------- 1 38.28 0.145 -0.27 \- \- \- \- -0.27 2 39.16 0.143 0.04 -0.27 \- \- \- -0.11 3 40.17 0.186 0.17 0.04 -0.27 \- \- -0.02 4 41.06 0.212 0.00 0.17 0.04 -0.27 \- -0.02 5 42.07 0.223 0.02 0.00 0.17 0.04 -0.27 -0.01 6 44.05 0.221 -0.36 0.02 0.00 0.17 0.04 -0.03 7 45.13 0.190 -0.28 -0.36 0.02 0.00 0.17 -0.09 8 46.05 0.149 0.52 -0.28 -0.36 0.02 0.00 -0.02 9 46.80 0.215 0.59 0.52 -0.28 -0.36 0.02 0.10 10 48.48 0.240 0.74 0.59 0.52 -0.28 -0.36 0.24 11 49.41 0.242 0.69 0.74 0.59 0.52 -0.28 0.45 12 54.95 0.320 0.49 0.69 0.74 0.59 0.52 0.61 13 56.09 0.215 0.30 0.49 0.69 0.74 0.59 0.56 14 57.54 0.152 0.39 0.30 0.49 0.69 0.74 0.52 15 59.00 0.195 0.41 0.39 0.30 0.49 0.69 0.45 16 60.05 0.252 0.39 0.41 0.39 0.30 0.49 0.40 17 62.41 0.222 0.32 0.39 0.41 0.39 0.30 0.36 18 67.55 0.135 0.43 0.32 0.39 0.41 0.39 0.39 19 68.26 0.259 0.47 0.43 0.32 0.39 0.41 0.40 20 69.76 0.216 0.57 0.47 0.43 0.32 0.39 0.43 :::	PubMed Central	2024-06-05T03:55:54.060625	2005-3-6	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554994/", "journal": "BMC Bioinformatics. 2005 Mar 6; 6:43", "authors": [ { "first": "Koji", "last": "Kadota" }, { "first": "Ryutaro", "last": "Fukumura" }, { "first": "Joseph J", "last": "Rodrigue" }, { "first": "Ryoko", "last": "Araki" }, { "first": "Masumi", "last": "Abe" } ] }
PMC554995	Background ========== Child labour covers all economic activities carried out by children regardless of their occupational status \[[@B1]\]. It has probably being in existence almost as long as the history of mankind. Economic activity is a broad concept that encompasses most productive activities of children. It includes both work that is permissible under the International Labour Organization\'s (ILO) conventions and that which is not \[[@B2]\]. Child labour is prevalent worldwide, occurring both in developing and developed countries \[[@B3],[@B4]\]. It is estimated that about 352 million children are engaged in some form of economic activity in the world \[[@B2]\]. Estimates of the ILO put the number of children fully at work in developing countries at 120 million and those working and schooling at 250 million \[[@B5]\]. Most of child labour takes place in Asia, the Pacifics and Africa \[[@B6]-[@B9]\]. In most parts of Africa the prevalence ranges from 20% to 54% \[[@B10]\]. The prevalence in Nigeria, Cote d\'Ivoire and Zambia ranges from 20% to 30% \[[@B10]\]. Very few Nigerian studies \[[@B6]\] provide information on children who both school and work. Some studies \[[@B11]\] from other parts of the world actually excluded schooling children from the definition of child labour. This study aims to document the prevalence and types of child labour among primary and junior secondary school pupils in Nigeria. Methods ======= This presentation is part of a broader study on child labour among school children in public primary and junior secondary schools in Sagamu Local Government Area of Ogun State, Nigeria. The study was conducted between October 1998 and September 1999 (inclusive). It was approved by the Ethical Committee of the Olabisi Onabanjo University Teaching Hospital Sagamu (OOUTH). Sagamu Local Government Area (SLGA) is one of the 15 Local Government Areas in the State. It is predominantly rural and semi-urban with a land space of 68.4 sq km. The estimated population for the year 1997 is 177,514. Administratively, SLGA is made up of 11 wards. The total school enrolment in the 50 primary and 16 junior secondary schools (JSS) for the year 1997 was 30,597, comprising 21,476 primary and 9,121 JSS pupils. Excluding 6,762 new entrants who did not have academic records for the preceding year left 23,835 pupils (17,891 primary and 5,944 junior secondary school pupils). Thus the ratio of primary to junior secondary school pupil population was about 3 to 1. This ratio was considered while selecting the subjects for the study. Sampling -------- The survey sample was drawn randomly from all 50 public primary and 16 secondary schools in the LGA. Using ballot papers, one primary and one secondary school were randomly selected from each administrative ward. However, one ward had no primary school and four wards had no secondary school. Thus, a total of ten primary and seven secondary schools were selected. There were five classes in each primary school and two in each junior secondary after excluding primary 1 and JSS1 pupils because they were new entrants in their respective schools and therefore did not have academic records for the preceding year. Each class was made up of at least two arms. One arm of each eligible class in the selected schools was randomly selected, also using ballot papers. Altogether, 64 classes were selected: 50 classes from 10 primary schools and 14 classes from 7 secondary schools. Thirty pupils were randomly selected from each class to form the study school. The pupils were interviewed one at a time between the hours of 8.00 am and 2.00 pm on Monday through Friday using a structured, close-ended questionnaire. The interviews were conducted by author MBF with the help of previously trained research assistants. Relevant background information on age, sex, family background, involvement or otherwise in after-school economic activities were obtained. Information on parental education, occupation/income was obtained through requests accompanying letters to parents and guardians asking for their consent. The occupation/income and educational attainments of the parents were used to determine socio-economic index scores of the children using modified criteria described by Oyedeji \[[@B12]\]. Each parent was given two index scores: one for occupation/income and the other for educational attainment. The scores were rated on a scale of 1 to 5, from the more educated and more highly placed occupation/income groups to the least advantaged. The mean of the four scores for both parents was calculated and the value became the assigned socioeconomic group of the family/child. Where the calculated mean was not a whole number, the next higher integer was used. Child labour was defined as any type of economic task, paid, unpaid, or exploitative, engaged in by a child less than 18 years of age, which places the interests of the beneficiary well above those of the child and is detrimental to the physical mental, social, educational and moral development of the child \[[@B1]\]. For the purpose of analysis the study children were grouped into two; those involved in child labour and those who were not. Chi-square analysis was used to compare proportions and probability; (p) values less than 5% (0.05) were accepted as statistically significant. Results ======= A total of 1675 day pupils were recruited into the study. The male/female ratio was 1:1.02. The majority (86.4%) of respondents were of Yoruba extract. Their ages ranged between 5 and less than 18 years. Those in primary schools were 1299 while 376 were in junior secondary schools as shown in Table [2](#T2){ref-type="table"}. One thousand and eighty pupils were involved in child labour giving an overall prevalence of 64.5%. The leading occupational activity was street trading (Figure [1](#F1){ref-type="fig"}). Three hundred and eighty seven girls were engaged in street trading compared to 308 boys (45.7% versus 37.2 %, X = 0.004). No child was involved in bonded labour or prostitution. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### General characteristics of the subjects (n = 1675). ::: ------------------------ ------------------------------------------ *Subjects* [No. of respondents (%)]{.underline} Sex distribution Male 828 (49.4) Female 847 (50.6) Tribe Yoruba 1448 (86.4) Igbo 111 (6.60) Hausa 35 (2.1) Others 81 (4.9) Age distribution 5 to 6 years 50 (3.0) 7 to 16 years 1594 (95.2) 17 years 31 (1.8) Level of education Primary 1299 (77.6) Secondary 376 (22.4) ------------------------ ------------------------------------------ ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Distribution of subjects involved in child labour by gender, education, family religion, custodian and number of children in the family. (n = 1675) ::: ----------------------------------- -------------------------- ------------------------------ -------------- ------- ----------- Involved in child labour Not involved in child labour Total Characteristics no (%) no (%) χ^2^ p-value Gender Male 514 (62.1) 314 (37.9) 828 (100.0) Female 566 (66.8) 281 (33.2) 847 (100.0) 3.91 0.048 Level of schooling Primary 891 (68.6) 409 (31.4) 1299 (100.0) Secondary 189 (50.3) 187 (49.7) 376 (100.0) 42.8 \< 0.0001 Religion Christianity 631 (61.9) 388 (38.1) 1019 (100.0) Islam 445 (68.3) 207 (31.7) 652 (100.0) 6.94 0.008\* Others 4 (100.0) 0 (0.0) 4 (100.0) Custodian Both parents 643 (65.8) 334 (34.2) 977 (100.0) Single father 73 (54.1) 62 (45.9) 135 (100.0) Single mother 196 (65.8) 102 (34.2) 298 (100.0) Other relatives 165 (64.7) 90 (35.3) 255 (100.0) Unrelated guardian 3 (30.0) 7 (70.0) 10 (100.0) 12.74 0.014 No. of children in the family 1 13 (46.4) 14 (53.6) 27 (100.0) 2 or 3 143 (58.6) 101 (41.4) 244 (100.0) 4 or 5 352 (62.7) 209 (37.3) 561 (100.0) 6 or 7 308 (68.6) 142 (31.4) 450 (100.0) 8 or 9 264 (67.2) 129 (32.8) 393 (100.0) 9.13 0.0025\# ----------------------------------- -------------------------- ------------------------------ -------------- ------- ----------- \Comparison was limited to Christians and Moslems because there were very few practitioners of other religions. \# Chi-square analysis for linear trend ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Pattern of child labour among study children. ::: ![](1472-698X-5-2-1) ::: As shown in Figure [2](#F2){ref-type="fig"}, child labour was most prevalent among children 9 to 14 years of age (10.2% to 13.0% of all the children involved in child labour). Table [2](#T2){ref-type="table"} shows the distribution of the children according to gender, educational level, family religion, custodian and number of children in the family. Girls, primary school pupils and Moslem children were all significantly involved in child labour than their counterparts (p = 0.048, 0.001 and 0.008 respectively). Comparison was limited to Christians and Moslems because there were very few practitioners of other religions. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Age distribution of children involved in child labour. ::: ![](1472-698X-5-2-2) ::: Over 80% of the study children lived with one or both parents, 15.2% with other relatives and very few (0.3%) with unrelated guardians. Children living with parents (both or single) and relatives had higher prevalence rates of child labour. The number of children living with unrelated guardians was relatively small but this group had less than 50% the prevalence of child labour of their counterparts living with relatives (χ^2^= 5.22, p \<0.025). The prevalence of child labour did not vary significantly among children living with different types of relatives (p \> 0.05). A significant rising trend of involvement in child labour with increasing number of children in the family (χ^2^for trend = 9.13, p = 0.0025) was observed. As many as 82.8% of working children were assigned economic tasks by one or both parents. Also 89.8% of the children performed these tasks in order to contribute to the family economy for feeding and for school fees. In Table [3](#T3){ref-type="table"} child labour decreased with increasing parental education. This trend was clearer with respect to maternal education. However, in Table [4](#T4){ref-type="table"}, child labour rates increased with decreasing parental socio-economic status. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Distribution of subjects involved in labour activities according to parental education. ::: Father Mother --------------------------------------------------------------------------------------------- ---------------------- ------------------------------ ---------------------- ------------------------------ [Parental level of education]{.underline}* [no (%)]{.underline} [Odds ratio]{.underline} [no (%)]{.underline} [Odds ratio]{.underline} University graduate or equivalent 30 (35.7) 1.00 14 (45.2) 1.00 Senior secondary school certificate/Teaching or other professional certificate 107 (54.6) 2.16 72 (46.2) 1.04 Junior secondary school certificate/Teacher\'s Grade II certificate/Equivalent 136 (68.7) 3.95 86 (54.8) 1.47 Junior secondary school education/Modern III certificate/Primary school leaving certificate 513 (68.5) 3.91 516 (68.3) 2.62 No formal education/Quranic school/Barely literate/dead parent 227 (67.2) 3.68 329 (68.1) 2.59 χ^2^for trend 29.33 32.86 p \< 0.00001 \< 0.00001 ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Distribution of subjects according to parental socio-economic status. ::: --------------------- -------------------------------- ------------------------------------ ------------------- -------------------- Socioeconomic class Involved in child labour n (%) Not involved in child labour n (%) Total respondents Odds ratio [I]{.underline} [18 (32.7)]{.underline} [37(67.3)]{.underline} [55]{.underline} [1.00]{.underline} II 78 (50.0) 78 (50.0) 156 2.06 III 403 (64.5) 222(35.5) 625 3.73 IV 442 (70.4) 186(29.6) 628 4.88 V 42 (70%) 18 (30%) 60 4.8 --------------------- -------------------------------- ------------------------------------ ------------------- -------------------- χ^2^for trend = 40.87, p \< 0.00001 OR = Odds ratio I = very high II = high III = medium IV = low V = very low ::: Discussion ========== Child labour is a topical issue of global concern but statistics on the subject are often underestimated partly because of practical difficulties and also because of differences in the design and implementation of surveys \[[@B5]\]. The observed prevalence rate of 64.5% found in this study confirms the existence of child labour as an important secondary activity of school children in Sagamu Local Government Area of Nigeria. It is in consonance with high rates reported in Nigeria \[[@B10]\] and other developing countries \[[@B7]\]. The finding is also in keeping with an earlier report from Nigeria \[[@B6]\] and various parts of the world, that children both school and work \[[@B4],[@B9],[@B13]\]. Results of surveys by the ILO in four developing countries found that two-thirds of children combined school with work \[[@B4],[@B15]\]. A rate of 51.9% was found in an earlier study carried out in Nigeria \[[@B16]\], while the rate was about 50 % in both Cairo and Bogotá \[[@B17]\]. The differences in prevalence rate may reflect differences in methodology and data collection. The present study focused on children within the compulsory age of schooling, which is also probably the peak age for child labour. Also, this study unlike previous local ones was school-based. The children were the primary respondents and interviews were conducted away from the influence or interference of parents. There was a higher representation of girls than boys among working children also in accordance with previous observations \[[@B7],[@B12]\]. It is attractive to conjecture that the finding reflects gender bias in upbringing aimed at preparing the girls for traditional roles of small-scale economic trades to boost family economy. It may also be speculated that girls in the traditional African society are more amenable and responsive to parental control and as such are assigned economic tasks more often. The present study considered the role of children in economic tasks within the family enterprise and in and around the home. These activities usually involve more girls than boys in the traditional Nigerian setting. Moslem children were more often involved in child labour than their Christian counterparts. Other studies \[[@B19],[@B20]\] had noted the influence of religious affiliation in the Moslem states of northern Nigeria and commented that wife seclusion (purdah) often puts a great economic burden on children. However, the practice of purdah is not a feature of southern Nigeria Islamism. Thus, the findings cannot be explained on that basis. It may however be speculated that the association of Moslem religion with child labour is related to family size. There was an increasing trend of engagement in child labour with increasing number of children within the family. This observation is consistent with other studies \[[@B21]\]. It may be, as has been suggested by other workers \[[@B22]\], that the desire for large family size is based on potential economic considerations. On the other hand, it may be validly argued, that a large family size, with the attendant spread of lean resources over many dependants, would force parents to engage their children in labour activities. Street trading was the dominant economic activity of working children in the study, agreeing with the findings from other urban and semi-urban settings in Nigeria \[[@B16],[@B17]\]. However in Enugu \[[@B23]\], elsewhere in the country house servants constitute about two-thirds of the work force. Enugu is a major regional headquarters and is predominantly inhabited by the Igbo tribe in contrast to Sagamu LGA with its rural/semi-urban status and predominantly Yoruba population. The social and economic pressures differ and may account in part, for the variations in major economic tasks performed by children. It is comforting that, in contrast to reports from some other developing countries \[[@B3],[@B18],[@B24]\], no child in the present series was bonded or involved in prostitution. These practices are strange to the Ijebu Yoruba, buttressing the well-known influence of culture and tradition \[[@B10]\] on prevalence of child labour. This influence of culture, with respect to child rearing might also partly explain the high prevalence rate of child labour herein reported. The Ijebu Yoruba are known to be very industrious. They get their children introduced to family economic activities rather early. The stress of rural and semi-urban economies might also have put pressure on parents to engage their children in some form of economic activity in order to enhance family income \[[@B3],[@B7],[@B13],[@B21],[@B25]-[@B27]\]. Most working children were assigned their roles by their parents, largely to supplement family income. The prevalence of child labour did not differ significantly among children living with one or both parents or with a related guardian, Children living with single mothers were, not surprisingly, also more affected. Harsh economic realities, the burden of paying school fees and catering for a large family size may be the underlying reason parents compel their children to engage in work activities. On the other hand, children living with non-related guardians were surprisingly significantly less involved in labour activities. This finding however should be interpreted with a lot of caution because the number of children living with non-relatives was small. It is noteworthy however, to the extent that children living with their biological parents should theoretically be better cared for than those in foster homes. It is conceivable, that affected children may have been sent to live with more affluent guardians to give such children better prospects for the future. The prevalence of child labour increased with decreasing parental education and socio-economic class. These findings agree with other studies \[[@B7],[@B8],[@B13],[@B21],[@B28]\]. The reasons for these observations are fairly obvious. Educated parents are more likely to understand the need for a growing child to concentrate on activities capable of enhancing realization of their full potential and the need to avoid potentially \'harmful\' activities. Also, better socio-economic standing removes the pressure on parents to put their children to work. Conclusion ========== There is a need to protect the girl child, advocate reduction in family size, and promote parental education/economic empowerment in order to reduce the urge on children to perform economic roles. There is also a need to study all the ramifications of child labour for proper planning and for the protection of school children if Nigeria is to improve on her child survival, developmental and protection indices in the near future. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= MBF conceived of the study, participated in its design, collection, statistical analysis and interpretation of data. FON contributed to the statistical analysis, interpretation of the data and has been involved in drafting and revising the manuscript. AOO participated in coordination of the study and revision 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/1472-698X/5/2/prepub> Acknowledgements ================ Our gratitude to resident Doctors and Medical students for their assistance in data collection and to Dr L.O.A. Thanni for his useful comments in preparation of this manuscript.	PubMed Central	2024-06-05T03:55:54.063512	2005-3-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554995/", "journal": "BMC Int Health Hum Rights. 2005 Mar 2; 5:2", "authors": [ { "first": "Bolanle M", "last": "Fetuga" }, { "first": "Fidelis O", "last": "Njokama" }, { "first": "Adebiyi O", "last": "Olowu" } ] }
PMC554996	After the publication of this work \[[@B1]\] it was brought to our attention that the concentrations of the reagents listed for the cell lysis buffer are those of the original stocksolutions and not the required final concentrations. The correct final concentrations for this buffer are as follows: HEPES 50 mM NaCl 100 mM NaF 10 mM EDTA 5 mM Na3VO4 0.5 mM NEM 2 mM Triton 0.1% Complete protease inhibitors (Roche). We regret any inconvenience that this inaccuracy may have caused, and thank Dr. Bräuer for bringing it to our attention.	PubMed Central	2024-06-05T03:55:54.066508	2005-3-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554996/", "journal": "BMC Biochem. 2005 Mar 3; 6:3", "authors": [ { "first": "C", "last": "Burnett" }, { "first": "P", "last": "Makridou" }, { "first": "L", "last": "Hewlett" }, { "first": "K", "last": "Howard" } ] }
PMC554997	Background ========== Breast pain is a common problem, which can affect up to 70% of women \[[@B1]\]. Breast pain or mastalgia can be cyclical or non-cyclical. The cyclical type of breast pain has been attributed to sex hormonal changes through the menstrual cycle that may increase the size of the breast tissue, which stretches the internal structures and causes pain or soreness. Numerous studies have demonstrated variation in pain perception during the menstrual cycle \[[@B2]-[@B5]\]. Heat sensitivity is increased in the luteal (17--22) phase of the menstrual cycle \[[@B6]\] and lowest in the periovulatory phase (day 12--16), but other studies have shown variation at other times in the cycle. Non-cyclical breast pain can be caused by hormonal influences particularly oestrogen, and other causes such as macromastia, local infection or inflammation; rarely, breast cancer can present as breast pain. Macromastia may cause areas of numbness in the breast and problems with nipple erectile function, which is thought to be related to the stretching of the nerve supply with increase in breast size \[[@B7]\]. Post-surgical breast pain is also a significant entity, with about 50% of women who undergo mastectomy suffering from chronic pain one year after their operation \[[@B8],[@B9]\]. The mechanisms of breast pain in the majority of women are not well understood at the cellular or molecular level. We hypothesized a relationship between clinical breast pain, nerve growth factor (NGF) and its regulated ion channels or receptors expressed by nociceptor fibres. Estrogens upregulate NGF receptor mRNA in sensory neurons \[[@B10]\], and enhance the proliferative effects of NGF \[[@B11],[@B12]\]. As NGF is a key molecule that determines the sensitivity of nociceptors in humans \[[@B13]\] and animal models \[[@B14]\], sex hormonal influences could be responsible for altered NGF activity during the menstrual cycle, leading to cyclical breast soreness or pain. NGF expression is also increased by inflammation, and this is responsible for the collateral nerve fibre sprouting and hypersensitivity of nociceptor fibres associated with inflammation. The hypersensitivity is, in part, mediated via the capsaicin or vanilloid receptor 1 (TRPV1), which is required for thermal hyperalgesia in rodents \[[@B15],[@B16]\], and is activated by heat pain. Thermal hyperalgesia can occur during the menstrual cycle and it is well known that the core body temperature alters during the cycle (this is a qualitative test for ovulation), and thus heat conductance and perception and tolerance of heat alters during the cycle \[[@B2],[@B6]\]. The TRPV1 receptor is activated also by the products of inflammation. We have therefore studied TRPV1-expressing nerve fibres and NGF in skin from women with and without breast pain and tenderness. The recently discovered vanilloid thermoreceptors TRPV3 and TRPV4, which are also expressed by sensory fibres and activated by warmth, were also studied \[[@B17],[@B18]\]. Methods ======= Patients -------- Eighteen patients were recruited (n = 12 breast reduction for macromastia; n = 6 breast reconstruction) at Chelsea and Westminster, Charing Cross, Ravenscourt Park Hospitals in London and Broomfield Hospital in Essex were recruited. Breast reduction patients had no previous surgery. The breast reconstruction patients had Latissimus dorsi flap reconstructions after previous mastectomies, and had implants. Patients below 18 years or above 70 years, with any local skin inflammation, infection or cancerous skin changes were excluded. The Research Ethics Committee of Hammersmith Hospitals Trust and Mid Essex Hospitals Trust gave ethical permission for the study. Informed consent was obtained prior to the clinical examination and questionnaire administration. ### Clinical pain assessment Age, parity, height, weight and menstrual data were collected. Details of current surgery, any previous breast surgery and breast disease were also recorded. A questionnaire which included questions on breast and period pain was administered, a diagram to indicate painful and tender areas and the 78 pain descriptors from the McGill Pain Questionnaire \[[@B19]\] were produced, along with a 10 cm unmarked visual analogue scale (VAS). The presence of breast pain was defined from the results of the Breast Pain Questionnaire using the total Pain Rating Index (PRI (total)) \[[@B19]\] and VAS scores marked by the patient in centimetres being more than zero to identify those patients with breast pain. Only two patients were taking simple analgesia for breast pain. Immunohistochemistry -------------------- Full thickness skin biopsies were collected from each patient along the incision line of about 2 mm depth. Samples were coded, frozen on site and stored at -70°C. The skin samples were mounted in embedding medium (Tissue-Tek OCT compound, Sakura Finetek, USA). Frozen tissue sections (10 μm) were collected onto poly-L-lysine-coated (Sigma Poole Dorset UK) glass slides and post-fixed in freshly prepared, 4% w/v paraformaldehyde in phosphate buffered saline (PBS; 0.1 M phosphate; 0.9% w/v saline; pH 7.3). After washing in PBS, endogenous peroxidase was blocked by incubation with 0.3% w/v hydrogen peroxide in methanol. After a further wash in PBS the tissue sections were incubated overnight with affinity purified antibodies to TRPV1 (polyclonal rabbit anti-TRPV1; GlaxoSmithKline, Harlow, UK; 1/5000; 1/10,000), TRPV3 (polyclonal rabbit anti-TRPV3; GlaxoSmithKline, Harlow, UK; 1/1000), TRPV4 (polyclonal rabbit anti-TRPV4; GlaxoSmithKline, Harlow, UK; 1/250; 1/1000), recombinant human NGF (polyclonal rabbit anti-NGF; Genentech, San Francisco, USA; 1/4000) or marker of large and some small calibre nerve fibres (mixed mouse monoclonal antibodies to neurofilaments 200 kD, 70 kD and 57 kD; DAKO cytomation Cambs., UK, 1/50,000; Novocastra, Newcastle upon Tyne, UK, 1/500). Methodological controls included omission of primary antibodies, or their replacement with pre-immune serum. Specificity of antibodies has been described in previous publications \[[@B18],[@B20]\]. Sites of antibody attachment were revealed using biotinylated goat anti- rabbit or biotinylated horse anti-mouse IgG (Vector Laboratories, High Wycombe, Bucks., U.K.) and nickel-enhanced, immunoperoxidase (avidin-biotin complex -- ABC elite; Vector Laboratories, High Wycombe, Bucks., U.K.). Nuclei were counterstained with 0.1% w/v aqueous neutral red. The intensity of NGF immunostaining was graded on a scale 0 -- 3 where 0 = negative or no immunoproduct, 1 = weak immunoproduct, 2 = intermediate intensity immunoproduct and 3 = intense immunoproduct. Intra-epidermal and sub-epidermal TRPV1-, TRPV4 or neurofilament -- positive fibres were counted and the length and thickness of the epidermis was measured using a calibrated microscope, eyepiece graticule. Similarly, fibres that extended through the epidermis were counted, along with arborising \"clusters\" of fibres. The two observers who performed the histological studies were blinded with regard to clinical pain scores. Statistical analyses -------------------- A non-parametric, two tailed test (Mann Whitney U) was used. Commercially available statistical software was used to perform the test (Prism 3™). Results ======= Clinical pain assessment ------------------------ Pain Rating Indices (PRI) and Visual Analogue Scores (VAS) were used to group patients with (n = 10) and without (n = 8) breast pain: pain group; PRI -Median (range) 12 (4--30); Mean (SEM) 13.44(3.07) and VAS -- Median (range) 5(3.7--6.7); Mean (SEM) 5.14(0.31)\]; in patients without breast pain, PRI and VAS were zero. Only 2 patients reported thermal pain descriptors (burning, hot), while most reported ache and tenderness. The numbers of breast reduction and reconstruction patients with pain were 7 out of 12 and 3 out of 6 respectively, and all reported pain of duration greater than 6 months. The presence (n = 8) or absence (n = 10) of dysmenorrhoea was also recorded. There was no association between breast pain and the presence of dysmenorrhoea. Immunostaining -------------- ### TRPV1 TRPV1-immunoreactive fibres were present mainly in the sub-epidermis in normal (pain free) skin (Fig. [1A](#F1){ref-type="fig"}). In breast pain, TRPV1-immunoreactive fibres appeared to be more abundant in the epidermis and frequently seen to pass along the junction of the stratum corneum (intra-epidermal fibres -- IEF; Fig. [1B, Ci, ii](#F1){ref-type="fig"}) often with multiple fibres (fibre \"clusters\"; Fig. [1Di, ii](#F1){ref-type="fig"}- arrows). Intra-epidermal fibres and fibre \"cluster\" counts/millimetre of epithelium were significantly higher in patients with breast pain (Table [1](#T1){ref-type="table"}; Fig. [2](#F2){ref-type="fig"}). This significance was maintained despite exclusion of patients with previous breast surgery (i.e. the breast reconstruction patients; Pain, n = 7; No Pain, n = 5; p = 0.0303). While some specimens from pain patients showed thinning of the epidermis, this was not so overall for the pain group (Table [1](#T1){ref-type="table"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### TRPV1-immunoreactive nerve fibres in breast skin. (A) Normal, control skin : TRPV1-immunoreactive nerve fibres (small arrows) in the sub-epidermis. (B) Painful skin (macromastia patient): intra-epidermal, TRPV1-immunoreactive nerve fibre (arrow) deriving from a large, sub-epidermal fascicle and extending to the stratum corneum. (Ci) Painful skin (breast reconstruction patient): TRPV1-immunoreactive intra-epidermal fibres passing along the junction between the epidermis and stratum corneum (Cii-enlarged area from Ci). (Di) Painful skin (macromastia patient): multiple branching, TRPV1-immunoreactive intra-epidermal nerve fibres (arrows) extending to the stratum corneum (Dii-enlarged area from Di). Large double arrows indicate relative epidermal thickness. Scale bars: A, B, C(i), D(i) = 50 μm; C(ii), D(ii) = 10 μm. ::: ![](1472-6874-5-2-1) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Histology results ::: Patients with no breast pain (N = 8) Patients with breast pain (N = 10) P-Mann Whitney Test ------------------------------ ------------------------------------------ ---------------------------------------- ------------------------- Intra-epidermal Median (range) Median (range) TRPV1 intra-epidermal fibres 0.69 (0.00 -- 1.27) 2.15 (0.77 -- 4.38) 0.0009\\ TRPVR1 fibre \"clusters\" 0.15 (0.00 -- 0.27) 0.37 (0.00--0.85) 0.0085\* Sub-epidermal TRPV1 fibres 2.98 (1.59--4.04) 3.79 (0.63 -- 5.92) 0.1457 Neurofilament fibres 3.38 (0.83 -- 5.92) 2.95 (0.94 -- 4.95) 0.7618 NGF staining 2.5 (1.0--3.0) 2.38 (1.0--3.0) 0.5726 Epidermal Thickness (mm) 0.04 (0.02 -- 0.12) 0.04 (0.03 -- 0.05) 0.9654 ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Quantification of TRPV1-immunoreactive, intra-epidermal fibres and breast pain. Scattergrams show TRPV1-immunoreactivity in (A) intra-epidermal fibres and (B) intra-epidermal fibre \"clusters\" in patients with and without breast pain. ::: ![](1472-6874-5-2-2) ::: ### TRPV3 TRPV3 immunoreactivity was detected in basal keratinocytes and occasional suprabasal cells throughout the epidermis. Quantification of immunostaining showed a significant (\P = 0.011) increase with breast pain (Fig. [3](#F3){ref-type="fig"}). TRPV3-immunoreactivity was not detected in skin nerve fibres in this study. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### TRPV3-immunoreactivity in breast skin. TOP PANELS: TRPV3-immunoreactive keratinocytes mostly in basal layer and graded from grade 0/negative (top left) to grade 3/strong staining (bottom right). Scale bar = 100 μm BOTTOM PANEL: Scattergram showing grading assessment and a significant increase (\P \< 0.05) of TRPV3 immunoreactivity in patients with pain. ::: ![](1472-6874-5-2-3) ::: ### TRPV4 Basal keratinocytes also displayed TRPV4 immunoreactivity in groups of cells which were particularly strong at the apex of dermal papillae, where immunoreactivity appeared most strong at the cell membrane junction (Fig [4](#F4){ref-type="fig"}). Quantification of immunostaining showed a significant (p \< 0.03) increase with breast pain (Fig. [4](#F4){ref-type="fig"}). TRPV4 immunoreactivity was also detected in fine nerve fibres scattered through the sub-epidermis, and showed a trend (p = 0.402) for increased frequency in painful subjects (Fig [4](#F4){ref-type="fig"}). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### TRPV4-immunoreactivity in breast skin. TOP FOUR PANELS: TRPV4-immunoreactivity in keratinocytes mostly at the cell membrane and graded from grade 0/negative (top left) to grade 3/strong staining (bottom right). Scale bar = 100 μm MIDDLE PANEL: Fine, sub-epidermal, TRPV4-immunoreactive fibres (arrows). Scale bar = 100 μm BOTTOM PANELS: Scattergrams showing grading assessment of sub-epidermal fibres (left panel) and keratinocytes (right panel) significantly increased (\P \< 0.05) in patients with pain. ::: ![](1472-6874-5-2-4) ::: ### NGF NGF immunoreactivity was present in basal keratinocytes in all samples (Fig [5Ai, ii](#F5){ref-type="fig"}) with little difference in intensity between pain and no pain specimens (Table [1](#T1){ref-type="table"}). In some pain specimens the epidermis appeared to be thinner, and there was evidence of NGF expression in suprabasal as well as basal keratinocytes (Fig [5Bi](#F5){ref-type="fig"} and [5ii](#F5){ref-type="fig"}, -- arrows), which correlated with the presence of TRPV1positive fibres (seen in a serial section from the same case shown in Fig [1B](#F1){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### NGF-immunoreactivity in breast skin. (Ai) Normal, control skin: NGF-immunoreactive basal keratinocytes. (Aii)-enlarged area from Ai showing NGF confined to single layer of keratinocytes. (Bi) Painful skin (macromastia patient): NGF-immunoreactive basal and supra-basal (Bii*-arrows) keratinocytes in skin with thin epidermis. ::: ![](1472-6874-5-2-5) ::: ### Nerve marker (neurofilaments) Neurofilaments showed nerve fibres in sub-epidermal and dermal regions only but no significant changes were detected between groups (Table [1](#T1){ref-type="table"}). Discussion ========== While breast pain and tenderness is a common problem, in the majority of women the mechanisms underlying breast pain are poorly understood. Our study focused on Nerve Growth Factor (NGF) and the expression of the capsaicin receptor 1 (TRPV1) in nociceptor fibres, as these are key molecules in pain and hypersensitivity. Our finding, that TRPV1-positive intra-epidermal fibres were significantly increased in patients with breast pain and tenderness, is both novel and important. The increased and abnormal \"clusters\" of intra-epidermal fibres were shown in patients who had no previous breast surgery, and no known episodes of mastitis. Hence this may be a surrogate marker for \"idiopathic\" or macromastia related breast pain in some patients. Studies are in progress to correlate changes in TRPV1 nerve fibres with quantitative sensory perception thresholds. The cause of the increased intra-epidermal fibres is not known. Given the trend to thinning of the epidermis, normally associated with denervation \[[@B21]\], in the biopsies from some patients with breast pain, the increased intra-epidermal fibres here may represent nerve fibre sprouts following cutaneous terminal damage. The intra-epidermal fibre morphology and \"clusters\" would be in keeping with this explanation, as fibres ran in unusual patterns. There was no overall change in the sub-epidermal fibre counts for TRPV1 or the structural nerve marker neurofilament. NGF-immunostaining intensity was similar in the different groups. However, in some patients, there appeared to be staining for NGF in the supra-basal epidermis in addition to the basal cells, which is usually associated with inflammation or denervation; increased NGF is known to cause collateral sprouting \[[@B22]\]. Further studies, using quantitative NGF assays and in situ hybridisation, need to be performed to address this issue (these studies would require more substantial skin biopsies). The menstrual cycle influences on NGF levels are also difficult to determine with the current sample size. Our recent studies have demonstrated increased TRPV1-immunoreactive nerve fibres in inflammatory bowel disease \[[@B23]\], and in the mucosal and sub-mucosal layers of patients with rectal hypersensitivity, where they correlated with thermal and mechanical hypersensitivity, suggesting increase of polymodal nociceptors \[[@B20]\]. We proposed that topical capsaicin or resiniferatoxin treatment, which reduces the numbers of TRPV1 positive fibres, may be a useful therapeutic approach in rectal hypersensitivity. Topical capsaicin has been reported to be useful for treatment for post-mastectomy chronic pain \[[@B24]\], but it is uncertain if it could substantially help breast pain or tenderness in macromastia, as some of the symptoms are likely to arise from deeper structures. Oral selective TRPV1 antagonists thus deserve consideration: both mechanical and thermal hyperalgesia may be reversed by capsazepine, a TRPV1 antagonist \[[@B25]\], again suggesting an effect on polymodal nociceptors. Thus patients may be helped with respect to mechanical symptoms, which predominate in comparison with thermal descriptors. Little is known of the roles of TRPV3 and TRPV4 in human pain pathophysiology and keratinocyte function. While we have previously demonstrated TRPV3 in human sensory neurons \[[@B18]\], no TRPV3 staining was observed in skin innervation in this study, presumably as the levels in the periphery were below the detection limit of our method. It may be speculated that increased TRPV3 and TRPV4 in observed in keratinocytes may alter keratinocyte expression of NGF and other molecules, which in turn may sensitise nociceptors. Conclusion ========== Breast pain and tenderness appears to be associated with abnormal intra-epidermal innervation. This may reflect re-innervation of skin following nerve stretch damage, and/or collateral sprouting. While further studies are necessary to establish functional links between the TRPV1, TRPV3 and TRPV4 immunohistological changes and breast pain, our findings indicate a path for increasing understanding and treatment of breast pain. List of abbreviations ===================== TRPV = transient receptor potential vanilloid; NGF = Nerve Growth Factor; VAS = Visual Analogue Score; PRI = Pain Rating Index Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= PG and EW recruited patients, collected biopsies and participated in immunohistology studies. PF participated in immunohistology and coordination of the study. AH participated in design of the study and recruitment of patients. JD, GS and CB provided antibodies and helped draft the manuscript. PA conceived the original study, its design and coordination, and helped with 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/1472-6874/5/2/prepub> Acknowledgements ================ We gratefully thank the surgeons at Hammersmith Hospitals NHS Trust, Chelsea and Westminster Hospital, London, and Broomfield Hospital, Chelmsford, UK, who provided the tissues for this study.	PubMed Central	2024-06-05T03:55:54.066925	2005-3-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554997/", "journal": "BMC Womens Health. 2005 Mar 8; 5:2", "authors": [ { "first": "Preethi", "last": "Gopinath" }, { "first": "Elaine", "last": "Wan" }, { "first": "Anita", "last": "Holdcroft" }, { "first": "Paul", "last": "Facer" }, { "first": "John B", "last": "Davis" }, { "first": "Graham D", "last": "Smith" }, { "first": "Chas", "last": "Bountra" }, { "first": "Praveen", "last": "Anand" } ] }
PMC554998	Background ========== Primary Sjögren\'s syndrome (pSS) is a common systemic autoimmune disease that mainly affects middle-aged women. The exact aetiology remains elusive, and appears multifactorial. The disease is characterised by a chronic periductal lymphocytic infiltration of the exocrine glands, predominantly of the lacrimal and salivary glands. The chronic inflammation is assumed to lead to glandular tissue destruction, resulting in keratoconjunctivitis sicca and hyposalivation and associated symptoms of ocular and oral dryness \[[@B1],[@B2]\]. The salivary dysfunction in pSS is of significant clinical importance and may cause chronic oral discomfort as well as compromised oropharyngeal functions \[[@B3]-[@B5]\]. Subsequently, the patients\' well-being and oral health-related quality of life are also impaired \[[@B5],[@B6]\]. Cross-sectional studies have shown that patients with pSS have a high caries experience with lesions usually located on the cervical surfaces of the teeth, as well as early dental loss due to caries \[[@B3],[@B7]-[@B10]\], despite good oral hygiene \[[@B11]\]. Dental caries, which is a local, but also a multifactorial disease, arises from a concerted action of the aetiological factor (dental plaque) and determinants including salivary factors, medication, systemic disease as well as behavioural factors \[[@B12]\]. However, the selective significance of salivary factors on dental caries in pSS remains an open question. In light of the impaired saliva formation in pSS it is noteworthy that an exact cut-off value for salivary flow and/or separate salivary constituents predicting the risk of developing dental caries has not yet been agreed on. In the diagnosis of Sjögren\'s syndrome (SS), an unstimulated whole saliva flow rate of ≤ 1.5 ml/15 min is currently considered pathological \[[@B13]\], but at this point the caries process may have been going on for years revealed by the presence of a high number of decayed, missed and filled teeth \[[@B3],[@B7]-[@B10]\]. The American-European Consensus Classification Criteria for pSS include evaluation of salivary gland dysfunction by means of parotid sialography, salivary gland scintigraphy, whole saliva sialometry and labial salivary gland biopsy as well as questionnaire concerning symptoms of oral dryness \[[@B13]\]. The purpose of this study was to evaluate parotid flow rate, whole and parotid saliva composition and dental caries as potential oral markers of disease severity in patients with pSS. Thus we hypothesised that patients with pSS exhibit specific changes in salivary flow and composition due to presence of focal lymphocytic infiltration in their labial salivary glands (FS) and/or serum autoantibodies to Ro (SSA) and/or La (SSB) antigens (AB) as compared to age- and gender-matched healthy controls with normal salivary gland function. We further hypothesised that in patients with pSS dental caries is related to impaired salivary flow and associated changes in saliva composition. Methods ======= The study included 20 randomly selected patients with pSS, who attended the Department of Oral Medicine, University of Copenhagen, for routine follow-up examinations. They were diagnosed according to the Copenhagen criteria, but also met the American-European Consensus Classification Criteria for pSS \[[@B13],[@B14]\]. All patients had complaints of dry eyes and dry mouth according to the questionnaire of the American-European classification criteria \[[@B13]\] as well as evidence of keratoconjunctivitis sicca. Eighteen patients had unstimulated whole saliva flow rate (UWS) ≤ 1.5 ml/15 min. Two patients had UWS \>1.5 ml/15 min, but otherwise fulfilled the criteria. Fourteen patients had a positive labial salivary gland biopsy with focus score ≥ 1 as well as presence of AB. Among the remaining 6 patients, 2 had FS, but not presence of AB, and 4 patients had presence of AB, but not FS. The labial salivary glands of the latter 4 patients displayed chronic sialoadenitis where inflammatory cells were observed but did not form any foci. In all 20 patients, estimation of the presence of serum autoantibodies was performed within the same timeframe as the saliva collections and the clinical assessments. In 16 of these patients labial salivary gland biopsies were performed at the same time. The remaining 4 patients refused a labial salivary gland rebiopsy, as they had their diagnosis of pSS for 25, 23, 16 and 10 years, respectively. In order to understand the underlying salivary pathophysiology in pSS, the study also included 20 healthy controls selected among persons attending the Institute of Odontology for dental treatment to obtain reference values of normalcy. The selection was based on the requirements that controls had no present or past medical history of systemic disease, did not take any medication including contraceptives and hormonal replacement, had no current use of tobacco products, and matched the pSS patients with regard to age. Clinical examination -------------------- One examiner (AMLP) conducted the whole saliva collections, interviews, and oral clinical examinations. About 2 hours after collection of whole saliva, another examiner (AB) conducted collections of parotid saliva. In order to minimize the influence of circadian cycle on salivary secretion and composition all procedures were carried out in the same order and at a fixed time of the day, i.e. between 9.00 and 11.45 a.m. The local Danish Ethical Committees approved the study protocol, and all participants completed an informed consent form according to the Declaration of Helsinki. The number of decayed, missing and filled surfaces (DMFS) was recorded excluding third molars as previously described in details \[[@B5]\]. Plaque and gingival indices and periodontal probing pocket depth were determined at four sites per tooth on six index teeth (16, 21, 24, 36, 41, and 44) \[[@B8]\]. Each subject underwent a standardised interview including inquiries on medical disease symptoms, dental history, dental visits, medication, tobacco smoking, dietary (especially regarding daily sugar intake) and oral hygiene habits. Furthermore, symptoms of oral dryness were assessed by means of a categorised questionnaire (based on Beck\'s inventory scale, item 9 \[[@B15]\]) with four degrees of severity (scores 0--3), as previously described \[[@B8]\]). Collection of whole and parotid saliva -------------------------------------- Unstimulated whole saliva (UWS) and paraffin-stimulated whole saliva (SWS) were sampled over a 15-min and 5-min period, respectively, as described previously \[[@B8]\]. Unstimulated parotid saliva (UPS) and stimulated parotid saliva (SPS) were sampled over an average period of 20 min and 5 min, respectively. A saliva collection set-up impermeable to CO~2~was used \[[@B16]\]. Stimulation of the parotid saliva was initiated by applying 1 ml of 1% citric acid to the dorsal part of the tongue every 15 sec. The flow rate, given with 2 decimal places, was determined by weighing the saliva-collecting cup, tube, and syringe before and after saliva collection, and expressed as ml/min \[[@B8]\]. The subjects were instructed to refrain from eating, drinking, smoking, and any oral hygiene for 2 h preceding the saliva sampling. Sialochemical analysis ---------------------- Concentrations (mM) of sodium, potassium and total calcium were determined by atomic absorption spectroscopy \[[@B16],[@B17]\]. Concentrations (mM) of chloride were measured by coulometric titration and total phosphate (mM) by the molybdic reaction \[[@B16],[@B17]\]. Total protein (μg/ml) was measured by the Coomassie reaction. Amylase activity (stated as the catalytic activity of the enzyme, i.e., kat = mole/s and given in μkat/l, where one μkat/l corresponds to 60 U/l) was measured by means of the Phadebas™ test kit. Saliva was diluted 1000 times more than usually recommended for plasma by the test kit. The volume of saliva required for complete sialochemical analysis was 240 μl corresponding to an average collection period for UPS of less than 20 min and for SPS less than 5 min for the pSS patients. Parotid saliva pH, P~CO2~, buffer capacity, and saturation with regard to hydroxyapatite Parotid saliva pH and P~CO2~were determined on an ABL 605 blood gas analyser (Radiometer™), and the HCO~3~^-^concentration was calculated from the pH and Pco~2~values as previously described \[[@B17]\]. The parotid saliva buffer capacity (β), which originated from the HCO~3~^-^and phosphate buffer systems, was calculated individually for each saliva sample at the pH value of the sample \[[@B18]\]. Briefly, the buffer capacity for each buffer system was calculated as: 2.3 \[C\] \* (\[C~B~\]/\[C\]) \* (1-(\[C~B~\]/\[C\])) Where (\[C\]) states the total concentration of the buffer system and (\[C~B~\]) states the concentration of the base in the buffer system. The concentrations of the bases was calculated as previously described \[[@B16]\] and the pK values used for the bicarbonate and phosphate buffer systems was 6.1 for carbonic acid \[[@B16],[@B17]\] and 6.8 for H~2~PO~4~^-^\[[@B19]\]. The sums of calculated β from both the HCO~3~^-^and phosphate buffer systems are in this study denoted as saliva β. At pH values above 5 (counting for all except one sample) the contribution from the protein buffer system, relative to the HCO~3~^-^and phosphate buffer systems, was considered negligible \[[@B16]\] and therefore not included in the saliva β. The degree of saturation (DS~HAP~) of saliva with respect to hydroxyapatite, i.e. the calcium phosphate salt and main mineral of the tooth tissues, was calculated according to the method described by Schmidt-Nielsen \[[@B19]\]. The negative logarithm of the ionic product of hydroxyapatite (pI~HAP~) was calculated from the saliva p \[Ca^2+^\], p \[HPO~4~^2-^\], and p \[H^+^\]. The negative logarithm of the solubility product of hydroxyapatite (pSP~HAP~) was calculated by the constant given by Schmidt-Nielsen \[[@B19]\] and the ionic strength of each saliva sample. Given the relatively low ionic strength of the saliva samples analysed (0.039 ± 0.020) we found this simple method for calculation of DS~HAP~acceptable. Finally, the critical pH of parotid saliva, i.e. the pH value at which saliva is saturated (i.e., neither super- nor undersaturated) with respect to hydroxyapatite (pI~HAP~equal to pSP~HAP~) was calculated. Statistical analysis -------------------- Differences in the salivary and clinical parameters between the patient and control groups were analysed by Wilcoxon rank-sum test (categorised variables) and two-sampled t-test (numerical variables). Fisher\'s test (less than ten in one category) and the chi-squared test (more than ten) were used for analysis of distributions between the two groups. Associations between variables were analysed by the Spearman rank order correlation analysis (r~s~). In order to find the best predictor for a given outcome, multiple regression analysis was used with stepwise backward elimination with the adjusted R-squared values given. Significance was selected at a level of P≤ 0.05. Results ======= Clinical findings ----------------- Table [1](#T1){ref-type="table"} summarises some of the anamnestic data and results of self-assessed oral dryness of the 20 female patients with pSS and 20 age-matched healthy females included in this study. Sixteen pSS patients were taking prescribed medicines on a regularly daily basis. Seven of these patients were taking medicines known to impair saliva secretion and/or cause compositional changes of saliva \[[@B20]\]. These included antihistamines and antidepressants that cause reduction in salivary flow due to inhibition of the muscarinic cholinergic receptors \[[@B21]\], and blockers of β-adrenoceptors that lead to impaired protein secretion \[[@B22]\]. However, none of these medicines are known to cause specific changes in the inorganic salivary composition. The 7 patients taking \"xerogenic\" medicines all had symptoms of oral dryness and/or were diagnosed as pSS with hyposalivation (n = 5) before their current medication was commenced. We therefore found no significant association between the intake of medicines, including the number of medicines, the salivary flow rates, focus scores, and serum autoantibodies. All the pSS patients complained of dry mouth. In pSS, a significant inverse correlation was found between scores of oral dryness and the SWS (r~s~= -0.50, P= 0.025), but only a tendency toward a relationship between the oral dryness and UWS, UPS and SPS. On the other hand, in both groups as a whole, displaying large variations in oral dryness and salivary flow rates, the subjective measures were significantly inversely correlated to UWS (r~s~= -0.83, P\< 0.001), SWS (r~s~= -0.81, P\< 0.001), UPS (r~s~= -0.74, P\< 0.001), and SPS (r~s~= -0.77, P\< 0.001). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Anamnestic data and results of self-assessed oral dryness of the female patients with primary Sjögren\'s syndrome (pSS) and the female healthy controls. Results are given as number of patients or scores (yes/no) and as means ± SD. ::: pSS (n= 20) Healthy controls (n= 20) P-value ----------------------------------------------- --------------- ---------------------------- ------------ Age (years) 60 ± 15 56 ± 13 0.437^a^ Duration of disease (years)\* 6 ± 7 0 NA Duration of symptoms (years) 10 ± 7 0 NA Xerogenic medicines (yes/no)^†^ 7/13 0/20 NA Smokers (yes/no)^§^ 5/15 0/20 NA Cigarettes per day (smokers only)^§^ 7 (2--20) 0 NA Tooth brushing (times per day) 3 ± 1 2 ± 1 0.001^a^ Dental floss and/or toothpicks daily (yes/no) 16/4 15/5 1.000^b^ Dental visits per year (number) 3 ± 1 2 ± 1 0.001^a^ Oral dryness (score 0/1/2/3) 0/3/4/13 20/0/0/0 \<0.001^b^ \* The time from established diagnosis to present examination. ^†^Patients were interviewed regarding their intake of medicines. Their intake of xerogenic medicines included antidepressants, antihistamines and β-blocking agents \[20\]. ^§^Patients were also interviewed regarding their smoking habits, since cigarette smoking has been shown to reduce the extent of labial salivary gland lymphocytic infiltration \[23\] and to have a negative impact on periodontal status. Smoking, however, has no impact on salivary flow or composition \[24\]. P-values obtained by a two-sample t-test (a) and Fisher exact test (b). NA; not analyzed. ::: The results of the clinical examination are shown in Table [2](#T2){ref-type="table"}. In spite of the fact, that the patients brushed their teeth with fluoride-containing toothpaste and visited their dentist more frequently than the healthy individuals (Table [1](#T1){ref-type="table"}), they had a significantly higher number of decayed, missed and filled teeth and a higher gingival index. No significant differences were found between the two groups in terms of plaque index (frequency distribution of scores), the periodontal probing pocket depth or the regular daily use of dental floss and/or toothpicks. Apart from avoiding acidic, spicy, crunchy and dry foods the patients\' dietary habits (including sugar intake) did not differ from those of the healthy controls. Neither the time since pSS diagnosis, nor the duration of disease symptoms or the age, were correlated to the flow rates. Moreover, plaque- and gingival indices as well as periodontal probing pocket depth were not correlated to the salivary flow rates, time since pSS diagnosis, duration of disease symptoms, DMFS, or age. In the groups as a whole, however, a positive association between the gingival index and age was found (r~s~= 0.33, P= 0.04). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Oral findings in the patients with primary Sjögren\'s syndrome (pSS) and the healthy controls. Results are given as number of patients or scores (yes/no) and in medians (ranges). ::: pSS (n= 20) Healthy controls (n= 20) P-value -------------------------------------------- ---------------- ---------------------------- ------------ No. of teeth 22 (6--28) 28 (0--28) 0.011^a^ DMFS\* 83.0 (25--140) 43.0 (10--140) 0.001^a^ Distribution of D/M/FS 23/745/1020 14/305/662 \<0.001^b^ Subjects with dentures (yes/no) 4/16 7/13 0.480^c^ Distribution of PI (no. of 0/1/2/3 scores) 245/179/54/2 244/152/60/0 0.272^b^ Distribution of GI (no. of 0/1/2/3 scores) 346/116/16/2 330/95/31/0 0.034^b^ Probing pocket depth (PPD, mm) 2 (1--4) 2 (1--3) 0.523^a^ \D/M/FS, decayed, missed, filled surfaces; PI, plaque index; GI, gingival index. P-values obtained by Wilcoxon rank sum test (a), Chi^2^- test (b), and Fisher exact test (c). ::: Salivary flow and composition ----------------------------- As shown in Tables [3](#T3){ref-type="table"} and [4](#T4){ref-type="table"}, the pSS patients had significantly lower UWS, SWS, UPS and SPS than the healthy controls. All pSS patients and one single healthy individual (without symptoms or clinical signs of local or systemic disease) had with UWS ≤ 0.10 ml/min and/or SWS ≤ 0.70 ml/min, which are the cut-off values for hyposalivation \[[@B13],[@B25]\]. In 8, 11 and 5 patients, UWS, UPS and SPS, respectively, were close to or equal to 0 ml/min. In both the patient and control group UWS and SWS were mutually correlated (r~s~= 0.79, P\< 0.001 and r~s~= 0.45, P\< 0.05, respectively), but only in pSS a mutual correlation was obtained between UPS and SPS (r~s~= 0.74, P\< 0.001). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Unstimulated (UWS) and stimulated (SWS) whole salivary flow rate and composition in the patients with primary Sjögren\'s syndrome (pSS, n= 20) and the healthy controls (n= 20). Results are given in median (range). ::: UWS SWS ------------------------------- ----------------------------- ----------------------------- --------- ------------------------------ ----------------------------- --------- pSS Healthy controls P-value pSS Healthy controls P-value Flow rate (ml/min) 0.02 (0.00--0.23) (n= 20) 0.39 (0.06--1.10) (n= 20) \<0.001 0.14 (0.01--1.66) (n= 20) 1.40 (0.54--2.82) (n= 20) \<0.001 Na^+^(mM) 12.0 (8.0--47.0) (n= 11) 8.0 (4.5--17.0) (n= 20) 0.008 16.0 (8.0--59.0) (n= 12) 10.3 (5.5--30.0) (n= 20) 0.020 K^+^(mM) 22.2 (5.1--48.0) (n= 11) 21.9 (6.8--33.4) (n= 20) 0.536 21.6 (15.2--34.6) (n= 12) 22.0 (11.5--26.5) (n= 20) 0.533 Total calcium (mM) 2.0 (1.3--3.2) (n= 7) 1.7 (0.5--2.8) (n= 19) 0.285 1.3 (0.7--1.9) (n= 12) 1.3 (0.8--2.5) (n= 20) 0.558 Cl^-^(mM) 25.8 (16.0--62.0) (n= 10) 18.3 (5.2--26.0) (n= 19) 0.003 22.6 (13.5--58.6) (n= 11) 16.1 (10.5--28.6) (n= 20) 0.037 Total phosphate (mM) 6.0 (1.2--15.0) (n= 9) 6.8 (2.4--11.7) (n= 19) 0.961 4.4 (2.4--8.5) (n= 11) 4.1 (1.6--11.5) (n= 20) 0.606 Total protein (mg/ml) 3.00 (1.52--8.84) (n= 8) 3.29 (1.36--6.25) (n = 19) 0.560 3.50 (0.421--8.93) (n= 12) 3.04 (1.23--5.54) (n= 20) 0.491 Total protein output (mg/min) 0.210 (0.06--0.80) (n= 8) 1.09 (0.50--2.38) (n= 19) \<0.001 0.95 (0.11--4.33) (n = 12) 3.88 (2.35--8.06) (n= 20) \<0.001 Amylase activity (μkat/l) 365 (0--4500) (n= 10) 980 (0--8940) (n= 19) 0.155 1490 (0--4080) (n= 12) 2280 (0--5890) (n= 20) 0.199 P-value obtained by Wilcoxon\'s rank sum test. ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Unstimulated (UPS) and stimulated (SPS) parotid salivary flow rates, composition including pH, P~CO2~, buffer capacity, degree of saturation with regard to hydroxyapatite, and critical pH in the patients with primary Sjögren\'s syndrome (pSS, (n= 20)) and the healthy controls (n= 20). Results are given in medians (range). ::: ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------- UPS SPS ------------------------------- ------------------------------- ----------------------------- --------- ----------------------------- ----------------------------- --------- pSS Healthy controls P-value pSS Healthy controls P-value Flow rate (ml/min/gland) 0.00\ (0.00--0.04) (n= 20) 0.04 (0.00--0.13) (n= 20) \<0.001 0.10 (0.00--0.87) (n= 20) 0.72 (0.17--1.57) (n= 20) \<0.001 pH 5.5 (4.9--6.2) (n= 7) 6.1 (5.4--6.6) (n= 14) 0.008 6.8 (5.3--7.6) (n= 13) 7.1 (6.8--8.0) (n= 19) 0.020 P~CO2~(KPa) 2.5 (2.1--4.8) (n= 7) 3.3 (0.5--7.9) (n= 14) 0.455 3.5 (1.4--7.5) (n= 13) 5.5 (0.3--10.0) (n= 19) 0.021 Na^+^(mM) 7.0 (0.0--111.0) (n= 9) 0.0 (0.0--8.5) (n= 19) 0.002 15.5 (3.5--93.0) (n= 15) 7.5 (0.0--65.0) (n= 20) 0.034 K^+^(mM) 24.5 (4.5--52.5) (n= 9) 29.0 (17.5--62.0) (n= 19) 0.209 26.5 (12.0--53.0) (n= 15) 23.0 (18.0--40.0) (n= 20) 0.689 Total calcium (mM) 1.0 (0.4--2.4) (n= 9) 1.0 (0.6--4.1) (n= 16) 0.887 1.1 (0.6--1.8) (n= 13) 0.8 (0.4--1.4) (n= 20) 0.008 Cl^-^(mM) 22.5 (5.0--100.5) (n= 9) 19.0 (12.0--43.0) (n= 19) 0.588 27.0 (12.5--94.0) (n= 14) 15.5 (9.0--59.0) (n= 20) 0.037 Bicarbonate (mM) 0.2 (0.0--0.8) (n= 8) 0.8 (0.1--3.5) (n= 14) 0.017 3.6 (0.1--26.8) (n= 12) 11.3 (3.8--43.9) (n= 19) 0.025 Total phosphate (mM) 5.6 (2.3--15.0) (n= 7) 9.3 (4.1--18.9) (n= 16) 0.066 6.1 (2.1--8.2) (n= 11) 5.2 (3.2--16.9) (n= 20) 0.577 Total protein (mg/ml) 1.11 (0.47--8.97) (n= 9) 2.02 (0.64--5.34) (n= 18) 0.316 1.33 (0.62--6.66) (n= 14) 1.60 (0.84--4.19) (n= 20) 0.589 Total protein output (mg/min) 0 (0--0.18) (n= 9) 0.07 (0--0.30) (n= 18) \<0.001 0.13 (0--1.38) (n= 14) 1.30 (0.29--5.42) (n= 20) \<0.001 Amylase activity (μkat/l) 7760 (3700--21200)\ 5815 (1670--27140)\ 0.193 5220 (560--17240)\ 5520 (710--14710)\ 0.624 (n= 7) (n= 18) (n= 13) (n= 20) β(mmol H^+^/l^.^pH unit) 1.2 (0.1--2.9) (n= 7) 4.0 (2.3--8.9) (n= 13) \<0.001 4.4 (0.5--6.7) (n= 10) 5.2 (0.7--10.1) (n= 19) 0.171 DS~HAP~ 1.4 (0.2--3.6) (n= 9) 3.1 (2.5--10.4) (n= 16) 0.001 7.3 (0.6--21.4) (n= 13) 10.0 (4.5--22.0) (n= 19) 0.372 Critical pH 5.4 (5.1--6.2) (n= 7) 5.3 (5.0--5.5) (n= 16) 0.413 5.5 (5.2--6.2) (n= 11) 5.7 (5.2--5.8) (n= 19) 0.611 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------- The buffer capacity (β) was calculated from the bicarbonate and phosphate concentrations individually for each saliva sample at its pH value, DS~HAP~denotes the degree of saturation with regard to hydroxyapatite. P-value obtained by a Wilcoxon\'s rank sum test. \* corresponds to mean value below 0.01 ml/min. ::: In 10, 9, 13 and 7 cases, respectively, volume of UWS, SWS, UPS and SPS samples from pSS patients were insufficient for full sialochemical assessment. In pSS, the concentrations of sodium were significantly higher in both whole and parotid saliva as compared to the healthy controls. Similar results were obtained for chloride concentrations (except for the UPS) (Tables [3](#T3){ref-type="table"} and [4](#T4){ref-type="table"}). Regarding the concentrations of the other electrolytes (potassium, total calcium and total phosphate) as well as the organic components (total protein and amylase activity) no statistically significant differences were found between the two groups. In order to test whether the changes in sodium and chloride concentrations were merely related to changes in salivary flow rates, the patients and healthy controls were matched by their UPS and SPS flow rates within the range of 0.01--1.00 ml/min. Figures [1A](#F1){ref-type="fig"} and [1B](#F1){ref-type="fig"} illustrate that the pSS patients may be divided into two subgroups. In one group of patients the salt reabsorption pattern was similar to that of the healthy controls, whereas another group displayed high concentrations of parotid sodium and chloride despite low parotid flow rates. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### shows the saliva composition as a function of the parotid saliva flow rate (both unstimulated and stimulated) in patients with pSS (bold circles) and healthy age-matched controls (open circles). Lines were fitted by linear (a and b) or non-linear regression (c-h) depending on the best obtainable fit judged from the R-squared values with unbroken lines representing pSS and dotted lines the controls. Figures 1a and b illustrates that the pSS patients can be divided into two subgroups with regard to salt reabsorption. One group of patients follow the reabsorption pattern of the healthy controls, whereas the other group display high sodium and chloride concentrations despite low parotid flow rates indicating differences in the degree of glandular affection by the disease of the patients studied. ::: ![](1472-6890-5-4-1) ::: The pH and bicarbonate concentration in parotid saliva were significantly lower in the pSS patients as compared to the healthy controls (Table [4](#T4){ref-type="table"}). Subsequently, the pSS patients also had a significantly reduced buffer capacity (β) of UPS and a lower degree of saturation with regard to hydroxyapatite (DS~HAP~) in their UPS than the controls. In pSS, the pH in UPS was in fact only 0.1 pH units from their critical pH. Overall, the salivary β and DS~HAP~were reduced due to low pH, bicarbonate and phosphate concentrations. In pSS, P~CO2~of SPS was lower than in that of the healthy controls (P= 0.02). Nevertheless, at corresponding parotid flow rates, be it stimulated or not, the relations between the flow rates, the saliva pH and the bicarbonate concentrations were similar to those of the healthy controls (Fig. [1C](#F1){ref-type="fig"} and [1D](#F1){ref-type="fig"}). Also the concentrations of potassium, total phosphate, total calcium and total protein (Fig. [1E, F, G](#F1){ref-type="fig"}, and [1H](#F1){ref-type="fig"}) were similar to those of the controls. Determinants of DMFS -------------------- As shown in Table [2](#T2){ref-type="table"} the pSS patients had significantly higher DMFS than the healthy controls (p \< 0.001) due to higher number of filled (51 ± 23) and missed (37 ± 35) tooth surfaces than the controls (33 ± 19 and 15 ± 35, respectively) (P\< 0.05). Also the prevalence of dental treatment, i.e. the number of filled surfaces relative to the total number of present tooth surfaces, was significantly higher in the patients (55 ± 27%) than in the controls (25 ± 14%) (P\< 0.001). However, no significant differences were obtained for the number of decayed surfaces between the patients and controls. Table [5](#T5){ref-type="table"} shows the explanatory power of the two sets of variables exhibiting the highest explanatory power on DMFS in the pooled patient and control groups, namely complaints of oral dryness and the actual salivary flow rates. The estimate states the theoretical expected increase or decrease in DMFS upon an integer increase of the explaining variables, i.e. one score of subjective oral dryness or one ml of saliva. Furthermore, the subjects\' age were added to the analyses due to the well-known effect of this variable upon the number of missing surfaces, which was also found to be significant in this study (r~s~= 0.63, P\< 0.001). As shown DMFS increases with increasing age (per year) and increasing complaints of oral dryness, but decreases with increasing salivary flow rates. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Explanatory power of age as well as subjective and objective salivary variables on DMFS in the patient group and control group all together (n= 40). ::: Estimate for DMFS SD for DMFS estimate Adjusted R-squared P-value ------------------------------- ------------------- ---------------------- -------------------- --------- Subjective model Age 1.4 0.4 0.26 \<0.001 \+ oral dryness questionnaire 11.1 2.3 0.53 \<0.001 Objective model Age 1.4 0.4 0.26 \<0.001 \+ UWS -57.4 20.7 0.37 \<0.001 \+ SWS -28.9 9.4 0.49 \<0.001 \+ UPS -499.2 173.0 0.57 \<0.001 Estimate, SD, adjusted R-squared, and P-value obtained by multiple regression analysis. The estimate states the increase or decrease in DMFS upon addition of the explaining variables (age + oral dryness, or + UWS, SWS, and UPS) by an integer (one year, score, or ml). Since complaints of oral dryness and salivary flow demonstrated strong interrelations they could not be included in the same analysis. As shown age in combination with complaints of oral dryness explained 53% of the variance in DMFS, and age combined with salivary flow rates explained 57%. ::: In the pSS patients, regardless of their age, the complaint of oral dryness, but not the actual salivary flow rate, was associated with dental status (DMFS). The lack of significant correlation between flow rate and DMFS may be ascribed to the narrow window of flow rates in pSS. Scores of oral dryness correlated significantly with the total DMFS (r~s~= 0.53, P\< 0.05) and the prevalence of dental treatment (r~s~= 0.51, P\< 0.05). In addition, the prevalence of dental treatment increased with the duration of pSS (r~s~= 0.49, P\< 0.05), independently of the patient\'s age. Salivary gland focus score, serum autoantibodies, salivary flow and composition ------------------------------------------------------------------------------- In the pSS group, the focus scores (which ranged from 0 to 4) were significantly inversely correlated to UWS (r~s~= -0.53, P\< 0.05), SWS (r~s~= -0.68, P\< 0.01), SPS (r~s~= -0.57, P\< 0.01), but not to UPS. Focus scores were not correlated to oral dryness, time since pSS diagnosis, and duration of disease symptoms. It is noteworthy, however, that the patient group exhibited large variability with regard to time since diagnosis and age (Table [1](#T1){ref-type="table"}). The number of focus scores and presence of serum autoantibodies were positively correlated to salivary concentrations of sodium and chloride in SPS (r~s~= 0.61, P\< 0.05 and r~s~= 0.71, P\< 0.01). Although highly increased sodium and chloride concentrations were found in UPS as well, the number of observations was too small to generate statistical significance due to the sample volumes that did not allow for sialochemistry (Table [4](#T4){ref-type="table"}). Results further revealed that pSS patients with the lowest UWS (i.e., 0--0.05 ml/min) also had the highest focus scores (mean focus score 2.3 vs. 0.7, respectively, P\< 0.05), presence of serum autoantibodies (n = 14), highest concentrations of salivary sodium and chloride, but lowest salivary concentrations of total phosphate compared to patients with UWS ≥ 0.05 ml/min. Pooling data of UPS and SPS for the pSS group allowed us to compare profiles of the patients with high sodium and chloride concentrations to those with low ones. The results of this analysis revealed that pSS with more than 40 mM sodium in their parotid saliva also had significantly higher focus scores as well as presence of serum autoantibodies, than pSS patients below this value (P\< 0.01). The former also tended to have lower parotid flow rates, more decayed tooth surfaces and to be younger than the other pSS patients. Discussion ========== Oral dryness and reduced salivary flow rates are some of the most predominant and troubling oral sequelae of pSS. This discussion focuses on the hypothesis if impaired salivary flow and/or composition and high caries experience may serve as potential markers of disease severity in pSS. Caries experience, oral hygiene, and salivary flow rates -------------------------------------------------------- The present study confirms the results of previous cross-sectional studies showing that pSS patients have a significantly higher DMFS compared to healthy controls, despite the fact that these patients often have a good oral hygiene and frequent dental follow-up visits \[[@B5],[@B9],[@B10]\]. When comparing SS patients with patients suffering from other immune diseases and patients with xerostomia of other origins, Boutsi et al. \[[@B26]\] found no significant differences in the number of decayed, missed or filled teeth. In accordance with our results, the SS patients were characterised by having lower salivary flow rates, better oral hygiene habits, slightly higher gingival scores, but similar plaque scores, compared to the other groups \[[@B26]\]. Regarding the other periodontal measures, our results support those of previous studies showing that presence of periodontal disease is not substantially increased in pSS \[[@B2],[@B26]\]. The slightly increased gingival scores could be explained by altered inflammatory response probably due to hormonal changes \[[@B27]\]. It has been reported that pSS patients harbour lower numbers of periopathogenic microorganisms than healthy controls \[[@B28]\]. In contrast, and despite a good oral hygiene, pSS patients appear to harbour higher numbers of cariogenic and acidophilic microorganisms such as Streptococcus mutansand Lactobacillusspecies than healthy controls \[[@B11]\]. Similar relationships between low salivary flow rates and increased Lactobacillus counts have also been observed in patients with hyposalivation of other origins \[[@B29]\]. Thus, impaired salivary flow and changes in saliva composition as seen in pSS are assumed to favour a more aciduric oral microflora manifested by an increased incidence of caries and fungal infections \[[@B28]\]. At low flow rates, the bicarbonate concentration, pH, and buffer capacity as well as the clearance of microorganisms and dietary sugars in the oral cavity generally decrease \[[@B29]\], thereby promoting an environment dominated by these oral pathogens and prolonged exposure of dietary sugars to the teeth. In pSS patients with severely reduced salivary flow rates the shift in the oral microflora appears to occur despite a good oral hygiene. In addition, the high number of microbial retention sites generated by dental restorations such as fillings, crowns and bridges found in pSS patients may contribute to the shift in oral microflora \[[@B28]\] and complicate the maintenance of sufficient oral hygiene procedures. In accordance with previous studies we found that the DMFS score is inversely correlated to salivary flow rates and especially the unstimulated whole saliva flow rate \[[@B8],[@B10]\]. However, it has not yet been possible to identify a cut-off value for salivary flow rate that can predict the risk of developing dental caries, as caries is a multifactorial disease. In this study, 90% of the patients had UWS flow rates below 0.10 ml/min, but as this value is merely a part of the classification criteria for pSS, it does not necessarily reflect the point at which caries is likely to develop. It has been suggested that salivary flow rates up to 0.16 ml/min, which include most pSS patients, may result in oral candidiasis \[[@B31]\] and increased development of experimental caries \[[@B29]\]. Meanwhile, the onset of increased caries activity in pSS remains unclear. It has been stated that in patients with SS, loss of teeth due to caries precede the first symptom of xerostomia by on average 9 years \[[@B10]\]. Changes in salivary flow and composition, and subsequently development of caries, appear to precede the symptom of oral dryness by several years. Increased caries activity that cannot be explained by changes in habits related to oral hygiene or diet may therefore represent a useful clinical feature to suspect early pSS in women without complaints of oral dryness, or intake of xerogenic medications. In contrast to the uncertainty pertaining to the onset of pSS and the initial consequences in terms of increased caries lesions, the future perspectives on the patients\' dental health seem gloomy without professional preventive intervention. In accordance with the observation that past caries experience is one of the best predictors for future caries \[[@B32],[@B33]\], the pSS patients in this study have a much higher risk of developing future caries and ultimately to loose teeth than the control group. Accordingly, these patients should receive an individual dental care programme in terms of oral hygiene instructions, professional oral hygiene regimens, fluoride treatment, dietary supervision and frequent dental follow-up visits in order to prevent accelerated caries development. Self-assessment of oral dryness ------------------------------- In healthy subjects, the sensation of oral dryness usually occurs when whole saliva flow rate is reduced with more than 50% \[[@B34]\]. In this study, all patients had complaints of dry mouth and also substantially decreased salivary flow rates, whereas the healthy controls had no dry mouth complaints and in general, salivary flow rates within the normal range \[[@B35]\]. Nonetheless, in pSS, the scores of oral dryness were only inversely correlated to SWS, which may reflect a larger span of SWS values than of UWS. We have previously found an inverse correlation between scores of oral dryness and UWS \[[@B8]\]. In the present study, the patients had symptoms of oral dryness median 10 years prior to the diagnosis of pSS. Symptoms of oral dryness in combination with the participants\' age could explain about half of the variance in DMFS (Table [5](#T5){ref-type="table"}). The questionnaire may therefore not only be helpful in assessing the intensity of oral dryness but also in identifying patients with high DMFS. Saliva pH and buffer capacity ----------------------------- The ability of human saliva to buffer acids is essential for maintaining pH values in the oral environment above the critical pH for hydroxyapatite (HAP), thereby protecting the teeth against demineralisation. The buffer systems responsible for the human saliva buffer capacity include the bicarbonate, phosphate and protein systems \[[@B17],[@B36]-[@B38]\]. In normalcy, where the pH ranges from 6.0 to 7.5, the bicarbonate and phosphate buffer systems are by far the dominant ones having optimal buffering capacity at their pK values of 6.1 and 6.8, respectively \[[@B16],[@B19]\], whereas the proteins have some effect on the buffer capacity at acidic pH values below 5 \[[@B16],[@B38]\]. In this study, the parotid saliva buffer capacity was calculated individually for each pSS patient and healthy control based on the saliva bicarbonate and phosphate concentrations at the respective saliva pH values. This calculation gives an estimate of the buffer capacity of the saliva at the time it is secreted from the parotid gland. In the pSS patients, the buffer capacity of UPS was significantly lower than in the healthy controls, mainly due to the low pH value and bicarbonate concentration in saliva caused by the low flow rates. Moreover, in pSS most of the pH values of UPS were below the relevant pK values of both the bicarbonate and phosphate buffer systems. As compared to healthy controls, the pSS patients will therefore experience far more abundant pH drops in their saliva if exposed to acidic challenges leading to a higher risk of tooth demineralisation. Apart from a low buffer capacity, and thereby impaired ability to maintain a non-acidic saliva pH, the pSS patients also had a significantly lower degree of saturation with respect to HAP in their saliva (Table [4](#T4){ref-type="table"}). Thus, in pSS, the mean pH of UPS was only one tenth of a pH unit above their mean critical pH. This implies that even a minor drop in pH will lead to undersaturation of their saliva with respect to HAP and result in either caries lesions or erosive damage to the teeth depending on the origin of the acidic challenge. Salivary bicarbonate and phosphate ---------------------------------- Despite the differences between pSS patients and healthy controls in sodium and chloride concentrations at comparable low flow rates, stimulated or not, the HCO~3~^-^concentration and saliva pH did not differ between the two groups (Fig. [1A--D](#F1){ref-type="fig"}). The transport of bicarbonate in the salivary glands is believed to occur via chloride/bicarbonate exchange mechanisms \[[@B39]\]. The concentration of bicarbonate in saliva is a consequence of the metabolic CO~2~-turnover in the salivary glands. CO~2~freely diffuses across the epithelial boundaries, and due to the presence of carbonic anhydrase, the partial pressure for CO~2~and pH in the glandular compartments governs how much of bicarbonate buffer system is present in form of HCO~3~^-^in the saliva. The duct epithelium has dual functions with respect to bicarbonate transport, since it can both reabsorb (at low secretion rates) and secrete (at high secretion rates due to increased metabolic turnover of the gland). This study demonstrated a tendency towards lower total phosphate concentrations in pSS patients than in healthy controls. Previous studies have found significantly reduced phosphate concentrations in stimulated parotid and SM/SL saliva of patients with SS compared to healthy controls and patients with conditions resembling SS \[[@B40]-[@B42]\]. It should be stressed that the mechanism behind phosphate transport in human salivary gland tissues has not yet been fully characterised. It probably includes an acinar secretion and/or a ductal reabsorption via sodium-phosphate co-transport mechanism as that observed in the renal proximal tubules \[[@B43]\]. Salivary sodium and chloride ---------------------------- Our sialochemical results are interpreted within the frame of the classical two-stage model of saliva formation \[[@B44]\]. Under normal physiological conditions, and in response to nervous stimuli, the acinar cells produce primary saliva, which has an ionic composition resembling that of plasma. As the primary saliva passes through the duct system it becomes modified by reabsorption of sodium and chloride (but without water due to the low water permeability) whereby the final saliva secreted into the oral cavity becomes hypotonic with sodium and chloride concentrations much below that of the original primary saliva. The composition of the final saliva secreted into the oral cavity strongly depends on the secretion rate in such a way that at low flow rates the saliva contains low sodium and chloride concentrations and as the flow rates increase the concentrations of sodium and chloride will rise. This normal physiological relation between parotid flow rate and sodium and chloride concentrations was seen in the healthy controls and in some of the pSS patients (0.01--1.00 ml/min) as well (Fig. [1A](#F1){ref-type="fig"} and [1B](#F1){ref-type="fig"}). However, for other pSS patients with flow rates within the same frame, another picture emerged, since their sodium and chloride concentrations were remarkably higher than normally whether stimulated or not. Thus, on a group basis the pSS patients have significantly higher concentrations of sodium and chloride than the healthy controls (Table [4](#T4){ref-type="table"}). This finding is in accordance with several previous studies on whole saliva, parotid and submandibular/sublingual saliva (SM/SL) \[[@B5],[@B40]-[@B42],[@B45],[@B46]\]. The concentrations of sodium and chloride have also been shown to be higher in SM/SL of patients with pSS and secondary SS compared to patients with clinical conditions resembling SS, i.e., sialoadenosis, sodium retention dysfunction syndrome and medication-induced xerostomia \[[@B42]\]. Overall, these compositional changes appear to be unique for some pSS patients. It has been stated that SM/SL glands are affected earlier by SS than the parotid glands due to an average reduction of stimulated SM/SL flow rate preceding that of the stimulated parotid flow rate \[[@B40],[@B42]\]. On the other hand, cut-off values for sodium, chloride and phosphate in SPS and stimulated SM/SL being predictive for SS demonstrated almost similar specificity (69 and 71%, respectively) and sensitivity (81%) \[[@B32]\]. Other salivary constituents --------------------------- Despite the low salivary flow rates seen in the pSS, the acinar transport mechanisms involved in the formation of primary saliva seem to be unaffected by the glandular lymphocytic infiltration. Accordingly, concentrations of potassium, total calcium, total protein and amylase activity in whole and parotid saliva did not differ from those of the healthy controls, which is in agreement with previous reports \[[@B40],[@B45]\]. Furthermore, it has been shown that the output of statherin and acidic proline-rich proteins, which reflect the secretion of selected parotid proteins, did not differ between pSS patients and healthy controls \[[@B5]\]. Normal concentrations of total calcium, total protein and levels of amylase activity indicate that the remaining functional acinar cells are capable of synthesis and secretion of primary saliva with normal composition despite the marked lymphocytic infiltration and structural changes. Impaired in ductal salt reabsorption reflects disease severity in pSS --------------------------------------------------------------------- The changes in salivary composition indicate that the duct epithelium, and mainly the striated duct epithelium, cannot effectively reabsorb the high concentrations of sodium and chloride of the primary saliva in some of the pSS patients, despite low salivary flow rates. The pSS patients who exhibited high concentrations of sodium and chloride were also characterised by having the lowest flow rates, the highest focus score (FS) and highest concentrations of serum autoantibodies (AB) in addition to a tendency of being younger than the pSS patients with normal salivary concentrations of sodium and chloride. Overall, the subgroup of pSS patients with the highest concentrations of salivary sodium and chloride and lowest total phosphate concentrations appeared to be more severely affected by pSS having more exocrine and non-exocrine disease manifestations than those without these salivary changes supporting previous observations \[[@B5]\]. It should be stressed that there was no relationship between reduced salivary flow and compositional changes and intake of medications. The question is whether the subgroup of patients with normal salivary concentrations of sodium and chloride and some preserved salivary gland function has a late onset of pSS or a \"milder\" glandular response to autoimmunity compared to the subgroup with high salivary sodium and chloride concentrations or salivary secretion close to or equal to 0 ml/min. Another relevant question is whether the latter subgroup of patients also has a greater risk of developing malignant lymphoma. The results of our study therefore need to be tested in a large prospective cohort study and compared with group of patients with non-immunological destruction of their salivary glands in order to validate the use of specific salivary changes and dental caries as markers of salivary gland dysfunctional severity/disease severity in pSS. At present there is no international expert consensus regarding measures for assessment of disease activity, severity, damage or outcome in pSS that can be used in the evaluation of clinical trials of new therapies and longitudinal observational studies. Recent reports, however, indicate that the development and evaluation of such measures has begun \[[@B47],[@B48]\]. In pSS, the salivary gland histopathology is characterised by periductal lymphocytic infiltration and acinar destruction. The duct epithelium, however, appears relatively unaffected by the lymphocytic infiltration, which contrasts the observed salivary changes in pSS. On the other hand, the discrepancy may be explained by morphological differences between the labial salivary glands, which predominantly consist of mucous acini and have a very short duct system, and the parotid glands, which comprise serous acini and have long duct system. Nevertheless, it has been stated that the histopathological changes of the labial salivary glands mimic those of the parotid glands \[[@B49],[@B50]\]. It has been suggested that Bcl-2 positive basal cells of striated/excretory ducts possess an extensive capacity for pluridirectional morphogenetic differentiation \[[@B51]\]. On this basis, it could be speculated that the duct cells in some pSS patients possess antigenic properties, which initiate an autoimmune response that could be associated with morphogenesis and cell differentiation of the salivary gland tissues. It has not yet been possible to identify a specific anti-salivary duct antibody in pSS that is capable of inhibiting cell differentiation. Our finding of more pronounced acinar and ductal tissue functional impairment in pSS patients with both FS and AB, than in patients with FS or AB, supports the idea that circulating autoantibodies or inflammatory mediators produced locally by the inflammatory cells, interfere with the neural release of neurotransmitter substances or interact with the binding of neurotransmitters to receptors on the cell surface, thereby impairing the acinar secretion and/or the ductal reabsorptive modification of saliva \[[@B2]\]. Along this line, an in vitrostudy on isolated human acini and duct segments from pSS patients have shown that these cells possess functional receptor systems and normal response in changes in the intracellular free calcium concentration upon maximal secretagogue stimulation \[[@B52]\]. The fact that the ductal uptake of sodium viaamiloride-sensitive epithelial sodium channel (ENaC) is regulated by circulating adrenal mineralocorticoids, e.g. aldosterone \[[@B53]\] could also indicate that some pSS patients have low levels of aldosterone. However, this still needs to be clarified. Conclusion ========== The results of this study indicate that specific sialometric and sialochemical glandular changes, particularly changes in sodium and chloride concentrations, may serve as oral markers of disease severity in pSS. The question arises whether pSS represents a continuum of patients with different stages of disease/affection of salivary glands or different diseases affecting the salivary gland tissues. There is great need of disease assessment and outcome markers in pSS. The hypotheses generated from our results on changes in salivary flow and composition as well as high caries experience as potential markers of the extent of autoimmune-mediated salivary gland dysfunction in pSS therefore need to be tested in a large prospective cohort study including patients with early to long-standing disease. In addition, the mechanisms underlying the impaired ductal salt reabsorption observed in the pSS patients with presence of both labial salivary gland focus score and serum-autoantibodies need to be further elucidated in future pathophysiological studies. Abbreviations ============= SS = Sjögren\'s syndrome; pSS = primary Sjögren\'s syndrome; FS = focal lymphocytic infiltration in the labial salivary glands; UWS = unstimulated whole saliva flow rate; SWS = stimulated whole saliva flow rate; UPS = unstimulated parotid saliva flow rate; SPS = stimulated parotid saliva flow rate; AB = presence of serum autoantibodies to Ro (SSA) and/or La (SSB) antigens; DMFS = decayed, missing and filled tooth surfaces; PI and GI = plaque and gingival indices; PPD = periodontal probing pocket depth; HAP = hydroxyapatite. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= All three authors participated in the design of the study and the statistical analysis. AMLP attended coordination of the study and conducted the whole saliva collections, interviews, oral clinical examinations and labial salivary gland biopsies. AB performed the parotid saliva collections and sialochemical analyses. 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/1472-6890/5/4/prepub> Acknowledgements ================ We would like to acknowledge skilled technical assistance by laboratory technician Mrs. Joan Lykkeaa and the support of the Danish Dental Association Research Foundation (DTF and FUT/Calcin), the Colgate Research Foundation, the Danish Medical Research Council and the Danish Research Foundation Sygekassernes Helsefond.	PubMed Central	2024-06-05T03:55:54.069055	2005-3-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554998/", "journal": "BMC Clin Pathol. 2005 Mar 1; 5:4", "authors": [ { "first": "Anne Marie Lynge", "last": "Pedersen" }, { "first": "Allan", "last": "Bardow" }, { "first": "Birgitte", "last": "Nauntofte" } ] }
PMC555463	Background ========== Osteoporosis is defined as \' . . .a skeletal disorder characterized by compromised bone strength predisposing a person to an increased risk of fracture. Bone strength reflects the integration of two main features; bone density and bone quality\' \[[@B1]\]. The presence of a vertebral or other fragility fracture is a strong predictor of risk of future fracture, and is a major indicator of the presence of osteoporosis \[[@B2]-[@B4]\]. A fragility fracture is defined as a low trauma fracture (eg. a fall from a standing height or less) \[[@B3]\]. Many studies indicate that fewer than 30% of patients with fragility fractures are being treated for osteoporosis. Rates of treatment appear particularly low following hospital discharge (\<10%) \[[@B5]-[@B7]\], suggesting that treatment recommendations are not being made to family physicians. Assessment of treatment rates 6 months to 2 years after fracture are somewhat higher (in the 20--38% range) \[[@B8]-[@B13]\]. Kiebzak et al. followed patients for 1--5 years after hip fracture. At follow-up, they found that 71% of women were receiving treatment, but the proportion of men receiving therapy remained relatively low \[[@B14]\]. Four Canadian studies of osteoporosis treatment following fragility fracture were identified. Two studies examined osteoporosis treatment among hip fracture patients after hospital discharge, and in both, treatment rates were relatively low (\< 10%) \[[@B6],[@B15]\]. In Ontario, Hajcsar et al. interviewed patients one year after fragility fracture, and found that the use of approved pharmaceutical agents such as bisphosphonates and hormone replacement therapy (HRT) remained low (7.4% and 16%, respectively) \[[@B13]\]. Khan et al. reported considerably higher treatment rates among patients after wrist fracture, with approximately 38% of patients receiving either a bisphosphonate or HRT at follow-up \[[@B9]\]. The purpose of this study was to examine osteoporosis treatment among older home care clients with a diagnosis of osteoporosis and/or prevalent fracture. As this population has relatively frequent contact with health care providers, we expected that osteoporosis treatment rates should be higher than previously reported. Methods ======= Participants were 330 older home care clients participating in a longitudinal study of medication adherence and related health outcomes. Details of the primary study can be found elsewhere \[[@B16]\]. Briefly, between April and June of 2000, 585 home care clients residing in two southern Alberta health regions were identified by random samples stratified by rural/urban residence. Rural clients represented those living on a farm, acreage or in a village or town (with a population less than 10,000) and residing greater than 35 km from a major urban centre (cities of Calgary or Lethbridge). Inclusion criteria were: currently receiving home care services under the jurisdiction of their respective health region, age 65 or greater and provision of informed consent from either the subject or a legal guardian. Of the original random sample of 585, 10 subjects had died and 10 had moved or were unavailable at the time of study recruitment. Further exclusions included subjects who were in hospital, transferred to long term or palliative care, or mentally incompetent without a legal guardian (n = 40), who required a translator (n = 6) or who posed a safety concern for the study nurses (n = 7). Of the remaining 512 subjects, 46 (9.0%) refused to participate and 136 (26.6%) were not contacted after the predetermined sample size (based on estimation of differences in adherence rates between rural and urban subjects) \[[@B16]\] had been achieved. Data on demographics, health and functional status and service utilization were collected with a standardized assessment tool, the Minimum Data Set for Home Care (MDS-HC) \[[@B17],[@B18]\], and several supplemental questions regarding medication use, smoking and tobacco use. Information on therapeutic substances was recorded from container labels for all substances used in the past 7 days. Therapeutic substances included prescribed, over-the-counter and complementary or alternative products. All drug data were entered into a database using the Anatomical Therapeutic Chemical classification system (ATC codes). Assessment of medication adherence was based on self-report data \[[@B16]\]. Four study nurses, trained in the administration of the MDS-HC and medication assessment, collected data during in-home interviews lasting approximately 1.5 hours. This study received ethics approval from the Health Research Ethics Board of the University of Calgary and the Ethics Review Committee of the Chinook Health Region. Fracture prevalence and diagnosis of osteoporosis were determined from the disease diagnoses section of the MDS-HC (Section J). The MDS-HC includes specific categories for charting of: \'hip fracture\', \'other fracture\', and \'osteoporosis\'. Prevalent fractures were defined as those that had resulted in a hospitalization (in last 90 days), currently required treatment and/or symptom management or were being monitored by a home care professional. Coverage for treatment was examined for anti-osteoporotic therapies approved for use in 2000 \[[@B19]\]. These included calcium with vitamin D, and the following pharmaceutical agents: etidronate, alendronate, hormone replacement therapy, raloxifene and calcitonin. Prevalence estimates of treatment for osteoporosis among subjects with a prevalent fracture and/or diagnosis of osteoporosis were reported. Descriptive bivariate comparisons were also conducted to examine potential variations between osteoporotic subjects who received treatment and those who did not. Fisher\'s exact test was reported for the bivariate comparisons. Due to the limited number of subjects with osteoporosis or fracture, multivariable analyses were not feasible. Results ======= Demographic and health status variables are summarized in Table [1](#T1){ref-type="table"}. The mean age of the total sample (n = 330) was 82 years (standard deviation = 7.8, range 65--101) and most clients were female (78.5%) and not married (70.6%). Almost half (41.8%) had completed at least high school education and a substantial proportion of subjects lived in a seniors\' lodge (36.7%). A total of 78 subjects (23.6%) had at least one potential indication for osteoporosis treatment. The demographic characteristics of these subjects were similar to the sample as a whole (Table [1](#T1){ref-type="table"}), with the exception that this group included a greater proportion of women (93.6%). Demographic data were only available for a sub-group of all non-respondents. Analyses of this sub-sample showed no significant differences between non-respondents and respondents in relation to age and sex. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Summary of demographic characteristics and health status among older home care clients. ::: Variable Total Study Population N = 330 % (n) Subjects with diagnosis of osteoporosis or fracture N = 78 % (n) --------------------------------- ------------------------------------------ ---------------------------------------------------------------------- [Demographics]{.underline} Age Mean (SD) 82 (7.8) 83 (8.3) (≥ 85) 43.3 (143) 42.3 (33) Female 78.5 (259) 93.6 (73) Not married 70.6 (233) 79.5 (62) Education (≥ High School) 41.8 (138) 44.9 (35) Living arrangements Seniors\' Lodge\* 36.7 (121) 34.6 (27) [Health Status]{.underline} Cognitive impairment (CPS\\ score \>1) 23.3 (77) 24.4 (19) \# Comorbid conditions (\>2) 71.8 (237) 71.8 (237) \* Versusprivate home \\ MDS-HC Cognitive Performance Scale ::: The prevalence of osteoporosis diagnoses and fractures are summarized in Table [2](#T2){ref-type="table"}. Forty-seven subjects (14.2%) had a diagnosis of osteoporosis, three of whom had at least one prevalent fracture. Prevalence of at least one fracture without diagnosis of osteoporosis was recorded for 31 (9.4%) subjects; 13 hip fractures (1 with \'other fracture\'), and 18 with \'other\' fractures. One client had only unlabelled medication bottles, thus medication data were available for 77 of these subjects. Approximately 50% of subjects with a diagnosis of osteoporosis or prevalent hip fracture received treatment, compared to only 28% of subjects with \'other\' fractures (p = 0.11). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Indications for treatment of osteoporosis among older home care clients. ::: Diagnoses N = 330 *Receiving treatment\ N = 77** ------------------------------- ----------------------- ---------------------------------- Diagnosis of osteoporosis\\ 14.2 (47) 50.0 (23/46^†^) Hip fracture^‡^ 3.9 (13) 53.8 (7/13) \'Other\' fracture^??^ 5.5 (18) 27.8 (5/18) Total 23.6 (78) 45.5 (35/77) \* Treatments included: calcium with vitamin D and/or drug therapy (bisphosphonate or hormone replacement therapy). \\ Three subjects also had at least one fracture. ^†^Medications unlabelled for one subject. ^‡^One subject also had \'other fracture\' ?? Type of fracture was available for 10/18 subjects: 3 wrist, 1 humerus, 2 ankle, 2 vertebral, 1 shoulder and 1 rib fracture. ::: Specific therapeutic interventions for osteoporosis among subjects with a diagnosis of osteoporosis and/or fracture are shown in Figure [1](#F1){ref-type="fig"}. No subjects were using calcitonin or raloxifene. In total, only 35 subjects (45%) were receiving at leastminimal treatment for osteoporosis (i.e. at least calcium with vitamin D). Only 30 subjects (39%) were receiving therapy with an approved prescription drug (bisphosphonate and/or hormone replacement therapy), with 5 subjects (6%) being treated only with calcium and vitamin D. Six subjects receiving prescription drug therapy were also taking calcium and vitamin D (not shown in Figure). Of the 42 subjects not receiving minimal treatment (i.e. drug therapy and/or calcium with vitamin D), 3 were taking calcium only, and 10 were taking a multivitamin that may have contained calcium and/or vitamin D. Bivariate comparisons of potential factors that may influence receipt of treatment are summarized in Table [3](#T3){ref-type="table"}. A greater proportion of nonadherent subjects were observed in the group that did not receive treatment (50% versus31%). The proportion of subjects with lower education levels was also relatively higher among the non-treated group. None of the 5 men among the osteoporotic group were receiving treatment. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Comparison of osteoporotic subjects by treatment status. (n = 77) ::: Variable No Treatment n = 42 % (n) Treatment n = 35 % (n) Fisher\'s exact p-value ---------------------------------------------- ------------------------------- ---------------------------- ----------------------------- Age (≥ 85) 42.8 (18) 42.8 (15) 1.00 Sex (male) 11.9 (5) 0 0.06 Education (≥ High School) 38.1 (16) 54.3 (19) 0.18 Residence (seniors\' lodge) 40.4 (17) 28.6 (10) 0.34 Cognitively impaired 23.8 (10) 22.8 (8) 0.60 Depression 14.3 (6) 14.3 (5) 0.75 Confined to wheelchair/bed 21.4 (9) 20.0 (7) 1.00 Fall(s)in past 90 days 31.0 (13) 22.9 (8) 0.45 On steroid medication 4.76 (2) 11.4 (4) 0.40 Nonadherent (with any prescribed medication) 50.0 (21) 31.4 (11) 0.11 ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Osteoporosis therapy among subjects with a diagnosis of osteoporosis and/or fracture. (HRT = hormone replacement therapy) No treatment (n = 29) Calcium only (n = 3) Multivitamin (n = 10) Bisphosphonate (n = 21) HRT (n = 7) Bisphosphonate + HRT (n = 2) Calcium + vitamin D only (n = 5) ::: ![](1471-2474-6-7-1) ::: Discussion ========== Home care clients are expected to have relatively frequent contact with health care providers. Among those with a diagnosis of osteoporosis or prevalent fracture (n = 78), 30% had weekly contact with nursing staff, 42% had at least one emergency or emergent care visit or hospitalization in the past 3 months, and the majority (67%) had a recent (\<6 months) review of their total medication regimen. Thus, more opportunity for intervention following fracture, and higher rates of treatment would be expected among this population. Although the proportion of home care clients receiving treatment was somewhat higher than reported in most previous studies (45% versus\<39%, respectively) \[[@B5]-[@B8]\], \[[@B10]-[@B13]\], the majority of subjects with a diagnosis of osteoporosis or fracture indicative of osteoporosis were still not receiving even minimal therapy (i.e. calcium and vitamin D). Consistent with previous reports \[[@B14]\], we also found that men were not receiving therapy following fracture. In this home care sample, none of the 5 men with indication for osteoporosis treatment were receiving therapy. Three of these men had fractures at sights other than at the hip, where lower intervention rates were observed even for women. However, 2 of these men had a charted diagnosis of osteoporosis and were still not receiving treatment. Our data also indicate that subjects who fracture at sites other than the hip may be less likely to receive treatment. While many of the fractures observed in this study (Table [2](#T2){ref-type="table"}) were typical of osteoporotic fractures with respect to fracture site \[[@B2],[@B4]\], our data are limited in that the MDS-HC does not include an assessment of the cause of the fracture. If some fractures were the result of trauma, our findings may overestimate the deficit in osteoporosis therapy. Conversely, the prevalence of osteoporosis may be underestimated in our study population, as the diagnosis was based on self-report information. The prevalence of osteoporosis in Canadian women aged 50+ years, based on BMD, has been estimated at 15% \[[@B20]\]. If diagnosis were based on BMD alone, a higher prevalence would be expected in the older population in this study. However, physicians\' diagnoses of osteoporosis may derive from factors other than BMD, and our findings are consistent with previous work that suggests that many patients with osteoporosis are not receiving appropriate therapy. We also examined several factors potentially associated with undertreatment. However, the small numbers of subjects with osteoporosis or prevalent fracture limit the interpretation of our findings. Although some trends are apparent, such as undertreatment of men and those with lower education, we cannot comment conclusively on these observations due to the limited sample size and cross-sectional nature of the study. Further evaluation of these factors utilizing a larger sample and appropriate multivariable analysis may provide further insight regarding the potential impact of specific determinants. Speculation regarding reasons for the apparent undertreatment of osteoporosis has focused on physician oversight. However, several factors may play a role in the choice to initiate treatment. A recent study indicated that some physicians have concerns about the proven efficacy of osteoporosis treatment among older populations and may be exercising judgment with respect to minimizing polypharmacy in this population \[[@B21]\]. Further, physicians are not the only factor involved in decisions to initiate, and/or continue with therapy. In a study conducted by McKercher et al., physicians reported that the choice to initiate osteoporosis treatment was also dependent upon acceptance of the therapy by patients and/or family members \[[@B21]\]. Interviews with patients following fragility fracture also indicate that some patients choose not to use osteoporosis therapy \[[@B8],[@B13]\] (e.g. concerns about side effects). The higher rate of nonadherence with medications among those not receiving therapy in this study, also suggests that lack of therapy may be due to patient choices. However, the assessment of adherence by self-report, as used in this study, only provided a measure of adherence with overall drug regimens, not specific medications. Collection of more detailed information on adherence with specific osteoporosis therapies in future studies may clarify this association. Despite limitations, our findings highlight the growing concern that many patients with osteoporosis are not receiving appropriate therapeutic interventions. The lower treatment rate among subjects with fractures at sites other than the hip suggests that physicians may not be recognizing the probability of underlying osteoporosis. While the diagnosis of osteoporosis by BMD measurement has been widely publicized, recent clinical practice guidelines have emphasized the importance of a history of fragility fracture in the identification of patients with osteoporosis \[[@B3]\]. Our findings suggest that improving recognition of osteoporosis among older persons presenting with fractures may be an important educational objective for practicing physicians. Conclusions =========== Four previous studies conducted in Canada have examined osteoporosis diagnosis and treatment interventions following fracture. Our data confirm low treatment rates among patients with fracture, and also indicate that even patients with a documented diagnosis may not be receiving therapy. The reasons for lack of treatment of osteoporosis are not yet clear. The reports of patient and physician concerns regarding side effects and polypharmacy warrant further investigation, and suggest that nonpharmacologic interventions may be more acceptable in certain patient populations. Considering the complexity of issues involved in decisions to initiate and continue with treatment, future studies focusing more on evaluation of physician and patient awareness of osteoporosis and factors influencing treatment decisions are needed. Evaluation of other patient-related factors, such as adherence and persistence with osteoporosis therapy, and their impact on health outcomes (e.g. fracture), are also relevant. Such studies may provide insight regarding specific interventions needed to reduce risk of morbidity and mortality associated with osteoporosis. Competing interests =================== Shelly Vik and Colleen Maxwell have no competing interests. David Hanley\'s competing interests include consultancies with, honoraria for speaking from, or involvement in research with, the following companies or organizations: Amgen, Astra-Zeneca, Aventis, the Dairy Farmers of Canada, Eli Lilly, Merck, Novartis, NPS Pharmaceuticals, Pfizer, Procter and Gamble, Roche, and Wyeth. Authors\' contributions ======================= Analysis of data, interpretation and the original draft were completed by Shelly Vik. Colleen Maxwell and David Hanley contributed to conception and interpretation, and provided critical evaluation of clinical and methodological content, and subsequent revisions. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2474/6/7/prepub> Acknowledgements ================ We are most grateful to the four home care nurses (Michelle Copeland and Sue Couchman from the Calgary Health Region and Bonnie Matson and Trudy Harbidge from the Chinook Health Region) for their data collection efforts and clinical input on this home care study. We wish to also acknowledge Chad Mitchell for his clinical input and assistance with drug coding. Special thanks is also given to the 330 home care clients and their caregivers (formal and informal) who graciously gave of their time to participate in this important study. The Resident Assessment Instrument-Home Care study/initiative was supported by an unrestricted grant from The Merck Company Foundation, the philanthropic arm of Merck & Co. Inc. Whitehouse Station, New Jersey, USA to the Institute of Health Economics. Dr. Maxwell is funded by a New Investigator Award from the Canadian Institutes of Health Research -- Institute on Aging and a Population Health Investigator Award from the Alberta Heritage Foundation for Medical Research (AHFMR). Shelly Vik is currently a PhD candidate (Epidemiology), and is funded by the Canadian Institute of Health Research (CIHR) Skeletal Health Training Program and the Alberta Provincial CIHR Training Program in Bone and Joint Health.	PubMed Central	2024-06-05T03:55:54.073836	2005-2-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555463/", "journal": "BMC Musculoskelet Disord. 2005 Feb 11; 6:7", "authors": [ { "first": "Shelly A", "last": "Vik" }, { "first": "Colleen J", "last": "Maxwell" }, { "first": "David A", "last": "Hanley" } ] }
PMC555464	Background ========== Ant Colony Optimisation (ACO) is a population-based stochastic search method for solving a wide range of combinatorial optimisation problems. ACO is based on the concept of stigmergy-- indirect communication between members of a population through interaction with the environment. An example of stigmergy is the communication of ants during the foraging process: ants indirectly communicate with each other by depositing pheromone trails on the ground and thereby influencing the decision processes of other ants. This simple form of communication between individual ants gives rise to complex behaviours and capabilities of the colony as a whole. From the computational point of view, ACO is an iterative construction search method in which a population of simple agents (\'ants\') repeatedly constructs candidate solutions to a given problem; this construction process is probabilistically guided by heuristic information on the given problem instance as well as by a shared memory containing experience gathered by the ants in previous iterations (\'pheromone trails\'). Following the seminal work by Dorigo et al.\[[@B1],[@B2]\], ACO algorithms have been successfully applied to a broad range of hard combinatorial problems, including the traveling salesman problem, the graph colouring problem, the quadratic assignment problem and vehicle routing problems (see, e.g.,\[[@B3]-[@B5]\]). The research presented in this paper builds on an ACO algorithm first proposed in \[[@B6]\] (and later improved in \[[@B7]\]) for ab-initioprotein folding under a widely studied abstract model -- the hydrophobic polar (HP) model. In particular, we extend our previous ACO algorithm to the 3D HP model and improve its performance by modifying the subsidiary local search procedure. The protein folding problem is one of the most challenging problems in computational biology, molecular biology, biochemistry and physics. Even under simplified lattice models, the protein folding problem is non-deterministic polynomial-time hard (NP-hard) \[[@B8]\]. The ab-initioprotein folding problem can be broken down into three sub-problems: 1) design of a model (with a desired level of accuracy); 2) definition of an energy function that can effectively discriminate between native and non-native states; and 3) design of a search algorithm that can efficiently find minimal-energy conformations. A number of search (or sampling) methods have been proposed in the literature to solve the protein folding problem, including Monte Carlo algorithms, Evolutionary Algorithms, Tabu Search and hybrid approaches. ACO, which has been very successfully applied to other combinatorial problems, appears to be a very attractive computational method for solving the protein folding problem, since it combines aspects of chain-growth and permutation-based search with ideas closely related to reinforcement learning. These concepts and ideas apply rather naturally to protein folding: By folding from multiple initial folding points, guided by the energy function and experience from previous iterations of the algorithm, an ensemble of promising, low-energy complete conformations is obtained. These conformations are further improved by a subsidiary local search procedure and then evaluated to update the accumulated pheromone values that are used to bias the generation of conformations in future iterations of the algorithm. In this paper, we ask and address the following questions: Is ACO a competitive method for solving the ab-initioprotein folding problem under the 2D and 3D HP models? How does its performance scale with sequence length? What is the role of the parameters of the ACO algorithm for the efficiency of the optimisation process? Which classes of structures (if any) are solved more efficiently by ACO than by any other known algorithms? Finally, it should be noted that our ACO algorithm for this problem is based on very simple design choices, in particular with respect to the solution components reinforced in the pheromone matrix and of the subsidiary local search procedure. We discuss which of the many design choices underlying our algorithm should be reconsidered in order to achieve further performance improvements. The hydrophobic polar model --------------------------- Due to the complexity of the protein folding problem, simplified models such as Dill\'s hydrophobic-polar (HP) model have become one of the major tools for studying protein structure \[[@B9]\]. The HP model is based on the observation that the hydrophobic force is the main force determining the unique native conformation (and hence the functional state) of small globular proteins \[[@B9],[@B10]\]. In the HP model, the primary amino acid sequence of a protein (which can be represented as a string over a twenty-letter alphabet) is abstracted to a sequence of hydrophobic (H) and polar (P) residues that is represented as a string over the letters H and P. The conformations of such an HP sequence are restricted to self-avoiding walks on a lattice. For the 2D HP model, a 2-dimensional square lattice is typically used, and the 3D HP model is generally based on a 3-dimensional cubic lattice. An example of a protein conformation under the 2D HP model is shown in Figure [1](#F1){ref-type="fig"}. The energy of a conformation is defined as the number of topological contacts between hydrophobic amino acids that are not neighbours in the given sequence. More specifically, a conformation cwith exactly nsuch H-H contacts has energy E(c) = n·(-1); for example, the 2D HP conformation shown in Figure [1](#F1){ref-type="fig"} has energy -9. The HP Protein Folding Problem can be formally defined as follows: Given an HP sequence s= s~1~s~2~\...s~n~, find an energy-minimising conformation of s, i.e.,find c\* ∈ C(s) such that E(c\) = min{E(c) \\| c∈ C}, where C(s) is the set of all valid conformations for s. It has been proved recently that this problem and several variations of it are NP-hard \[[@B8]\]. Existing 2D and 3D HP protein folding algorithms ------------------------------------------------ A number of well-known heuristic optimisation methods have been applied to the 2D and 3D HP Protein Folding Problem, including Evolutionary Algorithms (EAs) \[[@B11]-[@B15]\] and Monte Carlo (MC) algorithms \[[@B16]-[@B22]\]. The latter have been found to be particularly robust and effective for finding high-quality solutions to the HP Protein Folding Problem \[[@B18]\]. Besides general optimisation methods, there are other heuristic methods that rely on specific heuristics that are based on intuitions or assumptions about the folding process, such as co-operativity of folding or the existence of a hydrophobic core. Co-operativity is believed to arise from local conformational choices that result in a globally optimal state without exhaustive search \[[@B23]\]. Among these methods are the hydrophobic zipper method (HZ) \[[@B23]\], the contact interactions method (CI) \[[@B24]\], the core-directed chain growth method (CG) \[[@B25]\], and the constraint-based hydrophobic core construction method (CHCC) \[[@B26]\]. The hydrophobic zipper (HZ) strategy developed by Dill et al.is based on the hypothesis that once a hydrophobic contact is formed it cannot be broken, and other contacts are formed in accordance with already folded parts of the chain (co-operativity of folding) \[[@B23]\]. The contact interactions (CI) algorithm by Toma and Toma \[[@B24]\] combines the idea of HZ with a Monte Carlo search procedure that assigns different conformational freedom to the different residues in the chain, and thus allows previously formed contacts to be modified according to their computed mobilities. The core-directed chain growth method (CG) by Beutler and Dill \[[@B25]\] biases construction towards finding a good hydrophobic core by using a specifically designed heuristic function and by approximating the hydrophobic core with a square (in 2D) or a cube (in 3D). The constraint-based hydrophobic core construction method (CHCC) by Yue and Dill \[[@B26]\] is complete, i.e.,always guaranteed to find a global optimum; it attempts to find the hydrophobic core with the minimal possible surface area by systematically introducing geometric constraints and by pruning branches of a conformational search tree. A similar, but more efficient complete constraint satisfaction search method has been proposed by Backofen et al.\[[@B27]\] for the more complex face-centred cubic lattice. An early application of Evolutionary Algorithms to protein structure prediction was presented by Unger and Moult \[[@B14],[@B15]\]. Their non-standard EA incorporates characteristics of Monte Carlo methods. Currently among the best known algorithms for the HP Protein Folding problem are various Monte Carlo algorithms, including the \'pruned-enriched Rosenbluth method\' (PERM) of Grassberger et al.\[[@B16],[@B18]\]. PERM is a biased chain growth algorithm that evaluates partial conformations and employs pruning and enrichment strategies to explore promising partial solutions. Other methods for solving protein folding problems include the dynamic Monte Carlo algorithm by Ramakrishnan et al.\[[@B21]\], which introduced long-range moves involving disconnection of the chain, and the evolutionary Monte Carlo (EMC) algorithm by Liang and Wong \[[@B19]\], which works with a population of individuals that each perform Monte Carlo optimisation; a variant of EMC also reinforces certain secondary structures (alpha-helices and beta-sheets). Finally, Chikenji et al.introduced the multi-self-overlap ensemble (MSOE) Monte Carlo method \[[@B17]\], which considers overlapping chain configurations. Other Monte Carlo methods that have been particularly useful in off-lattice protein folding include generalised ensemble methods, such as umbrella sampling \[[@B28]\] (with replica exchange sampling \[[@B29],[@B30]\] being the most common variant) and multi-canonical (entropic) sampling \[[@B30],[@B31]\]. Replica exchange Monte Carlo (parallel tempering) has also been applied to the off-lattice HP model \[[@B32]\]. Currently, when applied to the square and cubic lattice HP model, none of these algorithms appears to completely dominate the others in terms of solution quality and run-time. Our ACO algorithm for the 2D and 3D HP protein folding problem -------------------------------------------------------------- In previous work, we have applied ACO to the 2D HP Protein Folding Problem \[[@B6],[@B7]\]; in the following, we briefly summarise the main features of our ACO algorithm and the improvements introduced in this work. Details on our ACO framework and the new ACO-HPPFP-3 algorithm developed in the context of this work are given in the \'Methods\' section. As usual, the ants in our ACO algorithm iteratively undergo three phases: the construction phase-- during which each ant constructs a candidate solution by sequentially growing a conformation of the given HP sequence, starting from a folding point that is chosen uniformly at random among all sequence positions; the local search phase-- when ants further optimise protein conformations folded during the construction phase; and the pheromone update phase-- when ants update the pheromone matrix (representing the collective global memory of the colony) based on the energies of the conformations obtained after the construction and the local search phases. A general outline of ACO is shown in Figure [2](#F2){ref-type="fig"}. The solution components used during the construction process, the local search phase and the pheromone update are local structure motifs (or relative folding directions) straight(S), left(L), right(R) in 2D, and straight(S), left(L), right(R), up(U), down(D) in 3D, which for each amino acid indicate its position on the 2D or 3D lattice relative to its direct predecessors in the given sequence (see Figure [3](#F3){ref-type="fig"}). In 3D, the relative folding directions are defined as in \[[@B33]\]: A local coordinate system is associated with every sequence position, such that Scorresponds to the direction of the xaxis, Lto the direction of the yaxis, and Uto the direction of the zaxis. Each local motif corresponds to a relative rotation of this coordinate system (for the forward construction: S = no rotation, L = 90° counter-clockwise around the zaxis, R = 90° clockwise around the zaxis, U = 90° clockwise around the yaxis, D = 90° counter-clockwise around the yaxis). Since conformations are rotationally invariant, the position of the first two amino acids can be fixed without loss of generality. Hence, we represent candidate conformations for a protein sequence of length nby a sequence of local structure motifs of length n- 2. For example, the conformation of Sequence S1-1 shown in Figure [1](#F1){ref-type="fig"} corresponds to the motif sequence LSLLRRLRLLSLRRLLSL. During the construction phase, ants fold a protein from an initial folding point by probabilistically adding one amino acid at a time based on the two sources of information: pheromone matrix values τ(which represent previous search experience and reinforce certain structural motifs) and heuristic function values η(which reflect current energy of the considered structural motif); details of this process are given in the \'Methods\' section. The relative importance of τand ηis determined by parameters αand β, respectively, whose settings are detailed in the \'Discussion\' section. Similar to other ACO algorithms known from the literature, our algorithm for the HP Protein Folding Problem incorporates a local search phase that takes the initially built protein conformation and attempts to optimise its energy further, using probabilistic long-range moves that are described in detail in the \'Methods\' section. Finally, the pheromone update procedure is based on two mechanisms: Uniform pheromone evaporation is modelled by decreasing all pheromone levels by a constant factor ρ(where 0 \<ρ≤ 1), and pheromone reinforcement is achieved by increasing the pheromone levels associated with the local folding motifs used in a fraction of the best conformations (in terms of energy values) obtained during the preceding construction and local search phase. Furthermore, to prevent search stagnation when all of the pheromone is accumulated on very few structural motifs, we introduce an additional renormalisation mechanism for the pheromone levels (controlled by a parameter θwhere 0 ≤ θ\< 1; details are given in the \'Methods\' section). Different from our previous ACO algorithms for the HP Protein Folding Problem, our new algorithm, ACO-HPPFP-3, supports the 3D HP cubic lattice model in addition to the 2D HP square lattice model. Furthermore, it uses a different iterative improvement strategy, a modified long-range move operator and a less restrictive termination criterion in its local search phase. ACO-HPPFP-3 was used in all ACO experiments described in the following. Results ======= To compare ACO-HPPFP-3 with algorithms for the 2D and 3D HP Protein Folding Problem described in the literature, we tested it on a number of standard benchmark instances as well as on two newly created data sets, one of which was obtained by randomly generating amino acid sequences with hydrophobicity value characteristic of globular proteins, while the other consists of biological sequences that were translated into HP strings using a standard hydrophobicity scale. (These new data sets will be described in more detail later in this section.) Results for standard benchmark instances ---------------------------------------- The 21 standard benchmark instances for 2D- and 3D-HP protein folding shown in Table [1](#T1){ref-type="table"} have been widely used in the literature \[[@B6],[@B12],[@B14]-[@B17],[@B19],[@B25]\]. Experiments on these standard benchmark instances were conducted by performing a number of independent runs for each problem instance (in 2D: 500 runs for sequence length n≤ 50, 100 runs for 50 \<n≤ 64, and 20 runs for n \>64; in 3D: 100 runs for each sequence). Unless explicitly indicated otherwise, we used the following parameter settings for all experiments: α: = 1, β: = 2, ρ: = 0.8 and θ: = 0.05. Furthermore, all pheromone values were initialised to 1/3 in 2D and to 1/5 in 3D, and a population of 100 ants was used, 50% of which were allowed to perform local search. The local search procedure was terminated when no improvement in energy had been obtained after between 1 000 (for n≤ 50) and 10 000 (for n\> 50) scans through the protein sequence. We used an elitist pheromone updating scheme in which only the best 1% of all ants was allowed to perform pheromone updates. The probability ![](1471-2105-6-30-i2.gif) of keeping the previous direction when feasible during the long-range mutation move was set to 0.5 (see \'Methods\' section). These settings were determined in a series of experiments in which we studied the influence of different parameter settings and will be further discussed later. All experiments were performed on PCs with 2.4 GHz Pentium IV CPUs, 256 Kb cache and 1 MB RAM, running Redhat Linux (our reference machine), and run-time was measured in terms of CPU time. Most studies of EA and MC methods in the literature, including \[[@B12],[@B14],[@B15],[@B19]\], report the number of valid conformations scanned during the search. This makes a performance comparison difficult, since run-time spent for backtracking and the checking of partial or infeasible conformations, which may vary substantially between different algorithms, is not accounted for. We therefore compared ACO to the best-performing algorithm from the literature for which performance data in terms of CPU time is available -- PERM \[[@B18]\] (we used the most recent implementation, which was kindly provided by P. Grassberger). We note that the most efficient PERM variant for the HP Protein Folding Problem uses an additional penalty of 0.2 for H-P contacts \[[@B34]\]. Since this corresponds to an energy function different from that of the standard HP model underlying our ACO algorithm as well as other algorithms developed in literature, we used the best performing variant of PERM \[[@B18]\] based on the standard energy function in our experiments. It may be noted that the chain growth process in PERM can start from the N- or C-terminus of the given HP sequence, and in many cases, this results in substantial differences in the performance of the algorithm. To capture this effect, we always ran PERM in both directions, and in addition to the respective average run-times, t~1~and t~2~, we report the expected time for solving a given problem instance when performing both runs concurrently, t~exp~= 2·(1/t~1~+ 1/t~2~)^-1^. For all runs of PERM, the following parameter settings were used: inverse temperature γ: = 26 and q: = 0.2. The results obtained on standard 2D benchmark instances (see Table [2](#T2){ref-type="table"}) indicate that ACO-HPPFP-3 is competitive with the EA and MC methods described in the literature; it works very well on sequences of sizes up to 64 amino acids and produces high quality suboptimal configurations for the longest sequences considered here (85 and 100 amino acids). On average, ACO requires less CPU time than PERM for finding best known conformations for Sequence S1-8; but PERM performs better for Sequences S1-6 and S1-7 as well as for the longer sequences of 85 to 100 residues (Sequence S1-9 to S1-11). Sequence S1-8 has a very symmetrical optimal state (see Figure [4](#F4){ref-type="fig"}), which -- as argued in \[[@B18]\] -- would be difficult to find for any chain growing algorithm. All algorithms from the literature which we are aware of have problems folding this sequence; ACO-HPPFP-3, on the other hand, is able to handle this instance quite well, since a number of ants folding from different starting points in conjunction with a local search procedure that involves large-scale mutations originating from different sequence positions can produce good partial folds for various parts of the chain. In comparison with other algorithms for the 2D HP Protein Folding Problem considered here (EA, EMC, MSOE), ACO-HPPFP-3 generally shows very good performance on standard benchmark instances. In case of the 3D HP Protein Folding Problem (see Table [3](#T3){ref-type="table"}), the majority of algorithms for which we were able to find performance results in the literature use heuristics that are highly specialised for this problem. Unlike HZ, CG and CI, ACO-HPPFP-3 finds optimal (or best known) solution qualities for all sequences. However, PERM (when folding from the N-terminus) and CHCC consistently outperform ACO-HPPFP-3 on these standard 3D HP benchmark instances, and CG reaches best known solution qualities substantially faster in many cases. We note that for Sequence S2-3 and S2-7, PERM\'S performance is greatly dependent on the folding direction. Result for new biological and random data sets ---------------------------------------------- To thoroughly test the performance of ACO-HPPFP-3, we created two new data sets of random and biological sequences of length ≈ 30 and ≈ 50 amino acids (ten sequences for each length; for details, see [additional data file 1](#S1){ref-type="supplementary-material"}). Random sequences were generated based on the observation that most globular proteins have a fairly uniform amino acid profile, and the percent of hydrophobic residues of majority of globular proteins falls in the range of 40--50% \[[@B35]\]. Thus, the probability of generating character Hat each position of a sequence was chosen to be 0.45, and in the remaining cases (i.e.,with probability 0.55), we generated a P. For the biological test-sets, ten sequences were taken from the PDBSELECT data set with homology \< 25% from the Protein Data Bank (PDB) in order to obtain a non-redundant representative set of proteins. These protein sequences were translated into HP strings using the hydrophobicity scale classification of RASMOL \[[@B36]\], according to which the following amino acids were considered hydrophobic: Ala, Leu, Val, Ile, Pro, Phe, Met, Trp, Glyand Tyr. Non-standard amino acid symbols, such as X and Z, were skipped in this translation. Figures [5](#F5){ref-type="fig"} and [6](#F6){ref-type="fig"} illustrate the performance of ACO-HPPFP-3 vsPERM in terms of mean CPU time over 10 runs per instance and algorithm; for practical reasons, each run was restricted to 1 CPU hour on our reference machine, and the lowest energies obtained in these runs (listed in [additional data file 1](#S1){ref-type="supplementary-material"}) are not necessarily optimal. As can be seen from these results, in 2D, ACO-HPPFP-3 performs roughly comparably to PERM (PERM\'S t~exp~was calculated as described in the previous subsection): ACO-HPPFP-3 reaches the same energies as PERM, but on some instances, particularly of length 50, requires more run-time. In 3D, ACO-HPPFP-3 generally requires a comparable amount of run-time on sequences of length 30 and outperforms PERM on one random sequences of length 30, but performs noticeably worse on sequences of length 50 and in some cases does not reach the same energy. We also generated longer sequences of length 75; for these, ACO-HPPFP-3 failed to reach the minimal energy values obtained by PERM in a number of cases. The run-times for both algorithms are reported in detail in [Additional file 1](#S1){ref-type="supplementary-material"}; we note that on some sequences, the performance of PERM depends significantly on the direction of folding. Interestingly, there is no significant difference in performance between the biological and random test-sets for either PERM or ACO-HPPFP-3. In summary, the performance of ACO-HPPFP-3 is comparable with that of PERM (the best known algorithm for the 2D and 3D HP Protein Folding Problem) on biological and random sequences of length 30--50, but worse on longer sequences. This scaling effect is significantly more pronounced in 3D than in 2D. We note that neither ACO-HPPFP-3 nor PERM were optimised for short sequences (n≤ 30), but by using parameter settings different from the ones specified earlier, the performance of both algorithms can be significantly improved in this case. Characteristic performance differences between ACO and PERM ----------------------------------------------------------- To further investigate the conditions under which ACO performs well compared to PERM, we visually examined native conformations found by both algorithms, paying special attention to conformations for which one of the two algorithms does not perform well (see Figures [7](#F7){ref-type="fig"} and [9](#F9){ref-type="fig"}). Based on our observations, we hypothesised that PERM usually performs well on sequences that have a structural nucleus in the native conformation at one of the ends of the sequence (particularly the end from which PERM starts folding the sequence); on the other hand, it has trouble folding sequences whose native conformations have structural nuclei in the middle of the sequence. In comparison, ACO is not significantly affected by the location of the structural nucleus (or multiple nuclei) in the sequence, since it uses construction from different folding points as well as the long-range mutation moves in local search, which can initiate refolding from arbitrary sequence positions. Here, we use the term \'structural nucleus\' to refer to a predominantly locally folded part of the chain that can be relatively easily folded sequentially based on local sequence information \[[@B37]\]. For most sequences considered in this study, we observed a single structural nucleus, which is not surprising, given their relatively short length; however, it is generally believed that longer sequences have multiple folding nuclei \[[@B37]\]. The left side of Figure [7](#F7){ref-type="fig"} shows an example of a relatively short biological sequence (B50-7, 45 amino acids) with a unique native hydrophobic core in the 2D HP model. (This is rare for HP sequences, which usually have a high ground state and hydrophobic core degeneracy: According to our observations, of the 11 standard benchmark instances in 2D, only Sequences S1-1, S1-3, S1-4 have a unique hydrophobic core; in 3D, none of the sequences studied here have a unique hydrophobic core.) This sequence has no structural nuclei at its ends; instead, the two ends interact with each other. ACO-HPPFP-3 outperforms PERM by a factor of 2 on this sequence in terms of CPU time: using a cut-off time of 1 CPU hour per run, PERM found the optimum with energy -17 in an average run-time of 284.06 CPU seconds (t~1~= 271 sec, t~2~= 299 sec), while using the same cut-off time and machine, ACO-HPPFP-3 found the optimum in an average run-time of 130 CPU seconds. We also designed two additional sequences, D-1 and D-2, of length 50 and 60, respectively, that have a unique native state in which both ends of the sequence interact with each other (see Figure [8](#F8){ref-type="fig"}). Sequence D-1 also has a structural nucleus near its C-terminus. When testing the performance of PERM and ACO-HPPFP-3 on these sequences, we found that on D-1, ACO-HPPFP-3 requires a mean run-time of 236 CPU seconds, compared to t~1~= 3 795, t~2~= 1, t~exp~= 2 CPU seconds for PERM (values are based on 100 successful runs). When this sequence was reversed, PERM started folding the sequence from the structural nucleus, and its mean run-time dropped to 1 CPU second. A result similar to that for sequence B50-7 was obtained for Sequence D-2, which has no structural nuclei at the ends, but a native state in which the ends interact with each other. Here, ACO-HPPFP-3 was found to require a mean run-time of 951 CPU seconds (again, mean run-times were obtained from 100 successful runs), compared to t~1~= 9 257, t~2~= 19 356, t~exp~= 12 525 CPU seconds for PERM; as expected, in this case, reversing the folding order of the sequence did not cause a decrease in PERM\'S run-time. We also analysed native conformations of sequences on which PERM outperforms ACO and observed that the end from which PERM starts folding is relatively compact and forms a structural nucleus in the resulting conformation. An example of a conformation with the structural nucleus at the beginning of the sequence (near the N-terminus, i.e.,residue 1) is shown in the right panel of Figure [7](#F7){ref-type="fig"}. For this biological sequence (B50-5, 53 amino acids), PERM finds an optimal conformation with an energy of -22 in t~1~= 5, t~2~= 118, t~exp~= 9 CPU seconds, while the average run-time for ACO-HPPFP-3 is 820 CPU seconds. Our ACO algorithm generally performs worse than PERM on sequences that have structural nuclei at the ends, because it tends to spend substantial amounts of time compacting local regions in the interior of the sequence, while PERM folds more systematically from one end. These observations also hold in 3D, as seen from two random sequences folded in 3D (see Figure [9](#F9){ref-type="fig"}). To further investigate our hypothesis, we studied differences between the distributions of native conformations found by ACO-HPPFP-3 and PERM, respectively. For this purpose, we introduced the notion of relative H-H contactorder, which captures arrangement of H residues in the core of the folded protein, and thus determines the topology of the conformation (the closely related concept of contact order was first defined in \[[@B38]\]). Relative H-H contact order is defined as follows: ![](1471-2105-6-30-i3.gif) where lis the number of H-H contacts, nis the number of H residues in the sequence, and iand jare interacting H residues that are not neighbours in the chain. Intuitively, CO~H-H~specifies the average sequence separation between H-H residues in contact per H in the sequence. Figure [10](#F10){ref-type="fig"} shows cumulative frequency distributions of relative H-H contact order values for sets of native conformations of a 2D (the left panel) and 3D (the right panel) standard benchmark instance, respectively, found by ACO-HPPFP-3 and PERM over 500 independent runs, each of which was terminated as soon as a native conformation had been found. These results show that the ACO algorithm finds a set of native conformations with a wider range of H-H contact order values than PERM; in particular, ACO-HPPFP-3 finds conformations with high relative H-H contact oder as compared to PERM (more distant parts of the chain interact; for example, relative CO~H-H~= 0.324 for Sequence S1-7 in 2D and relative CO~H-H~= 0.75 for Sequence S2-5 in 3D are not found by PERM; similar results were obtained for other sequences), which further supports our hypothesis that both, in 2D and 3D, PERM is biased toward a more restricted set of native conformations. We performed analogous experiments for the case where PERM is allowed to keep certain statistics from one run to another as in \[[@B18]\] (runs are no longer independent) and found no significant differences in the set of conformations obtained (data not shown). To further examine the topological differences between ensembles of native conformations found by the two algorithms, we also looked at the hydrophobic solvent accessible area (defined as SA~H-H~: = ∑~h~E~h~, where E~h~is the number of unoccupied lattice sites around H residue h), the number of H-H contacts, and the H-H contact order as a function of the length of the sequence prefix (starting from the N-terminus of the sequence -- where PERM starts folding). In this analysis, we calculated the properties of interest mentioned above for the native conformations found in 100 independent runs by ACO-HPPFP-3 and PERM, and plotted the mean values of the respective quantities as functions of the sequence prefix length (see Figures [11](#F11){ref-type="fig"}, [12](#F12){ref-type="fig"} and [13](#F13){ref-type="fig"}). As seen in Figure [11](#F11){ref-type="fig"}, ACO-HPPFP-3 is less greedy than PERM, both in 2D (left side) and in 3D (right side), and it tends to leave more lattice sites around H residues accessible for future contacts with other H residues that appear later in the chain. This is also reflected in the mean number of H-H contacts formed when folding prefixes of increasing length; ACO-HPPFP-3 tends to form fewer H-H contacts than PERM for short and medium size prefixes (see Figure [12](#F12){ref-type="fig"}). By examining the dependence of absolute H-H contact order (defined as ![](1471-2105-6-30-i4.gif), the average sequence separation per H-contact) on prefix length, we furthermore observed that different from PERM, ACO-HPPFP-3 realises the bulk of its local H-H interactions in the middle of the given sequence (see Figure [13](#F13){ref-type="fig"}). This further confirms that ACO is capable of finding native conformations with structural folding nuclei that are not located at or near the end of a given protein sequence. The results illustrated in Figures [11](#F11){ref-type="fig"}, [12](#F12){ref-type="fig"} and [13](#F13){ref-type="fig"} are typical for all 2D and 3D HP instances we studied. Discussion ========== Although conceptually rather simple, our ACO algorithm is based on a number of distinct components and mechanisms. A natural question to ask is whether and to which extent each of these contributes to the performance reported in the previous section. A closely related questions concerns the impact of parameter settings on the performance of ACO-HPPFP-3; further details concerning parameters can be found in the \'Methods\' section. To address these questions, we conducted several series of experiments. In this context, we primarily used three standard test sequences: Sequence S1-7 of length 60 and Sequence S1-8 of length 64 (long sequences) in 2D, as well as Sequence S2-5 of length 48 in 3D (all standard benchmark sequences for 3D are 48 amino acids in length); these sequences were chosen because the CPU time required to find the best known solutions was sufficiently small to perform a large number of runs (100--200 per instance). Following the methodology of Hoos and Stützle \[[@B39]\], we measured run-time distributions (RTDs) of our ACO algorithm, which represent the (empirical) probability distribution over the run-time required to reach (or exceed) a given solution quality; the solution qualities used here are the known optima or best known energies for the respective sequences. Pheromone values and heuristic information ------------------------------------------ Two important components of any ACO algorithm are the heuristic function, which indicates the desirability of using particular solution components during the construction phase, and the pheromone values, which represent information learned over multiple iterations of the algorithm. Three parameters control the influence of the pheromone information versus heuristic information on the construction of candidate solutions: the relative weight of the pheromone information, α; the relative weight of the heuristic information, β; and the pheromone persistence, ρ(see also \'Methods\' section). In the first experiment, we investigated the impact of pheromone (α) and heuristic information (β), and their relative importance for the performance of our ACO algorithm. As can be seen from the results shown in Figure [14](#F14){ref-type="fig"}, both the pheromone values and the heuristic information are important in 2D and 3D; when ignoring either of them (α: = 0 or β: = 0, respectively), the algorithm performs worse, particularly for longer 2D sequences (n\> 50; for short 2D sequences with n≤ 50, the pheromone matrix does not appear to play a significant role, since sequences are generally easily solved by the subsidiary local search procedure alone). The optimal settings for αand βfor most problem instances seem to be around α= 1 and β= 2, as shown in Figure [14](#F14){ref-type="fig"}. It should be noted that in 3D, pheromone information appears to be less important than in 2D, which suggests that the specific solution components used in our algorithms are somewhat less meaningful in 3D. The goal of the next experiment was to further explore the role of experience accumulated over previous iterations in the form of pheromone values. To this end, we varied the pheromone persistence, ρ, while keeping other parameters constant. The results shown in Figure [15](#F15){ref-type="fig"} show that in 2D, it is important to utilise past experience (i.e.,to choose ρ\> 0), but also to weaken its impact over time (i.e.,to use ρ\< 1). At the same time, closer examination revealed that for ρ\> 0, attrition, i.e.,the construction of inextensible partial conformations, is a major problem, which is a result of the accumulation of pheromone from multiple conformations. This is why the backtracking mechanism described in the \'Methods\' section is extremely important for the performance of our algorithm in 2D. In 3D, for the previously stated reasons and because of the fact that the attrition problem is much less severe, the impact of the persistence parameter is generally smaller than in 2D. Ant colony size and length of local search phase ------------------------------------------------ During the initial empirical evaluation of our algorithm, we observed that ant colony size (i.e.,the number of ants used in each iteration) and the duration of local search (expressed as a number of non-improving search steps we are willing to consider before terminating the local search procedure) are correlated and significantly affect its performance. To further investigate this phenomenon, we conducted additional experiments in which we fixed the ant colony size and varied the maximal number of non-improving steps during local search, and vice versa. In this series of experiments, different colony sizes were considered, from a single ant up to a population of 5 000 ants, and the number of non-improving steps in local search was varied from 100 to 10 000. The results, shown in Figure [16](#F16){ref-type="fig"}, indicate that there is an optimal colony size of about 100 ants for both, 2D and 3D; ACO-HPPFP-3 is quite robust with respect to colony size, but performance decreases for very small or very large colony sizes. Intuitively, this is the case because the use of a population of ants provides diversification to the search process, which enables it to explore different regions of the underlying search space; very small populations provide insufficient diversification, and the search stagnates easily, while for very large populations, the additional time required for running the search phases for each ant on the same sequential machine is not amortised any longer by increased efficiency of the overall search process. Our results also indicate that the performance of ACO-HPPFP-3 is more sensitive to the number of non-improving steps than to ant colony size. The optimal value for the maximum number of non-improving steps tolerated (per ant) before the local search phase terminates was found to be around 1 000 for short 2D sequences (n≤ 50) and around 10 000 for long 2D sequences (n\> 50); the latter value also appeared to be optimal for all 3D sequences considered here. This observation follows the intuition that more degrees of freedom, as present for longer sequences and in higher dimensions, require more time for local optimisation, since for any conformation, improving neighbours tend to be rarer and hence harder to find. Selectivity and persistence of local search ------------------------------------------- As described in the \'Methods\' section, our ACO algorithm uses selective local search, i.e.,local search is only performed on a certain fraction of the lowest energy conformations. We observed that ACO-HPPFP-3 is fairly robust with respect to the fraction of conformations to which local search is applied; good performance was obtained for local search selectivity values between 5% and 50%, but performance was found to deteriorate when local search is performed by all ants. Intuitively, similar to colony size, local search selectivity has an impact on search diversification. If too few ants perform local search, insufficient diversification is achieved, which typically leads to premature stagnation of the search process. On the other hand, if local search is performed by too many ants, the resulting substantial overhead in run-time can no longer be amortised by increased search efficiency. Similarly to selective local search, pheromone update was performed only by a certain fraction of so-called \'elitist ants\' whose solution quality after the local search phase is highest within the population. As in the case of local search selectivity, ACO-HPPFP-3 shows robustly high performance for elitist fractions between 1% and 50% (results are not shown here), but performance deteriorates markedly when all ants in the colony are allowed to update the pheromone matrix. In a final experiment, we studied the impact of the persistence of local search, i.e.,of the probability ![](1471-2105-6-30-i2.gif) of retaining (feasible) previous relative directions during long-range mutation moves. As can be seen in Figure [17](#F17){ref-type="fig"}, good performance is generally obtained for ![](1471-2105-6-30-i2.gif) values between 0.3 and 0.7. Both extreme cases, ![](1471-2105-6-30-i2.gif) = 0, which corresponds to an extremely H-contact greedy mutation operator, and ![](1471-2105-6-30-i2.gif) = 1, in which refolding always follows previous directions when feasible, result in a substantial decrease in performance. When ![](1471-2105-6-30-i2.gif) = 0, the decrease of performance in 3D is smaller than in 2D, because there is no severe attrition as in 2D, where greedy placement of H residues leads to early formation of very compact partial conformations, which often cannot be extended into valid complete conformations. The performance decrease for high ![](1471-2105-6-30-i2.gif) values is due to insufficient ability of the chain to fold into a new conformation that accommodates well the local change in structure which triggered the refolding. Conclusions =========== In this work, we have shown that ant colony optimisation (ACO) can be applied in a rather straight-forward way to the 2D and 3D HP Protein Folding Problems. Even though our ACO-HPPFP-3 algorithm is based on very simple structure components (single relative directions) and a simple subsidiary local search procedure (iterative first improvement), it performs fairly well compared to other algorithms and specialised heuristics on the benchmark instances considered here, particularly in 2D. The only non-specialised algorithm that typically performs better than our ACO algorithm, both in 2D and 3D, is PERM. We observed that, particularly in 3D, the run-time required by ACO-HPPFP-3 for finding minimum (or best known) energy conformations scales worse with sequence length than PERM. However, our results show that our ACO algorithm finds a different ensemble of native conformations compared to PERM, and has less difficulty folding sequences whose native states contain structural nuclei located in the middle rather than at the ends of a given sequence, as well as sequences with structures in which the ends interact. We found that two major components of ACO-HPPFP-3 -- the pheromone values, which capture experience accumulated over multiple iterations of the search process and from multiple conformations, as well as the heuristic information that provides myopic guidance to the folding process -- play a significant role for longer 2D sequences and, to a lesser extent, for 3D sequences, which suggests that in 3D, it may be preferable to associate pheromone values with more complex solution components. We also found that the subsidiary local search procedure is crucial for the performance of the algorithm; in particular, to ensure that high-quality conformations are obtained, it is very important to allow the local search procedure to run sufficiently long. In an earlier version of our algorithm \[[@B7]\], we used substantially more stringent termination criteria, which forced us to additionally use non-greedy local search (probabilistic iterative improvement, which accepts worsening steps) in addition to the greedy local search procedure used here. The results presented in this study indicate that by using a new and simpler local search procedure, ACO-HPPFP-3 achieves better performance; this is probably due to the fact that the new local search procedure is based on a type of long-range move that leads to a larger effective search neighbourhood. We have shown that all components of our ACO algorithms contribute to its performance. In particular, its performance is affected by the following components and parameters (listed in the order of decreasing impact): pheromone values, termination criterion for local search, persistence of long-range moves, ant colony size, pheromone persistence, heuristic function, selectivity of local search, and selectivity of pheromone update (i.e.,fraction of elitist ants). Because of its ability to find more balanced ensembles of minimum (or close to minimum) energy conformations, our new ACO algorithm can greatly facilitate investigations of the topology and location of structural nuclei, which we plan to undertake in future work. Finally, while HP protein folding problems are of considerable interest because of their conceptual simplicity, ultimately, most applications of protein folding algorithms require the use of more realistic models of protein structure. Since it does not rely on heuristics and properties that are specific to the HP model and yet performs very well on this restrictive, but not entirely unrealistic abstract model, we believe that relatively straight-forward extensions of our ACO algorithm to more complex and realistic models of protein structure hold significant promise. Methods ======= Our new ACO algorithm, ACO-HPPFP-3, iterates construction, local search, and pheromone update phases until a termination condition is satisfied; in the context of this work, we mostly terminated the algorithm upon reaching a given energy threshold. In the following, we describe the three search phases in detail. Construction phase, pheromone and heuristic values -------------------------------------------------- During the construction phase of ACO-HPPFP-3, each ant first determines a starting point within the given protein sequence; this is done by uniform random choice. From this starting point, the sequence is folded in both directions, adding one residue at a time. Each ant performs probabilistic chain-growth construction of the protein conformation, where in every step, the structure is extended either to the left or to the right, such that the ratio of unfolded residues at each end of the protein remains (roughly) unchanged. Here, we assume that folding is performed in 3D (the 2D case is handled analogously by considering three relative directions {S, L, R} instead of five {S, L, R, U, D}, see also \[[@B6]\]). The relative directions in which the conformation is extended in each construction step are determined probabilistically based on a heuristic function η~i,d~and pheromone values τ~i,d~, according to the formula: ![](1471-2105-6-30-i5.gif) The pheromone values τ~i,d~indicate the desirability of using the local structure motif with relative direction d∈ {S, L, R, U, D} at sequence position i. Initially, all τ~i,d~are equal, such that local structure motifs are chosen in an unbiased way; but throughout the search process, the pheromone values are updated to bias folding towards the use of local motifs that occur in low-energy structures (the updating mechanism will be described in more detail later). The heuristic values η~i,d~are based on the energy function E. They are defined according to the Boltzman distribution as η~i,d~: = ![](1471-2105-6-30-i6.gif), where γis a parameter called the inverse temperature (as in \[[@B18]\]), and h~i,d~is the number of new H-H contacts achieved by placing amino acid iat the position specified by direction d. During construction, it may happen that the chain cannot be extended without running into itself. This situation is called attrition, and our algorithm overcomes it as follows: First, starting at the end at which attrition occurred, half of the sequence that has been folded up to this point is unfolded. Then, this segment of the chain is refolded; the first residue (i.e.,the last one that was unfolded) is placed such that its relative direction differs from what it had been when attrition occurred, while all of the subsequent residues are folded in a feasible direction that is chosen uniformly at random. This backtracking mechanism is particularly important for longer protein sequences in 2D, where infeasible conformations are frequently encountered during the construction phase. Local search ------------ The local search phase is based on a long-range mutation move that has been designed to avoid infeasible conformations. It also has a number of important advantages over the more commonly used point mutation moves or Monte Carlo moves (i.e.,the end, crankshaft and corner moves \[[@B40]\]): It is easy to implement; it decreases the number of infeasible conformations encountered, even when the protein is very compact (at high densities); it considers a larger neighbourhood that subsumes the single point mutation neighbourhood; and it has some validity in terms of the physical processes taking place during the protein folding process. Similar attempts have been previously undertaken, but these involved disconnection of the chain \[[@B21]\]. From studies of protein folding dynamics, it is known that proteins display a broad range of motions that range from localised motions to slow large-scale movements \[[@B37]\]. Inspired by this complex process, we designed a long-range mutation move that starts by selecting a residue whose relative direction is randomly mutated and then adapts the rest of the chain by probabilistically changing relative directions starting from this initial position \[[@B7]\]. During this adaptation, for each residue, with a probability ![](1471-2105-6-30-i2.gif) (0 ≤ ![](1471-2105-6-30-i2.gif) ≤ 1) its previous relative direction, if it is still feasible, is left unchanged, and otherwise (i.e.,with probability 1 - ![](1471-2105-6-30-i2.gif), or if the previous direction has become infeasible), a different relative direction is chosen, where the probability for each direction dis proportional to the corresponding heuristic value η~i,d~. Formally, this can be written as follows: ![](1471-2105-6-30-i7.gif) where P\[d~i~: = ![](1471-2105-6-30-i8.gif)\] is the probability of choosing direction ![](1471-2105-6-30-i8.gif) as the relative direction d~i~at sequence position i. Unlike in our previous implementation \[[@B7]\], the local search phase of our new ACO algorithm is a simple iterative first improvement procedure that is based on this long-range mutation move. The outline of this local search procedure is shown in Figure [18](#F18){ref-type="fig"}. Iterative first improvement accepts a new conformation generated via long-range mutation only if the solution quality of a new conformation c\' improves over the current solution quality (energy) of c.This search process is greedy in the sense that it does not allow worsening steps, and it is terminated when no improving steps have been found after a specific number of scans through the chain (this number is a parameter of the algorithm). Since this local search procedure has a relatively high time-complexity, in each iteration of ACO-HPPFP-3 it is only applied to a certain fraction of the highest-quality conformations constructed by the ants in the preceding construction phase. Update of the pheromone values ------------------------------ After each construction and local search phase pheromones are updated according to τ~i,d~:= ρ·τ~i,d~, (4) where 0 \<ρ≤ 1 is the pheromone persistence, a parameter that determines how much of the information gathered in previous iterations is retained. Subsequently, selected ants with low-energy conformations update the pheromone values according to τ~i,d~:= τ~i,d~+ Δ~i,d,c~, (5) where Δ~i,d,c~is the relative solution quality of the given ant\'s candidate conformation cif that conformation contains local structure motif dat sequence position i, and zero otherwise. As a further mechanism for preventing search stagnation, we use an additional renormalisation of the pheromone values that is conceptually similar to the method used in MAX - MIN* Ant System \[[@B41]\]: After the standard pheromone updates according to Equations 3 and 4, all τvalues are normalised such that ∑~d∈{S,L,R,U,D}~τ~i,d~= 1 for every residue i; additionally, whenever for a given sequence position ithe minimal normalised pheromone value (min~d∈{S,L,R,U,D}~τ~i,d~)/(∑~d∈{S,L,R,U,Dr}~τ~i,d~) falls below a threshold θ(which is a parameter of the algorithm), the minimal τ~i,d~value is set to θ, while the maximal τ~i,d~value is decreased by θ- min~d∈{S,L,R,U,D}~τ~i,d~. (If there is more than one minimal τ~i,d~value, all of these are increased to θ, and if there is more than one maximal τ~i,d~value, one of them is chosen uniformly at random.) This guarantees that the probability of selecting an arbitrary local structure motif for the corresponding sequence position does not become arbitrarily small, and hence ensures the probabilistic approximate completeness of our algorithm (see \[[@B42]\]). Implementation details and availability --------------------------------------- ACO-HPPFP-3 has been implemented in C++ and compiled using gcc (version 3.3.3) for the Linux operating system; a Linux executable is available from <http://www.cs.ubc.ca/labs/beta/Projects/ACO-HPPFP>. Authors\' contributions ======================= Both authors contributed to the development of ideas, design of experiments, analysis and interpretation of results, and the writing of the paper. AS implemented the proposed method and performed the computational experiments. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Additional information on biological and randomly generated HP sequences.This file (in .pdf format) contains tables providing additional information on our new test sets of biological and randomly generated HP sequences and the results from our computational experiment with ACO and PERM. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This work has been supported by an NSERC Postgraduate Scholarship (PGS-A) held by AS and by HH\'s NSERC Individual Research Grant \#238788. We thank Peter Grassberger for kindly providing us with his implementation of PERM and for very useful feedback on earlier versions of this paper. We also thank the anonymous reviewers for helpful suggestions. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### A sample protein conformation in the 2D HP model. The underlying protein sequence (Sequence S1-1 from Table 1)is HPHPPHHPHPPHPHHPPHPH; black circles represent hydrophobic amino acids, while white circles symbolise polar amino acids. The dotted lines represents the H-H contacts underlying the energy calculation. The energy of this conformation is -9, which is optimal for the given sequence. ::: ![](1471-2105-6-30-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### ACO outline. Generic outline of Ant Colony Optimisation (for static combinatorial problems). ::: ![](1471-2105-6-30-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Local structure motifs. The local structure motifs which form the solution components underlying the construction and local search phases of our ACO algorithm in 3D. ::: ![](1471-2105-6-30-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### The 2D native state of the standard Sequence S1-8. The native conformation of Sequence S1-8 from Table 1 (64 amino acids; energy -42), found by ACO-HPPFP-3 in an average CPU time of 1.5 hours and by PERM in t~1~= t~2~= t~exp~= 78 hours. ::: ![](1471-2105-6-30-5) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Performance comparison of ACO-HPPFP-3 and PERM on biological and random instances in 2D. Mean CPU time (natural log transformed) required by ACO-HPPFP-3 vsPERM for reaching the best solution quality, as observed over 10 runs with a cut-off time of 1 CPU hour for sequences of length 30 and 50 in 2D. The left and right plots show the results for the biological and random test-sets, respectively. Performance results for instances of size 30 are indicated by circles, while stars mark results for instances of size 50. The dashed lines indicate the band within which performance differences are not statistically significant. Mean run-times were obtained from 10 runs per instance and algorithm, and we only show data points for the runs where the best known solution quality was reached at least in some runs out of 10 by both algorithms (when unsuccessful runs were present, the expected time was calculated as in \[43\]); results for both successful and unsuccessful runs are given in the [Additional file 1](#S1){ref-type="supplementary-material"}. ::: ![](1471-2105-6-30-6) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Performance comparison of ACO-HPPFP-3 and PERM on biological and random instances in 3D. Mean CPU time (natural log transformed) required by ACO-HPPFP-3 vsPERM for reaching the best solution quality, as observed over 10 runs with a cut-off time of 1 CPU hour for sequences of length 30 and 50 in 3D. The left and right plots show the results for the biological and random test-sets, respectively. Performance results for instances of size 30 are indicated by circles, while stars mark results for instances of size 50. Mean run-times were obtained from 10 runs per instance and algorithm, and we only show data points for the runs where the best known solution quality was reached at least in some runs out of 10 by both algorithms (when unsuccessful runs were present, the expected time was calculated as in \[43\]); results for both successful and unsuccessful runs are given in the [Additional file 1](#S1){ref-type="supplementary-material"}. ::: ![](1471-2105-6-30-7) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Illustration and comparison of difficult structures for PERM and ACO-HPPFP-3 in 2D. Left side: Lowest energy conformation of a biological sequence (B50-7, 45 amino acids, energy -17) that is harder for PERM (t~1~= 271, t~2~= 299, t~exp~= 284 CPU seconds) than for ACO-HPPFP-3 (t~exp~= 130 CPU seconds; cut-off time 1 CPU hour). Right side: Lowest energy conformation of a biological sequence (B50-5, 53 amino acids, energy -22) that is much harder for ACO-HPPFP-3 than for PERM; within a cut-off time of 1 CPU hour, both ACO-HPPFP-3 and PERM reached this energy in 10 out of 10 runs in t~avg~= 820 and t~1~= 5, t~2~= 118, t~exp~= 9 CPU seconds on average, respectively. ::: ![](1471-2105-6-30-8) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Performance of ACO-HPPFP-3 and PERM on designed sequences in 2D HP. Left side: Unique minimal energy conformation of a designed sequence, D-1 (length 50, energy -19); ACO-HPPFP-3 reaches this conformation much faster than PERM when folding from the left end (mean run-time over 100 successful runs for ACO-HPPFP-3: 236 CPU seconds, compared to t~1~= 3 795, t~2~= 1, t~exp~= 2 CPU seconds for PERM). Right side: Unique native conformation of another designed sequence, D-2 (length 60, energy -17); ACO-HPPFP-3 finds this conformation much faster than PERM folding from either end (mean run-time over 100 successful runs for ACO-HPPFP-3: 951 CPU seconds, compared to t~1~= 9 257, t~2~= 19 356, t~exp~= 12 524 CPU seconds for PERM). ::: ![](1471-2105-6-30-9) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Illustration and comparison of difficult structures for PERM and ACO-HPPFP-3 in 3D. Left side: Lowest energy conformation of random sequence R50-9 (50 amino acids, energy -30), which is harder for PERM when folding from the left end than for ACO-HPPFP-3; with a cut-off time of 1 CPU hour, ACO-HPPFP-3 reached this energy in 10 out of 10 runs with t~exp~= 1000 CPU seconds, while PERM failed to find a conformation with this energy in 7 out of 10 runs when folding from the left end (t~1~= 9 892, t~2~= 2, t~exp~= 3 CPU seconds). Right side: Lowest energy conformation of random sequence R50-7 (50 amino acids, energy -38), which is much harder for ACO-HPPFP-3 than for PERM; with a cut-off time of 1 CPU hour, PERM reached this energy in two out of 10 runs when folding from the left and in 10 of 10 runs when folding from the right end in t~1~= 15 322, t~2~= 46, t~exp~= 92 CPU seconds, while the lowest energy reached by ACO-HPPFP-3 over ten runs was -37. ::: ![](1471-2105-6-30-10) ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### Comparison of distributions of H-H contact order of native structures found by ACO-HPPFP-3 and PERM in 2D and 3D. Distributions of H-H contact order for 500 conformations of Sequence S1-7 from Table 1 (60 amino acids) in 2D (left side) and Sequence S1-5 from Table 1 (48 amino acids) in 3D (right side) found by ACO-HPPFP-3 and PERM. ::: ![](1471-2105-6-30-11) ::: ::: {#F11 .fig} Figure 11 ::: {.caption} ###### *Plot of mean hydrophobic solvent accessible area, ACO-HPPFP-3 vsPERM in 2D and 3D. Mean hydrophobic solvent accessible area as a function of prefix length for a biological sequence (B50-4, 50 amino acids) in 2D (left side) and Sequence S2-6 from Table 1 (48 amino acids) in 3D. Crosses and circles represent mean values for an ensemble of 100 native structures found by ACO-HPPFP-3 and PERM, respectively. ::: ![](1471-2105-6-30-12) ::: ::: {#F12 .fig} Figure 12 ::: {.caption} ###### Plot of mean number of H-H contacts, ACO-HPPFP-3 vsPERM in 2D and 3D. Mean number of H-H contacts as a function of prefix length for a biological sequence (B50-4, 50 amino acids) in 2D (left side) and Sequence S2-6 from Table 1 (48 amino acids) in 3D. Crosses and circles represent mean values for an ensemble of 100 native structures found by ACO-HPPFP-3 and PERM, respectively. ::: ![](1471-2105-6-30-13) ::: ::: {#F13 .fig} Figure 13 ::: {.caption} ###### Plot of mean H-H contact order, ACO-HPPFP-3 vsPERM in 2D and 3D. Mean H-H contact order as a function of prefix length for a biological sequence (B50-4, 50 amino acids) in 2D (left side) and Sequence S2-6 from Table 1 (48 amino acids) in 3D. Crosses and circles represent mean values for an ensemble of 100 native structures found by ACO-HPPFP-3 and PERM, respectively. ::: ![](1471-2105-6-30-14) ::: ::: {#F14 .fig} Figure 14 ::: {.caption} ###### Impact of parameter settings on ACO-HPPFP-3 performance in 2D and 3D: relative weights of pheromone and heuristic information. Effect of the relative weights of pheromone information, α, and heuristic information, β, on the average CPU time required for obtaining minimal energy conformations of Sequence S1-8 in 2D (length 64, left side) and Sequence S2-5 in 3D (length 48, right side). ::: ![](1471-2105-6-30-15) ::: ::: {#F15 .fig} Figure 15 ::: {.caption} ###### Impact of parameter settings on ACO-HPPFP-3 performance in 2D and 3D: pheromone persistence. Effect of the pheromone persistence parameter, ρ, on the average CPU time required for obtaining minimal energy conformations of Sequence S1-8 in 2D (length 64, left side) and Sequence S2-5 in 3D (length 48, right side). ::: ![](1471-2105-6-30-16) ::: ::: {#F16 .fig} Figure 16 ::: {.caption} ###### Parameter settings influence on ACO-HPPFP-3 performance in 2D and 3D: ant colony size and maximum number of non-improving local search steps. Mean CPU time required for finding minimum energy conformations of Sequence S1-7 in 2D (length 60, left side) and Sequence S2-5 in 3D (length 48, right side), as a function of ant colony size and the maximum number of non-improving local search steps. ::: ![](1471-2105-6-30-17) ::: ::: {#F17 .fig} Figure 17 ::: {.caption} ###### Parameter settings influence on ACO-HPPFP-3 performance in 2D and 3D: probability of retaining previous directions in local search. Mean CPU time required for finding minimum energy conformations of Sequence S1-8 in 2D (length 64, left side) and Sequence S2-5 in 3D (length 48, right side), as a function of the probability of retaining previous directions (![](1471-2105-6-30-i2.gif)) during long-range mutation moves. ::: ![](1471-2105-6-30-18) ::: ::: {#F18 .fig} Figure 18 ::: {.caption} ###### Outline of the subsidiary local search procedure. The iterative first improvement local search procedure that is performed by selected ants after the construction phase. ::: ![](1471-2105-6-30-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### 2D and 3D HP standard benchmark instances. Benchmark instances for the 2D and 3D HP Protein Folding Problem used in this study with optimal or best known energy values E\. Most instances for 2D and 3D HP can also be found in \[44\]; Sequence S1-9 (2D) is taken from \[45\], and the last two instances (2D) are from \[21\]. H~i~and P~i~indicate a string of iconsecutive H\'s and P\'s, respectively; likewise, (s)~i~indicates an i-fold repetition of string s. ::: ID Length E\* Protein Sequence --------- -------- ------- ------------------------------------------------------------------------------------------------------------------------------------------------------ 2D HP S1-1 20 -9 (HP)~2~PH~2~PHP~2~HPH~2~P~2~HPH S1-2 24 -9 H~2~(P~2~H)~7~H S1-3 25 -8 P~2~HP~2~(H~2~P~4~)~3~H~2~ S1-4 36 -14 P~3~H~2~P~2~H~2~P~5~H~7~P~2~H~2~P~4~H~2~P~2~HP~2~ S1-5 48 -23 P~2~H(P~2~H~2~)~2~P~5~H~10~P~6~(H~2~P~2~)~2~HP~2~H~5~ S1-6 50 -21 H~2~(PH)~3~PH~4~PH(P~3~H)~2~P~4~H(P~3~H)~2~PHPH~4~(HP)~3~H~2~ S1-7 60 -36 P~2~H~3~PH~8~P~3~H~10~PHP~3~H~12~P~4~H~6~PH~2~PHP S1-8 64 -42 H~12~(PH)~2~(P~2~H~2~)~2~P~2~HP~2~H~2~PPH~2~P~2~HP~2~(H~2~P~2~)~2~(HP)~2~H~12~ S1-9 85 -53 H~4~P~4~H~12~P~6~(H~12~P~3~)~3~HP~2~(H~2~P~2~)~2~HPH S1-10 100 -50 P~3~H~2~P~2~H~4~P~2~H~3~(PH~2~)~2~PH~4~P~8~H~6~P~2~H~6~P~9~HPH~2~PH~11~P~2~H~3~PH~2~PHP~2~HPH~3~P~6~H~3~ S1-11 100 -48 P~6~HPH~2~P~5~H~3~PH~5~PH~2~P~4~H~2~P~2~H~2~PH~5~PH~10~PH~2~PH~7~p~11~H~7~P~2~HPH~3~P~6~HPH~2~ 3D HP S2-1 48 -32 HPH~2~P~2~H~4~PH~3~P~2~H~2~P~2~HPH~2~PHPH~2~P~2~H~2~P~3~HP~8~H~2~ S2-2 48 -34 H~4~PH~2~PH~5~P~2~HP~2~H~2~P~2~HP~6~HP~2~HP~3~HP~2~H~2~P~2~H~3~PH S2-3 48 -34 PHPH~2~PH~6~P~2~HPHP~2~HPH~2~(PH)~2~P~3~H(P~2~H~2~)~2~P~2~H~P~HP~2~HP S2-4 48 -33 PHPH~2~P~2~HPH~3~P~2~H~2~PH~2~P~3~H~5~P~2~HPH~2~(PH)~2~P~4~HP~2~(HP)~2~ S2-5 48 -32 P~2~HP~3~HPH~4~P~2~H~4~PH~2~PH~3~P~2~(HP)~2~HP~2~HP~6~H~2~PH~2~PH S2-6 48 -32 H~3~P~3~H~2~PH(PH~2~)~3~PHP~7~HPHP~2~HP~3~HP~2~H~6~PH S2-7 48 -32 PHP~4~HPH~3~PHPH~4~PH~2~PH~2~P~3~HPHP~3~H~3~(P~2~H~2~)~2~P~3~H S2-8 48 -31 PH~2~PH~3~PH~4~P~2~H~3~P~6~HPH~2~P~2~H~2~PHP~3~H~2~(PH)~2~PH~2~P~3~ S2-9 48 -34 (PH)~2~P~4~(HP)~2~HP~2~HPH~6~P~2~H~3~PHP~2~HPH~2~P~2~HPH~3~P~4~H S2-10 48 -33 PH~2~P~6~H~2~P~3~H~3~PHP~2~HPH~2~(P~2~H)~2~P~2~H~2~P~2~H~7~P~2~H~2~ ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Performance comparison of various algorithms for the 2D HP Protein Folding Problem. Comparison of the solution quality obtained in 2D by the evolutionary algorithm of Unger and Moult (EA) \[14\], the evolutionary Monte Carlo algorithm of Liang and Wong (EMC) \[19\], the multi-self-overlap ensemble algorithm of Chickenji et al.(MSOE) \[17\], the pruned-enriched Rosenbluth method (PERM) and ACO-HPPFP-3. For EA and EMC, the reported energy values are the lowest among five independent runs, and the values in parentheses are the numbers of valid conformations scanned before the lowest energy values were found. Missing entries indicate cases where the respective method has not been tested on a given instance. The CPU times reported in parentheses for MSOE have been determined on a 500 MHz CPU, and those for PERM and ACO-HPPFP-3 are based on 100 -- 200 runs per instance on our reference 2.4 GHz Pentium IV machine. The energy values shown in bold face correspond to currently best known solution qualities. ::: ID E GA EMC MSOE PERM t~1~ PERM t~2~ PERM t~exp~ ACO ------- --------- ------------------ ------------------ ----------------- ---------------------------- ------------------- ------------------- ----------------------------- S1-1 -9 -9(30 492) -9(9 374) -9(\< 1 sec) -9(\< 1 sec) -9(\< 1 sec) -9(\< 1 sec) S1-2 -9 -9(30 491) -9(6 929) -9(\< 1 sec) -9(\< 1 sec) -9(\< 1 sec) -9(\< 1 sec) S1-3 -8 -8(20 400) -8(7 202) -8(6 sec) -8(\< 1 sec) -8(2 sec) -8(\< 1 sec) S1-4 -14 -14(301 339) -14(12 447) -14(\< 1 sec) -14(\< 1 sec) -14(\< 1 sec) -14(4 sec) S1-5 -23 -23(126 547) -23(165 791) -23(3 min) -23(\< 1 sec) -23(2 sec) -23(1 min) S1-6 -21 -21(592 887) -21(74 613) -21(3 sec) -21(3 sec) -21(3 sec) -21(15 sec) S1-7 -36 -34 (208 781) -35 (203 729) -36(7 sec) -36(3 sec) -36(4 sec) -36(20 min) S1-8 -42 -37 (187 393) -39 (564 809) -39 -42 (78 hrs) -42 (78 hrs) -42 (78 hrs) -42(1.5 hrs) S1-9 -53 -52 (44 029) -53(64 sec) -53(60 sec) -53(1 min) -53(20% of runs 1 days) S1-10 -50 -50(50 hrs) -50(50% of runs 1 hrs) -50(20 min) -50 -49 (12 hrs) S1-11 -48 -47 -48(9 min) -48(7 min) -48(8 min) -47 (10 hrs) ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Performance comparison of various algorithms for the 3D HP Protein Folding Problem. Comparison of the solution quality obtained in 3D by the hydrophobic zipper (HZ) algorithm \[23\], the constraint-based hydrophobic core construction method (CHCC) \[26\], the core-directed chain growth algorithm (CG) \[25\], the contact interactions (CI) algorithm \[24\], the pruned-enriched Rosenbluth method (PERM) and ACO-HPPFP-3. For CI, only the best energies obtained are shown. For HZ, CHCC and CG, the reported CPU times are taken from \[25\]; these are the expected times for finding optimal solutions on a Sparc 1 workstation. In the case of HZ, the reported CPU times are based on an extrapolation from the measured times required for finding suboptimal conformations with the energy values listed here. The CPU times for PERM and ACO-HPPFP-3 were determined on our reference 2.4 GHz Pentium IV machine based on 50 -- 100 runs per instance. The energy values shown in bold face correspond to currently best known solution qualities. ::: ID E HZ CHCC CG CI PERM t~1~ PERM t~2~ PERM t~exp~ ACO ------- --------- ----------------- ------------------ ------------------ --------- ------------------- ------------------- ------------------- ------------------ S2-1 -32 -31(4 hrs) -32(30 min) -32(9.4 min) -32 -32(0.1 min) -32(0.5 min) -32(0.2 min) -32(30 min) S2-2 -34 -32 (18 hrs) -34(2.3 min) -34(35 min) -33 -34(0.3 min) -34(48 min) -34(0.6 min) -34(420 min) S2-3 -34 -31 (23 hrs) -34(30 min) -34(62 min) -32 -34(0.1 min) -34(4 days) -34(0.2 min) -34(120 min) S2-4 -33 -30 (19 days) -33(71 min) -33(29 min) -32 -33(2 min) -33(4 min) -33(3 min) -33(300 min) S2-5 -32 -30 (1.3 days) -32(32 min) -32(12 min) -32 -32(0.5 min) -32(19 min) -32(1 min) -32(15 min) S2-6 -32 -29 (2.1 days) -32(80 min) -32(460 min) -30 -32(0.5 min) -32(0.1 min) -32(0.2 min) -32(720 min) S2-7 -32 -29 (2.5 days) -32(110 min) -32(64 min) -30 -32(0.5 min) -32(2 days) -32(1 min) -32(720 min) S2-8 -31 -29 (4 hrs) -31(530 min) -31(38 min) -30 -31(0.3 min) -31(8 min) -31(0.6 min) -31(120 min) S2-9 -34 -31(4.5 hrs) -34(8.3 min) -33 -32 -34(5 min) -34(10 min) -34(7 min) -34(450 min) S2-10 -33 -33(1.1 hr) -33(4.8 min) -33(1.1 min) -32 -33(0.01 min) -33(0.01 min) -33(0.01 min) -33(60 min) :::	PubMed Central	2024-06-05T03:55:54.075713	2005-2-14	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555464/", "journal": "BMC Bioinformatics. 2005 Feb 14; 6:30", "authors": [ { "first": "Alena", "last": "Shmygelska" }, { "first": "Holger H", "last": "Hoos" } ] }
PMC555532	Background ========== Aggregation of Dictyostelium discoideumproceeds by an oriented migration of the amoebae towards a source of the attractant cAMP which is synthesized and released periodically by cells in the center of the aggregate. Stimulation with cAMP activates liberation of stored Ca^2+^and extracellular Ca^2+^-entry \[[@B1]\] leading to a \[Ca^2+^\]~i~-transient \[[@B2]-[@B4]\]. Chemotaxis proceeds in the presence of extracellular EGTA but not in the presence of intracellular Ca^2+^buffers, so a \[Ca^2+^\]~i~-elevation is necessary and release of stored Ca^2+^is sufficient for oriented migration \[[@B5]\]. On the other hand, the view that a \[Ca^2+^\]~i~-increase is essential for chemotaxis was called into question by analysis of a cell line where the iplAgene was disrupted by homologous recombination \[[@B6]\]. The iplAgene is the only gene known in the Dicyosteliumgenome so far that shares homology with IP~3~-receptors of higher eukaryotes. However, whether its protein product indeed constitutes a functional IP~3~-receptor and its cellular localization are not known. The iplA^-^mutant was found to aggregate and to form fruiting bodies although neither cAMP-activated ^45^Ca^2+^-entry nor a \[Ca^2+^\]~i~-elevation were detected \[[@B6]\]. From these results the authors concluded that the iplAgene product is central to the regulation of \[Ca^2+^\]~i~and that its presence and thus the presence of an agonist-activated \[Ca^2+^\]~i~-increase is not necessary for proper chemotaxis and development. However, agents that interfere with IP~3~-receptor mediated signaling such as XestosponginC \[[@B7]\] were found to influence not only cAMP-induced Ca^2+^-fluxes but also the chemotactic response and aggregation of Dictyostelium\[[@B8]\]. In this study we aimed to clarify these conflicting findings and analyzed both, capacitative and chemoattractant-induced Ca^2+^-fluxes and the effect of their disturbance on chemotaxis and differentiation of the iplA^-^mutant. Mn^2+^-influx was used to determine the rates of ion fluxes into cells with filled and emptied stores and related to Ca^2+^-electrode recordings in cell suspensions. We found that ion fluxes, chemotaxis and differentiation were sensitive towards alteration of the Ca^2+^-homeostasis. Capacitative Ca^2+^-entry was normal in the mutant and upon stimulation with agonist Ca^2+^- and Mn^2+^-fluxes occurred, yet to a considerably reduced extent. Spontaneous motility and chemotactic performance of mutant amoebae was strongly impaired by the intracellular presence of a Ca^2+^-chelator. Results ======= Extracellular \[Ca^2+^\] affects development and chemotaxis of wild type and iplA^-^ -------------------------------------------------------------------------------------- As iplA^-^cells formed fruiting bodies, albeit somewhat smaller in size, it was concluded that chemotactic aggregation and differentiation was normal \[[@B6]\]. We analyzed development of the mutant in parallel with wild type at various conditions. When cells differentiated on H5-agar plates (control situation) we consistently found a delay in the onset of aggregation by 1--2 h in the mutant; the smaller size of fruiting bodies was due to breaking of aggregation strands yielding smaller mounds (Fig. [1](#F1){ref-type="fig"}). Next we asked whether the absence or presence of Ca^2+^affects development. Differentiation on EGTA-containing agar plates and thus the steady reduction of internal Ca^2+^-levels dose dependently resulted in a delay of aggregation and a decrease in the size of aggregates and fruiting bodies in both strains. Doses of 5--10 mM EGTA in the agar did not significantly alter the time point of aggregation which is in accordance with previous data \[[@B9]\] showing requirement of additional multiple washing of amoebae with EGTA in order to affect aggregation. At concentrations of 15--20 mM EGTA however, aggregation occurred at later time points (Fig. [2](#F2){ref-type="fig"}), on average at 13 ± 3 h in wild type and at 19 ± 2 h in iplA^-^cells (mean ± s.e.m. from 5 experiments); despite the daily variations in aggregation timing the mutant strain was delayed as compared to the wild type in each of the experiments performed. On the other hand, the presence of Ca^2+^in the agar and therefore the continuous loading of cells with Ca^2+^\[[@B10]\] resulted in stronger impairment of aggregation in the wild type. The delayed aggregation of wild type cells in the presence of Ca^2+^was not due to inhibition of chemotaxis (see below). Now the formation of aggregates was observed consistently at earlier time points in the iplA^-^strain than in wild type (Fig. [3](#F3){ref-type="fig"}; on average 7 ± 0.5 h vs. 15 ± 5 h until aggregate formation in iplA^-^and wild type amoebae, respectively, at 20 mM CaCl~2~in 6 independent experiments); indeed, under this condition differentiation of iplA^-^cells resembled that of wild type observed in the control situation. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### iplA^-^cells have an altered pattern of development. Differentiation of the mutant and the wild type strain was assayed in parallel on agar plates. Cells at different time points of development on H5-agar are shown. Wild type amoebae aggregated at t~7~, whereas aggregation of the mutant strain was delayed and aggregation strands broke (t~10~); therefore, smaller fruiting bodies were formed as compared to the wild type. The full width of the image corresponds to 12.5 mm. ::: ![](1471-2121-6-13-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Development of iplA^-^cells is impaired by depletion of internal Ca^2+^-stores due to EGTA-treatment. Differentiation of the wild type and the mutant on plates containing 20 mM EGTA is shown. Aggregation was delayed in both strains till t~15~and t~18~in wild type and iplA^-^cells, respectively. The size of the aggregates and the fruiting bodies were much smaller than under control conditions. The full width of the image corresponds to 12.5 mm. ::: ![](1471-2121-6-13-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### In the presence of external Ca^2^aggregation is accelerated in iplA^-^cells. Differentiation of the mutant and the wild type strain was assayed in parallel on agar plates supplemented with 20 mM CaCl~2~. Aggregate formation occurred earlier in iplA^-^cells (at t~7~) than in wild type (starting at t~19~) in the presence of Ca^2+^. The full width of the image corresponds to 12.5 mm. ::: ![](1471-2121-6-13-3) ::: Then we analyzed the effect of treatment with either EGTA or Ca^2+^on basal cell motility. We found that under control conditions the general morphology of the cells as well as extension of pseudopods was practically identical in both strains (Fig. [4 A, B](#F4){ref-type="fig"}, \[see [Additional file 1](#S1){ref-type="supplementary-material"}\]). Preincubation with 10 mM EGTA for 60 min led to strong rounding of wild type and mutant amoebae (Fig. [4 C, D](#F4){ref-type="fig"}) with reduced extension of small pseudopods \[see [Additional file 2](#S2){ref-type="supplementary-material"}\]. By contrast, pretreatment with 10 mM CaCl~2~did not affect the morphology (Fig. [4 E, F](#F4){ref-type="fig"}) or the extension of pseudopods \[see [Additional file 3](#S3){ref-type="supplementary-material"}\] in both strains. Next we tested chemotaxis of amoebae towards a cAMP-filled glass capillary. Under control conditions cells of both strains oriented and migrated towards the tip of the capillary (Fig. [5 A, B](#F5){ref-type="fig"}); the average chemotactic speed was not different between wild type and the mutant strain (10.7 ± 2.1 vs. 11.0 ± 0.7 μm/min; mean ± s.e.m. of 20 wild type and 43 mutant amoebae analyzed in 3 and 4 independent experiments, respectively). Incubation in 10 mM EGTA for 60 min abolished chemotaxis in most of the wild type and the iplA^-^amoebae: small pseudopods were extended randomly and the cells did not approach the capillary tip (Fig. [5 C, D](#F5){ref-type="fig"}). Only rarely, cells of both strains exhibited an oriented but highly reduced migration towards the cAMP source (3% and 5% of 33 wild type and 19 mutant cells analyzed in 3 independent experiments, respectively). Thus the loss of Ca^2+^from stores impairs both, orientation and migration also in the absence of the iplAgene product. By contrast, when amoebae were incubated in 10 mM CaCl~2~for 60 min to load stores, they oriented and migrated towards the cAMP capillary (Fig. [5 E, F](#F5){ref-type="fig"}). The chemotactic speed of wild type cells (9.9 ± 1.2 μm/min; mean ± s.e.m. of 17 cells tested in 3 independent experiments) was comparable to that under control conditions whereas mutant amoebae chemotaxed significantly faster (13.6 ± 1.7 μm/min; mean ± s.e.m. of 15 mutant amoebae analyzed in 3 independent experiments) than wild type cells in the presence of 10 mM CaCl~2~(Mann-Whitney rank sum test, p = 0.041). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### General morphology of wild type and iplA^-^cells under control conditions (A, B), in the presence of 10 mM EGTA for 60 min (C, D) or in the presence of 10 mM CaCl~2~for 80 min (E, F). In H5-buffer or in the presence of 10 mM CaCl~2~the morphology was not significantly different between wild type and mutant amoebae. However, in the presence of EGTA the cells of both strains were rounded. Photographs were taken at t~5~. Basal motility under these conditions can be viewed in the accompanying movies. ::: ![](1471-2121-6-13-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Chemotaxis of wild type and mutant amoebae at different experimental conditions. The tracks of individual cells (in red) migrating during chemotactic stimulation (position of the tip of the cAMP-filled capillary: green star) are shown. In H5-buffer (A, B) both cell types migrated in an oriented manner towards the capillary tip, albeit not always in a straight line. After preincubation with 10 mM EGTA for 60 min and in its continued presence during the chemotaxis assay (C, D) the cells remained stationary with random pseudopod extension. Preincubation of amoebae with 10 mM CaCl~2~(E, F) did not impair chemotaxis; rather, the cells of both strains migrated towards the capillary tip. Chemotaxis experiments were done at t~6~. ::: ![](1471-2121-6-13-5) ::: Buffering of intracellular \[Ca^2+^\] impairs chemotaxis -------------------------------------------------------- The observation that aggregation occurred in the mutant cell line although a cAMP-activated increase in \[Ca^2+^\]~i~was not detectable resulted in the conclusion that \[Ca^2+^\]~i~-changes were not necessary to accomplish chemotaxis \[[@B6]\]. We used the mobile buffer approach originally described by Speksnijder et al. \[[@B11]\] which allows to analyze the requirement of a \[Ca^2+^\]~i~-gradient for a given response. If in Dictyosteliuma \[Ca^2+^\]~i~-increase was necessary for chemotaxis, the presence of a Ca^2+^-chelator in the cytosol should impair orientation and/or migration. In a previous study, we had introduced the Ca^2+^-chelator BAPTA and its derivatives into the cytosol of wild type amoebae which indeed had inhibited chemotactic migration and reduced chemotactic orientation \[[@B5]\]. Here we used the Ca^2+^-indicator Fura2-dextran to clamp \[Ca^2+^\]~i~and loaded the indicator into wild type and mutant cells in the absence of external CaCl~2~. The treatment affected chemotactic performance of wild type as well as iplA^-^amoebae. Lack of extracellular CaCl~2~during the loading process induced strong rounding of the amoebae and loss of migration. Figure [6](#F6){ref-type="fig"} shows that the capacity to orient chemotactically and to extend pseudopods towards the capillary tip was reduced by 58% in wild type (93 cells tested in 4 independent experiments). Inhibition was also evident in iplA^-^cells (Fig. [6](#F6){ref-type="fig"}) showing sensitivity of the mutant towards buffering of intracellular Ca^2+^-levels and eradication of \[Ca^2+^\]~i~-changes: the fraction of pseudopods extended in direction of the cAMP-source was reduced by 75% (74 cells tested in 3 independent experiments). These results show that not only in wild type but also in the iplA^-^cell line the ability to orient and to migrate in fact depends on an agonist-activated \[Ca^2+^\]~i~-elevation. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Chemotaxis of iplA^-^cells is impaired in the intracellular presence of a Ca^2+^-buffer. Wild type and mutant amoebae were loaded with Fura2-dextran and their ability to protrude pseudopods towards a cAMP-filled glass capillary was compared to that of untreated cells. In both strains the presence of the chelator in the cytosol led to a decrease in the fraction of cells extending pseudopods and migrating towards the cAMP source. ::: ![](1471-2121-6-13-6) ::: Analysis of Ca^2+^-fluxes ------------------------- Our findings that differentiation was sensitive towards depletion of Ca^2+^or loading of the cells with Ca^2+^and that chemotaxis was blocked by intracellular Ca^2+^-buffers led us to investigate Ca^2+^-fluxes in the mutant cell line. We used a Ca^2+^-sensitive electrode in cell suspensions to measure Ca^2+^-fluxes, an approach different from that of Traynor et al. \[[@B6]\] who had studied ion fluxes by ^45^Ca^2+^-measurements. First we tested whether the coupling of stores to the plasma membrane, i.e. capacitative Ca^2+^-fluxes without prior stimulation with agonists were altered. In Dictyosteliuminduction of capacitative Ca^2+^-influx requires active intracellular Ca^2+^-pumps. Their inhibition by either thapsigargin or 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) does not evoke influx; rather, stores have to be emptied by treatment with EGTA \[[@B12]\]. Capacitative Ca^2+^-fluxes were studied early during differentiation (t~2~--t~4~). At this time Ca^2+^-influx and Ca^2+^-efflux are at an equilibrium which held true for both, wild type and mutant cells (not shown); in suspensions of cells at later stages of development influx strongly exceeds efflux \[[@B1]\]. In iplA^-^and in wild type cells emptying of storage compartments via preincubation of amoebae with EGTA induced capacitative Ca^2+^-entry (Fig. [7 A](#F7){ref-type="fig"}) which was blocked by addition of 1 mM NaN~3~(Fig. [7 B](#F7){ref-type="fig"}). The characteristics of influx were comparable in wild type and mutant cells. These data show that capacitative Ca^2+^-influx does not depend on the product of the iplAgene. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Recordings of Ca^2+^-fluxes in iplA^-^and wild type amoebae. \[Ca^2+^\]~e~was measured in cell suspensions with a Ca^2+^-sensitive electrode. (A) Treatment of amoebae with 5 mM EGTA for 30 min activated capacitative Ca^2+^-influx (one out of 12/6 determinations in 4/3 independent experiments is shown for iplA^-^and wild type, respectively). (B) Capacitative influx was blocked by the addition of 1 mM NaN~3~(one out of 5/4 determinations in 3/3 independent experiments). Measurements were done at t~3~. ::: ![](1471-2121-6-13-7) ::: On the other hand, agonist-activated ^45^Ca^2+^-entry had been reported to be absent in the mutant strain; in their study, Traynor et al. had stimulated cells with cAMP in the presence of 0.1 mM CaCl~2~\[[@B6]\]. The use of a Ca^2+^-sensitive electrode allows to measure much lower levels of extracellular Ca^2+^to analyze Ca^2+^-fluxes, in the range of approximately 1 μM Ca^2+^. Indeed, we found that under this condition reversible Ca^2+^-entry occurred after addition of 1 μM cAMP (Fig. [8 A](#F8){ref-type="fig"}) that amounted to 10.2 ± 4.3 pmol Ca^2+^/10^7^cells (mean ± s.d. from 9 experiments). The level of influx represented roughly 5% of wild type influx (Fig. [8 B](#F8){ref-type="fig"} and \[[@B13],[@B14]\]). Ca^2+^-influx was delayed in the mutant and the time to reach the maximum was longer than in wild type cells \[[@B13],[@B14]\]. In addition, challenge with arachidonic acid (AA) induced influx (Fig. [9 A](#F9){ref-type="fig"}). Again, the mutant was less sensitive and higher concentrations were required than those reported to evoke Ca^2+^-entry in wild type cells (Fig. [8 B](#F8){ref-type="fig"}, [9 C](#F9){ref-type="fig"} and \[[@B13]\]). Neither 10 nor 20 μM AA were effective; in the wild type 10 μM AA activates influx of 190 ± 58 pmol Ca^2+^/10^7^cells \[[@B14]\]. At 60 μM AA entry occurred in the mutant strain which amounted to an average of 107 ± 21 pmol Ca^2+^/10^7^cells (mean ± s.d. from 7 experiments). Preincubation of cells with the SERCA-type Ca^2+^-ATPase inhibitor BHQ reduced AA-induced influx by 82 % (Fig. [9 B](#F9){ref-type="fig"}) to an average of 21 ± 18 pmol Ca^2+^/10^7^cells (mean ± s.d. from 3 experiments). These data show that Ca^2+^-fluxes across the plasma membrane do occur in iplA^-^cells as well but at a reduced level. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Agonist-activated Ca^2+^-fluxes in suspensions of iplA^-^and wild type amoebae. cAMP elicited reversible Ca^2+^-influx in the mutant (A) and in wild type ((B) and see \[14\]); measurements were done at t~7~--t~7.5~. Note the different doses of CaCl~2~added for calibration. The time points of cAMP-addition (1 μM) and of AA-addition (6 μM) in the wild type are indicated by arrows. ::: ![](1471-2121-6-13-8) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Fatty acids activate Ca^2+^-fluxes in iplA^-^amoebae. (A) 60 μM AA evoked a transient decrease in \[Ca^2+^\]~e~representing Ca^2+^-influx; measurement was done at t~6~(B) After preincubating amoebae with the SERCA-type Ca^2+^-ATPase blocker BHQ (100 μM) for 20 min to inhibit uptake of Ca^2+^into internal storage compartments, the AA-activated response was absent; measurement was done at t~7.5~. Results of measurements with wild type (C, D) stimulated with 6 μM AA are shown for comparison. ::: ![](1471-2121-6-13-9) ::: Next we tested whether the mutant strain was able to release stored Ca^2+^when stimulated with cAMP or AA. Fluxes were measured in suspensions of cells with permeabilized plasma membranes; any change in \[Ca^2+^\]~e~thus reflects efflux of Ca^2+^from storage compartments. Both, cAMP and arachidonic acid activated release of stored Ca^2+^(Fig. [10 A](#F10){ref-type="fig"}). On average, addition of 1 μM cAMP released 7.3 ± 3.4 pmol Ca^2+^/10^7^cells and 16.3 ± 7.2 pmol Ca^2+^/10^7^cells were liberated after stimulation with 3 μM AA (mean ± s.d. from 10 and 3 experiments, respectively). The amount of Ca^2+^-efflux from stores after cAMP stimulation was 61% of that found in wild type cells (Fig. [10 B](#F10){ref-type="fig"} and \[[@B15]\]) whereas release upon AA-challenge was in the range of 5--10% of wild type (see Fig. [2](#F2){ref-type="fig"} in \[[@B14]\]). In addition to the Ca^2+^-electrode recordings, we studied Ca^2+^-fluxes in suspensions of partially purified storage compartments fluorimetrically. ATP induced Ca^2+^-sequestration (Table [1](#T1){ref-type="table"}) was of similar magnitude and rate as in wild type stores. This result indicates that the decreased release of Ca^2+^from the stores measured with the Ca^2+^-electrode is not due to a lack of storage capacity. Moreover, the addition of AA evoked release of Ca^2+^from stores as did inhibition of Ca^2+^-pump(s) by thapsigargin. XestosponginC that inhibits Ca^2+^-uptake and activates Ca^2+^-release in wild type \[[@B8]\] and the ionophore ionomycin also resulted in substantial Ca^2+^release in the mutant cell line (Table [1](#T1){ref-type="table"}). All values were in the same range as those for wild type. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Determination of Ca^2+^-fluxes in partially purified storage compartments of the iplA^-^mutant and of wild type. Ca^2+^-sequestering vesicles were prepared as outlined in Methods. Measurements were performed with the pellet and supernatant fraction. ATP-induced uptake and release activated by different agents is given as nmol Ca^2+^-uptake/min and mg of protein and pmol Ca^2+^-release/tube, respectively (mean ± s.d.). In release experiments 60--75 μl of pellet and 120--140 μl of supernatant fraction were used per tube. Numbers in brackets give number of experiments; n.d.: not determined. ::: iplA^-^ Wt ----------------------- ----------------- ----------------- ----------------- ----------------- Uptake (nmol/min\mg) 1 mM ATP 1.96 ± 0.55 (4) 0.28 ± 0.15 (4) 1.87 ± 0.74 (3) 0.38 ± 0.16 (3) Release (pmol/tube) 10 μM AA 360 ± 227 (8) 761 ± 218 (6) 396 ± 122 (6) 961 ± 374 (2) 40 μM Thapsigargin 570 ± 250 (6) 170 ± 73 (6) 582 ± 123 (5) 265 ± 135 (2) 6 μM XestosponginC 201 ± 55 (4) n.d. 276 ± 46 (3) n.d. 2 μM Ionomycin 447 ± 147 (5) 147 ± 41 (4) 580± 173 (3) 193 ± 76 (2) ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### cAMP and arachidonic acid elicit Ca^2+^-release from internal stores. (A) \[Ca^2+^\]~e~was recorded at t~7~in iplA^-^cells with permeabilized plasma membranes. Amoebae were challenged with 1 μM cAMP and 3 μM AA, respectively. (B) The response of permeabilized wild type stimulated with 1 μM cAMP at t~6~is shown for comparison. ::: ![](1471-2121-6-13-10) ::: Mn^2+^-quenching experiments ---------------------------- The \[Ca^2+^\]~e~-recordings in suspensions of cells as described above detect the sum of Ca^2+^-influx and efflux. Therefore, a complementary approach which monitors influx only was pursued, by using the Mn^2+^-quenching technique in single intact amoebae. This method is based on the fact that many Ca^2+^-channels are permeable to Mn^2+^\[[@B16]\] and that the Ca^2+^-indicator Fura2 binds Mn^2+^with high affinity. Fluorescence of the indicator is quenched upon binding \[[@B17]\]. We compared quenching of Fura2-dextran fluorescence activated by addition of Mn^2+^alone or in combination with 1 μM cAMP in wild type and mutant cells. Higher concentrations of MnCl~2~were required to quench Fura2-dextran fluorescence in iplA^-^amoebae (Fig. [11](#F11){ref-type="fig"}, Table [2](#T2){ref-type="table"}). Reduction of fluorescence occurred at 1 μM Mn^2+^and 1 μM Mn^2+^/cAMP in wild type (Fig. [11 A, B](#F11){ref-type="fig"}; see also \[[@B12]\]) but not in the mutant (Fig. [11 C, D](#F11){ref-type="fig"}) where addition of 100 μM Mn^2+^/cAMP was necessary; 100 μM Mn^2+^alone was not effective (Fig. [11 C, D](#F11){ref-type="fig"}) but started at 200 μM Mn^2+^(not shown). These data show that the reduction in ion fluxes in the mutant were indeed due to an alteration in entry mechanisms. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Rate of basal and cAMP-induced Mn^2+^-influx. Amoebae were challenged with 1 or 100 μM Mn^2+^either with or without 1 μM cAMP. Cells were preincubated with 0.1 mM EGTA as outlined in Methods. Mn^2+^quenching of fura-2-dextran fluorescence was tested in H5-buffer and is expressed as decrease in fluorescence units/sec (mean ± s.e.m.). Numbers in brackets give number of cells tested and number of experiments. ::: Stimulation Preincubation ------------------------- ------------------- -------------------- 1 μM Mn^2+^ 0 (121/5) 0.37 ± 0.2 (142/3) 1 μM Mn^2+^/1 μM cAMP 0 (135/5) 1.4 ± 0.1 (207/4) 100 μM Mn^2+^ 0 (109/3) 100 μM Mn^2+^/1 μM cAMP 1.0 ± 0.2 (292/9) ::: ::: {#F11 .fig} Figure 11 ::: {.caption} ###### Basal and cAMP-induced Mn^2+^-influx. Influx was assayed by quenching of Fura2-dextran fluorescence. (A, B) The response of wild type amoebae is shown for comparison; 1 μM Mn^2+^± 1 μM cAMP was added. iplA^-^cells in nominally Ca^2+^-free buffer were challenged with 100 μM Mn^2+^± 1 μM cAMP at t~7~(closed symbols); when 1 μM Mn^2+^was added (open symbols) no influx was detected (C, D). After preincubation with EGTA influx was observed at 1--2 μM Mn^2+^± 1 μM cAMP (E, F). Fluorescence intensity at 360 nm excitation is shown as mean ± s.e.m ::: ![](1471-2121-6-13-11) ::: In principle, the iplAgene product could form a channel in the plasma membrane or in membranes of internal stores. The lack of the iplAgene product in the stores might impair their coupling to the plasma membrane. As we had observed capacitative Ca^2+^-entry in the mutant we asked whether manipulation of the filling state of the stores altered ion fluxes. First we tested the effect of emptying of stores on Mn^2+^-influx. When cells were preincubated with EGTA, the requirement for high doses of Mn^2+^to quench fluorescence was abrogated. Now capacitative and also agonist-activated Mn^2+^-influx occurred at concentrations of MnCl~2~comparable to those used under control conditions in wild type, in the range of 1--2 μM (Fig. [11 E, F](#F11){ref-type="fig"}). This result renders the possibility that the plasma membrane is altered in the mutant unlikely. Yet, the rate of Mn^2+^-influx observed in EGTA-treated mutant amoebae was still less than in wild type cells with respect to both basal and cAMP-activated fluxes (53 and 58% of wild type \[[@B12]\], respectively). \[Ca^2+^\]~i~-determination --------------------------- In wild type cells treatment with EGTA augments responsiveness and cAMP-elicited \[Ca^2+^\]~i~-transients are detected at low extracellular \[Ca^2+^\] \[[@B12]\]. However, an agonist-induced \[Ca^2+^\]~i~elevation was not observed in iplA^-^cells under these conditions. On the other hand, when stores were loaded in the continued presence of CaCl~2~, we observed that differentiation of the mutant resembled the wild type as described above. This led us to compare the \[Ca^2+^\]~i~-response of wild type and mutant amoebae after pretreatment with CaCl~2~. In wild type cells preincubation with 1 mM CaCl~2~for 10--15 min and its continued presence during the \[Ca^2+^\]~i~-imaging experiment is required to activate a \[Ca^2+^\]~i~-transient after challenge with cAMP (Fig. [12 A](#F12){ref-type="fig"} and see \[[@B12]\]); in nominally Ca^2+^-free medium or at very low extracellular \[Ca^2+^\] a cAMP-activated \[Ca^2+^\]~i~-elevation is not observed \[[@B12]\]. In accordance with the data of Traynor et al. \[[@B6]\] this condition (Fig. [12 B](#F12){ref-type="fig"}) and even increasing the concentration of CaCl~2~to 20 mM resulted in no detectable \[Ca^2+^\]~i~-increase in the mutant strain (not shown). However, we found that the basal \[Ca^2+^\]~i~level in iplA^-^amoebae was significantly lower than in wild type and amounted to an average of 36 ± 3 nM (mean ± s.e.m. of 8 determinations in 3 independent experiments) as compared to 50 ± 2 nM in wild type (mean ± s.e.m. of 29 determinations in 9 independent experiments; Mann-Whitney rank sum test, p = 0.002). ::: {#F12 .fig} Figure 12 ::: {.caption} ###### \[Ca^2+^\]~i~-recordings in cells preincubated with CaCl~2~in order to load stores. (A) The response of wild type upon stimulation with 1 μM cAMP (arrow) at standard conditions, i.e. after preincubation with 1 mM CaCl~2~for 10--15 min and stimulated in the presence of 1 mM CaCl~2~is shown. Values give mean ± s.e.m. of 7 cells. (B) When iplA^-^cells were stimulated with 1 μM cAMP at standard conditions (as outlined in (A)), no response was observed. Mean ± s.e.m. of 6 cells is shown. (C) Wild type was preincubated with 1 mM CaCl~2~for 4--5 h; after washing cells were incubated in H5-buffer supplemented with 1 μM CaCl~2~and challenged with 1 μM cAMP (arrow). Values give mean ± s.e.m. of 16 cells. (D) iplA^-^incubated for 3 h with 20 mM CaCl~2~were washed and subsequently \[Ca^2+^\]~i~-imaging was done in buffer containing 1 mM CaCl~2~. Arrow indicates the time point when 1 μM cAMP was added. Mean ± s.e.m. of 10 cells is shown. ::: ![](1471-2121-6-13-12) ::: For stronger loading of Ca^2+^-stores, we preincubated cells with 1 mM CaCl~2~for 4 h. After this treatment a \[Ca^2+^\]~i~-elevation upon addition of cAMP was detected in 60% of wild type amoebae even at low extracellular \[Ca^2+^\] levels, i.e. when the buffer used to wash the cells had been supplemented with only 1 μM CaCl~2~(Fig. [12 C](#F12){ref-type="fig"}). Starting from a basal level of 48 ± 4 nM, the height of the \[Ca^2+^\]~i~-transient amounted to 23 ± 2 nM (mean ± s.e.m. of 12 determinations in 8 independent experiments). Again, these conditions were not effective in iplA^-^cells; rather, the sensitivity of the mutant was shifted to higher Ca^2+^concentrations as had been found with Mn^2+^-quenching experiments. We preincubated iplA^-^amoebae with 20 mM CaCl~2~for 3 h; after washing thoroughly, a cAMP-induced \[Ca^2+^\]~i~-elevation in the presence of 1 mM CaCl~2~was observed (Fig. [12 D](#F12){ref-type="fig"}) in 28% of the cells; its average height amounted to 67 ± 11 nM (mean ± s.e.m. of 15 determinations in 4 independent experiments) starting from a basal level of 39 ± 2 nM. In the course of these experiments we once observed a response also under standard conditions, i.e. at 1 mM \[Ca^2+^\]~e~without prior incubation in 20 mM CaCl~2~; the height of the increase amounted to 54 ± 6 nM (mean ± s.e.m.). Yet, this was a rare event (once in 31 determinations). Discussion ========== The role of cAMP-activated \[Ca^2+^\]~i~-changes for chemotaxis has been questioned by Traynor et al. \[[@B6]\] who reported results obtained with the iplA^-^mutant cell line favouring insignificance of \[Ca^2+^\]~i~for the chemotactic response. The authors had observed formation of fruiting bodies even though neither ^45^Ca^2+^-fluxes nor an agonist induced \[Ca^2+^\]~i~-elevation were detectable. The discrepancy between our view that a \[Ca^2+^\]~i~-elevation is necessary for a proper chemotactic response \[[@B5]\] and the conclusion of Traynor et al. prompted us to analyze chemotaxis, differentiation and the \[Ca^2+^\]-regulation of the iplA^-^mutant in detail. In particular, we tested not only basal and cAMP-activated ion fluxes but also capacitative Ca^2+^-entry which is induced by emptying internal Ca^2+^-stores via preincubation of amoebae with EGTA \[[@B12]\]. Aggregation and development of wild type and mutant cells on agar plates was sensitive towards continuous emptying or loading of Ca^2+^-stores. These effects are not necessarily caused by altering chemotactic migration. It is conceivable that other Ca^2+^-dependent processes were affected, e.g. that the timing or pattern of gene expression or the establishment of cell contacts was altered. Although incubation of mutant amoebae for 2 h with 20 mM CaCl~2~or of wild type with 1 mM CaCl~2~for 4--5 h or with 1 mM EGTA for 1 h \[[@B12]\] did not significantly increase or lower basal \[Ca^2+^\]~i~it is possible that the continued presence of 10 mM EGTA or CaCl~2~for many hours affects basal levels of \[Ca^2+^\]~i~which in turn might mediate effects on gene expression as was shown for prolonged incubation of cells with BHQ \[[@B18]\]. During development Dictyosteliumcells form Ca^2+^-dependent and EDTA/EGTA-sensitive cell-cell contacts that are mediated by gp24 and DdCAD-1 (\[[@B19],[@B20]\]; for review see \[[@B21]\]). Therefore, chelation of extracellular Ca^2+^might also inhibit cell adhesion. However, mutant cells whose gene encoding DdCAD-1 had been disrupted show normal chemotaxis and cell streams. Furthermore, mound formation was accelerated and only culmination was delayed by about 6 hours \[[@B20]\]. If only Ca^2+^-dependent cell adhesion was affected in our development assay in the presence of EGTA we would expect a similar phenotype. However, aggregation was clearly delayed. This argues for additional Ca^2+^-dependent processes during aggregation. When we tested the influence of the presence of EGTA or CaCl~2~on spontaneous motility and chemotaxis we found that in both strains motility in general was strongly impaired and that chemotaxis of the amoebae towards the cAMP-filled glass capillary was virtually abolished upon depletion of internal stores by the extracellular presence of EGTA. This effect is time dependent; after 30 min of incubation with 10 mM EGTA the behaviour of wild type amoebae was found to be unaltered and only after treatment for 0.5--1 h rounding and reduction of pseudopod elongation towards the capillary tip occurred \[[@B5]\]. Prolonged incubation for more than 1 h as carried out in this study completely inhibited the chemotactic response. These results strengthen the view that Ca^2+^has a necessary role in chemotaxis in wild type and in the mutant as well. When the cellular Ca^2+^content falls below a critical value Ca^2+^-dependent cytoskeletal rearrangements \[[@B22],[@B23]\] that are necessary for both, random pseudopod extension during spontaneous motility and oriented pseudopod formation after chemotactic stimulation no longer take place correctly. On the other hand, the presence of 10 mM CaCl~2~induced no alteration of basal cell motility in wild type or mutant amoebae. Yet, during chemotactic stimulation the average speed of migration towards the capillary tip was higher in mutant than in wild type cells. In this respect it is of importance that the basal level of \[Ca^2+^\]~i~was significantly lower in the former. At standard conditions the reduced basal \[Ca^2+^\]~i~does not impair the capacity of the mutant to chemotax. Therefore, this particular mutant strain represents the \"minimal solution\" with respect to the concentration of cytosolic Ca^2+^necessary to accomplish cytoskeletal rearrangements and extrusion of a pseudopod correctly. However, in the presence of 10 mM extracellular Ca^2+^during cAMP-stimulation Ca^2+^-fluxes are enhanced allowing more efficient formation of pseudopods. We had shown previously that a small global elevation of \[Ca^2+^\]~i~activates the extension of pseudopods all over the cell\'s circumference whereas a larger increase induces contraction of the amoebae \[[@B24]\]. In our view, the strongest evidence that a \[Ca^2+^\]~i~-transient is necessary for the extension of pseudopods rests upon the experiment where a Ca^2+^-chelator was introduced into the cytosol of the amoebae. This treatment led to rounding of the amoebae and a general reduction of pseudopod formation (see also \[[@B5]\]). Upon stimulation with a cAMP-filled capillary, the extension of oriented pseudopods was greatly reduced and migration towards the capillary tip was abolished. As Speksnijder et al. \[[@B11]\] had pointed out the fact that the presence of a chelator has an effect shows that a \[Ca^2+^\]~i~-gradient is essential for a given response. In summary, these data support the notion that an elevation of \[Ca^2+^\]~i~is required to extend pseudopods; suppression of the \[Ca^2+^\]~i~-elevation inhibits motility in general. Upon chemotactic challenge with cAMP this \[Ca^2+^\]~i~-gradient has to be established in a locally restricted fashion in order to allow local, oriented pseudopod formation (see \[[@B2],[@B25],[@B26]\]); otherwise pseudopods would be extended in all directions (see above, \[[@B24]\]). Our results imply that in iplA^-^cells such a \[Ca^2+^\]~i~-gradient occurs as well, either nonrestricted allowing extension of pseudopods at random sites during spontaneous motility or restricted locally after chemotactic stimulation leading to oriented pseudopod formation. The fact that in the mutant cell line cAMP-activated \[Ca^2+^\]~i~-changes were practically undetectable under our standard condition argue for a \[Ca^2+^\]~i~-increase that is either smaller and/or more restricted to distinct domains within the cell than in wild type amoebae. Indeed, in only one out of roughly 30 determinations did we observe a cAMP-activated \[Ca^2+^\]~i~-transient under standard conditions. These results imply a crucial role but not an absolute necessity of the iplAgene product for the regulation of cAMP-induced \[Ca^2+^\]~i~-changes. By using a Ca^2+^-sensitive electrode in cell suspensions, we analyzed which aspects of \[Ca^2+^\]~i~are controlled by the iplAgene product. Besides studying agonist-induced Ca^2+^-fluxes we also investigated capacitative Ca^2+^-entry and found that this type of influx was similar in mutant and wild type cell suspensions. We obtained equivalent results by testing Mn^2+^-quenching of Fura2-dextran fluorescence which showed that capacitative entry is independent of the iplAgene product. On the other hand, using the Ca^2+^-sensitive electrode, we found that in the iplA^-^mutant the agonist cAMP and also AA did activate Ca^2+^-entry into intact cells. The difference between the data published by Traynor et al. \[[@B6]\] and our results is most likely due to the experimental conditions: the magnitude of the Ca^2+^-fluxes that we observed was considerably lower than in wild type cells and detectable at low extracellular \[Ca^2+^\] only. The ^45^Ca^2+^-flux studies had been performed at 100 μM external CaCl~2~; so the fraction of ^45^Ca^2+^entering the cells was presumably too low to be detected reliably. Moreover, we found cAMP- and AA-induced Ca^2+^-release from stores in cells with permeabilized plasma membranes. These data show that cAMP-induced Ca^2+^-release from stores in iplA^-^cells is functional. However, much like the Ca^2+^-influx, agonist-activated liberation from stores was smaller than in wild type amoebae. In line with these results are the findings using Mn^2+^-quenching to assay ion fluxes in intact single cells: higher doses of Mn^2+^were necessary to detect influx. There are several interpretations for the results above. (i) There are two types of channels responsible for Ca^2+^-influx: one type being activated by emptying of the stores and sustaining capacitative Ca^2+^-entry which is unaffected in iplA^-^cells and the other one mediating agonist-induced Ca^2+^-fluxes, the latter being under the control of the iplAgene product. The view that there are two strictly separated ion channels seems unlikely as under conditions of emptied stores cAMP-activated Mn^2+^-quenching occurred in the mutant as well. (ii) The same channel(s) mediate capacitative and agonist-activated fluxes but upon stimulation with agonists it cannot be addressed properly when iplAis disrupted. This implies a role of the protein in the liberation of Ca^2+^from the stores which is a prerequisite for the triggering of Ca^2+^-entry \[[@B12]\]. In the mutant this cannot proceed normally so subsequent activation of Ca^2+^-influx and the generation of a full \[Ca^2+^\]~i~-increase is impaired. The results of the experiments where stores were strongly loaded with Ca^2+^prior to stimulation support this notion. In this situation release from stores should be augmented. Indeed, in both, wild type and mutant cells, cAMP-activated \[Ca^2+^\]~i~-elevations occurred at an extracellular \[Ca^2+^\] (see Fig. [12](#F12){ref-type="fig"}) where without pretreatment no increase was observed. Presumably, release of Ca^2+^from the filled stores contributed to the observed \[Ca^2+^\]~i~-increase to a greater extent than under standard conditions. The requirement for 20 fold higher concentrations of CaCl~2~during preincubation to elicit an agonist-induced \[Ca^2+^\]~i~-elevation in iplA^-^cells are most likely due to the reduction in Ca^2+^-entry which necessitates a higher concentration gradient across the plasma membrane to fill the stores efficiently. An as yet unresolved issue is the mechanism that induces Ca^2+^-entry upon liberation of Ca^2+^from the stores. From our data we conclude that in Dictyosteliumthese signals are different when the stores are emptied by EGTA or by agonist-activated signaling cascades. Otherwise one cannot explain normal capacitative Ca^2+^- and Mn^2+^-influx induced by EGTA-treatment and a requirement for 100 fold higher ion concentrations to induce Mn^2+^-entry by cAMP. If indeed the iplAgene product constitutes an IP~3~-receptor like channel that is located on membranes of stores the physical coupling of the receptor to channels in the plasma membrane as a mechanism to activate extracellular Ca^2+^-entry \[[@B27]\] should be missing in the mutant. On the other hand, emptying of stores by EGTA-treatment influences not only the IP~3~-sensitive store but also other stores and thus exerts a much more general effect on the cells. Studies using microarrays should reveal whether the expression of other genes is affected by the absence of iplAand thus might give a clue how \[Ca^2+^\]~i~is regulated in the mutant although one type of Ca^2+^-store is malfunctional. Conclusion ========== Our results show that Ca^2+^fluxes and regulation of Ca^2+^homeostasis take place in the iplA^-^mutant and that chemotaxis and development of the mutant are sensitive to disturbance of the Ca^2+^homeostasis. In wild type cells and in cells lacking the iplAgene changes in \[Ca^2+^\]~i~are necessary to orient and to migrate chemotactically; their abolition causes loss of chemotaxis towards a cAMP source. The iplAgene product exerts a crucial role in the control of basal \[Ca^2+^\]~i~and of agonist induced Ca^2+^-fluxes. It is not required to activate capacitative Ca^2+^-influx. Thus the mechanisms responsible for capacitative and agonist-activated Ca^2+^-fluxes are different. Methods ======= Materials --------- Fura2-dextran and Fura2 were purchased from MobiTec; cAMP was from Boehringer. Cell culture ------------ D. discoideumwild type strain Ax2 and the iplA^-^cell lines HM1049 and HM1038 (kindly provided by Dr. D. Traynor) were cultured as described \[[@B14]\] in the absence or presence of 10 μg/ml Blasticidin S, respectively. There was no difference between the two mutant strains with respect to the assays performed; therefore, results of measurements with either HM1038 or HM1049 are shown. Cells were washed by repeated centrifugation and resuspension in cold Sørensen phosphate buffer (17 mM Na^+^/K^+^-phosphate, pH 6.0). Amoebae were shaken at 2 × 10^7^cells/ml, 150 rpm and 23°C until use. The time, in hours, after induction of development is designated t~x~. \[Ca^2+^\]~e~-electrode recordings ---------------------------------- \[Ca^2+^\]~e~in cell suspensions was recorded as described elsewhere \[[@B14]\]. Cells at t~5~--t~8~were washed by repeated centrifugation and resupended at 5 × 10^7^cells/ml in 5 mM Tricine, 5 mM KCl, pH 7.0. Permeabilization was done by addition of filipin (15 μg/ml) to cell suspensions exactly as outlined in \[[@B15]\]. Capacitative Ca^2+^-influx was analyzed in cells with emptied storage compartments \[[@B12]\]: amoebae at t~2~--t~4~were incubated with 5 mM EGTA for 30 min before washing in the above buffer. \[Ca^2+^\]~i~-determination and Mn^2+^-quenching experiments ------------------------------------------------------------ Cells were loaded with Fura2-dextran (5 mg/ml + 1 mM CaCl~2~) at t~4~--t~5~as described \[[@B12]\]. Aliquots (2--5 μl) of washed cells in H5-buffer (5 mM Hepes, 5 mM KCl, pH 7.0) were placed on glass coverslips and incubated in a humid chamber. 10--15 min prior to the experiment, 85--88 μl of H5-buffer + 1 mM CaCl~2~were added. In a series of experiments to load stores, wild type and iplA^-^cells were incubated with 1 mM CaCl~2~for 4--5 h and with 20 mM CaCl~2~for 2--3 h, respectively. Then they were thoroughly washed exactly as described previously \[[@B12]\] and incubated either in H5-buffer supplemented with 1 μM CaCl~2~(wild type; free \[Ca^2+^\] in the solution was measured to be 2--2.5 μM, see also \[[@B12]\]) or in H5-buffer +1 mM CaCl~2~(iplA^-^); final volume was 90 μl. Single cell \[Ca^2+^\]~i~-imaging was performed at t~7~--t~8~as described \[[@B14]\]; stimulation was done by adding 10 μl of cAMP (10 μM). For Mn^2+^-quenching assays, washed cells were incubated in H5-buffer and challenged with Mn^2+^or Mn^2+^/cAMP. In order to study fluxes in cells with partially emptied internal storage compartments cells were preincubated with EGTA (10 μl of H5-buffer plus 0.1 mM EGTA for 1--2 h). 10--15 min prior to the experiment this solution was carefully removed and 100 μl of H5-buffer was added. This was repeated three times; final volume was 90 μl. Fluorescence quenching was measured at 360 nm excitation; influx rates are given as decrease of fluorescence units/sec. Measurement of Ca^2+^-fluxes in partially purified storage compartments ----------------------------------------------------------------------- Analysis of vesicular Ca^2+^-fluxes was done as described \[[@B8]\]. In brief, 3 ml of cells at t~1~--t~6~(2 × 10^8^cells/ml) in 20 mM Hepes, pH 7.2, were lysed by passage through Nuclepore filters. A final concentration of 3 % sucrose, 50 mM KCl, 1 mM MgCl~2~, 20 μg/ml leupeptin, 1 μg/μl aprotinin, 2.5 mM dithiothreitol and 1 μM microcystin were added; unbroken cells were removed by centrifugation at 3000 g for 5 min. The supernatant was centrifuged again at 12000 g for 20 min. The sediment (P) was resuspended in 1 ml of the above buffer. The rate of uptake and release was determined in the pellet and supernatant fraction by measuring the extravesicular \[Ca^2+^\] with Fura2. Chemotaxis assays ----------------- Cells were analyzed for chemotaxis towards a capillary filled with 0.1 mM cAMP \[[@B28]\]. 250 μl of 1 × 10^5^cells/ml in H5-buffer were pipetted onto a glass coverslip and allowed to settle for 60 min. Chemotaxis was recorded on a video recorder for 30--45 min. Chemotaxis was also assayed in the presence of EGTA or CaCl~2~; then amoebae were incubated in the respective agents for 60 min before they were challenged with cAMP. Images were digitized and the behaviour of the cells was analyzed using a computer program written for this purpose. For determination of cell velocity, a square area of interest (AI) of variable size (usually roughly 1/3 of the area of the cell) was placed at the perimeter of the cell in the first image digitized at the beginning of the assay. In the next image (images were digitized at a 2--4 sec time interval) the program analyzed an area larger than the AI (this area was defined by adding a given number of pixels on each side of the AI) for a pattern that resembled that of the AI; when such a pattern was found then the AI was placed on this new spot. The difference between the position of the AI in the first image to that in the second image was expressed as a vector of a given length. The changes in cell shape during migration were compensated by updating the pattern within the AI for every consecutive image analyzed. Calibration of the system allowed to convert the sum of the vector lengths to the distance in μm that the cells had migrated at the end of the experiment and to calculate the velocity of the amoebae. To test the effect of the intracellular presence of a Ca^2+^-buffer on chemotaxis, cells were loaded with Fura2-dextran (5 mg/ml in the loading solution) by electroporation in the absence of added external Ca^2+^. The amount of indicator present in the cytosol is in the range of 2--5% of the concentration present during electroporation \[[@B29]\]. 20 min after loading, cells were stimulated for 3--4 min by placing the cAMP-filled glass capillary at a distance of 10--20 μm of the cells and the number of cells that extended oriented pseudopods and thus elongated towards the capillary tip within this time period was counted. We had shown previously that loading of amoebae with FITC-dextran as a control does not alter chemotaxis as compared to untreated cells \[[@B5]\]. Analysis of differentiation --------------------------- Time lapse recordings of the development of Ax2 and iplA^-^cells on 1.5 % agar in H5-buffer (H5-agar) were done by placing 4 × 10^6^cells each on one half of a petri dish (∅ 35 mm) at t~1~. The two populations were separated from each other by a thin plastic disc that had been inserted in the melted agar during cooling. Only after removal of fluid and slight drying of the plate the disc was removed which resulted in a thin rim separating the strains. Differentiation was recorded by capturing an image of the plate every 30 min using a stereo microscope (Stemi 2000, Zeiss) equipped with a CCD camera (AVT Horn) under the control of the AxioVision software package (Zeiss). In addition, development was assessed at various levels of extracellular CaCl~2~. Then H5-agar contained either 5--20 mM EGTA or 5--20 mM CaCl~2~. List of abbreviations ===================== Cytosolic free Ca^2+^concentration: \[Ca^2+^\]~i~ 2,5-di-(t-butyl)-1,4-hydroquinone: BHQ Arachidonic acid: AA Area of interest: AI Authors\' contributions ======================= RS recorded extracellular \[Ca^2+^\] in cell suspensions and participated in the design of the study. DFL participated in the recordings of extracellular \[Ca^2+^\] and the design of the study. KBR analyzed chemotaxis and differentiation of wild type and mutant cells. KH performed \[Ca^2+^\]~i~measurements. DM analyzed fluxes in partially purified storage compartments and was involved in the design of the study. CS participated in \[Ca^2+^\]~i~measurements, did Mn^2+^-flux studies, participated in the design of the study and wrote the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Spontaneous cell motility of wild type and iplA^-^cells in H5-buffer. Images of cells at t~5~were captured every 15 sec for 20 min. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Basal cell motility after preincubation of wild type and mutant amoebae with 10 mM EGTA for 60 min (t~4~--t~5~). Images of cells at t~5~were captured every 15 sec for 20 min in the continued presence of EGTA. Cells were rounded and extended smaller pseudopods than under control conditions. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 Cell motility of wild type and mutant cells after preincubation with 10 mM CaCl~2~for 80 min (t~4~--t~5.3~) is shown. Images of cells at t~5.3~were captured every 15 sec for 20 min in the continued presence of CaCl~2~. The behaviour of treated cells was not different from control amoebae. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ The authors wish to thank Dr. D. Traynor and Prof. R. Kay for generously providing the iplA*^-^strain and Rupert Mutzel and Gerd Knoll for many helpful discussions and critical reading of the manuscript. We are indebted to Georg Heine and Williams Pascual from the Wissenschaftliche Werkstätten of the University of Konstanz for computer programming and to Katja Drews, Cyrus Nassiri and Frank Zucchetti for performing initial Ca^2+^-electrode measurements. This work was supported by the Deutsche Forschungsgemeinschaft.	PubMed Central	2024-06-05T03:55:54.082077	2005-3-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555532/", "journal": "BMC Cell Biol. 2005 Mar 11; 6:13", "authors": [ { "first": "Ralph H", "last": "Schaloske" }, { "first": "Daniel F", "last": "Lusche" }, { "first": "Karen", "last": "Bezares-Roder" }, { "first": "Kathrin", "last": "Happle" }, { "first": "Dieter", "last": "Malchow" }, { "first": "Christina", "last": "Schlatterer" } ] }
PMC555533	Background ========== Notch (N) and Delta (Dl) are cell surface proteins that are required for differentiation of almost all tissues in the fruit fly Drosophila melanogaster. They are evolutionarily conserved, functioning similarly in animals from worms to humans \[[@B1],[@B2]\]. The best-known instance of their function is the process of lateral inhibition that initiates differentiation of the neuronal and epidermal tissues from proneural cells that are predisposed to making the neuronal tissue. Proneural cells express high levels of the neuronal transcription co-factors from the Achaete Scute Complex (ASC) or related genes \[[@B3],[@B4]\]. These factors require their partner Daughterless (Da) to activate transcription of the neurogenesis genes \[[@B5]-[@B7]\]. Da is expressed at low levels in all Drosophila cells \[[@B8]\] and up regulated in proneural cells specified to differentiate the neurons \[[@B5]\]. Whether or not the up regulation of Da expression is part of lateral inhibition is not clear in Drosophila. In Caenorhabditis elegans, however, the differential accumulation of the Da homolog HLH-2 is the earliest detectable difference between the cells taking up alternate fates during lateral inhibition \[[@B9]\]. As N and Dl are known to regulate Da expression \[[@B10]\], it is very possible that Da expression is regulated during lateral inhibition in flies as well. When N expressed on one proneural cell binds Dl expressed on the neighboring proneural cell, N is proteolytically cleaved to release the Notch intracellular domain (N^intra^) from the plasma membrane. N^intra^translocates to the nucleus and, in association with the transcription factor Suppressor of Hairless (SuH), activates transcription of the Enhancer of split Complex(E(spl)C) genes. Cells that express a high level of E(spl)CRNA suppress their neuronal predisposition, become the epidermal precursor cells (EPCs), and differentiate the epidermis. Cells that express a low level of E(spl)CRNA and a high level of Da protein become the Neuronal Precursor Cells (NPCs) and differentiate the nervous system \[[@B1],[@B2],[@B5],[@B11]\]. From here onwards, we refer to this SuH dependent N activity that promotes expression of E(spl)CRNA as SuH/N^intra^signaling. A 1.5 to 2-fold difference in the level of SuH/N^intra^signaling is sufficient to initiate specification of the EPCs and the NPCs \[[@B11]\]. This difference is amplified by subsequent activities of N and Dl, or activities of other genes responding to the initial difference in the level of SuH/N^intra^signaling. The lateral inhibition process described above is repeatedly used during development for differentiation of various tissues with minor variations or changes in target genes. Scabrous (Sca) is a secreted factor that is produced at high levels in the NPCs and functions non-autonomously to promote specification of the EPCs during differentiation of the compound eye and the bristle organ \[[@B12],[@B13]\]. In its absence, lateral inhibition is not abolished but is reduced in strength or becomes imprecise indicating that Sca only refines the process. Sca binds N and stabilizes it. These actions promote formation of sharp boundaries between neuronal and non-neuronal cells during development of the compound eye \[[@B14]\]. The possibility that Sca might bind Dl as well is suggested by the observation that simultaneous over expression of Sca almost completely blocks the effect of Dl over-expression on wing margin development but hardly modifies the effect of N over-expression \[[@B15]\]. Dl and Sca have also been observed to co-localize in intracellular vesicles in vivo \[[@B13]\]. The observations that Sca can promote N activity \[[@B14]\] but block Dl activity are paradoxical as SuH/N^intra^signaling is very much dependent on the activities of both N and Dl. One explanation for this paradox could be, that Sca promotes lateral inhibition by having one effect through N and a different one through Dl. Therefore, we addressed the following questions in this study. Does Sca bind Dl? If yes, does it affect any Dl activity? Are there Dl activities independent of its activity as a ligand of N? Is Sca capable of activating N in the absence of Dl? N and Dl are expressed in almost all cells in vivoand N receptor activities in response to Dl binding are widely used during development. In developmental instances where Sca is present, the expression data suggest that both N and Dl will have access to Sca. Thus, it is very difficult to separate in vivothe activities of N alone, Dl alone, N on Dl, Dl on N, Sca on N, Sca on Dl, and Sca on N and Dl together. Therefore, we addressed the questions posed above in an in vitromodel system based on Drosophila Schneider (S2) cells. S2 cells do not express the endogenous N, Dl, or Sca \[[@B14],[@B16]\]. S2 cells expressing N (S2-N cells) mixed with S2 cells expressing Dl (S2-Dl cells) reproduce all aspects of lateral inhibition \[[@B16]-[@B22]\]. Using these cells and the medium prepared from S2 cells expressing Sca into the medium \[[@B14]\], we show that Sca binds Dl, Dl has activity independent of its activity as a ligand of N, Sca can affect this activity of Dl, and Sca can activate N in the absence of Dl. These observations would be useful for undertaking the challenging task of determining how the various activities of N, Dl, and Sca are integrated during tissue differentiation. Results ======= Sca associates with Dl ---------------------- Although N and Sca complexes could be immuno-precipitated \[[@B14]\], we, and others \[[@B23]\], had failed to detect Sca on S2-N cells. We suspected that some factor present in the tissue culture medium was washed away when the cells were processed for immuno-fluorescent detection of Sca. To overcome such problems, we made Sca-GFP and established stable S2 cells expressing it (S2-Sca-GFP cells). S2-Sca-GFP cells produced the Sca-GFP protein of the expected size (as determined by western blotting) and both Sca and GFP antibodies recognized this protein (data not shown). We concluded that S2-Sca-GFP cells expressed the expected Sca-GFP protein and used the conditioned medium from these cells to treat live S2-N, S2-Dl, and S2 cells. Live S2-Dl cells showed the strongest GFP signals, followed by live S2-N cells, and then live S2 cells (Fig. [1A--C](#F1){ref-type="fig"}). The signals were so strong on the S2-Dl cells that the signals on S2-N cells were not obvious at the same brightness/contrast settings. When cells were simultaneously fixed and rinsed with 1X PBS, the signals were comparable at the same settings (insets in Fig. [1A--C](#F1){ref-type="fig"}). Signals could not be detected on S2-N or S2-Dl cells after three 5-minute washes with 1X PBS, confirming our suspicion that the standard immuno-fluorescence procedure is inappropriate for detecting Sca binding on S2-N or S2-Dl cells. Secreted GFP did not bind the surfaces of any of these cells (data not shown). This indicated that the Sca part of Sca-GFP fusion protein bound the S2-N and S2-Dl cell surfaces. In all experiments conducted to determine the activity of Sca, N, or Dl, that are described below, we used only S2 cells expressing the wild type Sca because (1) we do not perform washes to remove non-specifically bound proteins and (2) we wanted to avoid possible GFP associated effects (stability, etc.). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Sca associates with Dl. A-C. Fluorescent photomicrographs of different cell lines treated with Sca-GFP medium for 30 minutes. Cells simultaneously fixed and rinsed in 4% paraformaldehyde/1X PBS are shown in the insets. Experiments were repeated three times. D. Western blots showing recovery of Dl in Sca immuno-precipitates from total protein extracts prepared from S2-Dl cells treated with S2-Sca cells. S2-Sca cells were used instead of Sca conditioned medium to maximize the ratio of bound to unbound Sca. Cross-linker = membrane insoluble and cleavable 3,3\'- Dithiobis (sulfosuccinimidylpropionate) (DTSSP), which cross-links proteins interacting at the cell surface. IP Ab = antibody used for immunoprecipitation; W Ab = antibody used on the western blot; ppt = immunoprecipitate; super = supernatant. Experiments were repeated two times. ::: ![](1471-213X-5-6-1) ::: We have previously shown that Sca and N form complexes \[[@B14]\]. To determine whether Sca forms complexes with Dl, we performed immuno-precipitation experiments with S2-Dl cells that were co-cultured with S2-Sca cells. Proteins interacting at the cell surfaces were either cross-linked or un-linked prior to cell lysis for protein extraction. Membrane insoluble cross-linkers improve recovery of cell surface complexes \[[@B18],[@B24]\]. Sca immuno-precipitation recovered Dl strongly in the presence of cross-linkers and relatively weakly in the absence of cross-linkers (Fig. [1D](#F1){ref-type="fig"}). No bands were observed when S2 cells were used instead of S2-Dl cells (data not shown). In the reverse experiments, Dl immuno-precipitations failed to recover Sca, possibly because there was too much unbound Dl in the extracts. Dl and Sca were not detected in the absence of immuno-precipitation antibodies (Fig. [1D](#F1){ref-type="fig"}, lanes 1 and 4) or in the absence of Scabrous (data not shown). We also recovered Sca in Dl immuno-precipitations and Dl in Sca immuno-precipitations from protein extracts of wildtype embryos (data not shown). These observations indicated that Sca associates with Dl. We explored the consequence of this association. Da expression in Dl cells is reduced in response to Sca ------------------------------------------------------- N promotes expression of E(spl)C m3gene in response to Dl \[[@B19],[@B20]\]. We examined whether Sca promoted expression of E(spl)C m3in S-N cells or S2-Dl cells and found that it was indeed the case with S2-N cells, but not with S2-Dl cells (Fig. [2A](#F2){ref-type="fig"}, lanes 1--6). S2-N cells showed a low level of E(spl)C m3expression when S2-Dl or S2-DlΔI cells were replaced with S2 cells, in the absence of Sca (Fig. [2A](#F2){ref-type="fig"}, lanes 1, 7--8); S2-Dl or S2-DlΔI cells mixed with S2 cells did not show any accumulation (Fig. [2A](#F2){ref-type="fig"}, lanes 13--16). The low level of E(spl)C m3RNA expression in S2-N cells in the absence of ligands is due to the low level of N^intra^produced upon induction of N expression in S2 cells \[[@B18]\]. This expression increases upon ligand treatment \[[@B18]\], resulting in increased expression of E(spl)C m3RNA expression (Fig. [2A](#F2){ref-type="fig"}, lanes 2, 10, 12). Numerous repetitions of the experiments indicated that Dl is a more potent ligand of N than Sca with respect to induction of E(spl)C m3expression (data not shown). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Dl down-regulates Daughterless protein expression, and N up-regulates E(spl)C m3gene expression, in response to Scabrous. A. Northern blots of total RNA from the indicated cell mixtures extracted at 0 or 45 minutes after treatment with medium containing Sca (+) or not (-). Gene probes used are shown on the right. m3 = E(spl)C m3and rp 49 = a ribosomal protein gene used to show the levels of total RNA in the lanes in all northern blots. Sca = conditioned medium prepared from the S2-Sca stable cell line in all experiments here onwards. The control medium used along side Sca medium (-) was prepared from heat shocked S2 cells. Experiments were repeated two times. For unknown reasons, the medium collected from heat shocked S2 cells (used in lanes 1, 3, and 5) produced higher background levels of E(spl)C m3RNA in S2-N cells (lane 1). B. Western blots showing the levels of Da and Dl in different Dl cell lines. S2-Dl, S2-Dl(1) and S2-Dl(2) are independently established hsDl cell lines. Ui = un-induced (i.e., not heat shocked). Hsp70 = heat shock 70 protein used to show the levels of proteins in the lanes of all western blots. Dl and DlΔI were detected with αDlEC. Da signals here (and the indicated signals elsewhere) were quantified relative to Hsp70 (western blots), rp49 (northern blots), or other indicated molecules, using the NIH Image 1.63 program. These experiments were repeated more than ten times. C. Western blots showing Da levels in the indicated cell mixtures, with (+) or without (-) Sca. These experiments were repeated five times. ::: ![](1471-213X-5-6-2) ::: E(spl)C m3expression appeared to be solely dependent on N activation and the Notch intracellular domain as it was promoted in S2-N cells treated with either S2-Dl cells or S2-DlΔI cells (Fig. [2A](#F2){ref-type="fig"}, lanes 7--12). As DlΔI lacks the intracellular domain, it is expected to behave only as a ligand of N and not generate any intracellular signal of its own in response to N binding. We observe comparable levels of SuH/N^intra^signaling with S2-Dl and S2-DlΔI cells (Fig. [2A](#F2){ref-type="fig"}, lanes 10, 12). This is not consistent with the in vivofindings that the Dl intracellular domain (lacking in DlΔI) is required for SuH/N^intra^signaling, possibly for promoting Dl internalization that results in exerting a \'pull\' on N and increased production of N^intra^\[[@B21],[@B25]-[@B29]\]. However, our results are consistent with other S2 cell studies showing that even fixed S2-Dl cells can promote production of SuH/N^intra^signaling in S2-N cells \[[@B19]\]. Thus, it is possible that that Dl internalization and pulling is not required for SuH/N^intra^signaling in S2 cells. In any case, in our S2 cell system, the S2-N and S2-Dl cells require shaking for formation of cell aggregates. As a consequence, we shake all cell mixtures, including those containing the secreted ligand Sca. This shaking might have simulated the pulling effect and overcome any deficiency DlΔI might have in this regard thereby resulting in a level of SuH/N^intra^signaling that is comparable to that produced by the full length Dl. We examined the expression of various proteins known to be involved in lateral inhibition to find out if Dl expression affected them. They were Numb, Dishevelled, Suppressor of Hairless, Wingless, Hairless, Hairy, Achaete, Da, and Armadillo. We found a relatively high level of Da protein in S2-Dl cells compared with the level in S2-DlΔI cells (Fig. [2B](#F2){ref-type="fig"}, lanes 1--2). Similar levels of Da were expressed in S2-DlΔI and S2 cells (data not shown). Two independently transfected S2-Dl cell lines also showed high levels of Da, and un-induced S2-Dl cells showed background levels of Da, indicating that Dl expression promotes Da expression (Fig. [2B](#F2){ref-type="fig"}, lanes 3--6). Increase in Da levels appeared to be specifically linked to Dl expression, as S2-N cells did not show an increase (Fig. [2C](#F2){ref-type="fig"}, compare lanes 1 & 3). Overall, Da expression in S2-Dl cells was 2.18X higher (+/- 0.37, p \< 0.05) than the level in S2 cells, sometimes more than 5X higher. Here and in all cases to follow, the blots shown in the figures are the most representative blots among replications. Graphs show quantification, relative to standards or other proteins (as indicated), of signals on the blots composing the figures as the response can be assessed only in comparison to the control lanes in the same experiment. Pooling data from all replications of an experiment obscured the response, or misrepresented the data, due to variation between different batches of cells. Therefore, we computed error variance for the degree of response over all replications of an experiment. These values for important responses are mentioned in the text. The number of repetitions of an experiment is indicated in the figure legends. We examined Da levels in S2-Dl and S2-N cells that were treated or not treated with Sca conditioned medium. We found that Sca treatment decreased the levels of Da in S2-Dl cells (Fig. [2C](#F2){ref-type="fig"}, lanes 3--4). The levels in S2-N cells were low and unaffected by Sca treatment (Fig. [2C](#F2){ref-type="fig"}, lanes 1--2). These experiments suggested that Sca blocks accumulation of Da in S2-Dl cells (2.81X, +/- 0.59, p \< 0.05). We also determined the levels of Da when S2-N and S2-Dl cells were together in the absence of Sca. The level of Da never increased (Fig. [2C](#F2){ref-type="fig"}, lanes 6--7). As N activation suppresses daughterlessRNA expression \[[@B18]\], it was possible that N activation suppressed Da expression in S2-N cells and masked an increase in S2-Dl cells. To determine if this was the case, we compared the level of Da in mixtures of S2-DlΔI cells and S2 cells with mixtures of S2-DlΔI cells and S2-N cells. As S2-Dl and S2-DlΔI cells activate N equally well (see Fig. [2A](#F2){ref-type="fig"}, lanes 9--12), any change in Da level would be due to N activation. We found comparable levels of Da in the two samples (Fig [2C](#F2){ref-type="fig"}, lanes 8--9). Thus, S2-Dl cells do not appear to increase Da expression in response to S2-N cells. When S2-N and S2-Dl cells were together in the presence of Sca, the levels of Da protein and E(spl)C m3RNA were very variable (data not shown). This was possibly due to the varying combinations of Sca effect on S2-Dl cells, Sca effect on S2-N cells, Dl effect on S2-N cells, and N effect on S2-Dl cells. Dl is processed to produce Dl intracellular domain (DlIC), constitutively, and the levels of DlIC increase upon N treatment \[[@B30]-[@B33]\]. Therefore, we examined the levels of DlIC following treatment of S2-Dl cells with S2-N cells or Sca medium. We found that the DlIC levels increased by 25 to 50% (relative to Dl levels) with both treatments (Fig. [3A](#F3){ref-type="fig"} and [3B](#F3){ref-type="fig"}). We examined the levels of Da in S2 cells expressing DlIC and DlTMIC (lacking the extracellular domain only and including the transmembrane domain). The levels of Da in S2-DlIC and S2-DlTMIC were always comparable to or lower than the level in S2 or S2-DlΔI cells (Fig. [3C](#F3){ref-type="fig"}). Also, we found Da levels to be negatively correlated with the accumulation of DlIC in time-course experiments (Fig. [3D](#F3){ref-type="fig"}). This negative correlation could be a direct consequence of the accumulation of DlIC or due to autoregulation of the dagene \[[@B34]\]. We examined the levels of Da in flies expressing heat shock induced Dl, N, or Sca, in flies heterozygous for the null alleles of N or Dl, and in flies homozygous for a null allele of Sca. We found that Da expression was strongly associated with Dl expression rather than with N expression, and inconsistently associated with Sca expression. These data are consistent with our findings in S2 cells but are not shown, as we cannot clearly separate the effects of N, Dl, and Sca, the way we can do in S2 cells. The experiments described in this section indicate that Da accumulation is promoted by the full-length Dl, not by the Dl intracellular domains (DlIC or DlTMIC), and Sca suppresses the activity of the full length Dl. The experiments also indicate that Sca promotes E(spl)C m3RNA expression in S2-N cells even in the absence of Dl. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### The levels of cleaved Dl intracellular domain is not associated with high levels of Da. A. Western blots (from a 8% SDS-PAGE) showing the level of Dl and DlIC in the indicated cell mixtures, with (+) or without (-) Sca. B. Western blots (from a 12% gel) showing the levels of Dl and DlIC in S2-Dl cells treated medium containing different levels of Sca. C. Western blots showing the levels of Da in the indicated cell mixtures. D. Western blots showing the levels of Da, Dl, and DlIC at different times following heat shock induction of Dl in S2-Dl cells. All experiments were repeated at least three times. ::: ![](1471-213X-5-6-3) ::: Dl regulates expression of fringeand pangolin ------------------------------------------------- To gather additional evidence for Dl activity independent of its activity as a ligand of Notch, we performed microarray experiments using the Affymetrix Drosophila GeneChip Arrays to compare gene expression in S2 cells and S2-Dl cells. Many genes relevant to known Dl functions responded in S2-Dl cells (at p \< 0.05, n = 3 × 2 pooled samples): axonal path finding genes (e.g., Gef64C, 39.38X,Up; Tenascin major, 6.77XUp), actin-based cell motility and kinases (Rho-Kinase, 15.08XUp; Rhophilin 3.4XUp; nemo 1.72XUp, basket 1.69XUp; pointed 2.2XUp), N signaling pathway genes (e.g., reaper, 2.26XUp; sanpodo, 1.91XUp), and oogenesis genes (e.g., swallow, 8.12XUp; sprouty, 3.58Xup). Expression of transformer, was also up (1.76X) and it is significant in the light of our observation that Dl promotes expression of Da: both Da and transformerare involved in sex determination. Expression of daRNA was not significantly increased in S2-Dl cells, possibly due to the negative part of the dagene autoregulation system \[[@B34]\]. The detailed analyses with validations will be published elsewhere. The experiment also identified fringe(fng) and pangolin(pan) as responding to Dl expression. fngis a glycosyl transferase that regulates the affinity of N for Dl \[[@B35]-[@B37]\], and possibly also the affinity of Dl for N \[[@B38]\]. panis a transcription factor functioning in the Wingless (Wg) pathway \[[@B39],[@B40]\]. Notch and Wg pathways interact closely at many differentiation events during development \[[@B24],[@B41]-[@B44]\]. Therefore, we chose fngand panfor further investigation. Northern blot analyses showed that the expression of fngand panwas higher in S2-Dl cells compared with S2-N or S2-DlΔI cells (Fig. [4A](#F4){ref-type="fig"}). DlIC and DlTMIC promoted expression of panand fngweakly, if at all (Fig. [4B](#F4){ref-type="fig"}). Two independently established S2-Dl cell lines also showed higher levels of fngand panRNAs (Fig. [4C](#F4){ref-type="fig"}). Sca treatment S2-Dl cells reduced the levels of fngand panRNA (Fig. [4D](#F4){ref-type="fig"}). This reduction was expected as Sca reduces the levels of the full length Dl (see Fig. [3](#F3){ref-type="fig"}). Thus, just as it was the case with Da expression, the full length Dl, not any of its parts, strongly promoted panand fngexpression. We examined the levels of fngand panRNA in flies expressing heat shock induced Dl, N, or Sca, in flies heterozygous for the null alleles of N or Dl, and in flies homozygous for a null allele of Sca. We found that fngand panRNA expression was strongly associated with Dl expression rather than with N expression, and inconsistently associated with Sca expression. These data are consistent with our S2-Dl cells data but are not shown, as we cannot clearly separate the effects of N, Dl, and Sca, the way we can do in S2 cells. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Dl promotes expression of fngand pan. A. Northern blots showing fngand panexpression in the indicated cell mixtures at 0 and 45 minutes after cell mixing. B. Northern blots showing fngand panexpression in the indicated cell lines two hours after induction of expression. C. Northern blots showing fngand panexpression in two other independently established S2-Dl cell lines. Cells used for lanes 1--2 were uninduced (ui); cells used for lanes 3--4 were heat shock induced. D. Northern blots showing fngand panexpression in S2-Dl cells that were either untreated or treated with Sca medium. All experiments were repeated at least three times. The fngband marked with an asterisk corresponds to the published mRNA \[35\]. Only this band was used for fngquantification. The panband shown is consistent with the information described in van de Wetering et al. \[40\] and Brunner et al. \[39\]. ::: ![](1471-213X-5-6-4) ::: Discussion ========== Our experiment addressed four questions. Does Sca bind Dl? If yes, does it affect any Dl activity? Are there Dl activities independent of its activity as a ligand of N? Is Sca capable of activating N in the absence of Dl? Results described in Figure [1](#F1){ref-type="fig"} show that Sca binds Dl. This binding is not dependent on N as S2-Dl cells do not express N. We have previously shown that Sca binds N \[[@B14]\]. It is possible that Sca binds N or Dl stronger when they are present together on the same cell or on neighboring cells. It would be possible to test this in the future using Atomic Force Microscopy that is best suited for determining binding strengths of cell surface proteins like N or Dl \[[@B21]\]. Results in Figure [2A](#F2){ref-type="fig"} show that Sca can promote SuH/N^intra^signaling through N in the absence of Dl, as S2-N cells do not express Dl. However, numerous repetitions of the experiment indicate that Sca is not as potent as Dl in this regard. This is consistent with the fact that lateral inhibition is blocked in the absence of zygotic Dl, which does not affect proneural cluster formation and thereby Sca expression. It would have been relatively easy to determine if over-expression of Sca in the absence of Dl rescues SuH/N^intra^signaling phenotypes, and the extent of this rescue, if Dl did not have any activity independent of N. Hopefully, it would be possible in the future, when we better understand this Dl activity and are able to circumvent it. Results in Figure [2A](#F2){ref-type="fig"} also show that the expression of E(spl)C m3 gene, a target of SuH/N^intra^signaling pathway, is responsive only to N indicating that this pathway is unlikely to be involved in mediating Dl activities. Results described in Figures [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"} and our microarray analysis show that Dl has activity independent of its activity as a ligand of N and Dl could be a receptor of Sca. This is clearly shown in experiments with S2-Dl cells that do not express N and we do not provide either N or Sca (Fig. [2B, C](#F2){ref-type="fig"}; [4A](#F4){ref-type="fig"}). The Dl activities we have described- promotion of expression of Da protein, fngRNA, and panRNA- can be detected in vivoas well although the interpretation here is not simple due to the many possible interactions among N, Dl, and Sca that cannot be easily sorted out. However, these data (which we do not show) strongly suggest that the Dl activities we have described in S2 cells represent the in vivoDl activities during development. The N independent Dl activity we have described is dependent on the full length Dl, not just on its intracellular domain or the extracellular domain (Figs. [2C](#F2){ref-type="fig"}; [3](#F3){ref-type="fig"}; 5A-B). This is different from the situation with N whose activity is based on the activity of its intracellular domain \[[@B45]-[@B47]\]. Accordingly, treatment with Sca, which promotes production of the Dl intracellular domain, suppresses Dl activity related to Da rather than promote it (Figs. [2C](#F2){ref-type="fig"}; [3](#F3){ref-type="fig"}). This observation also indicates that Sca is able to affect Dl activities. A clean dissection of Sca effects through N from its effects through Dl would require identification of Sca binding sites on Dl, and N and Dl binding sites on Sca. We know that Dl and Sca bind different regions of N \[[@B14],[@B48],[@B49]\]. It would not be too surprising if Dl bound N and Sca in different regions, and if Sca bound N and D in different regions. With that knowledge and suitable mutants, we might be able to determine whether N, Dl, and Sca activities function in a mutually exclusive or combinatorial manner in vivo. Dl activity that is independent of its N ligand activity has been speculated for some time. Efforts to identify it have intensified since the discovery that Dl gets proteolytically processed in the same manner as N \[[@B30]-[@B33]\]. However, it is extremely difficult to separate these two activities of Dl. The proof that the Dl activity we have identified actually functions during development in the expected manner, the details of the mechanisms underlying this function, and a better integration of the known functions of N, Dl, and Sca, will have to await more work which is neither quick nor simple. We hope that this work stimulates more efforts towards this task and makes this task a bit easier by identifying the potential of Sca as a regulator of Dl activity and the possibility that the full length Dl might be important for Dl activity independent of N, or Sca. Sca could also serve as a great tool for in vivodissection of Dl response to N, as Sca and N appear to have a similar effect on Dl. It is quite likely that our experiments did not pick up Dl receptor activity in response to N or Sca. In any case, the potential developmental significance of our findings is briefly discussed below. Da is a widely expressed protein and cells requiring its function show only a modest increase in its levels \[[@B5],[@B6],[@B8]\] indicating that, just like N^intra^/SuH signaling, small changes in Da levels might be sufficient for initiating or augmenting NPC specification and promoting neuronal differentiation. Small changes in Da levels might also be imposed by the built-in autoregulation of the dagene \[[@B34]\]. According to the well-accepted lateral inhibition model in the field, Dl activity as a ligand of N is postulated to increase in the NPCs and N receptor activity in response to Dl is postulated to increase in the EPCs \[[@B11]\]. Accordingly, Dl expression has been observed to increase in the NPCs or their equivalent cell types in certain instances involving N and Dl functions \[[@B50],[@B51]\]. Our data suggest that an increase in Da levels in these instances could be due to the accumulation of the full length Dl, not any its parts such as DlIC, DlΔI, or DlTMIC. The requirement for the intracellular and the extracellular domains to be linked might mean that we have detected Dl activity requiring Dl\'s presence at the membrane or in the cytoplasm. This is consistent with the report that the cellular transformation ability of Jagged 1, a mammalian Dl homolog, requires an intact protein containing both the extracellular and the intracellular domains \[[@B52]\]. It is possible that DlIC, in the nucleus \[[@B31]\], promotes other activity that is different from the one described here. It is also possible that Da, fng, or panmight not be the direct target of the full length Dl activity. Our microarray data indicate that many other genes (including some in the RAS or EGFR signaling pathways) are strongly up regulated in Dl expressing cells. It is possible that one of these genes is the primary target. It is also possible that Da, fng, or panaccumulation is significant only in the context of these other genes. We will have to await validation of other putative targets of Dl activity, and evaluation of their role in lateral inhibition or other activities involving N and/or Dl, to determine if Da, fng, or panare typical or atypical targets of Dl activity. N/Dl binding and SuH/N^intra^signaling are strongly affected by the functions of glycosyl transferases such as fng. The possibility that Dl, and not N, regulates fngRNA expression might explain some of the very complex functions of these glycosyl transferases and the complex interactions between N and Dl during lateral inhibition. As N and Dl activities are known to strongly interact with the Wg signaling pathway, it is interesting that Dl promotes panexpression. It is possible that Dl activity independent of N accounts for some of the interactions between the N and the Wg pathways. So far, these interactions have been considered only from the perspective of N receptor activity. Lastly, our data suggest interesting interactions among Dl, N, and Sca in instances of lateral inhibition and tissue differentiation when their functions overlap. The full length Dl promotes Da accumulation, not any of its parts that might result from processing in response to N or Sca binding. Thus, both the processed N and Dl might promote EPC specification- processed N through E(spl)CRNA and processed Dl through suppression of Da expression. Consequently, lateral inhibition might initiate with symmetrical actions of N and Dl promoting EPC specification in all proneural cells. Sca might boost N and Dl processing in the incipient EPCs while suppressing them or not affecting them in the incipient NPCs. Thus, it is possible that Sca or Sca-like molecule have a role in breaking the symmetrical actions of N and Dl during certain lateral inhibition instances. It is also possible that Sca mediates long range N signaling during differentiation of some other tissues, either alone or in association with Dl, as proposed by Renaud and Simpson \[[@B13]\]. By extending our results, it might be possible to develop strong hypotheses for testing in vivo, cleanly sort the different activities of N, Dl, and Sca, and understand the fascinating in vivodevelopmental mechanisms involving N, Dl, and Sca. Conclusion ========== Sca binds Dl and suppresses a Dl activity that is independent of Dl\'s activity as a ligand of N. This Dl activity requires the full length Dl and is not enhanced by expression of just the Dl intracellular domain, which is different from the mechanism underlying Notch activity. Da protein, fngRNA, and panRNA responds positively to the N independent Dl activity we have discovered. These could be direct or indirect targets. Our microarray analysis has identified many more putative targets of N independent Dl activity that can be explored for a better understanding of the complex interactions among Dl, Sca, and N during Drosophila development. Methods ======= DNA constructs -------------- Sca-Gfp: The stop codon of scawas replaced with a glycine codon and fused in-frame with GFP to obtain Sca-GFP. A Bam HI-KpnI fragment containing this scasequence was cloned into pEGFP vector (Clontech). The XbaI fragment containing Sca-GFP coding fragment was cloned into the pCaSpeR-hs vector. DlΔI: A stop codon and a XbaI restriction site was introduced after the transmembrane domain using PCR. The PCR product was checked for mutations and used to replace the BstEII-BcgI fragment in the Dl cDNA. An Eco RI-XbaI fragment from this construct (Dl amino acid 1 to 620) was cloned into the pCaSpeR-hs vector. DlIC: The Dl intracellular region (codon 619 to the stop codon 881) was PCR amplified, checked for errors, and cloned into the BglII-XbaI sites in the pCaSpeR-hs vector. Cell lines and conditioned medium --------------------------------- S2-N, S2-Dl, and S2-Sca cells have been previously described \[[@B14],[@B20],[@B49]\]. Other cell lines were established using the standard calcium phosphate transfection procedure and hygromycin selection. Conditioned medium was produced as described in Powell et al. \[[@B14]\], using serum-free or serum-containing Shields and Sang\'s M3 medium. For experiments, cells were heat shocked at 37°C for 30 minutes in a water bath, allowed to synthesize proteins for 2 hours, washed in culture medium without serum, mixed with the appropriate cell lines, and shaken gently in 14 ml falcon tubes for two hours or the indicated time. See Wesley and Mok \[[@B20]\] for more details. Immunoprecipitations, western blotting, northern blotting, RNA in situ, and protein staining -------------------------------------------------------------------------------------------- Procedures described in Lieber et al. \[[@B43]\], Wesley \[[@B24]\], Wesley and Saez \[[@B18]\], and Wesley and Mok \[[@B20]\] were followed. Eight per cent SDS-PAGE systems were used for western blotting, unless otherwise indicated; 1% formaldehyde-MOPS agarose system for northern blottings. fringecDNA (from Dr. Ken Irvine), rp49 cDNA, and rt-PCR amplified pangolincDNA were used to prepare probes for northern blots. Incubation times with ligands were two hours for western blots and 45 minutes for all northern blots; it was three hours for fngand pannorthern blot showing the effect of Sca (Fig. [4D](#F4){ref-type="fig"}). Antibodies: αSca (mAb sca1) and αDlEC (C594.9B) were obtained from the Developmental Studies Hybridoma Bank; αGFP (G-6539) and αHsp70 (H-5147) from Sigma; αDlIC (GPC2) from Dr. Marc Muskavitch, αDlIC (dC-19) from Santa Cruz Biotechnology, αDa (DAM 109-10) from Dr. Claire Cronmiller; and αNI from Dr. Toby Lieber. Microarray analysis ------------------- Heat shocked S2 and S2-Dl cells were treated with Sca or non-Sca medium for 45 minutes before extracting RNA. GeneChip Drosophila Genome Arrays from Affymetrix were used. RNAs were extracted, checked, and processed for hybridization according to procedures suggested by Affymetrix. We pooled RNA from two independent experiments and used three such pooled samples as replicates for each treatment. The MicroArray Core Facility at the University of Vermont prepared the probes, hybridized the chips, and statistically analyzed the data (using the GeneSifter program). We used the Microarray Suite program to examine the data. Authors\' contributions ======================= LPM and TQ designed and carried out many of the experiments in cultured cells and flies; BB carried out the immuno-precipitation experiments with cultured cells and helped in interpretation of data; MLC made the Sca GFP construct and assisted in many experiments; AH performed some experiments in cultured cells and prepared and maintained cell lines; FA assisted in statistical analyses and interpretation of data; and CSW conceived the study, designed experiments, and performed or participated in many of experiments in cultured cells and flies. LPM, BB, and MLC helped CSW in drafting the manuscript. All authors read and approved the final manuscript. Acknowledgements ================ We thank Drs. Claire Cronmiller, Nick Baker, Toby Lieber, and Matt Rand for materials; Dr. Matt Rand and Uma Wesley for critical comments on the manuscript. We also thank the two reviewers and a member of the Editorial Board for helpful comments and advice. The work was supported by the grant R 01 NS43122-03 to C. S. Wesley.	PubMed Central	2024-06-05T03:55:54.086404	2005-3-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555533/", "journal": "BMC Dev Biol. 2005 Mar 10; 5:6", "authors": [ { "first": "Lee-Peng", "last": "Mok" }, { "first": "Tielin", "last": "Qin" }, { "first": "Boris", "last": "Bardot" }, { "first": "Matthew", "last": "LeComte" }, { "first": "Asal", "last": "Homayouni" }, { "first": "Francois", "last": "Ahimou" }, { "first": "Cedric", "last": "Wesley" } ] }
PMC555534	Background ========== Upper urothelial carcinomas (UUC) represent about 5% of the urinary tract tumors, with transitional cell carcinomas of the renal pelvis and the ureter being the most common \[[@B1]\]. Exogenous agents such as smoking and occupational exposures to e.g. acrylamines constitute risk factors that are estimated to cause up to half of the tumors \[[@B2]\]. Hereditary factors also contribute to the development of UUC with a 2-fold increased risk among first-degree relatives \[[@B3]\]. The familial cases develop due to site-specific inheritance as well as within the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome \[[@B1],[@B3]-[@B5]\]. Individuals with HNPCC are at increased risk for several types of cancer, with the highest life-time risks for colorectal cancer (80%), endometrial cancer (40--60%), ovarian cancer (10--15%), cancer of the small intestine and upper urothelial cancer \[[@B4]\], and the revised Amsterdam criteria for the diagnosis of HNPCC consider these tumor types to be associated with the syndrome \[[@B6]\]. Although HNPCC-patients have a 14 to 75-fold increased risk of UUC, with the highest risks reported for carriers of mutations in MSH2, the absolute lifetime risk for mutation carriers to develop UUC is \<10% \[[@B7]-[@B9]\]. HNPCC is caused by a germline mutation in a DNA mismatch-repair (MMR) gene, most commonly affecting either of the genes MLH1(40%), MSH2(50%) or MSH6(10%) \[[@B10],[@B11]\]. Over 95% of the HNPCC-tumors are characterized by wide-spread microsatellite instability (MSI) and 90% by loss of immunohistochemical expression of the MMR protein affected \[[@B12]\]. Hence, these analyses are used in the clinical diagnosis of suspected HNPCC cases. However, somatic MMR defects occur in a subset of certain sporadic tumor types, e.g. in 15--20% of gastrointestinal and endometrial cancer, and are in most of these tumors caused by somatic hypermethylation of the MLH1promoter \[[@B13],[@B14]\]. Studies of the contribution of defective MMR to the development of urothelial carcinomas, assessed using MSI analysis, loss of MMR protein expression, and MMR gene mutations, have found a low frequency (\<10%) of MMR defects in urothelial carcinomas of the urinary bladder \[[@B15]\], but have indicated a high frequency (15--45%) of MMR defects in UUC \[[@B16]-[@B19]\]. Since data on the frequency of MMR defects in UUC are scarce and in order to characterize the contribution of the different MMR proteins to development of UUC, we assessed MSI and immunohistochemical expression of MLH1, MSH2, and MSH6 in a population-based series of UUC. Methods ======= Patient Material ---------------- In Sweden a population-based national Cancer Registry was started in 1958 and applies mandatory registration by both clinicians and pathologists in order to achieve maximal coverage (estimated to be 98%). We applied the southern Swedish part of the registry, which currently contains about 300.000 entries, to identify all carcinomas of the upper urothelial tract that had developed between 1992 and 1999. We identified 262 patients with a median age of 70 (range 34--90) years and a male:female ratio of 1.8:1. For further analyses, 27 patients were excluded because of lack of tumor blocks, and 19 because of autopsy-based diagnosis with autolysis that prevented good quality immunostaining. Hence, 216 patients with a median age of 69 (range 34--89) years were analyzed. Tumor location was as follows for the whole material (cases analyzed within parenthesis): renal pelvis 173 (154), ureter 75 (60) and an unspecified tumor location in 14 (2) patients. Data on family history of cancer or blood samples for mutation analysis were not available since the study was retrospective and register-based. Ethical approval for the study was obtained from the ethics committee at Lund University. Analysis of microsatellite instability (MSI) -------------------------------------------- Representative tumor blocks containing at least 20% tumor tissue were selected and DNA was extracted from 3 × 10-μm sections of formalin-fixed, paraffin-embedded tissue through incubation of the samples in EDTA-Tris-buffer with Proteinase K at 65°C for at least 2 hours, followed by boiling, centrifugation, and removal of the aqueous phase, which was stored at 4°C. MSI was assessed with the National Cancer Institute (NCI) panel; BAT25, BAT26, BAT34, BAT40, D2S123 and D5S346 \[[@B20]\]. These markers identify MSI with high accuracy in colorectal cancer, but the sensitivity of individual markers may vary between different tumor types \[[@B21]\]. The primer sequences used have been reported previously \[[@B22]\]. The markers were fluorescencely-labeled as follows: NED™ (yellow) for BAT25, 6-FAM™ (blue) for BAT26, BAT34C4 and D2S123, and HEX™ (green) for BAT40 and D5S346. The DNA microsatellite sequences were amplified by PCR according to the following programme; 94°C for 7 minutes, 10 × (94°C for 15 seconds, 45°C (BAT 25) / 50°C (other markers) for 15 seconds, and 72°C for 15 seconds), 23 × (89°C for 15 seconds, 45°C / 50°C for 15 seconds, and 72°C for 15 seconds), 72°C for 7 minutes, followed by a final cooling step at 4°C. 0.5--2 μl PCR product was mixed with 12 μl deionized formamide (Hi-Di Formamide, Applied Biosystems) and 0.5 μl ROX™ 500 Size Standard (Applied Biosystems), denatured at 95°C for 2 minutes, chilled on ice, and separated in Performance Optimized Polymer-4 (POP-4™) on the ABI PRISM™ 3100 Genetic Analyzer (Applied Biosystems) for fragment analysis. MSI was defined by the presence of extra peaks demonstrating altered length of the repetitive sequence. Data from at least three markers were required for the classification of tumors as microsatellite stable (MSS). The tumors were regarded as MSI-high if at least two microsatellites showed instability and as MSI-low if only one marker showed instability. All cases with suspected MSI were verified through repeated analysis. Immunohistochemistry -------------------- Immunohistochemical staining was performed using 4-μm sections of formaline fixed, paraffin-embedded tissue, which were mounted on DAKO ChemMate Capillary Gap Microscope Slides (DAKO A/S BioTek Solutions, USA) and dried at room temperature overnight followed by incubation at 60°C for 1--2 hours. The tissue sections were deparaffinized in xylol and rehydrated through descending concentrations of alcohol. Antigen retrieval was achieved by microwave-treatment in 1 mM EDTA, pH 9.0, at 900 W for 8 minutes followed by 15 minutes at 350 W. The slides were then allowed to cool for at least 20 minutes in the EDTA-solution. Immunohistochemical staining was performed in an automated immunostainer (TechMate 500 Plus, DAKO), according to the manufacturers instructions. The main steps were as follows: Mouse monoclonal IgG antibodies to MLH1 (clone G168-15, dilution 1:100, PharMingen, San Diego, CA, USA) MSH2 (clone FE-11, dilution 1:100, Oncogene research products, Boston, MA, USA), MSH6 (clone 44, dilution 1:1000, BD Transduction Laboratories) and PMS2 (clone:A16-4, dilution 1:500, BD Pharmingen) were applied and the sections were incubated at room temperature for 25 minutes. Thereafter, the slides were incubated with biotinylated anti-mouse antibody (DAKO) for 25 minutes (for MLH1 and MSH2) or with rabbit anti-mouse immunoglobulins (DAKO, dilution 1:400) for 20 min (for MSH6 and PMS2). Endogenous peroxidase activity was blocked in Peroxidase-blocking solution (DAKO) for 3 × 2,5 minutes. This was followed by incubation with streptavidin-horseradish peroxidase for 25 minutes for MLH1 and MSH2, whereas EnVision™/HRP rabbit/mouse (DAKO) incubation for 25 min was used for MSH6 and PMS2. Finally, the tissue sections were treated with diaminobenzidine (DAB) for 3 times 5 min, counterstained with hematoxylin for 1 min, rinsed in running tap water for 10 min, dehydrated in ascending concentrations of alcohol and mounted. After each step, the sections were rinsed in Tris buffered saline, pH 7.4, and Tween-20. In order to block nonspecific protein binding, bovine serum albumin was added to the buffer before the antibody incubation steps in the MLH1 and MSH2 stainings. A detailed protocol is available from the authors upon request. Two of the authors (K.E. and M.N.), who were blinded regarding the MSI status, independently evaluated all stained sections. Sections without nuclear staining in the tumor cells, in the presence of normal nuclear staining in lymphocytes and normal epithelial or stromal cells in the same section, were considered to have a lost expression (Fig. [1](#F1){ref-type="fig"}). The expression was classified as present, absent or non-evaluable without grading of the staining intensity. Results ======= Microsatellite analysis ----------------------- For the MSI analysis of the 216 cases, 16 tumors were excluded because of small tumor size or less than 20% tumor tissue in the samples, and 6 tumors were excluded because of lack of information from at least 3 MSI markers, which left 194 tumors successfully analyzed. A MSS phenotype was identified in 180 tumors, MSI-low in 5, and MSI-high in 9 tumors (table [1](#T1){ref-type="table"}, figure [1](#F1){ref-type="fig"}). Immunohistochemistry -------------------- Immunohistochemical staining for the MMR proteins gave evaluable results for MLH1 in 211 tumors, MSH2 in 216, MSH6 in 200 tumors and PMS2 in 215 tumors. Of the 180 MSS tumors, 177 showed retained expression for all evaluable proteins, as did also the 5 MSI-low tumors. One MSS tumor that was not assessed for MSI due to a small amount of tumor material, showed loss of MSH2 and MSH6 expression, one MSS tumor showed loss of MLH1 and PMS2, one MSS tumor showed loss of MSH6 expression, and one MSS tumor showed loss of MSH2 expression. Among the 9 MSI-high tumors, 5 showed a concomitant loss of expression of MSH2 and MSH6, 1 tumor showed loss of expression of MSH6 and 1 tumor showed loss of expression of MLH1 and PMS2. Retained expression of all four proteins was found in 2 MSI-high tumors of the renal pelvis (table [1](#T1){ref-type="table"}, figure [2](#F2){ref-type="fig"}). Synchronous and metachronous tumors ----------------------------------- Eleven patients had developed synchronous tumors of the upper urinary tract, and these cases were all analyzed. In one patient with synchronous urethral tumors the tumor tissue showed MSI and loss of expression of MLH1 in both tumors (U2-229), and the other patients with synchronous tumors had MSS tumors all of which showed retained expression of all three MMR proteins (table [1](#T1){ref-type="table"}). Metachronous UUC occurred in 3/262 patients, 2 of whom were included in the series analyzed, and these tumors were MSS and MSI-low, respectively, but both tumors showed retained MMR protein expression. In the whole series, 122 (97 among the analyzed cases) patients had been diagnosed with another malignancy, which was bladder cancer in 67 cases (54 among the cases analyzed). Among the cases with MSI and/or immunohistochemical MMR protein loss, 8 metachronous tumors developed and 5/5 analyzed (a leiomyosarcoma, a colon tumor, an endometrial cancer and 2 bladder tumors) displayed MSI and immunohistochemical loss of the concordant MMR protein (table [1](#T1){ref-type="table"}). Discussion ========== Urothelial carcinomas of the upper and the lower urothelial tract share many clinical and epidemiological traits. However, the UUC have specifically been associated with HNPCC, and in line with this observation the contribution of defective MMR has been reported to differ between these tumor types. In bladder cancer, a MSI-high phenotype has been found in 3--10% of the tumors \[[@B15],[@B23]\], whereas elevated microsatellite alterations at selected tetranucleotides (EMAST) has been described at a higher rate in bladder cancer and the latter phenomenon is being perused as a tumor marker in urine \[[@B24]\]. Higher MSI rates have been reported in UUC with 13--31% of the tumors showing MSI \[[@B16]-[@B19]\]. A similar anatomical specificity has been described in the ventricle with a higher number of MSI tumors in the antrum, and in the colorectum with 20% MSI tumors in the cecum and \<5% in the rectum \[[@B25]-[@B27]\]. We applied the population-based southern Swedish Cancer Registry to assess the contribution of defective MMR to the development of UUC. The results are based on 216/262 (82%) of the tumors that occurred in the southern Sweden health care region between 1992 and 1999. A MSI-high phenotype was found in 9/216 (4%) patients and a MSI-low phenotype in 5/216 (2%). In 11/216 cases synchronous tumors occurred within the urothelial tract and 1 patient (U2-229) had synchronous MSI-high tumors, all of which displayed a concordant immunohistochemical loss of MLH1. Thus, the vast majority of synchronous UUC does not display MMR defects and does not develop within the HNPCC syndrome. The overall frequency of MSI tumors detected in our study, 4%, is lower than the 13--31% previously reported (table [2](#T2){ref-type="table"}) \[[@B16]-[@B19]\]. Possible reasons for the discrepancy include that our study was unselected and population-based. Furthermore, Müller et al. \[[@B28]\] have suggested that microsatellite instability analysis should optimally be performed by using microdissection, where analysis is made on DNA extracted from tumor cells without dilution of DNA from normal cells. However, this was not available at our institution at the time the study. The marker selection is probably not the cause of discrepancy since the NCI marker panel for MSI analysis has proven effective in several extracolonic tumor types such as endometrial, ovarian and gastric cancer \[[@B20],[@B29]\]. Hartmann et al. \[[@B18]\] identified BAT40 (93% detection rate) and BAT25 (53%) as the best markers for the detection of MSI also in UUC and indeed reported that using a combination of the markers BAT40, BAT25, and BAT26 allowed identification of all MSI tumors. Whereas our finding of 5% MMR defects in renal pelvis tumors is in accordance with the 5--8% previously reported, we identified MMR defects in a lower (4%) fraction of the urethral tumors than the 25--41% previously reported \[[@B16]-[@B19],[@B23]\]. Loss of immunostaining was in our series detected in 7/9 MSI-high tumors, in one tumor biopsy that was to small to allow MSI analysis, and in 2 MSS tumors (figure [2](#F2){ref-type="fig"}). The immunohistochemical expression loss affected MSH2/MSH6 in 6 cases, MSH2 in one, MSH6 in two, and MLH1/PMS2 in two cases. Concordant loss of the same MMR protein was observed in the patient who had developed multiple synchronous tumors (table [1](#T1){ref-type="table"}). This frequency of immunohistochemical loss of expression in MSI-high tumors is similar to that previously reported (table [2](#T2){ref-type="table"}) and thus demonstrates that loss of immunostaining for at least one of the MMR proteins investigated is found in about 85% of MSI-high UUC \[[@B18],[@B23]\]. Regarding histological grade and stage among the tumors with MSI and/or MMR protein expression loss, the majority of the tumors were moderate differentiated (WHO-grade 2--3) and of early stages (table [1](#T1){ref-type="table"}). Synchronous/metachronous tumor development is common in urothelial cancer, mainly through intraepithelial migration or intraluminal dispersion of tumor cells \[[@B30]\]. In our series, 54/216 (25%) patients had developed metachronous bladder tumors. An increased incidence of metachronous tumors has been observed in patients with MMR defective UUC \[[@B19]\], and synchronous UUC is found in 1--2% of UUC patients \[[@B31]\]. Of the 8 patients with MMR defective tumors in our study, 5 had developed other malignant tumors, including two cancers of the urinary bladder, one colon cancer, one rectal cancer, one endometrial cancer, one cervical cancer, one soft tissue sarcoma, and one patient who had developed myelofibrosis (table [1](#T1){ref-type="table"}). Among these tumors, 5 could be retrieved and were immunohistochemically stained. The leiomyosarcoma, the colon tumor, the endometrial cancer and 2 bladder tumors showed loss of expression for MSH2/MSH6, which suggests an association with HNPCC. About 1/3 of HNPCC patients develop metachronous primary tumors, and the concordant MSI and loss of MMR protein expression in these cases strongly suggests HNPCC, although mutation analysis was not performed. The lifetime risk of developing UUC in HNPCC mutation carriers is estimated to be 4--10%, and UUC is in the revised Amsterdam criteria considered to be a HNPCC-associated tumor type, and screening for UUC is generally recommended in HNPCC-families (<http://www.insight-group.org>), with sonogrophy and urinary analysis. None of the patients in this series with MSI and/or IHC loss of MMR protein expression are previously known HNPCC patients in our health care region. Mutation analysis is not planned. A higher frequency of extraintestinal tumors has been reported in families with germline mutations in MSH2, and from the data available, MSH2seems to play a predominant role also in UUC; loss of MSH2 expression has been reported in 33--60% of MSI-high UUC tumors \[[@B18],[@B19],[@B32],[@B33]\]. Although our data suggest that MMR defects represent a minor tumorigenic pathway in the development of UUC, the high frequency of MSH2/MSH6 loss in MMR-defective tumors should caution clinicians to obtain an individual and a family history of cancer in patients with carcinomas of the renal pelvis and the ureter. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= KE conceived of the study, carried out the MSI analysis, performed immunohistochemical validation and drafted the manuscript. AI also carried out the MSI analysis. BI performed immunohistochemical validation. MN conceived of the study, and participated in its design and coordination and helped to draft the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/5/23/prepub> Acknowledgements ================ We would like to thank Eva Rambech and Kristina Lövgren for technical assistance and Britta Halvarsson for pathological review. The Swedish Cancer Society, the Nilsson Cancer Research Foundation, the Kamprad Research Foundation, the Gustaf V Foundation, and the Lund University Hospital Cancer Funds financially supported this study. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Microsatellite instability in case RP8-626 where the MSI-curves show instability with 1--2 additional peaks for the markers BAT25, BAT26 and BAT34 and larger size variation for the markers BAT40, D2S123 and D5S346. ::: ![](1471-2407-5-23-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Immunohistochemical staining for the MMR proteins shows loss of nuclear expression for MLH1 and PMS2 and retained expression of MSH2 and MSH6 in case U2-229, and loss of MSH2 and MSH6 with retained expression of MLH1 and PMS2 in case RP8-626. ::: ![](1471-2407-5-23-2) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Clinicopathological data of MMR-defective tumors ::: Case no. Sex / Age Tumor location (Synchron./Metachron.) WHO-Grade Stage Instable markers Immunohistochemical expression ---------- ----------- --------------------------------------- ----------- ------- ------------------ -------------------------------- ---- ---- ---- U1-113 F/54 ureter G2 pTX NE \+ \- \- \+ 53 endometrium (M) 4/6 \+ \- \- \+ U2-229 F/57 ureter G2 pT1 4--5/6 \- \+ \+ \- 57 ureter (S) 4--5/6 \- \+ \+ \- U3-821 M/61 ureter\* G2 pT1 2--3/6 \+ \- \- \+ RP1-101 M/60 renal pelvis G2 pT3 3/5 \+ \- \- \+ 43 sarcoma (M) 3/4 \+ \- \- \+ 53 colon (M) 1/6¤ \+ \- \- \+ RP2-131 M/75 renal pelvis G3 pT4 2/6 \+ \+ \+ \+ RP3-119 F/69 renal pelvis G3 pTa 0/6 \- \+ NE \- RP4-267 F/67 renal pelvis G2 pT1 5/5 \+ \- \- \+ 70 urinary bladder (M) 3/4 \+ \- \- \+ RP5-401 M/56 renal pelvis G2 pTa 0/6 \+ \+ \- \+ RP6-528 F/87 renal pelvis G2 pT1 2/5 \+ \+ \+ \+ 87 urinary bladder (S) 0/6 \+ \+ \+ \+ RP7-613 F/57 renal pelvis\^ G2 pT1 3/6 \+ \+ \- \+ 54 urinary bladder (M) 4/6 \+ \- \- \+ RP8-626 M/66 renal pelvis G3 pT2 6/6 \+ \- \- \+ RP9-701 F/49 renal pelvis\# G2 pTa 0/5 \+ \- NE \+ RP10-809 F/82 renal pelvis G3 pT4 4/4 \+ \- \- \+ ¤Positive for BAT26 \* Myelofibrosis at the age of 67 years. \^Cervical cancer at the age of 33. \# Rectal tumor at the age of 38. G2 moderately differentiated G3 poorly differentiated ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Summary of MMR studies in UUC ::: Reference Sex (male: female) Median age total/MSI-H Total no. of patients (renal pelvis/ureter/multifocal) Fraction MSI-high IHC loss ----------------- -------------------- ------------------------ -------------------------------------------------------- ------------------- ---------- Hartmann et al. 2.2:1 66 / 62 73 (40/22/11) 0.21 0.87 Catto et al. 2.0:1 68 / 66 71 (44/25/2) 0.13 0.78 Blaszyk et al. 2.8:1 70 / - 114 (61/53/-) 0.31 ND Roupert et al. 3.4:1\# \- / 70 164 (ND) 0.16 0.6\* Amira et al. 1.6:1 68 / 72 24 (19/4/1) 0.25 ND Ericson et al. 1.8:1 67 / 67 216 (154/60/2) 0.04 0.78 \* Only MSH2 assessed, ND: not determined \#Applies to subset with MSI :::	PubMed Central	2024-06-05T03:55:54.089933	2005-3-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555534/", "journal": "BMC Cancer. 2005 Mar 1; 5:23", "authors": [ { "first": "Kajsa M", "last": "Ericson" }, { "first": "Anna P", "last": "Isinger" }, { "first": "Björn L", "last": "Isfoss" }, { "first": "Mef C", "last": "Nilbert" } ] }
PMC555535	Background ========== Viruses survive in their hosts either by evading or countering host defenses. Viral evasion is a passive mechanism by which viruses overwhelm host defenses, or invade organs or cells where the host defenses cannot reach them. The ability of a virus to counter host defenses requires an active mechanism to either bypass or disarm the host machinery. Viruses invading vertebrate hosts produce virokines and viroceptors which interact with immune response molecules to inhibit or modulate their anti-viral activities \[[@B1],[@B2]\]. Recent studies have shown many viruses infecting a wide range of eukaryotic hosts encode proteins that suppress the RNA silencing, anti-viral defense response \[[@B3]-[@B6]\]. Silencing suppressors encoded by viruses limit degradation of viral RNAs by the RNA silencing machinery. Among plant viruses, some silencing suppressor proteins also affect symptom development and increase virus titer. The Cucumber mosaic virus(CMV) 2b, the Tobacco etch virus(TEV) HC-Pro, and the Tomato bushy stunt virus(TBSV) P19 \[[@B7]-[@B10]\] proteins are among the best studied silencing suppressors that are also pathogenicity determinants. The TBSV P19 protein was unique because it affects disease severity in a host specific manner \[[@B11],[@B12]\]. Little is known about the evolution and phylogenetic relationships of silencing suppressor proteins. In particular, viruses belonging to the genera Furo-, Hordei-, Tobra-, Peclu-, Beny-, Carla-, and Pomovirusencode small cysteine-rich proteins (CRPs) near the 3\' ends of their genomes, and some have been identified as both silencing suppressor proteins and pathogenicity factors. For example, the Barley stripe mosaic virus(BSMV; a hordeivirus) gamma b protein and the Peanut clump virus(PCV; a pecluvirus) 15K protein suppress RNA silencing, modulate symptom severity, and systemic virus accumulation \[[@B13]-[@B16]\]. The Tobacco rattle virus(TRV; a tobravirus) 16K CRP has been described as a pathogenicity factor and suppresses RNA silencing \[[@B17]\]. In complementation studies, the Soilborne wheat mosaic virus(SBWMV; a furovirus) 19K CRP, the BSMV gamma b protein, and the CMV 2b (which is not a CRP) protein functionally replaced the 16K CRP of TRV \[[@B15]\]. Since deletion of the TRV 16K CRP ORF reduced virus accumulation in plants, functional replacement by these heterologous viral ORFs indicates that these CRPs share some common function. Characterizing the functional similarities among these CRPs is crucial to understanding their evolutionary relationship. Until now the phylogenetic relationships among these CRPs are unclear \[[@B18]\]. This study was undertaken to characterize the SBWMV 19K CRP. SBWMV is a bipartite RNA virus and is the type member for the genus Furovirus\[[@B19]\]. RNA1 encodes the viral replicase and putative viral movement protein (MP). The viral replicase is encoded by a single large open reading frame (ORF) and is phylogenetically related to the Tobacco mosaic virus(TMV) replicase \[[@B20]\]. The 3\' proximal ORF of RNA1 encodes a 37K MP that shares sequence similarity with the dianthovirus MP \[[@B21],[@B22]\]. SBWMV RNA2 encodes four proteins. The 5\' proximal ORF of RNA2 encodes a 25K protein from a nonAUG start codon \[[@B23]\] and its role in virus infection is unknown. The coat protein (CP) ORF has an opal translational termination codon and readthrough of this codon produces a large 84K protein \[[@B23]\]. The CP readthrough domain (RT) is required for plasmodiophorid transmission of the virus \[[@B24]\]. The 3\' proximal ORF of RNA2 encodes a 19K CRP. To gain insight into the role of the SBWMV 19K CRP in virus infection, amino acid sequence comparisons were conducted to determine the relatedness of the SBWMV 19K CRP to other viral CRPs. The Potato virus X(PVX) infectious clone was used to express the SBWMV CRP and to study its role in virus pathogenicity and suppressing RNA silencing. Results ======= SBWMV 19K protein is a conserved CRP ------------------------------------ The Pfam Protein Families Database reports a family of CRPs with similar sequences which includes proteins from BSMV, PSLV, PCV and SBWMV (Pfam 04521.5). Since there are viruses not included in the Pfam report that encode CRPs, this study was undertaken to determine if there is a larger CRP family containing related viral proteins. Further examination in this study reveals that the CRPs encoded by all known hordei-, peclu- and furoviruses share significant sequence similarity (Fig. [1](#F1){ref-type="fig"}). Efforts to find similarity between these proteins and CRPs encoded by pomo-, beny- and potyviruses were not successful. Whether these other plant viral CRPs are also suppressors of silencing can not be concluded at this point for two reasons: insufficient study and only weak sequence similarity relationships. Sequences of CRPs that affect virus replication and are encoded by members of other virus genera were also determined to be unrelated \[[@B25]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Amino acid sequence alignment of the CRPs encoded by furo-, peclu-, tobra- and hordeiviruses. The positions of amino acids are numbered above the alignment. The secondary structure prediction is shown directly above the alignment. Cys and His residues are bold uppercase letters. The leucines of leucine zippers are in bold face. The placement of residues that differ from Pfam are underlined. Vertical bars at the bottom represent where the Pfam family starts and stops. The genus for each virus is indicated on the right of the sequence. Abbreviations and accession numbers for the 33 aligned viruses are used (those displayed are underlined): [LyRSV]{.underline}, Lychnis ringspot virusgi\_1107721; [CWMV-2]{.underline}, Chinese wheat mosaic virusgi\_14270345; CWMV, gi\_9635448; OGSV Oat golden stripe virus, gi\_9635452; SBWMV-NE88 gi\_9632360; SBWMV-NE gi\_1449160; [SBWMV OKl-1]{.underline}, gi\_1085914; SBWMV-NY, gi\_21630062; SBCMV-Ozz, Soilborne cereal mosaic virusgi\_12053756; SBCMV-Fra, gi\_9635249; [SBCMV-O]{.underline}, gi\_6580881; SBCMV-G, gi\_6580877; SBCMV-C, gi\_6580873; JSBWMV, Japanese soilborne wheat mosaic virusgi\_7634693; [SCSV]{.underline}, Sorghum chlorotic spot virusgi\_21427644; [PSLV]{.underline}, Poa semilatent virusgi\_321642; [BSMV-PV43]{.underline}, Barley stripe mosaic virusgi\_19744921; BSMV-RUS, gi\_94465; BSMV-JT, gi\_808712; BSMV-ND18, gi\_1589671; [PCV]{.underline}, Peanut clump virusgi\_20178597; [IPCV]{.underline}, Indian peanut clump virusgi\_30018260; TRV-PpK20, Tobacco rattle virus, gi\_20522121; TRV-ORY gi\_2852339; TRV-Pp085 gi\_42733086; TRV-PSG, gi\_112699; TRV-PLB, gi\_465018; TRV-CAN, gi\_1857116; TRV-FL, gi\_3033549; TRV-RSTK, gi\_6983830; [TRV-TCM]{.underline}, gi\_112701; [PepRSV]{.underline}, Pepper ringspot virus, gi\_20178602; [PEBV]{.underline}, Pea early browning virus, gi\_9632342. ::: ![](1743-422X-2-18-1) ::: The SBWMV 19K protein is a CRP because it contains nine Cys residues \[[@B20]\]. Seven of these Cys residues are conserved in all furovirus proteins and are located in the N-terminal half of the protein. Five of these residues are within the block of sequences designated as protein family Pfam04521.5 and three of the conserved Cys residues are also conserved in the hordeiviral and pecluviral proteins. A selection from this alignment was corrected for several misplacements of short peptide sequences and is shown in Figure [1](#F1){ref-type="fig"}. The alignment represents the entire length of these proteins, although the termini are aligned with less confidence than the core. Examination of the tobraviral CRP sequences revealed sufficient similarity to justify their alignment with the Pfam04521.5 sequences. The alignment resulted in a significance score between 6 and 7, suggesting that the tobraviral proteins belong to this family. The multiple sequence alignment of 33 CRPs from furo-, tobra-, peclu-, and hordeiviruses (Fig. [1](#F1){ref-type="fig"}) revealed three absolutely conserved residues: Cys70, Cys112, and His116 (numbering based on the aligned sequences). Gly113 was conserved in all viruses (except TRV-CAN) and is contained within a Cys-Gly-Xaa-Xaa-His motif in which one of the two Xaa residues is Lys or Arg. There is a Cys residue at position 7, 8 or 9 which is conserved in all except PCV and IPCV (pecluvirus) amino acid sequences. Alignment of the N-terminus is not exact since the PCV and IPCV proteins are N-terminally truncated. Within the N-terminal half, there are additional positions containing Cys residues that are conserved for some but not all viruses. For example, Cys9 is conserved among hordei-, tobra-, and some furoviruses; Cys at position 32 and 33 is conserved among all but pecluviruses; Cys36 is conserved among hordei- and furoviruses; Cys45 is conserved among furo and tobraviruses; Cys76 is conserved among furo and tobraviruses (except for SCSV; the pecluvirus PCV, but not IPCV, also has Cys76); Cys80 is conserved among all viruses except PeRSV and PEBV. Lys at position 52 and Arg at position 54 or 55 (Lys-Xaa-Arg or Lys-Xaa-Xaa-Arg) are conserved among all except PSLV. Gly at position 77 is conserved among all except tobraviruses. The secondary structure prediction derived from the multiple sequence alignment is a long helical region extending from or slightly beyond the Cys-Gly-Xaa-Xaa-His motif to within 20 residues of the C-terminus. The furoviral proteins have spacings of conserved Leu residues from positions 89 to 106 consistent with a leucine zipper structure (which was not apparent in the original Pfam 04521.5). The N-terminal halves of the aligned amino acid sequences, containing most of the Cys residues, have a mixture of extended, helical and loop predicted structures. The pecluviruses PCV and IPCV, and the hordeiviruses BSMV, LyRSV, and PSLV proteins contain a Ser-Lys-Leu sequence at the C-terminus. This tripeptide was shown for PCV to be a peroxisomal targeting signal \[[@B16]\]. This signal is not present in CRPs of furo- or tobraviruses. SBWMV 19K CRP aggravates PVX-induced symptoms --------------------------------------------- The tobravirus and hordeivirus CRPs have been described as pathogenicity determinants that regulate symptom severity in infected plants \[[@B15]\]. Since the SBWMV 19K protein is a similar CRP, experiments were conducted to determine if it also has an effect on symptom expression. The SBWMV 19K ORF was inserted into the PVX genome and PVX.19K infectious transcripts were used to inoculate N. benthamiana, N. clevelandii, C. quinoa, and C. amaranticolorleaves (Fig. [2](#F2){ref-type="fig"}). As a control, plants were also inoculated with PVX.GFP, which has the green fluorescent protein (GFP) gene inserted into the viral genome. The spread of PVX.GFP expression was monitored using a handheld UV lamp to monitor GFP expression and verify systemic virus accumulation (data not shown). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Plants infected with PVX.GFP or PVX.19K at 21 dpi. (A)N. benthamianaplants infected with PVX.GFP (left) and PVX.19K (right). (B, D)PVX.19K-infected N. benthamianaand N. clevelandiiplants, respectively, at 21 dpi show systemic necrosis. (C)PVX.GFP-infected N. clevelandiiplants. (E, F)C. quinoaand C. amaranticolorleaves infected with PVX.19K (left both panels) and PVX.GFP (right in both panels). ::: ![](1743-422X-2-18-2) ::: Symptoms were first observed in plants inoculated with PVX.GFP and PVX.19K between 10 and 14 dpi. By 21 dpi, systemic necrosis was evident in N. benthamianaand N. clevelandiiplants inoculated with PVX.19K (Fig. [2A, B](#F2){ref-type="fig"} and [2D](#F2){ref-type="fig"}) while PVX.GFP infected plants showed systemic mosaic symptoms (Fig. [2A](#F2){ref-type="fig"} and [2C](#F2){ref-type="fig"}). N. benthamianaplants infected with PVX.19K were clearly stunted in comparison to plants infected with PVX.GFP (Fig. [2A](#F2){ref-type="fig"}). The PVX.19K infected N. clevelandiileaves collapsed by 21 dpi (Fig. [2D](#F2){ref-type="fig"}). Immunoblot and northern analyses were conducted to verify PVX accumulation in the upper leaves of N. benthamianaplants. Immunoblot analysis was conducted using anti-PVX CP serum. High levels of PVX CP was detected in plants that were systemically infected with PVX.GFP (Fig. [3A](#F3){ref-type="fig"} lanes 1--4) and PVX.19K (Fig. [3A](#F3){ref-type="fig"} lanes 5--8). The SBWMV 19K CRP had no obvious effect on PVX accumulation in upper noninoculated leaves. Viral RNA accumulation was analyzed by northern blot and high levels of genomic RNA was detected in the upper leaves of PVX.GFP (Figure [3B](#F3){ref-type="fig"} lanes 2--4) and PVX.19K (Fig. [3B](#F3){ref-type="fig"} lanes 5--8) inoculated plants. Thus, the SBWMV 19K CRP did not seem to have a deleterious effect on PVX accumulation. RT-PCR was used to verify that the SBWMV 19K ORF was maintained in the PVX genome in systemically infected plants. RNA samples taken from the upper leaves of N. benthamianaplants which were used for northern analysis, were also used in RT-PCR reactions to verify the presence of the SBWMV 19K ORF in the PVX genome. In all samples it appeared that the SBWMV 19K CRP was stably maintained in the PVX genome (data not shown). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Immunoblot and northern analyses of the PVX infected N. benthamianaplants. (A)Immunoblot analysis conducted using PVX CP antiserum show similar levels of PVX.GFP virus (lanes 1--4) and PVX.19K virus (lanes 5--8). Lane 9 contains extract of non inoculated plants. (B)Northern analysis of RNA isolated from a healthy plant (lane 1), upper noninoculated leaves of PVX.GFP infected plants (lanes 2 -- 4) and upper noninoculated leaves of PVX.19K infected plants (lanes 5 -- 8). Blots were probed with a GFP sequence probe. The bottom image is the ethidium bromide stained gel showing ribosomal RNAs. Abbrev.: g, genomic RNA. ::: ![](1743-422X-2-18-3) ::: PVX.19K produced large necrotic lesions in the C. quinoaand C. amaranticolorleaves. Local lesions were detected in plants inoculated with PVX.GFP or PVX.19K between 5 and 7 dpi. PVX.19K-inoculated C. quinoaplants showed severe necrotic local lesions (Fig. [2E](#F2){ref-type="fig"}). The necrotic lesions gradually merged and the infected tissue eventually collapsed. PVX.19K-inoculated C. amaranticolorleaves showed enlarged chlorotic lesions advancing to necrotic lesions over time (Fig. [2F](#F2){ref-type="fig"}). PVX.GFP-inoculated C. quinoaleaves showed small chlorotic and necrotic local lesions while PVX.GFP-inoculated C. amaranticolorleaves showed mild flecks (Fig. [2F](#F2){ref-type="fig"}). Association of PVX.GFP with the local lesions was verified using a hand held UV lamp (data not shown). SBWMV 19K CRP is a suppressor of RNA silencing ---------------------------------------------- In this study we employed a widely used \"reversal of silencing assay\" to determine if the SBWMV 19K CRP is a suppressor of RNA silencing in plants \[[@B28]\]. In this assay, GFP-expression in the 16C transgenic N. benthamianaplants (Fig. [4B](#F4){ref-type="fig"}) was silenced by infiltrating young leaves with a suspension of Agrobacteriumexpressing GFP. The progression of GFP silencing was viewed first locally and then systemically using a hand held UV lamp. Within two weeks, the spread of GFP silencing was viewed systemically (Fig. [4C](#F4){ref-type="fig"}) and by three weeks, the only visible fluorescence is red fluorescence due to chlorophyll (Fig. [4D](#F4){ref-type="fig"}). At this time, the silenced plants were inoculated with PVX.19K. As PVX.19K viruses spread locally and then systemically, there was no change in GFP expression in the inoculated leaves or in the upper leaves (Fig. [4E](#F4){ref-type="fig"}). However, GFP expression was observed in the emerging leaves (Fig. [4F -- H](#F4){ref-type="fig"}). The SBWMV 19K CRP prevented RNA silencing only in emerging leaves where RNA silencing had not developed prior to virus infection. As a control, plants were also inoculated with PVX.GUS following infiltration with Agrobacterium. There was no evidence of GFP expression in the inoculated, mature, or new emerging leaves. The silencing phenotype was unaffected by PVX.GUS. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Evidence for RNA silencing suppression by the SBWMV 19K CRP. (A)nontransgenic N. benthamianaunder a UV lamp exhibits red fluorescence due to chlorophyll. (B)GFP-transgenic N. benthamiana(line 16C) exhibits green fluorescence under a UV lamp. (C)GFP was systemically silenced in the 16C transgenic N. benthamianafollowing infiltration with Agrobacterium. Here in the upper most leaves GFP silencing is vein centric. Systemic GFP silencing is detected initially within 2 weeks. (D)Within 3 weeks, GFP expression is completely silenced in the upper leaves. (E)GFP silenced plant inoculated with PVX.GUS. Emerging tissues of the infected plant remain silenced. (F, G, and H)GFP expression was observed in the emerging tissues of plants that were inoculated with PVX.19K. (I)Northern analyses of total RNAs from nontransgenic tissues (lanes 1, 2) and GFP transgenic tissues (lanes 4 -- 7) probed with a labeled GFP sequence probe. Lane 3 is blank. Lanes under the northern blot show ribosomal RNAs on an ethidium bromide stained gel. (J)Northern analysis of total RNAs from 16C plants infiltrated with Agrobacterium containing GFP constructs and probed with a labeled GFP sequence probe. Lanes 1--4 are RNA samples taken from plants that were also inoculated with PVX.19K. Lanes 5--8 are RNA samples taken from plants inoculated with PVX.GUS. Lanes under the northern blot show ribosomal RNAs. ::: ![](1743-422X-2-18-4) ::: Northern analyses was conducted to confirm RNA silencing in the upper leaves of Agrobacterium-infiltrated leaves and in the plants inoculated with PVX.GUS (Fig. [4I](#F4){ref-type="fig"} and [4J](#F4){ref-type="fig"}). GFP specific RNAs were detected in transgenic plants (Figure [4I](#F4){ref-type="fig"} lanes 4--7) and emerging leaves of plants injected with Agrobacteriumand inoculated with PVX.19K (Figure [4J](#F4){ref-type="fig"} lanes 1--4). GFP specific RNAs were not detected in untreated nontransgenic plants (Figure [4I](#F4){ref-type="fig"} lanes 1--3) or in plants that were injected with Agrobacteriumand inoculated with PVX.GUS (lanes 4--8). RNA samples collected from non silenced and silenced plants were also tested by Northern analysis to confirm the systemic accumulation of PVX.GUS or PVX.19K (data not shown). Since, GFP expression was restored in plants systemically infected with PVX.19K but remained silenced in plants inoculated with PVX.GUS, it is likely that the SBWMV 19K ORF is a suppressor of RNA silencing. Discussion ========== Many viruses encode proteins that suppress RNA silencing but the phylogenetic relatedness of these proteins is poorly understood. In this study, one class of viral CRPs, which were described as suppressors of RNA silencing and/or viral pathogenicity determinants, were shown to be phylogenetically related. These CRPs have a conserved Cys-Gly-Xaa-Xaa-His motif in which one of the two Xaa residues is Lys or Arg. The N-terminus has several conserved Cys residues that likely comprise a zinc finger motif. In fact, the ability of the gamma b protein of BSMV to bind Zn(II) was recently demonstrated \[[@B25]\]. Prior to 1999, SBWMV, BNYVV, PCV, and PMTV belonged to the genus Furovirus. As sequence data from different furoviruses have become available, it became clear that there are significant differences in the genome organization of these viruses, and therefore furovirus classification was revised in 1999 \[[@B19]\]. The genus Furovirusnow consists of viruses similar in genome organization to SBWMV \[[@B29]\]. These viruses are bipartite and have a single MP that is phylogenetically related to the tobamovirus and dianthovirus MPs \[[@B20],[@B22]\]. BNYVV, PCV, and PMTV were reclassified into the genera Benyvirus, Pecluvirus, and Pomovirus, respectively, for two reasons \[[@B19],[@B29]\]. First, the MPs of these viruses are phylogenetically distinct from SBWMV. BNYVV, PCV, and PMTV each possess a cluster of three slightly overlapping ORFs known as the \"triple gene block\", which has been shown for BNYVV \[[@B30]\] to mediate viral cell-to-cell movement. Second, benyviruses and pomoviruses differ from furoviruses in the number of genome segments. BNYVV has four or five genome segments while PMTV has three genome segments \[[@B31]\]. Pecluviruses like furoviruses have two genome segments, thus the primary difference between these virus genera is the MP ORFs \[[@B32]\]. This is significant because the initial amino acid sequence comparisons of CRPs from furo-, hordei-, tobra-, and carlaviruses included BNYVV as the type-member of the genus Furovirusand concluded that these small CRPs were unrelated \[[@B33]\]. Reclassification of the BNYVV as a member of the genus Benyvirusand inclusion of new members into the genus Furovirusled us to reexamine the relatedness of the viral CRPs. Based on the most recently defined taxonomic structure, the current amino acid sequence comparison presented in Figure [1](#F1){ref-type="fig"} indicates that the CRPs derived from viruses of the genera Furo-, Hordei-, Peclu-, and Tobravirusare phylogenetically related. On the other hand, these proteins are so different from CRPs encoded by Pomo-, Beny-and Carlavirusesthat the latter ones could not be included in the alignment (Fig [1](#F1){ref-type="fig"}). The present study shows that the SBWMV 19K CRP, when expressed from the PVX genome, functions as a pathogenesis factor and a suppressor of RNA silencing. The SBWMV 19K CRP, when it was expressed from the PVX genome, induced systemic necrosis on Nicotiana benthamiana, N. clevelandii, C. quinoa, and C. amaranticolor. These symptoms are distinct from the symptoms associated with PVX infection in these hosts, and from symptoms induced by SBWMV in its natural hosts. In systemic hosts, both PVX and SBWMV typically cause mosaic symptoms that range from mild to severe. In C. quinoaand C. amaranticolorboth PVX and SBWMV cause mild chlorosis. Severe necrosis and ultimate collapse of the tissue has been reported for other unrelated viral proteins that are pathogenicity factors and suppressors of RNA silencing. This include the Poa semilatent virus(PSLV) gamma b, TBSV P19, Tobacco etch virusHC-Pro, and the Rice yellow mottle virusP1 proteins\[[@B7],[@B11],[@B14],[@B34]\]. When we introduced the SBWMV 19K ORF into the TBSV vector and inoculated it to N. benthamiama, N. tabacum, C. quinoa, and C. amaranticolor(data not shown) plants, the SBWMV 19K CRP did not have any effect on symptomology (data not shown). However, it was reported previously that protein expression levels from the TBSV vector might be too low to test the effects of heterologous proteins on symptom severity \[[@B35]\]. Since an antibody to the SBWMV 19K CRP is unavailable, the levels of protein expression from PVX or TBSV vectors could not be analyzed to determine if gene dosage or protein expression levels contribute to symptom severity. In a related study, the SBWMV 19K and the BSMV gamma b CRPs could substitute for the TRV 16K CRP within the TRV genome, promoting virus replication and systemic accumulation \[[@B15]\]. The ability of the SBWMV 19K and the BSMV gamma b CRPs to induce severe symptoms when expressed from the PVX genome is reminiscent of phenomena described in relation to viral synergisms. The best studied viral synergism is between Tobacco etch virus(TEV) and PVX in which the TEV HC-Pro protein enhances accumulation and disease severity of PVX \[[@B34]\]. HC-Pro promotes infection of PVX by suppressing the anti-viral RNA silencing defense mechanism that would normally act on PVX to reduce virus infection. HC-Pro has the ability to increase PVX accumulation in the same way the SBWMV 19K CRP and the BSMV gamma b proteins were shown previously to enhance accumulation of TRV in infected plants \[[@B15]\]. Conclusion ========== The phylogenetic relatedness of the hordei-, peclu-, and furovirus CRPs is further substantiated by evidence that these proteins are all capable of suppressing RNA silencing in emerging leaves. This was demonstrated in the present and related studies using the same reversal of silencing assay used in this study. The SBWMV 19K CRP, the BSMV and PSLV gamma b CRPs, and the PCV 15K CRPs were each unable to change GFP expression in leaves where GFP was silenced prior to virus infection. However in each case, GFP expression occurred in newly emerging leaves \[[@B14],[@B16]\]. Thus, members of this family of CRPs similarly act on the RNA silencing machinery to block spread of the silencing signal into newly emerging leaves. In each case, the silencing suppressor activities of these CRPs have been compared to CMV and potyviruses in preventing onset of RNA silencing in new growth \[[@B14],[@B16]\]. While there is no evidence that the hordei-, peclu- and furovirus CRPs are related to the CMV or potyvirus silencing suppressors, it seems that the mode of action might be conserved among diverse viruses. Methods ======= Amino acid sequence comparisons ------------------------------- Related protein sequences were identified and retrieved from the NCBI data bank using PSI-BLAST. A PSI-BLAST search was launched with the amino acid sequence of the 19K CRP of Chinese wheat mosaic virus(CWMV, a furovirus). A similar search began with the amino acid sequence of BSMV gamma b, a sequence recovered in the CWMV search. Both searches converged at the second iteration and retrieved the same set of 22 sequences. This set contained CRPs derived from furo-, peclu- and hordeiviruses and contained the conserved P18 PFAM domain (\"protein family\"URL reference <http://pfam.wustl.edu/>\[[@B36]\]. A preliminary alignment of the retrieved proteins sequences was performed using the multiple sequence alignment mode of ClustalX. These twenty two furovirus and hordeivirus sequences were aligned using ClustalX alignments suggested in the BLAST outputs and PFAM. The tobraviral CRPs were not recovered by the above procedure, but upon manual inspection, appeared to have Cys residues in a linear arrangement that was similar to the set of 22 proteins. Eleven tobraviral protein sequences, exclusive members of a conserved domain in the Conserved Domain database <http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml> were aligned using ClustalX \[[@B37]\]. This tobraviral amino acid sequence alignment and the alignment of the 22 amino acid sequences sequences were assembled by ClustalX in profile mode, followed by manual adjustment. Amino acid sequences of aligned furo- and hordeiviral proteins were aligned with tobraviral amino acid sequences in profile mode of ClustalX (a total of thirty three sequences were aligned). A total of 33 amino acid sequences were aligned. In all cases, adjustments to the alignments were made using Se-Al \[[@B38]\]. Significance scores for the alignment of the two groups of protein sequences were calculated as previously described, using a structural conservation matrix, SCM2, for scoring \[[@B39]\]. Plasmids and bacterial strain ----------------------------- All plasmids were used to transform Escherichia colistrain JM109 \[[@B40]\]. The plasmids pPVX.GFP is an infectious viral clone and contains a bacteriophage T7 promoter \[[@B39]\]. The pPVX.GFP plasmid contains the PVX genome and the GFP adjacent to a duplicated CP subgenomic promoter. The plasmid pHST2-14 contains the TBSV genome and a mutation in the TBSV P19 ORF eliminating expression of a protein that suppresses RNA silencing \[[@B10],[@B42]\]. The plasmid pTBSV.GFP contains GFP inserted into the TBSV genome replacing the viral CP ORF \[[@B10]\]. The SBWMV 19K CRP ORF was inserted into the PVX.GFP genome, replacing the GFP ORF. The 19K CRP ORF was reverse transcribed and PCR amplified from purified SBWMV RNA using a forward primer (GCG GGG [ATC GAT]{.underline} ATG TCT ACT GTT GGT TTC CAC) containing added sequences encoding a ClaI restriction site (underlined) and a reverse primer (CGC [GTC GAC]{.underline} TCA CAA AGA GGA TAT CTT CTT TGG C) containing sequences encoding a SalI restriction site (underlined). PCR products and pPVX.GFP plasmids were digested with ClaI and SalI and then were ligated to prepare pPVX.19K. In vitro transcription and plant inoculations --------------------------------------------- In vitrotranscription reactions contained: 0.25 μg of linearized DNA, 5 μl of 5X T7 transcription buffer, 1.0 μl of 0.1 M DTT, 0.5 μl of SUPERase·In™ ribonuclease inhibitor (20 U/ μl) (Ambion, Austin, TX), 2.5 μl of an NTP mixture containing 5 mM ATP, CTP, UTP, and GTP (Pharmacia-Pfizer, Mississauga, Ontario, Canada), 0.7 μl of T7 polymerase (Ambion), and nuclease-free water to a final volume of 25 μl. The reactions were incubated for one and a half hour at 37°C \[[@B10]\]. Nicotiana benthamiana, N. clevelandii, Chenopodium quinoa, and C. amaranticolorplants were inoculated with infectious transcripts to study disease severity. Four plants, two leaves per plant, were inoculated in each experiment. Experiments were repeated at least three times. Ten μl of undiluted PVX.GFP or PVX.19K transcripts were rub-inoculated to each plant. The transgenic N. benthamianaline 16C was used to study RNA silencing. This line is homozygous for the GFP transgene at a single locus \[[@B44]\]. Plants were inoculated with transcripts following infiltration with Agrobacterium(see below). Agrobacterium infiltration of leaves ------------------------------------ Agrobacterium tumefaciensstrain C58C1 (pCH32) carrying a binary plasmid expressing GFP from a Cauliflower mosaic virus(CaMV) 35S promoter was used to silence GFP expression in N. benthamianaline 16C. Agrobacteriumcultures were grown overnight at 28°C in 5 ml of L-broth medium containing 5 μg/ml of tetracycline and 50 μg/ml of kanamycin. This 5 ml culture was used to inoculate 50 ml L-broth and grown overnight in medium containing 5 μg/ml tetracycline, 50 μg/ml kanamycin, 10 mM MES, and 20 μM acetosyringone. Cultures of Agrobacteriumcontaining GFP were pelleted by centrifugation and resuspended in a solution containing 10 mM MgCl~2~, 10 mM MES, and 150 μM acetosyringone. The final concentration of Agrobacteriumwas 0.5 OD~600~. The suspension was left at room temperature for 2--3 hours and then loaded into a 2 ml syringe. The syringe was used to infiltrate the suspension into the underside of the leaf. Visualization of GFP -------------------- A hand-held model B-100 BLAK-RAY long wave ultraviolet lamp (Ultraviolet Products, Upland, CA) was used to monitor GFP expression in 16C plants infiltrated with Agrobacteriumand in PVX.GFP inoculated plants. GFP fluorescence was recorded with a Sony Digital Still Camera model DSC-F717 (Sony Corporation of America, New York City, New York). In all plants analyzed, GFP expression was monitored every 3 days for up to 21 days post inoculation (dpi) or post infiltration with Agrobacterium. Immunoblot analysis ------------------- Immunoblot analyses were conducted according to \[[@B40]\]. Total protein from uninfected and infected N. benthamianaleaves was extracted in 1:10 (w/v) grinding buffer (100 mM Tris-HCl pH 7.50, 10 mM KCl, 5 mM MgCl~2~, 400 mM sucrose, 10% glycerol, and 10 mM β-mercaptoethanol). Extracts were centrifuged at 10,000 g for 10 min. Equal volumes of supernatants and protein loading buffer (2 % SDS, 0.1 M dithiothreitol, 50 mM Tris-HCl pH 6.8, 0.1% bromophenol blue, and 10 % glycerol) were mixed and boiled for 5 min. SDS-PAGE was carried out for 1 h at 200 V using 30 μl of each sample and 12.5% SDS -PAGE and the Biorad Mini-Protean 3 system (Biorad Laboratories, Hercules, CA). Proteins were transferred to PVDF membranes (Amersham Biosciences Corp., Piscataway, NJ) at 4°C overnight using protein transfer buffer (39 mM glycine, 48 mM Tris base, 0.037% SDS, and 20% methanol, pH 8.3) and a BioRad Trans-Blot system (BioRad Laboratories). Immunoblot analyses were conducted using the ECL-Plus Western Blotting Detection Kit (Amersham Biosciences Corp.). PVX CP antiserum (1:200) (Agdia, Elkhart, IN) was used. Northern analysis ----------------- Northern analyses were conducted according to \[[@B40]\]. For analyses of PVX infected plants and GFP expressing transgenic plants, a radiolabeled DNA probe was prepared using Rediprime II Random Prime Labeling System (Amersham Biosciences Corp.). Labeling was conducted using PCR products corresponding to either the GFP or PVX CP ORFs. For detection of TBSV.GFP and TBSV.19K in infected plant extracts, a DNA probe was labeled with digoxigenin (DIG). TBSV.GFP plasmids were digested with NcoI and SalI and a 614 nt fragment was gel eluted and labeled using Dig High Prime kit (Roche Applied Science Inc. Indianapolis, IN). The CSPD DIG Luminescence Detection Kit (Roche Applied Science Inc.) was used for chemiluminescence detection of DIG-labeled probes. Special thanks to Wenping Qui at Southwest Missouri State University for assistance with studies using TBSV to express the SBWMV 19k. The p26SBE-2 plasmid was obtained from Kay Scheets at Oklahoma State University and contains the 26S ribosomal RNA gene in pBluescript. This plasmid was used to prepare a DNA probe for membrane detection of rRNA \[45\]. The p26SBE-2 plasmid was digested with BamHI and EcoRI and a 1 kb fragment corresponding to the 26S rRNA was recovered and labeled using the Dig High Prime DNA labeling system (Roche Applied Science Inc.). Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= Jeannie Te did all cloning, plant inoculation experiments, gene silencing experiments. Ulrich Melcher did the amino acid sequence alignments and phylogenetic comparisons. Amanda Howard did some gene silencing experiments, photography. Jeanmarie Verchot-Lubicz conceived the study, did some molecular analysis, and wrote the paper. Special thanks to Wenpiny Qiu at Southwest Missouri State University for assistance with studies using TBSV to express the SBWMV 19k. Acknowledgements ================ Support for this project was provided by the Oklahoma Wheat Research Foundation, the USDA NRI Program Award OKLO-2470, and the Oklahoma Agriculture Experiment Station under the project H-2371.	PubMed Central	2024-06-05T03:55:54.092495	2005-3-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555535/", "journal": "Virol J. 2005 Mar 1; 2:18", "authors": [ { "first": "Jeannie", "last": "Te" }, { "first": "Ulrich", "last": "Melcher" }, { "first": "Amanda", "last": "Howard" }, { "first": "Jeanmarie", "last": "Verchot-Lubicz" } ] }
PMC555536	Background ========== DNA microarrays have become a widely used biotechnology for assessing expression levels of tens of thousands of genes simultaneously in a single experiment \[[@B1],[@B2]\]. Whether microarrays are being used for global tissue profiling or for differential expression studies, data normalization is an essential preliminary step before statistical analysis methods can be applied. The purpose of all normalization techniques is to transform the data to eliminate sources of variability stemming from experimental conditions, leaving only biologically relevant differences in gene expression for subsequent analysis. Normalization can be divided into two stages, intra-array normalization and inter-array normalization. Intra-array normalization deals with variability within a single array caused by factors such as differences in print-tip characteristics, channel differences in two-dye systems, and spatial heterogeneity across the array surface \[[@B3]-[@B5]\] and should be carried out using accepted methods before inter-array normalization is applied. This paper assumes intra-array normalization has been performed and presents an inter-array normalization method for comparison of gene intensity levels between multiple microarrays to deal with variation caused by such factors as differences in RNA isolation efficiency, labeling efficiency, hybridization conditions, exposure times, and detection efficiencies. It is now clear that simple inter-array normalization techniques, such as simple scaling to housekeeping genes or normalizing to a global mean, are not adequate for microarray data \[[@B6]\]. Housekeeping genes have been found to be more susceptible to modulation than previously thought \[[@B7]\]. Along with others \[[@B5]\], this paper underscores the potentially serious drawbacks of the global mean and other such methods. Recent literature has thus provided a plethora of more sophisticated normalization and analysis techniques as researchers struggle to cope with the task of microarray data analysis, some of which include maximum likelihood analysis \[[@B5]\], centralization \[[@B6]\], principal component analysis \[[@B8]\], analysis of variance \[[@B9]\] and Bayesian network analysis \[[@B10]\]. Analysis of publicly available large-scale SAGE gene expression data sets \[[@B11],[@B12]\] and an intra-phyletic survey of genome wide Affymetrix microarray experiments \[[@B13]\] have indicated that the large majority of expressed genes exhibited power-law distributions, while some microarray expression data exhibit a more log-normal distribution \[[@B14]\]. Our normalization procedure was inspired by the observation that the intensities measured on our microarray system also followed a power law distribution and can therefore be described by a simple mathematical model. Zipf\'s law \[[@B15]\] is a power law function that states that the magnitude of an intensity measurement (y) is inversely proportional to the rank (r) of that data point in the data set, y∝r^c^ (1) where cis a coefficient close to -1. Our microarray data can be classified as a generalized form of Zipf\'s law because the coefficient (c) is not always close to -1 and, in fact, varies between individual microarrays, making simple linear normalization procedures, such as global normalization to the same mean, inappropriate. However, the normalization procedure proposed here demonstrates that by taking Zipf\'s law into account, it is possible to apply a simple intra-array normalization procedure such that all filters have the same coefficient cand proportionality. We demonstrate the Zipf\'s law based normalization technique on microarray data sets representing both single channel and two channel technologies. In the single channel category, we produced two radio-labeled, nylon membrane based cDNA data sets, one commercial and one generated \"in-house\". Both systems contain a selection of genes chosen without regard to functional or pathway considerations, which make them especially appropriate for normalization using Zipf\'s law. These data sets were also normalized to a global mean (the mean of all microarrays) \[[@B16]\], and the quantile normalization method \[[@B17]\]. In addition we produced a two channel, fluorescently labeled, glass slide, oligo-based microarray data set generated \'in-house\'. This microarray can be classified as a \'boutique\' microarray because it consists of a selection of genes involved in apoptosis. This data set was normalized with a variant of the Zipf\'s law normalization method that uses a subset of the distribution as a proxy for normalizing the entire microarray. A comparison was then conducted against a variant of the loess normalization method that uses an a prioriselection of \'housekeeping\' genes as a proxy for normalization. The finding that our microarray data distributions conform to a power law distribution agrees with predictions based on genome wide gene expression studies \[[@B11]-[@B13]\], however Hoyle, et. al.\[[@B14]\] observed that microarray distributions were log normally distributed with possible power law tails. To investigate this discrepancy, and to verify that our normalization technique could be useful in the normalization of data sets from other microarray systems, we also surveyed publicly available data sets from the NCBI Gene Expression Omnibus \[[@B18]\]. The two assumptions upon which the normalization method are based are the same as those used in other normalization methods \[[@B5],[@B6]\], namely that in comparisons between similar tissues or cell lines under different experimental conditions i) most genes are not, or only moderately, regulated, and ii) approximately equal numbers of genes are up regulated as down regulated. Systems which conform to these two assumptions will be referred to as \'well-behaved\' in this paper. While these assumptions probably hold for microarrays derived from a diverse sampling of genes, for example an EST library survey, they may not hold for microarrays containing genes specifically selected based on function or pathway (so called \'boutique\' microarrays) as it is likely that most genes will be affected by the experimental treatments. One way to circumvent the restrictions resulting from these assumptions is to use a subset of data, or proxy, from the boutique array data set which fulfils the \'well-behaved\' criteria. In developing a boutique microarray normalization technique, Wilson et. al. \[[@B4]\] have devised a method for selecting a subset of genes within a microarray data set that have low variation between arrays and are well representative of the spectrum of intensities measured on the microarray. They term this a prioriselected subset \'housekeeping\' genes, however it should not be confused with the a posterioriset of genes typically envisioned when the term is used. Another possible proxy that could meet the \'well-behaved\' criteria are control spots which are included on the microarray during it\'s manufacture. We tested our normalization method on data from a two channel boutique microarray experiment using two types of control spots as proxies for normalization (Positive and negative internal controls, and housekeeping genes). The Zipf\'s law normalization methods were then compared with the variant of the loess method developed by Wilson et. al. \[[@B4]\] using housekeeping genes. Results ======= Verifying Zipf\'s Law --------------------- Before applying the described normalization method, the adherence of the reference curve (the median gene intensity data versus rank) to Zipf\'s law was verified. The most common method of verifying conformity to Zipf\'s law is a linear regression on the log~e~-log~e~transformed data set. Our regression showed a good fit, with a correlation coefficient of -0.98 and a slope of -0.56 for microarrays representing human colon (Figure [1a](#F1){ref-type="fig"}, Figure [6A](#F6){ref-type="fig"}, Table [1](#T1){ref-type="table"} set A), a correlation coefficient of -0.99 and a slope of -0.78 for rat brain microarrays (Figure [6B](#F6){ref-type="fig"}, Table [1](#T1){ref-type="table"} set B), and a correlation coefficient of -0.99 and a slope of -0.60 for the mouse apoptosis microarrays (Figure [6H](#F6){ref-type="fig"}, Table [1](#T1){ref-type="table"} set H). It should be noted that while the low ranking intensities may show a marked deviation from the regression line, this data typically accounts for a very small proportion of the total data and does not have a large affect on the regression curves. Normalization results -- single channel microarrays --------------------------------------------------- A comparison of the Zipf\'s law normalization method to the simple method of setting all arrays to a global mean (the mean of all microarrays) and to the quantile method was conducted on the single channel microarray data sets. Five human Unigene microarrays from the panel of thirty-two microarrays used in the sigmoidal colon experiments were selected to represent the greatest variability in pre-normalized data observed in the experiment (Figure [1b](#F1){ref-type="fig"}). Normalization to a global mean (Figure [1c](#F1){ref-type="fig"}) yielded data sets that displayed a higher variability in the coefficient cof the Zipf\'s power function (formula 1) than that observed after normalization by the Zipf\'s law method (Figure [1e](#F1){ref-type="fig"}) or the quantile method (Figure [1d](#F1){ref-type="fig"}). The Zipf\'s method showed the lowest variation in the Zipf\'s exponent and had the lowest spread of the data around the ln(rank) vs. ln(intensity) line. Results of an identical log~e~intensity versus log~e~rank plot comparison in Clontech rat microarrays showed little difference between the quantile and Zipf\'s methods \[see [Additional file 1](#S1){ref-type="supplementary-material"}\]. However it should be mentioned that this method of data plotting provides one view of the data which is especially favorable to the Zipf\'s law normalization method. Next we examine the results of the MA-plots, a technique that is especially favorable to the quantile normalization method. In order to access the effectiveness of the normalization method, pairwise comparisons using MA-plots (sometimes called RI plots, or log ratio vs. log mean intensity plots) \[[@B19]\] were carried out on the raw data, and data normalized with the global mean method, quantile normalization and Zipf\'s law on both data set A & B (Figure [2](#F2){ref-type="fig"} &[3](#F3){ref-type="fig"} respectively). With the raw data, the distribution of log-intensity ratios is not centered around zero which is as expected in an un-normalized data set. There is a noticeable intensity dependent effect, sometimes described as a \'banana\' curve, which is characteristic of many microarray data sets. Normalization with the global mean method results in a shift of the center of the log-intensity ratio distribution closer to zero, one important criterion for well normalized data, however, especially in the low log mean range, the bulk of the data points still deviate appreciably from zero. The intensity dependent effect is evident, with the low intensity end of the loess fit curving away from the zero axis. The intensity dependent effect is removed using the quantile method. The log intensity ratios of the data distributions normalized using Zipf\'s law are well centered around zero, but the intensity dependent effect is still apparent. In this case however, the bulk of the data lies very close to zero on the log-ratio scale. \[see [Additional file 2](#S2){ref-type="supplementary-material"}\] This is due to the fact that Zipf\'s law normalization not only shifts the data distribution on the log ratio scale, but also rotates the whole distribution in log-ratio log-intensity space. The Kolmogorov-Smirnov test is often used to determine whether data distributions differ significantly and provides a test statistic that measures the proportion of overlap between distributions which ranges from 0 (in the case of identical distributions) to 1 (for non-overlapping distributions) \[[@B20]\]. Mean Kolmogorov-Smirnov values (Table [2a, b](#T2){ref-type="table"}) showed the expected trend, with the high values for raw, unnormalized data decreasing when global median normalization was applied, decreasing again after Zipf\'s law normalization, and reaching zero for both data sets under quantile normalization. It should be noted that the Kolmogorov-Smirnov test statistic will always be zero after quantile normalization because this method forces the data distributions of all microarrays to be identical. Normalization Results -- Two Channel Boutique Microarray -------------------------------------------------------- Plots of log~e~intensity versus log~e~rank fitted with linear regressions show that the Zipf\'s law normalization based on internal controls (Figure [4a](#F4){ref-type="fig"}) and on selected housekeeping genes (Figure [4c](#F4){ref-type="fig"}) have relatively similar coefficients caccording to Zipf\'s power function (formula 1) as evidenced by the similarity in slopes of the regression lines. Loess normalization using selected housekeeping genes (Figure [4b](#F4){ref-type="fig"}) showed slightly more variation in ccoefficients. The unnormalized raw data is also depicted (Figure [4d](#F4){ref-type="fig"}) along with two other normalization results, the loess method (Figure [4e](#F4){ref-type="fig"}) and the quantile method (Figure [4f](#F4){ref-type="fig"}). These are provided for reference only. Neither method can be validly applied to boutique arrays because both rely on the \'well-behaved\' genes assumption. It should be noted that much of the variation in ccoefficients under the various normalization regimes is due to one channel (Cy3) on one microarray which had low median intensity and high variance due to low labelling efficiency (depicted in black in Figure [4](#F4){ref-type="fig"}). When normalized with the loess techniques (Figure [4c](#F4){ref-type="fig"} and [4f](#F4){ref-type="fig"}) the second channel (Cy5) on this array is adjusted to have a similar median intensity and variance, possibly skewing the results in favour of the Zipf\'s normalization techniques. To make the normalization method comparison unbiased, we eliminated this array from the analysis \[see [Additional file 3](#S3){ref-type="supplementary-material"}\]. The Zipf\'s normalization based on internal controls (a) showed the lowest variation in ccoefficients, the methods based on selected housekeeping genes (b, c) performed approximately equally well. Here again, raw (d), quantile normalized (e), and loess normalized (f) plots are provided for reference only. We generated MA plots for each of the normalization methods we compared (Figure [5](#F5){ref-type="fig"}). Typically, MA plots are produced from data from each channel of a single microarray. In addition to these \'within-array\' plots (the first three rows of graphs in Figure [5](#F5){ref-type="fig"}), we also examined \'between-array\' plots to evaluate the potential of the normalization methods to allow us to perform across array comparisons. The Zipf\'s using internal controls was slightly more well centered around the zero log ratio axis than the methods using selected housekeeping genes, especially in between-array plots. The raw and loess normalized plots are provided for reference only. Finally, to quantify the differences between distributions after normalization, pairwise Kolmogorov-Smirnov values were computed for both the complete boutique array data set (Table [2c](#T2){ref-type="table"}) and after eliminating the array which contained a low median intensity and high variance due to low labelling efficiency (Table [2d](#T2){ref-type="table"}). In addition to computing the Kolmogorov-Smirnov values for all possible between-array pairwise combinations, we also summarized just the within-array pairwise comparisons (in parenthesis in Table [2](#T2){ref-type="table"}). Of the normalization methods which can be applied to boutique microarrays, the Zipf\'s method using internal controls produced the most similar data distributions when all possible between-array comparisons are taken into consideration. When only within-array comparisons are considered, the Zipf\'s method using internal controls was better after the low labelling efficiency array was eliminated. The Zipf\'s method using selected housekeeping genes did not perform as well as the other methods in within-array comparisons, and was the middle performer when all possible between-array comparisons were computed. Kolmogorov-Smirnov values were computed from the global mean, Zipf\'s general, quantile, and loess normalization methods and are provided for reference only. Microarray platform comparison ------------------------------ In a survey of seventeen microarray data sets, Hoyle et. al. \[[@B14]\] reported that microarray data follow a log-normal distribution with power-law tails. The three data sets presented in this paper exhibited distinct power-law distributions (Table [1](#T1){ref-type="table"}, data sets A, B and H). To ascertain whether the data sets we used were unusual, we surveyed nine additional data sets (Table [1](#T1){ref-type="table"}, data sets C-G, I-K) to determine their conformity to Zipf\'s law and the log-normal distribution respectively. The microarray data sets fell into two broad categories, power law distributions (Figure [6](#F6){ref-type="fig"}, data sets A-E) and log normal distributions (Figure [6](#F6){ref-type="fig"}, data sets I-K). Of the six power law data sets, two (B and C) clearly followed Zipf\'s law distributions. The remaining four (data sets A, D, E, and H), while still power-law distributed, showing noticeable deviations from the distribution at the lower rank (higher intensity) portion of the distribution. Of the platforms that where recognizably log normal in distribution, two fluorescent dye labeled, oligo-based Affymetrics platforms (data sets K and L) followed the distribution most closely and two dye labeled, cDNA systems (data sets I and J) were perceptibly log normal. The two SAGE experiments (data sets F and G) which were included for comparison purposes, exhibited Zipf\'s law distributions. Coefficients of determination (r^2^) of the log mean intensity vs. log rank are a measure of conformation to a power-law distribution and ranged from 0.9968 to 0.7773 for microarray data sets, 0.9982 and 0.9978 for the SAGE experiments (Table [1](#T1){ref-type="table"}). Discussion ========== Zipf\'s law is based on observations made by linguist George Kingsley Zipf that the frequency of word occurrences in natural languages is proportional to the negative power of the rank order of the word. Beside the original findings in natural languages \[[@B15]\], Zipf\'s law has been found to apply to a plethora of natural phenomena, from the populations of cities to the impact factors of scientific journals as well as a variety of biological data, of which a review made available by Wentian Li \[[@B21]\] is an excellent online resource. It is important to point out, that being a phenomenological principle, Zipf\'s law does not imply that there is a universal underlying physical process at work. However, in much the same way that the Gaussian-Normal distribution occurs naturally in data and can be used to statistically test or otherwise manipulate the data, the fact that microarray data conforms to Zipf\'s law can be adapted for the purpose of microarray normalization. Zipf\'s law is a power law function that states that the magnitude of an intensity measurement is inversely proportional to the rank of that data point in the data set, where c is a coefficient close to -1. Ranking is a method common in statistics, which has previously been used to analyze microarray data. Hoyle el. al. \[[@B14]\] used ranking as a method for evaluating microarray data and proposed the use of several statistics including χ^2^to quantify the agreement of the distribution to Benford\'s Law \[[@B22]\], and σ^2^as a quality control measure to detect such factors as low signal to background ratio, or mRNA probes extracted from mixed cell types. Ranking also figured prominently in the evaluation of a survey of inter-array normalization methods \[[@B23]\] where the statistics \'absolute rank deviation\' and \'relative rank deviation\' were used to select the method that produces the most \'well-normalized\' data. The normalization procedure described in this paper is the first to combine these two ideas, namely that ranking can be used to judge the effectiveness of a normalization method, and that microarray data conforms to Zipf\'s law. We evolved these ideas into a novel and easily applicable normalization method and compared this method with existing methods to eliminate non-biological variation from microarray data sets. In order to implement an appropriate data normalization technique, it is important to know the distribution of a given data set. Several publications have examined the data distributions that typically result from microarray experiments. In a survey of seventeen microarray data sets, sixteen of which were fluorescent dye labeled, Hoyle et. al.\[[@B14]\] reported that microarray data were found to have a log normal distributions with power law tails. More recent publications have reported that the abundance of expressed genes exhibit power-law distributions \[[@B11],[@B13],[@B24]\]. Results from our own data sets and a subsequent survey of publicly available data sets from both radioactively and fluorescently labeled platforms suggest that both types of distributions can be manifested in microarray data. Comparisons between the Zipf\'s law and quantile normalization methods using MA plots showed that the quantile method effectively removes intensity dependant effects, sometimes referred to as \'banana\' curves, from microarray data sets, while the Zipf\'s law method has no effect on the curved nature of the intensity dependent effect. This is not altogether unexpected as the quantile method was specifically designed to remove such effects. While the Zipf\'s method does not remove the curve from the intensity dependent effect, it does minimize negative consequences by rotating the data distribution such that the maximal number of data points lie on the zero of the log ratio axis. In this respect, the Zipf\'s law normalization technique can be considered inferior to the quantile method, however, it may still be a useful tool where the quantile method cannot be applied. One such case, in which quantile normalization is inappropriate, is with so called \'boutique\' microarrays where the genes spotted on the array represent a selected set of genes, for example from a specific pathway or those involved with a particular biological process or disease state. In such systems, most genes are expected to be differentially regulated when control and experimental samples are compared and the expected data distribution of control samples may be significantly different than that of experimental samples (in mean intensity for example). The quantile normalization method would effectively remove this difference by replacing the data distribution of each microarray with the mean distribution of all arrays. In contrast, the principle of normalization according to Zipf\'s law can also apply to arrays of this type if a group of control spots are included on the microarray. These control spots could be an external reference probe which hybridises to a concentration gradient of matching spots on the array, or internal positive (highly expressed genes) and negative (spotting buffer) control spots on the microarray, or an a prioriselected set of housekeeping genes using a method such as that described by Wilson et. al. \[[@B4]\] or Schadt et. al. \[[@B25]\]. A linear model can be fitted to the control spots alone, and the normalization procedure can then be applied using the control spots as a proxy for the entire data distribution. The critical assumption in using control spots in normalization is establishing their relationship to the experimental spots. The results of our comparison between methods which are designed to normalize boutique microarray data show that Zipf\'s law normalization using internal control spots results in a relatively well normalized data set when compared to Zipf\'s law normalization using selected housekeeping genes and the modified loess method using selected housekeeping genes. In addition, the Zipf\'s law method produced data distributions which are more similar between arrays allowing for between-array comparisons which are advantageous in terms of both cost, because of the reduced number of microarrays that need to be run, and, statistical power, by allowing for greater numbers (n), experimental design permitting. Conclusion ========== In summary, we examined the applicability of using Zipf\'s law as the basis for a novel normalization technique, which is applicable to both one channel microarray data and two channel microarrays. This method is shown to out-perform such methods as global normalization to the mean but would appear to be inferior to quantile normalization. The quantile method was superior to Zipf\'s law in removing intensity dependent effects commonly seen in microarray data. While the latter method cannot be applied to boutique arrays, we show that the Zipf\'s normalization method used with internal positive and negative controls or with selected housekeeping genes normalizes boutique arrays as well as currently existing methods. Additionally, data normalized with the Zipf\'s method using internal control spots seems more amenable to between-array gene intensity comparisons when compared to other methods. Methods ======= Data acquisition ---------------- Data set A (Table [1](#T1){ref-type="table"}) was generated using a global genome-wide cDNA clone set (Human UniGene clone set RZPD 1 Build 138, NCBI \[[@B26]\]), which consisted of \~33,792 cDNA clone inserts spotted in duplicate onto membranes \[[@B16]\]. These microarrays (n = 31) were hybridized with ^33^P-labeled cDNA derived from total RNA extracted from biopsy material from the sigmoidal colon of normal (control, n = 11), and patients with Crohn\'s disease (condition A, n = 10) and ulcerative colitis (condition B, n = 10). To emphasize that our normalization technique can be used to normalize other array systems, the second array set used was a smaller, but widely used, commercially available microarray system. Data set B (Table [1](#T1){ref-type="table"}) was generated by using Atlas Rat cDNA microarrays (Clontech, 588 genes) probed with rat brain tissue, from control (cerebellum n = 10, olive n = 10) and harmaline treated (cerebellum n = 10, olive n = 9) animals. A third microarray data set, data set H (Table [1](#T1){ref-type="table"}) was included to demonstrate the normalization method on two channel fluorescent based (Cy3/Cy5) oligonucleotide systems. These custom produced boutique microarrays (n = 5) contained 1024 spots, and were used in a study to identify differences in apoptotic mechanisms in two different mouse cell lines. Microarrays were probed according to established protocols and exposed to imaging plates overnight (BAS-MS 2325) and scanned at a 50 μm resolution on a FLA-3000G phosphoimager (Raytest, Germany). Image gridding was carried out using VisualGrid^®^software \[[@B27]\], and intensity data was stored in a relational database and normalized and analyzed using database stored procedures and Perl scripts. All data was normalized from raw data, no background subtraction or other inter-array normalization was performed. Plots were generated using the Grace software package \[[@B28]\]. Normalization ------------- Normalization was accomplished by transforming the data such that the coefficient cand proportionality of the Zipf\'s power function (formula 1) are identical for all microarrays. This is easily achieved using a regression model on the log~e~intensity versus log~e~rank transformed data, which has the general form, ln (y) = a+ bln (r) + e (2) where yis the intensity, ris the rank, ais the regression constant (corresponding to proportionality in Zipf\'s power function), bis the regression coefficient (corresponding to the coefficient cin Zipf\'s power function), and eis an error coefficient, which is assumed to be normally distributed. The first step in this three step procedure was to compute the median intensity of each gene over all microarrays to establish ranks, which were used as the \'reference\' to which all microarrays were normalized. This was done by taking the median intensity (y~med~) of each gene, over all microarrays on which it was measured, and sorting the resulting list of medians to obtain their median ranks (r~med~). The regression model (2) is applied to the log~e~median intensities and their ranks to estimate a~med~and b~med~using the least squares method, ![](1471-2105-6-37-i1.gif) The ranking of genes by their median intensities effectively groups genes of similar overall expression level along the log rank axis. Under the assumptions that most genes are not differentially expressed, the reference curve generated from the median intensities should have an identical regression coefficient and constant to that of each individual microarray plotted using the ranks determined by the medians. For the genes which are differentially expressed, the median value represents a \'center\' around which expression levels on each individual array may vary, and the neighbouring (by rank) genes, which do not (or only slightly) vary, act to stabilize the regression line and allow normalization to be performed. In the second step of the normalization procedure, the regression model was applied individually to each microarray using the same ranking as the reference curve, ![](1471-2105-6-37-i2.gif) This results in a set of coefficients a~k~and b~k~which are estimated individually for each array using the least squares method, where k is equal to the number of microarrays in one channel systems, and equal to 2 time the number of microarrays (one for each channel) in two channel systems. Data from two channel arrays were treated in the same way as one channel systems, i.e. each channel was treated independently. In the third step, the difference between the expected gene intensity value on the kth array and that of the reference curve was applied as the normalization factor, ![](1471-2105-6-37-i3.gif) A scaling factor was applied to the raw data before normalization such that the values y~k~, ![](1471-2105-6-37-i4.gif) and ![](1471-2105-6-37-i5.gif) were always greater than one to avoid negative values after log transformation. After normalization, the same scaling factor was applied to the data to back transform to their original magnitude. For example, if the smallest raw value in the data set was 0.1, the unlogged raw data was multiplied by a scaling factor of 10 before normalization, and the unlogged normalized data was divided by the same scaling after normalization. In the special case of our third microarray data set (see Methods: Data Acquisition) which was a boutique array, the same procedure as described above was applied with the following modifications. Each microarray contained 32 spots each of internal positive controls (GAPDH, glyceraldehyde-3-phosphate\_dehydrogenase) and internal negative controls (spotting buffer). The medians of all gene intensities were computed (including internal positive and negative controls), and median ranks were assigned as described. However, only the medians of the 64 internal control spots were used to estimate a~med~and b~med~, and only the 64 internal control spots from each array were used to estimate a~k~and b~k~. In both cases, the ranks generated from the entire data set, were used. The normalization factor was then applied over the entire data set as described above. An alternative to the used of internal control spots for the normalization of boutique microarrays was also explored. Wilson, et. al. \[[@B4]\] described a method wherein a set of \'housekeeping\' genes is selected a priorifrom the data set by virtue of their low variance in intensity and such that the entire range of intensities observed on the microarrays is uniformly represented. We also applied the Zipf\'s law normalization technique to our boutique microarrays using the set of housekeeping genes selected using the method of Wilson, et. al. In addition to the normalization method based on Zipf\'s law, all data sets were normalized to a global mean (the mean of logged intensities from all microarrays) and the quantile method. The quantile method is applied by ranking the genes in each array by intensity, taking the median intensity at each rank, and replacing each gene intensity with the median intensity corresponding to the same rank. All normalization methods were compared to each other and to the raw data distribution using box plots and MA plots (pairwise array comparisons of the log-intensity ratio (M) to the mean log-intensity (A)). The two channel boutique microarray data set allowed further normalization methods not possible on one channel array systems to be applied. We normalized this data set using the popular loess method \[[@B19]\], and a modified Loess method specifically designed for boutique arrays using selected housekeeping genes described by Wilson, et. al. \[[@B4]\]. Software -------- The Zipf\'s normalization procedure was initially implemented as an SQL stored procedure in a relational database. However, because this is not easily transferable to other systems, we provide two further implementations, a Perl script and an Excel macro \[see Additional files [4](#S4){ref-type="supplementary-material"}, [5](#S5){ref-type="supplementary-material"}\]. Implementations are available for download from our website \[[@B29]\] and as additional files accompanying this paper. Both the Perl script and Excel macro implement matrix algebra style computation, using either built-in functions or the Perl PDL module \[[@B30]\]. Normalization of two channel arrays with the loess method was performed using the marray package from R\'s Bioconductor \[[@B4]\]. Loess normalization using selected housekeeping genes and the selection of the housekeeping genes themselves was done with the tRMA package \[[@B19]\] which is publicly available for download on the internet. Sample data sets are also provided with this paper \[see Additional files [6](#S6){ref-type="supplementary-material"}, [7](#S7){ref-type="supplementary-material"}, [8](#S8){ref-type="supplementary-material"}\]. Normalization method comparison ------------------------------- To compare and evaluate the effectiveness of the various normalization methods applied in this paper, several well established methods were used along with some less common techniques. MA plots \[[@B19]\] are a convenient way to examine differences in fluorescent marker efficiency and other dye effects in two channel microarray systems. In addition to the standard practice of generating within-array MA plots, we apply them additionally to one channel systems and between arrays in two channel systems to evaluate the extent to which a normalization procedure allows for multiple pairwise comparisons between microarrays. Plots of log~e~intensity versus log~e~rank fitted with linear regressions are a way to visually evaluate the normalization procedure according to the criteria of the Zipf\'s Law normalization. Specifically, all arrays have identical coefficients cand proportionality for the Zipf\'s power function when the slops and y-intercepts of the regression lines are identical. Finally, to quantify the similarity between microarray distributions after normalization, the mean Kolmogorov-Smirnov value was calculated over all possible pairwise combinations of microarrays within an experiment. In the case of two channel arrays, the mean of within-array Kolmogorov-Smirnov values was also computed (n = the number of arrays). It should be emphasized that even though the Kolmogorov-Smirnov values are technically a test statistic, no statistical test is performed. The values are here used only as a measure of similarity between microarray distributions. Microarray platform comparison ------------------------------ The underlying premise of the Zipf\'s normalization method is that microarray data distributions follow a power law distribution such that the relationship between the log intensities and the log ranks is clearly linear. While this assumption holds true for the three data sets we present in this paper, to evaluate the general applicability of the method we also examined eight publicly available data sets (Table [1](#T1){ref-type="table"}, data sets C-G, I, K-L) from the NCBI Gene Expression Omnibus \[[@B18]\], and one unpublished data set from an independently maintained website \[[@B31]\] (Table [1](#T1){ref-type="table"}, data set J). The survey contains a variety of microarray system types (cDNA vs. Oligo based, radioactivity vs. dye labeled systems, academic vs. commercially produced) and two SAGE experiments for comparison. Two plots were generated for each data set to ascertain the conformity to the Zipf\'s power law distribution and the log normal distribution respectively. For each data set, a representative array was constructed by ranking the intensities within each array, and then mean over ranks were taken. To determine how well data sets follow the Zipf\'s power law distribution, log intensity vs. log rank plots were constructed and linear regressions were performed. Data distributions, which were very linear in form, closely follow the power law distribution. A second plot of the distribution of (log y -- μ) / σ, where y is the mean intensity over ranks, and μ and σ^2^are the mean and variance, was made for each data set to visualize the conformity to log normal distribution. List of abbreviations ===================== EST -- Expressed Sequence Tag MA -- log ratio (M) vs. mean log intensity (A) NCBI -- National Center for Biotechnology Information RZPD -- Deutsches Ressourcenzentrum für Genomforschung GmbH SAGE -- Serial analysis of gene expression SQL -- Structured Query Language Authors\' contributions ======================= TL conducted the data analysis and implementation of algorithms, participated in the development of the normalization method and is principle author of this manuscript. CMC generated the Unigene and Clontech microarray data set, participated in the development of the normalization method and participated in manuscript preparation. PJPC conceived of and participated in the development of the normalization method. RH participated in the generation of microarray data sets and participated in the development of the normalization method. GD conceived of and coordinated neurology related aspects of this study. SS conceived of and coordinated gastrointestinal related aspects of this study. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Clontech microarray log plotsFive rat Clontech microarrays from the panel of thirty-nine microarrays probed with rat-brain tissue. Upper left to lower right: a. Log~e~median gene intensity vs. log~e~rank -- conformity to Zipf\'s law is demonstrated by the linear regression line (in red) b. Five microarrays chosen to maximize pre-normalization variability, each plotted according to the gene ranks determined by their median gene intensity levels. c. The same five microarrays, normalized to a global median, with regression lines. d. The same five microarrays, normalized with the quantile method, with regression lines. e. The same five microarrays normalized taking Zipf\'s law into account, with regression lines. For plots b-d, a sub-sample of 50% of the data points are plotted for readability. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Mean of squared log ratios from MA plots inFigure [2](#F2){ref-type="fig"} In Figure [2](#F2){ref-type="fig"}, it is difficult to see that the distribution of the Zipf\'s normalized data is more closely centered around zero on the log ratio axis than the Globally normalized data. To quantify this, the mean of squared log ratios was computed for each MA plot. The positions of the values in this table correspond exactly to the positions of the plots in Figure [2](#F2){ref-type="fig"}. In 6 out of 8 cases, the mean of squared log ratio is smaller in the Zipf\'s normalized data than in the corresponding Globally normalized data. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 Boutique microarray log plotsFour mouse apoptosis boutique microarrays used in the mouse cell line experiments. This is the same data set as shown in Figure [4](#F4){ref-type="fig"}, with the array containing one channel with low expression intensities and high variability removed. Upper left to lower right: Log~e~median gene intensity vs. log~e~rank -- a. Normalized according to Zipf\'s law, using internal positive and negative controls as proxies for the whole data set. b. Normalized with a loess curve fit using a selected set of housekeeping genes as proxies (see Methods). c. Normalized according to Zipf\'s law, using the same selected set of housekeeping genes as in b. as proxies d. The raw data. e. For comparison purposes only, normalized using the quantile method. f. For comparison purposes only, normalized using the standard loess method. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 4 Requires: Microsoft Excel (Does not handle missing data values.) ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 5 Requires: Perl (which runs on many platforms), the PDL perl module (Handles missing data values if PDL is compiled correctly.) ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 6 Microarray type: Filter based cDNA from the RZPD Number of genes: 33,792 Number of microarrays: 31 Probed with: Total RNA from human sigmoidal colon. Within microarray normalization: None ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 7 Microarray type: Clonetech Atlas Rat cDNA 7738-1 Number of genes: 558 Number of microarrays: 33 Probed with: Total RND from rat cerebellum and olive. Within microarray normalization: None ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 8 Microarray type: custom made glass slide Number of genes: 1024 Number of microarrays: 5 Probed with: Total RND from mouse cell lines. Within microarray normalization: None ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ The authors wish to thank Alexander Zein and Carl Manaster for critical input on data analysis techniques. We would like to express our appreciation to the clinicians and volunteers who provided tissue samples, and Hans Moises and Henrik Wilms for rat brain samples. We gratefully acknowledge the technical assistance of Brigitte Mauracher, and the invaluable assistance of the Max-Planck Institute for Molecular Genetics in Berlin, in particular Hans Lehrach, Holger Eickhoff and Elke Rohlfs. We also thank Sandra Freitag for advice on formulating the equations. This research was supported in part by a Training and Mobility of Researchers (TMR) grant, as well as grants from the German National Genome Research Program, the National Genome Research Network (NGFN) and the DFG (FOR423). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Unigene microarray log plots.Five human Unigene microarrays from the panel of thirty-one microarrays used in the sigmoidal colon experiments. Upper left to lower right: a. Log~e~median gene intensity vs. log~e~rank -- conformity to Zipf\'s law is demonstrated by the linear regression line (in red) b. Five microarrays chosen to maximize pre-normalization variability, each plotted according to the gene ranks determined by their median gene intensity levels. c. The same five microarrays, normalized to a global mean, with regression lines. d. The same five microarrays, normalized with the quantile method, with regression lines. e. The same five microarrays, normalized taking Zipf\'s law into account, with regression lines. For plots b-d, a sub-sample of 10% of the data points are plotted for readability. ::: ![](1471-2105-6-37-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Unigene microarray MA plots.MA plots of Raw Unigene data compared to data normalized with the Global mean, Zipf\'s, and Quantile methods (columns). Each row of plots represents one pairwise comparison, only 8 of the possible 10 pairwise comparisons of the 5 microarrays used in figure 1are shown. Lowess curves are plotted in red. ::: ![](1471-2105-6-37-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Clontech microarray MA plots.MA plots of Raw Clontech Rat data compared to data normalized with the Global mean, Zipf\'s, and Quantile methods (columns). Each row of plots represents one pairwise comparison, only 8 of the possible 10 pairwise comparisons of the 5 microarrays used in [Additional file 1](#S1){ref-type="supplementary-material"} are shown. Lowess curves are plotted in red. ::: ![](1471-2105-6-37-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Boutique microarray log plots.Five mouse apoptosis boutique microarrays used in the mouse cell line experiments. Upper left to lower right: Log~e~median gene intensity vs. log~e~rank -- a. Normalized according to Zipf\'s law, using internal positive and negative controls as proxies for the whole data set. b. Normalized with a loess curve fit using a selected set of housekeeping genes as proxies (see Methods). c. Normalized according to Zipf\'s law, using the same selected set of housekeeping genes as in b. as proxies d. The raw data. e. For comparison purposes only, normalized using the quantile method. f. For comparison purposes only, normalized using the standard loess method. ::: ![](1471-2105-6-37-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Boutique microarray MA plots.MA plots of the boutique data set comparing (in columns) Zipf\'s normalization using controls (Zipfs), Zipf\'s normalization using housekeepers (Zipfs HK), loess normalization using housekeepers (loess HK), raw data, and, for comparison purposes only, the standard loess normalization. Each row of plots represents one pairwise comparison, only 6 of the possible 45 pairwise comparisons of the 5 microarrays used in figure 4 are shown. The top three rows show within-array comparisons, and the bottom three rows show between-array comparisons. Lowess curves are plotted in red. ::: ![](1471-2105-6-37-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Data set comparison.Eleven microarray data sets (A-K) exhibiting varying degrees of conformation to power law and log normal distributions. On the left for each data set is a log mean intensity vs. log rank plot of the entire data set. Each array was sorted independently by intensity, and mean intensities for each rank over all arrays are plotted. A linear regression line is shown in red. Data sets with a linear distribution adhere well to a power law distribution. On the right for each data set is the distribution (ln(i) -- μ) / σ of the mean intensities used in the left hand plots, where i is the mean measured intensity for each rank and μ and σ are the mean and variance of i respectively. The standard normal curve N(0,1) is shown in red for comparison. Data sets that display a standard normal distribution adhere well to a log normal distribution. ::: ![](1471-2105-6-37-6) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Data set comparison ::: Set Microarray Platform Number of Data Points Number of Expts R2 GEO platform GEO experiment Array type --------- ------------------------------------ --------------------------- --------------------- -------- ------------------ -------------------- ---------------- A. Human Unigene RZPD 1 34560 31 0.9877 GLP284 GSE1510 cDNA, membrane B. Clontech Atlas Rat cDNA Expression 588 39 0.9968 GPL158 GSE1509 cDNA, membrane C. Clontech Atlas Human 1.2 (I & II) 1176 10 0.9903 GPL127, GPL128 GSE751 cDNA, membrane D. Clontech Atlas Mouse 1.2 1159 12 0.9460 GPL144 GSE565 cDNA, membrane E. Clontech Atlas Human Cancer 1.2 1160 36 0.9109 GPL158 GSE796 cDNA, membrane F. NlaIII: Rattus norvegicus 76790 1 0.9982 GPL23 GSM1679 SAGE G. NlaIII: Homo sapiens 101677 1 0.9978 GPL4 GSM14771 SAGE H. Mouse Apoptosis 1024 5 × 2 0.994 \-- \-- cDNA, glass I. Caltech 16K cDNA mouse 908 58 0.8892 na na cDNA, glass J. Stanford Human Unigene 908 24 0.9081 na na cDNA, glass K. Affymetrix GeneChip Rat Genome 8799 24 0.8538 GPL85 GSE776 Oligo, glass L. Affymetrix GeneChip Human Genome 12625 24 0.7773 GPL91 GSE803 Oligo, glass Eleven microarray data set comparison. Raw intensities, without background subtraction, were used. Controls and blanks were excluded. For Affymetrix chips (K and L), MM/PM ratios were used. For data set B two different Atlas arrays were analyzed together, when analyzed separately they gave similar results. For two channel array systems (I and J), each channel was treated as a separate array. For set I, only the cyanine-3 channel (spleen sample control) was used and for set J, both channels were used for analysis. Reference for data set J: Ross et. al. \[31\]. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Kolmogorov-Smirnov values ::: Microarray Platform Pairwise Combinations (within array) Raw Global Median Zipfs Quantile Loess Loess HK Zipfs Control Zipfs HK ---- ------------------------------------ ------------------------------------------ --------------- ------------------- ------------------- -------------- ------------------- --------------- ------------------- --------------- a. Clontech Atlas Rat cDNA Expression 465 0.539 0.484 0.119 0 na na na na b. Human Unigene RZPD 1 703 0.662 0.225 0.060 0 na na na na c. Mouse Apoptosis 45 (5) 0.548 (0.631) 0.340 (0.318) 0.149 (0.167) 0 (0) 0.471 (0.042) 0.487 (0.172) 0.182 (0.179) 0.303 (0.296) d. Mouse Apoptosis Subset 28 (4) 0.568 (0.667) 0.303 (0.287) 0.111 (0.129) 0 (0) 0.317 (0.038) 0.341 (0.190) 0.145 (0.128) 0.315 (0.291) Three microarray data sets presented in this paper and seven normalization techniques were compared by computing the mean Kolmogorov-Smirnov values of all possible pairwise combinations of arrays within a data set. In the case of the two channel mouse apoptosis microarray, within-array pairwise comparisons were also computed and are shown in parenthesis (here n = the number of arrays, as each array has 2 channels). The symbol \'na\' indicates that the normalization techniques which can only be carried out on two channel (loess) or boutique (loess HK, Zipfs Control, Zipfs HK) arrays were not performed on single channel arrays. Values in bold typeface were computed for reference purposes only -- these normalization methods cannot be validly applied to boutique microarrays. :::	PubMed Central	2024-06-05T03:55:54.095779	2005-2-23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555536/", "journal": "BMC Bioinformatics. 2005 Feb 23; 6:37", "authors": [ { "first": "Tim", "last": "Lu" }, { "first": "Christine M", "last": "Costello" }, { "first": "Peter JP", "last": "Croucher" }, { "first": "Robert", "last": "Häsler" }, { "first": "Günther", "last": "Deuschl" }, { "first": "Stefan", "last": "Schreiber" } ] }
PMC555537	Background ========== The use of complementary and alternative medicine (CAM), defined by the United States National Center for CAM as a group of diverse medical and health care systems, practices, and products that are not presently considered to be a part of conventional medicine, has grown tremendously in the United States \[[@B1]-[@B4]\]. A recent national survey reported that four out of every ten Americans used at least one form of CAM, and one out of five used prescription medications together with CAM \[[@B3]\]. The prevalence is even higher in patients with chronic medical problems (for example, 28 to 90% in patients with arthritis, 11--56% in those with cancer, 60% in patients with asthma and 67.8% in patients with human immunodeficiency virus) \[[@B5]-[@B11]\]. Biological based therapies (BBT) is an important type of CAM and is defined by the National Center for CAM as use of substances found in nature, such as herbs, foods, and vitamins. BBT is the second most commonly utilized CAM, with the first being prayer therapy \[[@B2]\]. Similar to other chronic medical conditions, patients suffering from a variety of cardiovascular diseases including coronary heart disease, congestive heart failure, stroke, arrhythmia and congenital cardiovascular defects, may also be looking to CAM to prevent or treat their illnesses. This is particularly likely since a number of BBT products including vitamin E, vitamin C, beta-carotene, fish oils, and coenzyme Q10 have been evaluated for prevention and/or treatment of cardiovascular diseases \[[@B12]-[@B25]\]. Despite a wide array of available BBT for cardiovascular conditions, studies evaluating the prevalence of usage of these agents are limited \[[@B21]-[@B25]\]. As CAM, in general, has become widely accessible to the public, and BBT may be purchased in pharmacies, health food stores and supermarkets, it is difficult to control patient usage of these products. In addition, the likelihood for adverse effects and interactions between conventional therapies and BBT places patients using such products at an increased risk of adverse drug events. It is, therefore, important to examine patient usage so as to advise and monitor them properly. Among the studies conducted to-date that included patients with a broad spectrum of cardiovascular diseases, few focused only on BBT. The studies examined different factors that may determine BBT use, but none of them examined the potential for side effects and drug interactions with other prescription and non-prescription medications that the patients were utilizing. Since patients with cardiovascular conditions consume many prescription medications with narrow therapeutic indexes and extensive drug interaction profiles \[[@B26],[@B27]\], it is important to look at the prevalence of use and potential drug interactions in a cohort of cardiovascular patients. The primary objective of this study was to determine the effect of the education level on the use of BBT in cardiovascular patients. The study also investigated the attitudes and beliefs towards BBT by patients with cardiovascular diseases. In addition, patient perceptions regarding the safety and efficacy of BBT, common BBT used and a list of potential BBT-drug interactions were reviewed. Methods ======= This is a cross-sectional, descriptive study utilizing structured interviews to assess the level of education, usage, beliefs and attitudes towards BBT among inpatients with cardiovascular diseases. The study was conducted in the Cardiac Care Center at the Mount Sinai Hospital, New York, USA from February 2001 to December 2002. Participants provided informed consent and were interviewed by one of the investigators. To maintain consistency of the interview and to prevent interviewer bias, a scripted letter was drafted for the investigators to invite the patients to participate in the study and to explain the process of the study. After a patient was enrolled, an investigator read the survey questions verbatim to the patient and tried not to elaborate whenever possible. Patients were included in the study if they had at least one of the following diagnoses: cardiovascular disease(s) including congestive heart failure, coronary heart disease, thromboembolic diseases, valvular heart disease, arrhythmia, vascular aneurysm, peripheral vascular disease, pulmonary hypertension, congenital heart disease and post heart transplant. Additional inclusion criteria included being 18 years of age or older, English speaking, no documented cognitive deficits precluding the patient from understanding the interviewer, and willingness to provide an informed consent. Prior to patient contact, the attending physicians of the eligible patients were contacted and informed about the study. If the attending physicians chose not to have their patients participate in the study, the patients were not included. Upon agreement of the physicians, subjects were invited to participate and were asked to sign an informed consent at their convenience prior to being interviewed. This study was approved by the Institutional Review Boards of Mount Sinai Hospital and Long Island University. Biological based therapy survey ------------------------------- Utilizing a structured instrument (see [Additional file 1](#S1){ref-type="supplementary-material"}), eligible subjects were interviewed by one of the investigators during their stay at the hospital. The BBT survey was modified and adapted from a previously published survey \[[@B6]\]. The participants could choose to answer or not to answer any question at their discretion, and could discontinue their participation in the study at any time during the interview. During the interview, demographic data, including age, gender, race, marital status, level of education, annual income, and working status, were collected. Additionally, history of cardiovascular and other medical conditions, and medications utilized were recorded. The definition of BBT in this study was similar to that defined by the United States National Center for CAM, which included all herbal supplements, vitamins and mineral supplements. The patients\' attitudes and beliefs towards BBT were assessed by asking them about their perceived safety and efficacy of BBT. The side effects and potential drug/food interactions listed by the patients were compared against those listed in the MicroMedex^®^Database \[[@B28]\]. The patients\' assessments of benefits of BBT as compared with conventional medicine were recorded. Additionally, the participants were asked whether they reported the use of BBT to their physicians, pharmacists, or other healthcare professionals. A review of patients\' medical records was performed to collect data about patients\' cardiovascular diseases and to confirm medications used. Although, the identity of the participants in this research study was kept confidential, patients were notified in the informed consent process that if potential BBT-prescription medication interactions were identified, their physicians would be notified. Statistical analysis -------------------- For this study to have an 80 percent power to detect a 20 percent clinically significant difference in determining factors of BBT use such as education level between the users and non-users of BBT, and establishing a p value of \< 0.05 as the level of statistical significance, approximately 200 patients (100 patients in each group) needed to be enrolled. For demographic parameters, continuous variables were compared between the two groups using Students\' t-test and categorical variables were compared using chi-square. Attitudes and beliefs regarding the safety and efficacy of BBT were compared between the two groups using a chi-square test for categorical data and Students\' t test for Likert-type scale questions. The BBT products used by cardiovascular patients were recorded and the likelihood of potential drug interactions between BBT and other medications the patients were taking were described. Statistical analyses were conducted using Statistical Product and Service Solutions program (SPSS^®^for Windows, Rel. 10.01 1999). Results ======= A total of 200 patients who were admitted to the Cardiac Care Center at Mount Sinai Hospital were enrolled into the study. All the physicians approached agreed to have their patients participate in the survey. Two of the patients were, eventually, excluded from data analysis due to incomplete survey data. Of the remaining 198 patients, 94 (47.5%) reported BBT use at some point in their lifetime and 84 (42%) reported using such products during the immediate 12 months prior to the survey. Of the 94 patients who used BBT, 32% reported using the products all the time. Demographic characteristics --------------------------- The demographic and socioeconomic characteristics of the total sample are presented in Table [1](#T1){ref-type="table"}. The mean age of surveyed participants was 60.0 ± 16.5 years, and their ages were normally distributed ranging from 19 to 102 years of age. The overall sample consisted of 118 (59.6%) men and 80 (40.4%) women. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Demographic Characteristics of Study Subjects ::: Users (%) Nonusers (%) p value ----------------------------- --------- --------------- ------------------ ------------- Total^1^ N = 198 94 (47.5) 104(52.5) Age^2^ 61.4 ± 16.7 58.7 ± 16.3 NSS Gender NSS Male 51(54) 67(64) Female 43(46) 37(36) Race NSS White 53(56) 46(44) Black 19(20) 27(26) Hispanic 12(13) 18(17) Other^3^ 10(11) 13(7) Education 0.0006 \< High school 12(13) 28(27) High school 23(24) 42(40) Some college 18(19) 12(12) College Graduate 28(30) 12(12) Graduate School 13(14) 8(8) Annual Household Income NSS \< \$10,000 17(27) 19(30) \$10,000--\$30,000 12(19) 15(23) \$30,000--\$50,000 8(13) 14(22) \$50,000--\$75,000 15(23) 7(11) \$75,000--\$100,000 4(6) 4(6) \> \$100,000 5(13) 5(8) ^1^lifetime use of BBT;^2^mean age ± standard deviation (range); ^3^Asian/Pacific Islander/Indian ::: Overall, education level significantly influenced the use of BBT, p = 0.0006. Among users, more patients had college degrees (28 \[30%\]) as compared with nonusers, (12 \[12%\], p = 0.003). In contrast, 42 (40%) nonusers finished high school versus 23 (24%) of users, p = 0.023. There were no significant differences between users and nonusers in other demographic variables. Cardiovascular diseases, other medical conditions and medications used ---------------------------------------------------------------------- The distribution of cardiovascular and noncardiovascular diseases reported by the sample is presented in Table [2](#T2){ref-type="table"}. The mean number of disease diagnoses carried by users and nonusers were 4 (range 1 -- 10) and 3 (range 1 -- 8), respectively, p = 0.723, while the mean number of cardiovascular diagnoses for users versus nonusers were 3 (range 1 -- 6) and 3 (range 1 -- 5), respectively, p = 0.134. The most common cardiovascular conditions diagnosed in users and nonusers were hypertension, 56 (24%) and 65 (27%), followed closely by coronary heart disease, 45 (19%) and 54 (18%), respectively. Distribution of other cardiovascular diseases between users and nonusers was similar as well. Similarly, the mean number of noncardiovascular diagnoses reported by users and nonusers of BBT were not significantly different: 2 (range 1 -- 5) and 1 (range 1 -- 5), respectively, p = 0.288. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Distribution of Cardiovascular and Noncardiovascular Diseases ::: Users Nonusers p value -------------------------------------------------- ----------- -------------- ------------- Average number of diseases 4 3 NSS Range 1 -- 10 1 -- 8 Average number of cardiovascular diseases 3 3 NSS Arrhythmia 25 32 Congestive Heart Failure 38 52 Coronary Heart Disease 45 54 Hyperlipidemia 31 43 Hypertension 56 65 Other^1^ 12 14 Post-heart Transplant Recipient 1 2 Thromboembolic Disease 13 16 Valvular Heart Disease 11 14 Average number of noncardiovascular diseases 2 1 NSS Arthritis 25 17 Cancer 3 4 Diabetes Mellitis 33 33 Gastrointestinal Disease 19 13 Hypothyroidism 5 11 Nervous System Disorder 8 9 Ocular Disease 6 6 Other^2^ 30 13 Pulmonary Disease 12 13 Renal Disease 7 12 ^1^aneurysm, peripheral vascular disease, infective endocarditis, pulmonary hypertension, congenital heart disease; ^2^allergic rhinitis, scleroderma, systemic lupus erythematosis, Raynaud\'s disease, benign prostatic disease, osteoporosis ::: The mean number of traditional prescription and nonprescription medications taken by the users of BBT (7 \[range 1--15\]) and nonusers (7 \[range 0--18\]) was not significantly different, p = 0.445. The most common cardiovascular medications prescribed for both users and nonusers were diuretics (71%, 73%), followed by aspirin (56%, 43%) and beta-blockers (49%, 50%). The cardiovascular and noncardiovascular medications taken by both groups were not different. Types and patterns of BBT use ----------------------------- The BBT products utilized by cardiovascular patients are presented in Table [3](#T3){ref-type="table"}. The mean number of BBT products utilized by cardiovascular patients was two. Vitamin E (41, \[44%\]) was the most commonly utilized BBT, followed by vitamin C, (30 \[32%\]). The prevalence of the remaining BBT is summarized in Table [3](#T3){ref-type="table"}. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### All Biological Based Therapies Utilized by Cardiovascular Patients (at any time) ::: Product Number Reasons for Use (per patient) ------------------------- ------------ ------------------------------------------------------------------------------------------------------------------------------------------------ AA\#5 (anti-arthritis) 1 treat arthritis Acidophillus 1 health Aloe vera 4 headache, stomach gas, skin pigmentation Atomic Drops 1 treat headache Bee Pollen 1 prevent cold Beta carotene 5 maintain good health, energy, improve heart contraction Bioflavinoid 1 bone health instructed by chiropractor Calcium 19 supplement, prevent osteoporosis, improve heart function Chamomile 2 stomach ache, improve heart condition Chromium picolante 2 supplement for heart condition, muscle strength Coenzyme Q 10 11 improve heart contraction, supplement to diet Dexatrim 1 weight lost DHEA^1^ 1 supplement Echinacea 5 prevent or treat cold, flu, stay healthy, boost immune system Fish Oil 12 decrease cholesterol, maintain circulation and good health, scleroderma Folic Acid 3 supplement, help with heart condition, sickle cell anemia Garlic 5 decrease cholesterol, help maintain good health Ginkgo biloba 5 antioxidant, enhance memory, energy Ginseng 4 increase energy, stamina, virility Glucosamine/chondroitin 10 treat arthritis, decrease joint pain Golden seal 1 body cleaner Grapeseed oil 1 preserve health Green tea 3 decrease cholesterol, improve circulation Mixed herbal tea 1 Sooth stomach upset, anxiety Insulin leaf tea 1 diabetes Iron supplement 4 anemia, increase energy Lecithin 2 improve heart condition and circulation, decrease cholesterol Alpha-Linolenic acid 1 improve heart condition Magnesium 8 supplement, antioxidant, improve metabolism, improve heart function Multivitamins 24 supplement, energy, sickle cell anemia Primrose oil 1 scleroderma Saint John\'s Wart 1 depression Saw palmetto 1 for prostate health Selenium 4 antioxidant, supplement, improve heart condition Strong bark 1 help with heart condition Valerian 4 decrease anxiety, improve sleep, to decrease blood pressure Vitamin B complex 17 supplement, improve heart condition, decrease leg cramps, energy Vitamin B12 2 supplement, anemia Vitamin C 30 antioxidant, supplement, help with heart condition, improve circulation, strengthen immune system, prevent or treat cold Vitamin E 41 antioxidant, supplement, help with heart condition, increase energy, decrease cholesterol, improve circulation, thin blood, treat hypertension Yohimbine 1 increase energy, stamina, virility Zinc 4 supplement ^1^dehydroepiandrosterone ::: The patients reported that their physicians were aware of their BBT use in 60% of the instances and pharmacists were aware in 32% of the cases. Only 33% of users reported that they were asked about BBT use during a history/physical examination by a health care professional. The patients were not surveyed about their pharmacists\' assessments of BBT use. Perceived benefits and attitudes towards BBT -------------------------------------------- A greater percentage of users (74.5%) of BBT reported that they believed these products to be safe substances as compared with nonusers (26.2%), p \< 0.001. Likewise, significantly more users believed that BBT was effective (70.2%) compared to nonusers (30.1%), p \< 0.001. More nonusers (72%) than users (45%) did not know whether BBT products work better, as well as, or worse than traditional medications, p \< 0.001. More users than nonusers believed that BBT works as well as traditional medications (30.9% versus 9.7%, p \< 0.001). Concerning adverse effects, more users (44.7%) of BBT than nonusers (17.5%) believed that BBT causes fewer side effects than traditional medications, p \< 0.001. At the same time, 32.2% of users and 62.1% of nonusers did not know whether BBT causes more or fewer side effects than traditional prescription medications, p \< 0.001. Potential drug-BBT interactions ------------------------------- Examination of patients\' prescription and nonprescription medication profiles and BBT utilized, revealed 42 potential drug-BBT interactions. The onset of the interaction and the degree of severity were classified according to that used by the MicroMedex HealthCare Series Integraded Index \[[@B28]\] and the published literature \[[@B29]-[@B43]\]. Suspected or potential interactions were communicated to the patients and their primary physicians. The most common interaction identified was coadministration of aspirin and vitamin E (16 cases) that could potentially result in an increased risk of bleeding due to additive inhibition of platelet aggregation \[[@B29]-[@B31]\]. Similar effects may result from coadministration of clopidogrel and vitamin E; this potential interaction was recognized in five cases \[[@B31]\]. Five instances of potential interaction between warfarin and vitamin E were identified and close monitoring of the International Normalized Ratio (INR) was recommended \[[@B32]-[@B34]\]. A list of other potential drug-BBT interactions that were identified is presented in Table [4](#T4){ref-type="table"}. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Potential Drug-Biological Based Therapies Interactions & Management ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Medication Alternative Pharmacotherapy Product Potential Interaction and Management Number of Patients Onset^28^\* Level of Severity^28^ ---------------- ----------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------ ----------------- --------------------------- Warfarin Green tea Green tea may antagonize warfarin effects. Monitor INR. 1 Delayed Moderate Warfarin Coenzyme Q 10 Coenzyme Q 10 may have procoagulant effects and may decrease response to warfarin.\ 2 Delayed Moderate Monitor INR. Warfarin Vitamin E Vitamin E may potentiate warfarin effects. Monitor INR and signs and symptoms of bleeding. 5 Delayed Moderate Warfarin Garlic Garlic has antiplatelet effects, and risk of bleeding may be increased.\ 1 Delayed Major Monitor INR and signs and symptoms of bleeding. Warfarin Ginkgo biloba Ginkgo inhibits platelet aggregation, risk of bleeding may be increased.\ 2 Delayed Major Monitor INR and signs and symptoms of bleeding Warfarin Fish oils Concomitant use of warfarin and fish oils may increase risk of bleeding.\ 1 Not specified Not specified Monitor INR and signs and symptoms of bleeding. Aspirin Vitamin E Coadministration of vitamin E and aspirin may increase the risk of bleeding. Monitor for signs and symptoms of bleeding 16 Not Specified Not specified Aspirin Garlic Additive antiplatelet effects may occur with coadministration of garlic and aspirin, risk of bleeding may be increased. Monitor for signs and symptoms of bleeding 1 Delayed Moderate Aspirin Fish oils Concomitant use of aspirin and fish oils may increase risk of bleeding.\ 3 Not specified Not specified Monitor for signs and symptoms of bleeding Aspirin Ginko biloba Ginkgo inhibits platelet aggregation; risk of bleeding may be increased.\ 1 Delayed Major Monitor for signs and symptoms of bleeding. Clopidogrel Vitamin E Concomitant use of vitamin E and clopidogrel may increase the risk of bleeding. Monitor for signs and symptoms of bleeding 5 Not specified Not specified Clopidogrel Garlic Additive antiplatelet effects may occur, risk of bleeding may be increased. Monitor for signs and symptoms of bleeding 1 Delayed Major Clopidogrel Coenzyme Q Coenzyme Q 10 may have procoagulant effects, may partially antagonize antiplatelet effect of clopidogrel. 1 Not specified Not specified Insulin Insulin leaf Enhanced hypoglycemic effects.\ 1 Delayed Moderate Patient was advised to discontinue insulin leaf while receiving insulin for glycemic control. Paroxetine Saint John\'s Wort Saint John\'s Wort induces cytochrome P450 3A4 enzyme and has mechanism of action similar to serotonin reuptake inhibitors.\ 1 Rapid Severe Administering it concomitantly with paroxetine will enhance the toxicity of paroxetine. Patient was advised to discontinue the use of Saint John\'s Wort ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ INR = International Normalized Ratio \* Delayed onset of interactions occurs after multiple doses of both agents. ::: Discussion ========== Despite a wide array of available BBT for cardiovascular conditions, studies evaluating the prevalence of their usage are limited. A review of the literature at the time of study initiation (January 2001) and more recently (June 2004) identified five studies in this area, and only a few included a broad spectrum of cardiovascular disease patients \[[@B21]-[@B25]\]. Wood et al. conducted a telephone survey in 107 patients randomly selected from the \"Improving Cardiovascular Outcomes in Nova Scotia\" database \[[@B24]\]. A majority of patients (64%) in this study utilized CAM. Ackman and colleagues evaluated the patterns of BBT use by patients with congestive heart failure (CHF) \[[@B21]\]. Out of 180 CHF patients, 59% used vitamins and minerals and 38% used herbal or health food products. Liu and associates evaluated the prevalence of CAM used in 263 patients undergoing cardiovascular surgery \[[@B22]\]. Seventy-five percent of respondents utilized CAM including vitamins (53.6%), nutritional therapy (17.1%) and herbs (9.9%). Compared with non-CAM users, users were older (p = 0.027), belonged to the Caucasian racial group, (p = 0.001) and had a higher level of education (p = 0.017). Additionally, the evaluation of attitudes towards the effectiveness of CAM revealed that users were more likely to believe that CAM would work in a complimentary manner with conventional medical treatments, p \< 0.05. Furthermore, more users than nonusers believed that CAM would promote general health and wellness, p \< 0.05. Of the patients surveyed, only 17% reported discussing CAM with their medical doctors. Another recent study evaluated the use of CAM among 246 patients attending a cardiac clinic prior to cardiac surgery \[[@B23]\]. A total of 182 (80.9%) patients used CAM, and 12.9% utilized megavitamins. Another study, conducted in Canada, focused on the use of over-the-counter medications and herbal products among patients with cardiac diseases, the majority of whom were diagnosed with coronary artery disease (74%) \[[@B25]\]. The authors reported that 23% of the patients used multivitamin or multivitamin/mineral products. Overall, the results of these five studies confirm a high prevalence of all kinds of CAM used, including BBT among patients with cardiovascular diseases. Biological based therapy used by cardiovascular patients -------------------------------------------------------- In our study, the lifetime prevalence of BBT use in the sample of 198 cardiovascular patients was 47.5%, which is very similar to the results reported by Eisenberg and colleagues in the general population (42%) \[[@B3]\]. Comparisons with other investigations of alternative medicine use in cardiovascular patients are difficult to make due to the various definitions of \"alternative medicine\" \[[@B21]-[@B25]\]. Some studies included items such as alternative procedures including acupuncture, while others included \"pharmacotherapy\" only (herbs, BBT, CAM and nutritional supplements). In addition, certain studies looked at only one cardiovascular disease while others looked at a broad range of patients with multiple diseases. Furthermore, different studies also used different measurements of incidence and prevalence; i.e., some investigators focused on lifetime use, others reviewed the previous 12-month history only, yet others limited their findings to BBT use in the 14 days prior to the investigation. Regardless, results indicated that a high percentage of patients with cardiovascular diseases are taking some kind of BBT (almost one out of every two patients). Health care professionals need to be aware of these findings and routinely inquire about BBT use by patients when taking a medication history. Similar to Ackman\'s study \[[@B21]\], the most frequently utilized BBT in the present study of cardiovascular patients was vitamin E, (41 \[43.6%\]). The popularity of vitamin E among cardiovascular patients is not surprising due to the alleged benefits of this vitamin in heart disease from early literature \[[@B12]-[@B14]\], and its relative availability in pharmacies, health food stores and supermarkets. As was the case with vitamin E, the use of vitamin C in the current investigation, (30 \[31.9%\]) was similar to previously reported results, being the second most common product utilized by cardiovascular patients \[[@B21]\]. Similar to other studies \[[@B1],[@B3],[@B6],[@B21]\], the current study confirmed that based on the patients\' reports, a high percentage of physicians (40%) and pharmacists (68%) were not aware of BBT used by cardiovascular patients. Use of BBT by the patients in this study was not routinely discussed with health care providers during medical history evaluation (33%). This is an alarming but not an isolated finding. On average, cardiovascular patients consumed 7 (range, 1 -- 18) prescribed medications, and 2 (range, 1 -- 12) BBT products. Considering the complex medical treatment received by cardiovascular patients, addition of unmonitored BBT to the patients\' regimens may place the users of these therapies at a greater risk for the development of adverse events and interactions with prescribed medications. As addressed by this analysis, 42 potential drug-BBT interactions were identified. Demographics impact on biological based therapy use in cardiovascular patients ------------------------------------------------------------------------------ Among the demographic variables collected in this analysis, no parameter other than the level of education had a significant impact on whether patients used BBT. The present investigation revealed that the education level among users (63% received some college or college and graduate school education) of BBT was higher than that of the nonusers (32%), p \< 0.001. This finding has also been reported in other studies \[[@B3],[@B6]\]. Higher level of education has been shown to significantly influence the use of alternative products and services \[[@B3]\]. Education level is sometimes directly related to economic status, thus the patients may have more resources to spend on BBT. Similarly, better-educated consumers may be more likely to be exposed to various less conventional forms of healthcare reading about their illnesses and treatment options. Educated patients also might be less inclined to accept their physicians\' knowledge and expertise, and may seek other treatment options. Perceived safety and efficacy of BBT ------------------------------------ The majority of BBT users (as compared to nonusers) in the current investigation believed that BBT products are safe, effective and cause fewer side effects as compared to prescription medications. These results are consistent with the findings by other investigators \[[@B6],[@B21]\], supporting a logical conclusion that patients who believe that BBT are safe and effective, are more likely to use them. Study limitations ----------------- Several limitations of the study need to be addressed. Those that are intrinsically related to survey data collection in general include potential bias of responders and nonresponders. Since participation in this study was voluntary, patients who chose to participate in the study may be more motivated and knowledgeable about the subject of BBT as compared to those who refused to participate. Therefore, the results of this study may not be applicable to the entire cardiovascular patient population. Also, a cross-sectional nature of this study precludes drawing any definitive conclusions regarding cause and effect relationships. For example, one cannot definitively conclude that more education will significantly effect one\'s decision to take BBT because other factors such as exposure to these types of products may have influenced more educated consumers to take these products. As with other surveys, recall bias cannot be eliminated as data collection relied on patients\' self-report, rather than objectively documenting BBT use. Additionally, with surveys there is always a possibility of misunderstanding the questions and responses, miscommunication between the interviewer and the patients, and inaccurate recording of the information. The majority of the survey collected factual information (i.e., demographics and BBT usage history). However, the questions that evaluated patients\' perception on the safety and efficacy of conventional medicine and BBT were not validated. This study was conducted in a large urban inner city hospital; the results may not be extrapolated to other cities or clinical settings. Another limitation may be the exclusion of non-English speaking patients, which may be particularly important since there are certain ethnic groups who are documented to use more BBT than are other ethnic groups (e.g., persons of or from Chinese descent utilize traditional Chinese herbal medications). Finally, terminology varies greatly in the published literature. Some authors use the term CAM in all instances, others differentiate CAM from BBT, etc. As a result, it is very difficult to compare published reports with certainty. Conclusion ========== In recent years, the interest of using BBT in disease management has increased dramatically in the medical and layman communities. The amount of valid scientific research in this area of therapy continues to increase. Yet, there are still many unknowns concerning BBT, especially in the area of adverse effects and drug interactions. The finding of a high prevalence of BBT (47.5%) use among cardiovascular patients and the lack of communication between patients and their physicians/pharmacists should be addressed by the health care community. Higher education level, as shown in the present study and other previous investigations \[[@B1],[@B3],[@B6],[@B22]\], is associated with an increased use of BBT, but it does not necessarily mean that these patients are aware of the potential detrimental effects of BBT, as demonstrated in the current study. In cardiovascular patients, the perceived effectiveness and safety of BBT, and assumed lack of side effects of these products as opposed to traditional medications, highlights an area for further education. A high incidence of potential drug-BBT interactions was also identified in this study (42 interactions in 94 users). Given that the use of BBT can have a direct effect on patient care, and users of these therapies do not always voluntarily report their use of these products to their providers, health care professionals need to inquire about BBT use routinely. Collecting complete patient histories and educating patients about potential dangers and possibilities of adverse effects and interactions between prescription medications and BBT (or other CAM) will lead to better overall patient care. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= LC participated in literature review, protocol development, collected and analyzed data, and submitted the manuscript for publication. DB collected the data. JWMC participated in study design, data analysis and preparation of manuscript. VR participated in design development and data analysis. HLK proposed the project and reviewed the manuscript. GCC, JM and BM participated in protocol implementation and manuscript review. All authors read and approved the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6882/5/4/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Appendix A -- Biological based therapy survey, It\'s a survey tool utilized in the study to collect patient data. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ The authors would like to acknowledge LilyAnn Jeu and Urania Rappo for their assistance in data collection.	PubMed Central	2024-06-05T03:55:54.100143	2005-3-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555537/", "journal": "BMC Complement Altern Med. 2005 Mar 3; 5:4", "authors": [ { "first": "Larisa", "last": "Chagan" }, { "first": "Diane", "last": "Bernstein" }, { "first": "Judy WM", "last": "Cheng" }, { "first": "Harold L", "last": "Kirschenbaum" }, { "first": "Vitalina", "last": "Rozenfeld" }, { "first": "Gina C", "last": "Caliendo" }, { "first": "Joanne", "last": "Meyer" }, { "first": "Bernard", "last": "Mehl" } ] }
PMC555538	Background ========== Desmoid tumours, which are frequently observed in Gardner\'s syndrome, are rare, slow-growing, histologically benign tumours caused by autosomal dominant gene mutation \[[@B1],[@B2]\]. They are, however, locally aggressive, compress surrounding structures and show frequent recurrences after surgical removal. Desmoid cells are characterized by abundant deposition of organic macromolecules in the extracellular matrix (ECM), by enhanced transforming growth factor β~1~(TGFβ~1~) gene expression and increased protein secretion \[[@B3]\]. Cell proliferation, angiogenesis and the accumulation of ECM macromolecules are all facilitated by tumour cell production of TGFβ~1~\[[@B3]-[@B6]\]. All components of ECM are degraded by matrix metalloproteinases (MMPs), a family of zinc-dependent neutral endopeptidases \[[@B7]\]. Two types of MMPs are required for dissolution of interstitial collagen: collagenases and gelatinases \[[@B8]\]. Collagenase-1 (MMP-1), collagenase-2 (MMP-8) and collagenase-3 (MMP-13) are the principal secreted neutral proteinases that initiate degradation of native fibrillar collagens of type I, II, III and V. They all cleave fibrillar collagens at a specific site, resulting in the generation of N-terminal 3/4 and C-terminal 1/4 fragments, which are further degraded by gelatinases \[[@B7],[@B9],[@B10]\]. Gelatinase-A (MMP-2) is expressed by several types of cells, especially fibroblasts, whereas gelatinase-B (MMP-9) is restricted to epithelial cells. MMP-2 and MMP-9 are thought to play major roles in the final degradation of fibrillar collagens after first cleavage by collagenases and denaturation \[[@B11]\]. MMP-2 also cleaves native type I collagen to N-terminal 3/4 and C-terminal 1/4 fragments which are identical to those generated by collagenases \[[@B12]\]. Several different tissue inhibitors of matrix metalloproteinases (TIMPs; TIMP-1 to TIMP-4) have been identified as the major natural inhibitors of MMPs \[[@B13]\]. TIMP-1 and TIMP-2 inhibit the activity of most MMPs \[[@B11]\]. Expression of TIMP-1 is up-regulated at the transcription level by various growth factors such as TGFβ~1~, whereas TIMP-2 is largely expressed constitutively by cultured cells \[[@B14]\]. Our previous studies showed desmoid fibroblasts enhanced deposition of organic macromolecules in the ECM and TGFβ~1~secretion \[[@B3]\]. Even if desmoid cells do not have estrogen receptors \[[@B3]\], adding toremifene, an antiestrogenic triphenylethylene derivate, decreased TGFβ~1~production and ECM macromolecule accumulation through a mechanism of action that still remains unclear \[[@B3],[@B6],[@B15],[@B16]\]. The present study investigates the rule of MMPs and TIMPs in the desmoid tumour and describes, for the first time, the effects of toremifene on MMPs and TIMPs. The results provided evidence that toremifene reduced ECM accumulation by decreasing collagen synthesis and increasing collagen degradation. Methods ======= Antiestrogen ------------ Toremifene (4-chloro-1,2-diphenyl-1-{4-\[2-(N,N-dimethylamino) ethoxy\] phenyl}-1-butene) citrate was purchased from Farmos (Farmos Group Ltd, Finland). Cell cultures ------------- Fibroblast cell lines were obtained from patients with Gardner\'s syndrome and were provided by NIGMS (Camden, N.J.). The GMO 6965 cell line was obtained from phenotypically healthy fibroblasts, and the GMO 6888 cell line was obtained from desmoid fibroblasts. All cell lines were cultured in Eagle\'s minimal essential medium (MEM) (Sigma, St. Louis, MO) supplemented with 20% fetal bovine serum (FBS) (GIBCO-Invitrogen, Basel, Switzerland), 2% non-essential amino acids (GIBCO), 2 mM L-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin in a humidified 5% CO~2~atmosphere at 37°C. Confluent cultures were obtained after 48 h of in vitro maintenance. The cells were cultured for 12 h in MEM. The medium was then discarded to avoid serum factor contamination. Toremifene was dissolved in ethanol and all the cultures were maintained in MEM containing ethanol or MEM containing toremifne in ethanol and treated as described below. Cell viability -------------- Normal (control) and desmoid fibroblasts were cultured for 24 h in MEM and ethanol or MEM containing 1 μM toremifene in ethanol (final concentration 0.1% v/v). Then 50 μl of sterile 0.4% trypan blue solution (final concentration 0.05%) was added to each culture well; cultures were incubated at 37°C for 15 min. Viable cells (trypan blue negative) and dead cells (trypan blue positive) were counted by a Burker chamber. Collagen synthesis ------------------ Confluent cultures of normal (GMO 6965) and desmoid fibroblasts (GMO 6888) were cultured for 3, 24 and 48 h in MEM without serum supplemented L-ascorbic acid (50 μg/ml), β-aminopropionitrile fumarate (50 μg/ml), 8 μCi/ml of ^3^H-proline (specific activity 35 Ci/mmole, Amersham, Freiburg, Germany) in the presence or absence of 1 μM toremifene. In a second set of experiments desmoid fibroblasts were cultured in MEM supplemented with L-ascorbic acid (50 μg/ml), β-aminopropionitrile fumarate (50 μg/ml) for 48 h with or without toremifene. ^3^H-proline was added for 48 h, for the last 24 and for the last 3 h. At the end of the labelling period collagen was extracted using the method of Webster and Harvey \[[@B17]\]. Samples were digested with pepsin (1 mg/ml) in mild agitation overnight at 4°C. Collagen was precipitated and redissolved in 500 μl cold acetic acid 0.5 M. Total radioactivity was counted in a liquid scintillation counter and expressed as cpm/μg protein. Northern blot analysis of procollagen α~1~(I) --------------------------------------------- Total RNA was isolated from confluent cultures of normal and desmoid fibroblasts maintained for 48 h in MEM alone or supplemented with 1 μM toremifene using the method of Chomczynski and Sacchi \[[@B18]\]. For Northern blot analysis equal amounts of total RNA (20 μg) were electrophoresed on 1% agarose gel containing 0,66 M formaldehyde and transferred on to nylon filters (Hybond N, Amersham). Before blotting, the gel was rinsed in water for 15 min at room temperature and then in 20X SSC (1 X SSC is 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7) for 10 min. Blots were pre-hybridised in 20 ml of a cocktail containing 1 mM EDTA pH 8, 0.25 M Na~2~HPO~4~pH 7.2 and 7% sodium dodecyl sulfate (SDS) for 4 h at 65°C. Probes were labelled with \[α-^32^P\] dCTP (3000 Ci/mM) by random priming (Amersham RPN 1601). Hybridisation was performed at 65°C overnight using 10^6^cpm/ml probe in the same buffer used for pre-hybridisation. After hybridisation, the nylon membrane was washed twice in 1 mM EDTA pH 8, 20 mM Na~2~HPO~4~pH 7.2 and 5% SDS at 65°C (5 min each) and then washed twice with 1 mM EDTA pH 8, 20 mM Na~2~HPO~4~pH 7.2 and 1% SDS at 65°C (5 min each). The filters were stripped and re-hybridised with a GAPDH probe to assess blot loading. For autoradiography the membranes were exposed to Kodak X-Omat film (Rochester, NY) at -80°C for 1 day. Autoradiographies were analysed by computerised scanning densitometry. Results are expressed as the ratio of procollagen α1(I)/control GAPDH densitometry signals. cDNA probes ----------- A 670 bp Eco RI-Hind III cDNA fragment from human pro-collagen α1(I) and a 1.3 kb PstI cDNA fragment from rat glyceraldehyde-3-phosphate dehydrogenase were used as probes in hybridisation \[[@B18]\]. Collagenase activity -------------------- Collagenase activity was determined using the method of Khorramizadeh et al., \[[@B19]\]. Confluent normal and desmoid fibroblasts were washed with MEM and cultured for 48 h in MEM or in MEM containing 1 μM toremifene. Proteins in the medium were precipitated by ammonium sulphate 65% w/v; precipitates were collected by centrifugation, dissolved in assay buffer (0.05 M Tris-HCl, 0.2 M NaCl, 5 mM CaCl~2~, 0.02% sodium azide, pH 7.4) and then dialysed overnight against the same buffer. The latent procollagenase was activated with trypsin (10 μg/ml) and then the trypsin was inactivated with soybean trypsin inhibitor (100 μg/ml). Acetic acid soluble type I collagen (25 μl of a solution 2 mg/ml) from bovine skin was incubated with the activated collagenase solution for 24 h. The products of the collagen digestion were separated by electrophoresis using 6% acrylamide gel containing SDS. The gels were stained with 0.25% Coomassie brilliant blue G-250 (50% methanol, 10% acetic acid), destained appropriately (40% methanol, 10% acetic acid) and fixed (5% methanol, 7.5% acetic acid). The digestion products were quantified with a computerised scanner. Preparation of conditioned media (CM) ------------------------------------- Confluent normal (GMO 6965) and desmoid (GMO 6888) fibroblasts were washed with 0.9% NaCl and cultured for 12 h in serum-free MEM. This medium was discarded to avoid contamination by serum factors and cells were cultured for the next 24 h in MEM and ethanol (control) or in MEM containing 1 μM toremifene in ethanol (final ethanol concentration 0.1% v/v). Conditioned media (CM) were collected and centrifuged for 10 min at 350 gto remove cell debris, dialysed against bidistilled water for 24 h, lyophilised and used for zymography and Western blot analysis as described below. Collagen and gelatin zymography ------------------------------- CM were analysed for gelatinases and collagenases by zymography. Samples were separated under non reducing condition on 6% polyacrylamide gels containing 1 mg/ml of gelatin (Sigma Chemical, St Louis; MO, USA) or 1 mg/ml collagen (Sigma) \[[@B20]\]. In one set of samples the proenzymatic forms were activated using 2 mM aminophenylmercuric acetate (APMA) for 1 h at 37°C. Samples were lyophilised and resuspended in Tris-HCl 0.4 M pH 6.8, SDS 5%, 20% glycerol and 0.03% bromophenol blue. Gels were loaded with 8 μg protein per sample or with 2 μg trypsin and run under Laemmli conditions \[[@B21]\]. After electrophoresis, gels were washed twice in 200 ml of 2.5% Triton X-100 (30 min each) under constant mechanical stirring and incubated in 50 mM Tris-HCl pH 7.5, 5 mM CaCl~2~, 0.02% Brij-35 and 200 mM NaCl at 37°C for 24 h. Gels were stained with Coomassie brilliant blue G-250. Proteinase activity, observed as cleared (unstained) regions, was converted to dark regions to better observation of bands. Western-blot analysis --------------------- CM were analysed for type I and type III collagen, MMP-1, MMP-2, MMP-9, TIMP-1 and TIMP-2 by Western blotting using specific monoclonal antibodies. Aliquots of CM, containing 50 μg of proteins, were separated on SDS-10% polyacrylamide gels under reducing conditions and transferred on to a nitrocellulose membrane. The membrane was blocked with blocking solution (5% w/v dried skimmed-milk powder in TBS 1X, 2 h at room temperature) and incubated with the specific monoclonal antibody in antibody solution (1% w/v dried skimmed-milk powder in TBS 1X, 2 h at room temperature). Bound antibody was detected with a sheep anti-mouse peroxidase-conjugated antibody in antibody solution. Western analysis was performed using chemiluminescence reagents from Amersham Pharmacia Biotech. Protein determination --------------------- Protein concentrations were determined by the Lowry assay \[[@B22]\] of aliquots of cell lysate. Statistical analysis -------------------- In some experiments, statistical analysis was performed using Student\'s t-test. Data are expressed as the means ± SD of four determinations. In other experiments, the results are reported as means ± SD of three separate experiments, each performed in quadruplicate. Statistical analysis was performed by Student\'s two-tailed t-test and by analysis of variance (ANOVA) followed by Sheffe F-test. Results ======= Cell viability -------------- The amount of dead cells and viable cells in normal fibroblasts, desmoid fibroblasts and desmoid fibroblasts plus toremifene was evaluated after 24 h of in vitro maintenance in the presence of trypan blue (Table [1](#T1){ref-type="table"}). Granted that the number of intact viable cells was high in all the experimental conditions, desmoid fibroblasts had the highest number of cells/culture and the lowest percentage of dead cells (0.0014%). Treatment of desmoid fibroblasts with toremifene enhanced the percentage of dead cells (0.011%) which, nevertheless, remained lower than in normal fibroblasts (0.025%.). Effects of toremifene on collagen synthesis ------------------------------------------- Collagen synthesis was evaluated after 3, 24 and 48 h of in vitro maintenance in the presence of ^3^H-proline (Table [2](#T2){ref-type="table"}). No significant difference was observed after 3 hours culture. After 24 and 48 h culture collagen production was significantly higher in desmoid than in normal fibroblasts, in both the cellular and extracellular compartments. The increase was 1.4 fold in the cells and 1.8 fold in the medium after 24 h; 1.3 fold in the cells and 1.8 fold in the medium after 48 h. Adding toremifene significantly decreased collagen synthesis at 24 and at 48 h. The reduction was greater after 48 h (42% in the cells and 38% in the medium). In a second set of experiments desmoid fibroblasts were cultured for 48 h with or without toremifene. The radiolabelled precursor was added for 48 h, in the last 24 h and in the last 3 h (Table [3](#T3){ref-type="table"}). Treatment with toremifene had an inhibitory effect at all times. The decrease in total collagen (cells + media) in desmoid fibroblasts treated with toremifene was 28% in the presence of ^3^H-proline for 48 h, 46% and 52% respectively in the presence of ^3^H-proline in the last 24 or 3 h of in vitro maintenance (Table [3](#T3){ref-type="table"}). Procollagen α~1~(I) mRNA expression ----------------------------------- Northern blots were performed to analyse procollagen α~1~(I) mRNA level in normal and desmoid fibroblasts (Fig. [1](#F1){ref-type="fig"}). Relative densitometric units were normalised to GAPDH mRNA levels. Normal and desmoid fibroblasts exhibited no significant differences in the steady-state mRNA levels for procollagen α~1~(I). Toremifene down regulated procollagen mRNA expression by 58% in desmoid cells. Western-blot analysis of type I and III collagen ------------------------------------------------ Media from normal and desmoid fibroblasts with or without toremifene were analysed by Western blotting to evaluate the presence of type I and III collagen using specific monoclonal antibodies (Fig. [2](#F2){ref-type="fig"}). Densitometric tracing of the autoradiograms quantified collagen secretion. Desmoid fibroblasts secreted much more type I (1.6 fold) and III (2.2 fold) collagen than normal cells. Toremifene reduced type I and III collagen by 31% and 18% respectively in desmoid fibroblasts. Collagenase activity -------------------- Collagenases, from ammonium sulphate-precipitated proteins of media of normal fibroblasts, desmoid fibroblasts and desmoid fibroblasts treated with toremifene, were incubated with soluble collagen and the digested products were evaluated by gel electrophoresis. Collagenases in the medium of normal fibroblasts digested more α~1~and α~2~chains of type I collagen into their corresponding 3/4 and 1/4 fragments than the collagenase in desmoid fibroblasts (Fig. [3](#F3){ref-type="fig"}). When band staining intensity was quantified by densitometry, the abundance of the 3/4 and 1/4 products of collagenase digestion was significantly greater in normal than in desmoid fibroblasts. Adding toremifene to desmoid fibroblasts markedly increased collagenase activity as shown by the increased amount of 3/4 and 1/4 fragments of α~1~and α~2~chains (Fig. [3](#F3){ref-type="fig"}). Collagen and gelatin zymography ------------------------------- Collagen and gelatin zymograms dosed the enzymatic activity of collagenases and gelatinases. Collagen zymogram, reported in Fig. [4](#F4){ref-type="fig"} (panel A and B), showed the samples produced a band of 52 kDa corresponding to MMP-1. Densitometric analysis of the counts, assuming the value of normal fibroblasts as 100%, demonstrated 2.3 fold increase in the 52 kDa collagenase activity in desmoid fibroblasts. When desmoid fibroblasts were treated with toremifene, the level of collagenase activity in the media was only minimally affected. No bands were present in trypsin (Fig. [4](#F4){ref-type="fig"}, panel A, line C), which can degrade gelatin but not collagen, confirming that collagen has been degraded in panel A. The gelatin zymogram (Fig. [4](#F4){ref-type="fig"}, panel C and D) showed two bands, one of 92 kDa corresponding to MMP-9, the other of 66 kDa corresponding to MMP-2. Desmoid fibroblasts produced the same amount of MMP-9, and larger (about 2 fold) amounts of MMP-2, than normal fibroblasts Adding toremifene to desmoid fibroblasts increased only MMP-2 activity by about 1.32 fold. To verify whether the bands detected in the collagen and gelatin zymography were due only to MMPs, two control gels were washed and incubated in buffers containing 10 mM EDTA. No bands were detected after this treatment, which indicated that the bands obtained in collagen and gelatin zymographies were entirely due to MMP activity. Toremifene addition to desmoid cells was accompanied by no changes in gelatinase activity. One set of samples in collagen and gelatin zymograms was treated with APMA to activate the proenzymes. Activation of the proenzymatic form had no significant effects on collagenase activity (Fig. [4](#F4){ref-type="fig"}, panel B), but enhanced gelatinase activity in desmoid fibroblasts (Fig. [4](#F4){ref-type="fig"}, panel D). Western-blot analysis of MMP-1, MMP-2, MMP-9 -------------------------------------------- The presence of MMP-1, MMP-2, MMP-9 in the media of normal fibroblasts, desmoid fibroblasts and desmoid fibroblasts plus toremifene was evaluated by Western-blot analysis using specific monoclonal antibodies (Fig. [5](#F5){ref-type="fig"}). Western blot analysis of MMP-1 (Fig. [5](#F5){ref-type="fig"}, panel A) showed that the amount of the protein was higher in desmoid (2 fold) and in desmoid than in normal fibroblasts (2 fold). Toremifene exhibited no significant increase of MMP-1 in desmoid cells (about 2.2 fold). MMP-2 (Fig. [5](#F5){ref-type="fig"}, panel B) showed two bands, the first due to the proenzymatic form (72 kDa) and the second to the active form (66 kDa). MMP-2 was significantly increased in desmoid fibroblasts (2.2 fold) and even more in desmoid fibroblasts plus toremifene (3.2 fold) compared with normal fibroblasts. No significant differences emerged in the production of MMP-9 (Fig. [5](#F5){ref-type="fig"}, panel C). Western-blot analysis of TIMP-1 and TIMP-2 ------------------------------------------ Western blot analysis showed that desmoid fibroblasts produced about 7.2 and 3.4 fold TIMP-1 (Fig. [6](#F6){ref-type="fig"}, panel A) and TIMP-2 respectively (Fig. [6](#F6){ref-type="fig"}, panel B) than normal fibroblasts. Adding toremifene to desmoid fibroblasts decreased TIMP-1 by 18%, but had no effect on TIMP-2. Discussion ========== Desmoid tumour is a benign non-invasive and non-metastasising neoplasm with an abnormal ECM macromolecule deposition which is stimulated by TGFβ~1~\[[@B3],[@B23],[@B24]\]. The regulation of extracellular matrix dynamics is clearly complicated, involving a balance between the deposition of structural components such as collagen and their degradation by MMPs, i.e. collagenases and gelatinases. MMP activity is itself regulated by a variety of mechanisms, including a requirement for enzyme modification to elicit maximal enzymatic activity and the activity of specific TIMPs \[[@B25]\]. There is now evidence that desmoid cells undergo dramatic clinical response to toremifene, implying the drug has a direct effect upon fibroblasts. Our previous studies showed that toremifene significantly inhibited TGFβ~1~activity which was six fold higher in desmoid than in normal fibroblasts \[[@B3]\]. As desmoid tumour is also associated with abnormal collagen production \[[@B26]\], in the present study we examined the rate of collagen synthesis and degradation in the presence or absence of toremifene. In our experimental conditions, type I and III collagen accumulation in the intra- and extra-cellular compartments showed no differences after 3 h of in vitro maintenance, but increased significantly more after 24 and 48 h in desmoid fibroblasts than in normal fibroblasts. No increase in collagen after 3 hours suggests its accumulation in desmoid fibroblasts is due to inhibition of degradation rather than to increased synthesis. The results are confirmed by procollagen α~1~(I) gene expression, which showed mRNA levels were only lower in desmoid cells treated with toremifene. Normal and desmoid fibroblasts expressed different amounts of MMPs. Several studies suggest that MMPs are over-expressed in malignant tumour progression and facilitate both local tumour invasion and metastasis \[[@B27],[@B28]\]. Different MMPs may play distinct roles at different stages of tumour development \[[@B29]\]. They may form a network, in which a single MMP is crucial for the cleavage of certain native or partially degraded matrix components and for the activation of other latent MMPs. MMP-1 plays a pivotal role in cancer progression and poor prognosis in colon-rectal, oesophageal and gastric cancer has been correlated with high MMP-1 expression \[[@B25],[@B30]\]. Nishiota \[[@B31]\] showed MMP-1 is expressed more strongly in the cancer front of invasion. MMP-2 is increased in cancer tissue and its over-expression is correlated with tumour-related basement membrane degradation and vascular invasion \[[@B32],[@B33]\]. Therefore inhibition of the expression or activity of only one MMP could potentially reduce peritumoural proteolytic activity and tumour invasion \[[@B34]\]. In this study we investigated the metalloproteinases most involved in type I collagen degradation, i.e. MMP-1 (collagenase-1), MMP-2 (gelatinase-A) and MMP-9 (gelatinase-B) and their natural inhibitors TIMP-1 and TIMP-2 \[[@B11]\]. Moreover TIMP-2 is 10-fold more potent than TIMP-1 against MMP-2 \[[@B11]\] which is involved either in the final degradation of native collagen or in the initial degradation cleaving native type I collagen to 3/4 and 1/4 fragments identical to those generated by MMP-1 \[[@B12]\]. Using Western blot we showed no differences in MMP-9 production, while MMP-1 and MMP-2 were higher in desmoid than in normal fibroblasts. Collagen and gelatin zymograms, in which the proteolytic enzymes were separated from TIMPs before the assay, proved the activities of collagenase MMP-1 and gelatinase MMP-2, as dosed in conditioned media, were higher in desmoid than in normal fibroblasts. However, collagenase activity, in the presence of TIMPs, was reduced in desmoid compared to normal fibroblasts as shown by the lower amount of 3/4 and 1/4 fragments of fibrillar collagen in desmoid cells. Together these results indicated the higher MMP-1 and MMP-2 activity in desmoid cells was masked by a 7-fold increase in TIMP-1 and a 3-fold increase in TIMP-2. TIMP-1 is a potent inhibitor of apoptosis in many cells types, its up-regulation protects the cells against apoptotic stimuli \[[@B35]\]; hence, greater number of viable cells in desmoid tumour. Upregulation in both inhibitors of MMPs may explain why the Desmoid tumour is characterised by an abundant deposition of ECM macromolecules and is neither malignant nor invasive. Toremifene addition to desmoid fibroblasts reduced the accumulation of collagen fibres but its mechanism of action remains unclear. Toremifene increased MMP-1 and MMP-2 activity by 8% and 25% respectively and decreased TIMP-1 by 18%. Despite these modest effects type I collagen degradation in 3/4 and 1/4 fragments increased almost 4-fold. Conclusion ========== Our previous studies showed that TGFβ~1~was 6-fold higher in desmoid than in normal fibroblasts and that toremifene significantly reduced TGFβ~1~activity and TGFβ~1~membrane-receptors \[[@B3]\]. So the effects of toremifene on MMPs and TIMPs could be linked to its effects on TGFβ~1~because the growth factor enhances organic macromolecule accumulation in the ECM via a reduction in MMP-1 and MMP-2 \[[@B36]\] and an increase in TIMP-1 \[[@B37]\], so favouring tumour mass growth through an inhibition of ECM macromolecule degradation. In the light of these data the reduction of organic macromolecules in the ECM in the presence of toremifene can be ascribed to its inhibition not only of collagen synthesis, but also of TGFβ~1~activity. Further studies on the regulation of MMP activities may clarify the role of toremifene on ECM degradation and provide important clues about pathogenesis of desmoid tumour. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= CB carried out collagen synthesis, collagenase activity and drafted the manuscript. CL and GB participated in the design of the study and carried out Northern blot analysis. LM and GG carried out RT-PCR, zimography and oestrogen receptor assay. AB and LC carried out Western blot analysis and performed the statistical analysis. PL conceived of the study, and participated in its 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/1471-2407/5/22/prepub> Acknowledgements ================ We thank Dr. G.A. Boyd for help with the English translation. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Expression of procollagen α~1~(I) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA in normal and desmoid fibroblasts.Panel A: NF: normal fibroblasts; DF: desmoid fibroblasts; DFT: desmoid fibroblasts plus toremifene. Similar results were obtained in four separate experiments. In panel B the absolute counts, obtained by densitometric analysis, were converted to percentages of control value, assuming the untreated level of normal fibroblasts as 100%. ::: ![](1471-2407-5-22-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Western blot analysis of type I and III collagen secreted into the medium by normal and desmoid fibroblasts.Panel A. The samples are as follow: NF normal fibroblasts; DF desmoid fibroblasts; DFT desmoid fibroblasts plus toremifene. Similar results were obtained in four separate experiments. Panel B. The absolute counts, obtained by densitometric analysis, were converted to percentages assuming the level of normal fibroblasts as 100%. ::: ![](1471-2407-5-22-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Collagenase activity in media derived from normal, desmoid fibroblasts and desmoid fibroblasts plus toremifene.Panel A. NF: normal fibroblasts; DF: desmoid fibroblasts; DFT: desmoid fibroblasts plus toremifene. Similar results were obtained in four separate experiments. In panel B the quantity of 3/4 and 1/4 fragments of digested collagen was determined by densitometric analysis. The absolute counts were converted to percentages assuming the level of normal fibroblasts as 100%. ::: ![](1471-2407-5-22-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Zymogram of media from normal fibroblasts, desmoid fibroblasts, desmoid fibroblasts plus toremifene.Collagen zymogram. One set of samples was treated with APMA to activate the proenzymatic forms. Panel A. NF: normal fibroblasts, DF: desmoid fibroblasts, DFT: desmoid fibroblasts plus toremifene. In the same zymogram an aliquot of samples was activated with APMA: NF: normal fibroblasts, DF: desmoid fibroblasts, DFT: desmoid fibroblasts plus toremifene, C: trypsin. Similar results were obtained in four separate experiments. Panel B. The absolute counts, obtained by densitometric analysis, were converted to percentages assuming the level of normal fibroblasts as 100%. Gelatin zymogram. One set of samples was treated with APMA to activate the proenzymatic forms. Panel C. NF: normal fibroblasts, DF: desmoid fibroblasts, DFT: desmoid fibroblasts plus toremifene. In the same zymogram an aliquot of samples was activated with APMA: NF: normal fibroblasts, DF: desmoid fibroblasts, DFT: desmoid fibroblasts plus toremifene. Similar results were obtained in four separate experiments. Panel D. The absolute counts, obtained by densitometric analysis, were converted to percentages assuming the level of normal fibroblasts as 100%. ::: ![](1471-2407-5-22-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Western blot analysis of MMP-1, MMP-2, MMP-9 secreted into the medium.Panel A: MMP-1; panel B: MMP-2; panel C: MMP-9. The samples are as follows: NF, normal fibroblasts; DF, desmoid fibroblasts; DFT, desmoid fibroblasts plus toremifene. Similar results were obtained in four separate experiments. Panel D. The absolute counts, obtained by densitometric analysis, were converted to percentages assuming the level of normal fibroblasts as 100%. ND = not determined. ::: ![](1471-2407-5-22-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Western blot analysis of TIMP-1 and TIMP-2 secreted into the medium.Panel A; TIMP-1. Panel B; TIMP-2. The samples are as follows: NF, normal fibroblasts; DF, desmoid fibroblasts; DFT, desmoid fibroblasts plus toremifene. Similar results were obtained in four separate experiments. Panel C. The absolute counts, obtained by densitometric analysis, were converted to percentages assuming the level of normal fibroblasts as 100%. ::: ![](1471-2407-5-22-6) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Cell viability ::: Viable cells Dead cells \% of dead cells ---------------------------------- ---------------------- ------------ ------------------ Normal fibroblasts 1,224,805 ± 56,280 308 ± 26 0.025 Desmoid fibroblasts 1,636,710 ± 84,105\* 29 ± 4 0.0017 Desmoid fibroblasts + toremifene 1,393,900 ± 86,400 161 ± 15 0.011 Fibroblasts were cultured for 24 h in MEM containing ethanol with or without toremifene. Number of viable and dead cells/culture after exposure to trypan blue for 15 min. The values are the means ± SD of five cultures. \Significance vs normal fibroblasts: P \< 0.01. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### ^3^H-proline incorporation into collagen fibres ::: Cells Media ---------------------------------- --------------- ------------------ 3 h Normal fibroblasts 1,060 ± 85 998 ± 94 Desmoid fibroblasts 885 ± 60 991 ± 39 Desmoid fibroblasts + toremifene 1,112 ± 134 837 ± 36 24 h Normal fibroblasts 2,313 ± 99 6,523 ± 388 Desmoid fibroblasts 3,340 ± 205\ 11,641 ± 421\* Desmoid fibroblasts + toremifene 2,286 ± 124°° 9,962 ± 644°° 48 h Normal fibroblasts 4,262 ± 280 17,955 ± 1,073 Desmoid fibroblasts 5,649 ± 103\* 32,988 ± 1,321\* Desmoid fibroblasts + toremifene 3,295 ± 261° 20,407 ± 980° Fibroblasts were cultured for 3, 24 and 48 h in MEM containing ^3^H-proline with or without toremifene. Total radioactivity value is expressed in cpm/mg protein and represents the mean ± SD of three determinations, each in quadruplicate. The statistical analysis was performed with analysis of variance (ANOVA). Differences vs normal fibroblasts: F-test significant at \99%; differences vs desmoid fibroblasts: F-test significant at °99%, °°95%. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### ^3^H-proline incorporation into collagen fibres ::: Cells Media Cells + Media ---------------------------------- --------------- ---------------- --------------- 48 h Desmoid fibroblasts 6,852 ± 290 33,915 ± 2,307 40,767 Desmoid fibroblasts + toremifene 4,243 ± 361\ 25,097 ± 814\* 29,340 24 h Desmoid fibroblasts 4,252 ± 416 15,423 ± 1,100 19,675 Desmoid fibroblasts + toremifene 1,432 ± 101\* 9,221 ± 381\* 10,653 3 h Desmoid fibroblasts 1,176 ± 48 1,498 ± 56 2,674 Desmoid fibroblasts + toremifene 534 ± 31\* 745 ± 74\* 1,279 Fibroblasts were cultured for 48 h in MEM with or without toremifene. ^3^H-proline was added for 48 h, for the last 24 h and for the last 3 h. Total radioactivity value is expressed in cpm/mg protein and represents the mean ± SD of three determinations, each in quadruplicate. The statistical analysis was performed with analysis of variance (ANOVA). Differences vs desmoid fibroblasts: F-test significant at \*99%. :::	PubMed Central	2024-06-05T03:55:54.105605	2005-3-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555538/", "journal": "BMC Cancer. 2005 Mar 1; 5:22", "authors": [ { "first": "Chiara", "last": "Balducci" }, { "first": "Cinzia", "last": "Lilli" }, { "first": "Giordano", "last": "Stabellini" }, { "first": "Lorella", "last": "Marinucci" }, { "first": "Giammario", "last": "Giustozzi" }, { "first": "Alessio", "last": "Becchetti" }, { "first": "Lucio", "last": "Cagini" }, { "first": "Paola", "last": "Locci" } ] }
PMC555539	Background ========== Colorectal cancer is the third most common cancer diagnosed both in men and in women in the United States \[[@B1]\]. The American Cancer Society estimates that 104,950 new cases of colon cancer (48,290 men and 56,660 women) and 40,340 new cases of rectal cancer (23,530 men and 16,810 women) will be diagnosed in 2005 \[[@B1]\]. It is estimated that in the United States colorectal cancer will cause about 10% of all cancer-related deaths during 2005 with 56,290 deaths (28,540 men and 27,750 women) \[[@B1]\]. For 1998--2000 the lifetime probability of men in the United States developing cancer of the colon and rectum was 1 in 17; for women it was 1 in 18 \[[@B2]\]. Genetic factors account for only 10% of colorectal cancers \[[@B3],[@B4]\] and thus environmental factors must also be involved in colon cancer development. Up to 80% of all colorectal cancer cases and deaths are attributable to diet \[[@B5]\] and such cases of colorectal cancer and related deaths may be prevented by dietary modifications \[[@B6]\]. Studies have suggested that a diet with an increased intake of fruit and vegetables correlates with a reduced risk of colorectal cancer \[[@B7],[@B8]\]. For this, the chemical components of plants known as phytochemicals may be crucially involved \[[@B9]\]. Particular phytochemicals characterized by their phenolic ring structures are termed polyphenols and the most abundant and widely distributed of these are the flavonoids. Quercetin is the most widely distributed flavonoid found in foods, and is most abundant in apples, onions, black tea and red wine \[[@B10]\]. Studies in vitroand in vivohave suggested that quercetin may have a protective role against breast \[[@B11]\], lung \[[@B12]\], liver \[[@B13]\], ovarian \[[@B14]\] and colon \[[@B15]-[@B17]\] cancers. Knowledge of how quercetin protects against cancer in general and colorectal cancer in particular could be gained by examining how quercetin affects the proteome of colon cancer cells. Specifically, the influence of quercetin on protein expression in the SW480 colon adenocarcinoma cell line could be instructive in elucidating the mechanisms underlying the protective role of the flavonoid against colon cancer. This would strengthen the scientific evidence for advocating a diet rich in fruit and vegetables to decrease the risk of developing colon cancer. Moreover, an identification of those proteins affected by quercetin could enhance understanding of the roles of these proteins in colon neoplasia. Use of proteomic techniques was therefore adopted in this study to determine the influence of quercetin treatment on protein expression in SW480 colon cancer cells. Quercetin treatment was found to result consistently in the decreased expression of three proteins and the increased expression of one protein. All, except one of the down-expressed proteins, were identified by mass spectrometry. Thus protein targets that could be the molecular basis of inhibition of colon cancer by quercetin were obtained. Methods ======= Cell culture ------------ SW480 human colon carcinoma cells (ATCC, Rockville, MD) were grown in Leibovitz\'s L-15 medium with 2 mM L-glutamine (ATCC, Rockville, MD), supplemented with 10% fetal bovine serum (Equitech-Bio Inc., Kerrville, TX). Cells were maintained in 75 cm^2^canted-neck flasks in 15 ml medium and incubated at 37°C without CO~2~. They were subcultured once per week at a ratio of 1:4. Quercetin treatment ------------------- When cells were about 90% confluent at seven days after passage the medium was discarded and replaced with medium containing 20 μM quercetin (Sigma, St. Louis MO). Cells were incubated for 48 h in the quercetin-containing medium at 37°C in air. Isolation of protein -------------------- Following incubation of the cells in quercetin for 48 h the medium was discarded and the cells washed three times for 1 min each time with phosphate-buffered saline. After complete removal of the final wash buffer, to each culture flask was added 240 μl boiling sample buffer I (0.3% sodium dodecyl sulfate (SDS), 200 mM dithiothreitol (DTT), 50 mM Tris-HCl, pH 8.0). The lysed cells were scraped together using a cell scraper (Fisher, Pittsburgh PA). The lysate from each culture flask was transferred to a 1.5 ml microfuge tube and heated for 5 min at 100°C. After chilling the tube on ice 24 μl (1/10 volume) of sample buffer II (50 mM magnesium chloride, 0.1% DNAse I, 0.025% RNAse A, 0.5 M Tris-HCl, pH 8.0) was added. Acetone was added to 80% (v/v) and the tube incubated on ice for 20 min. After centrifugation at 12,000 rpm for 10 min the supernatant was discarded and the pellet resuspended in 240 μl freshly prepared immobilized pH gradient (IPG) sample buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% DTT and 2% pH 3--10 ampholytes). The protein concentrations of the suspensions were measured using the method of Bradford with Bio-Rad reagent and bovine plasma gamma globulin as standard (Bio-Rad, Hercules, CA). Immobilized pH gradient electrophoresis --------------------------------------- As the first dimension of two-dimensional electrophoresis Bio-Rad pH 3--10 immobilized pH gradient (IPG) strips were used to separate the cell proteins according to their isoelectric points. A volume of the cell protein solution containing 2 mg protein was mixed with rehydration buffer (6 M urea, 2 M thiourea, 2% CHAPS, 0.4% DTT and 0.5% pH 3--10 ampholytes) for a final volume of 300 μl per IPG gel. The IPG strips were loaded with the protein sample and the strips passively rehydrated for 16 h. Isoelectric focusing of the strips was at 250 V for 15 min followed by rapid ramping to10,000 V for 3 h and maintenance at the peak voltage for another 60,000 Vh. Current was limited to 50 μA per gel. Second dimension slab gel ------------------------- The IPG strips were equilibrated with 6 M urea, 30% glycerol, 2% SDS, 1.55 M Tris, 2 mM tributylphosphine (TBP) and 0.00125% bromophenol blue (BPB) for 25 min with shaking at room temperature. The strips were rinsed in cathode buffer and applied to the top of 12% acrylamide slab gels. The cathode buffer contained 50 mM Tris, 384 mM glycine and 6.9 mM SDS. Low-melting point agarose (1% in cathode buffer) was layered over the strips. The anode buffer contained 25 mM Tris, 192 mM glycine and 3.5 mM SDS. The buffers were chilled to 4°C prior to use. A cooling plate maintained the system (Genomic Solutions, Ann Arbor, MI) at low temperature. The gels (22 cm × 22 cm × 1 mm) were run at 20 W per gel until the BPB dye front had migrated to within 1 cm from the bottom of the gel. The gels were stained with Coomassie blue. Quantification of protein spots ------------------------------- Second dimension gels were analyzed by Phoretix software (Nonlinear Dynamics, Newcastle, UK). Comparison between gels was achieved using normalized spot volumes. To begin with, differences greater or equal to 1.5-fold were considered to be significant. If these differences were subsequently reproduced in additional experiments, despite possibly being of lower magnitude, they were held to be consistent. Mass spectrometry ----------------- Differentially expressed protein spots were excised from the gels and digested with trypsin as reported previously \[[@B18]\]. Each gel plug was incubated with 150 μl of 50 mM ammonium bicarbonate containing 12.2 M acetonitrile. The gels were then dried and rehydrated with 5 μl of 50 mM ammonium bicarbonate containing 0.5 μg trypsin. This solution was added in 5 μl aliquots until the gel piece regained its original size. The gel was then covered with 50 mM ammonium bicarbonate and incubated overnight at 30°C. After the addition of 1.5 μl of 880 mM trifluoroacetic acid the peptides were extracted with 50 mM ammonium bicarbonate containing 14.6 M acetonitrile. The supernatants were dried to approximately 10 μl. The samples were each mixed with MALDI matrix solution prepared as 5 mg/ml α-cyano-4-hydroxycinnamic acid in a solution containing 1:1:1 acetonitrile, ethanol and 0.1% trifluoroacetic acid (pH 2.0). Each mixture was spotted on a MALDI target and allowed to air-dry. Peptide masses were then obtained by MALDI-TOF MS (Applied Biosystems 4700 Proteomics Analyzer). Calibration was performed with the same procedure using mixtures of peptides with known molecular masses. MS/MS ions produced by MALDI-TOF from the tryptic peptides and the results of a Mascot search <http://www.matrixscience.com/> were used to determine protein identification. The NCBInr database, i.e. the non-identical nr protein database of the National Center for Biotechnology Information, was utilized for the search. Results ======= Two-dimensional gel electrophoresis ----------------------------------- Fig. [1](#F1){ref-type="fig"} demonstrates a gel obtained from two-dimensional gel electrophoresis of protein extracted from SW480 cells. More than 500 protein spots could be observed. The gels obtained from two-dimensional gel electrophoresis of quercetin-treated and untreated (control) SW480 cell proteins were compared (Fig. [2](#F2){ref-type="fig"}). Two mg protein was loaded in each case. Four proteins were revealed to have undergone a consistent change in expression following treatment with 20 μM quercetin for 48 h. Three proteins had a decreased and one protein an increased expression. The changes in these particular proteins were consistent between four separate experiments, i.e. four separate quercetin treatments coupled in each case with an untreated control. Two experiments were run concurrently and then another two on a different occasion. The proteins with altered expression following quercetin treatment, consistent between different experiments, are labeled 1--4 in Fig. [2](#F2){ref-type="fig"}. With observation solely by eye some of the differences may not be readily apparent, but they were verified by analysis of the gels using Phoretix 2-D analysis software (Table [1](#T1){ref-type="table"}). The results in Table [1](#T1){ref-type="table"} are the means from analysis of the gels from two separate experiments conducted concurrently. In parentheses are the means from a second set of two experiments run concurrently at a different time to the first two experiments. The variation between the two sets of results can be attributed to the cells having been subcultured several times between the first and second sets of experiments. Furthermore, the cells could have been in a stage of growth for the second set of experiments different from that for the first. Despite these sources of variation, a consistent effect of quercetin in all four experiments was searched for. From these results proteins 1, 2 and 4 each had decreased expression following quercetin treatment of the cells. Protein 3 had increased expression. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Two-dimensional gel of protein from SW480 colon adenocarcinoma cells.SW480 cells were cultured as described in Methods. In this representative case the cells were not treated with quercetin and were thus a control. Cells were harvested; protein was extracted and subjected to two-dimensional electrophoresis as described in Methods. The gel was stained with Coomassie blue. The protein spots indicated were those subsequently determined to be significant in the effects of quercetin. ::: ![](1475-2891-4-11-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Two-dimensional gels of protein from control and quercetin-treated cells.After 7 days in culture SW480 cells were incubated for 48 h in medium containing A. 0.2% dimethylsulfoxide (DMSO), since DMSO was the vehicle in which stock quercetin had been dissolved, and B. 20 μM quercetin and 0.2% dimethylsulfoxide (DMSO). Cells were harvested, protein extracted and subjected to two-dimensional electrophoresis as described in Methods.The gels represent protein obtained from the control and quercetin-treated cells of one of four separate treatment groups. Treatment was identical for each group. The indicated protein spots differed in levels consistently for each of these treatment groups. ::: ![](1475-2891-4-11-2) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effect of quercetin treatment of SW480 cells on the volume of protein spots on 2-D gels. The 2-D gels of protein from the untreated (control) and quercetin-treated SW480 cells were analyzed with Phoretix software. Normalized spot volumes were computed, averaged for two separate experiments run concurrently, and compared between the control and quercetin-treated gels. The 2-D gels obtained from one of the experiments are presented in Fig. 2. Results are the means, from the two experiments, for protein spots that differed in volume consistently in these two experiments and a subsequent two experiments. Alongside these results in parentheses are the mean results from the second set of two experiments. ::: Protein spot number Control volume Quercetin volume Increase/Decrease Fold change ------------------------- -------------------- ---------------------- ----------------------- ----------------- 1 6.61 (1.65) 2.89 (1.35) Decrease -2.29 (-1.22) 2 5.91 (4.18) 2.06 (3.26) Decrease -2.87 (-1.28) 3 0.118 (0.036) 0.243 (0.108) Increase +2.06 (+3.00) 4 0.411 (0.102) 0.088 (nd) Decrease -4.67 (-high) ::: Mass spectrometry ----------------- The protein spots that were differentially expressed following quercetin treatment were excised from the gels and digested with trypsin. The tryptic digests were subjected to matrix-assisted laser desorption and ionization-time of flight (MALDI-TOF) mass spectrometry. The spectra were analyzed and the data entered into appropriate databases to identify the proteins. Table [2](#T2){ref-type="table"} lists the protein identities together with their functions. Protein number 1 could not be identified. Due to its proximity on the 2-D gels to protein number 2, i.e. type II cytoskeletal 8 keratin, it is possible that it is the same protein, but differing in its modification. It is known that this keratin is phosphorylated on serine residues; a process which is enhanced during epidermal growth factor stimulation and mitosis. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Proteins differentially expressed following quercetin treatment of SW480 cells. Protein spots determined to be present at different levels on gels following quercetin treatment of the SW480 cells were excised from the gels, digested with trypsin and subjected to MALDI-TOF mass spectrometry as described in Methods. From the mass spectra the identities of the proteins were determined using the appropriate databases. ::: Number from gel Protein Function --------------------- ------------------------------------------------ ------------------------------------------------------------------------------- 2 Keratin, type II cytoskeletal 8 Cytoskeletal structure, angiogenesis-related 3 Annexin family Ca^2+^and phospholipid binding, regulation of exocytic and endocytic pathways 4 NADH dehydrogenase (ubiquinone) Fe-S protein 3 Transfer of electrons from NADH to the respiratory chain ::: Discussion ========== Four proteins were found to have their expression consistently altered on quercetin treatment of SW480 colon carcinoma cells. Three of these proteins were down-regulated and one was up regulated following exposure to quercetin. Using mass spectrometry three of the proteins were identified. Those that were down-regulated were type II cytoskeletal 8 keratin and NADH dehydrogenase (ubiquinone) Fe-S protein 3. The up-regulated protein belonged to the annexin family. Type II cytoskeletal 8 keratin (keratin 8) in another proteomic study has been found to have a significantly lower abundance in the normal mucosa compared with the adjacent colon tumor for one specific patient for two out of three gel protein features identified as keratin 8 \[[@B19]\]. For the same patient, keratins 18 and 19 also had significantly lower concentrations in the normal mucosa compared with the adjacent tumor. Similarly, with paired samples of normal colon mucosa and adenocarcinomas derived from 27 patients, proteomic analysis revealed that keratin 18 was at a significantly lower level in the normal mucosa compared with the tumor tissue \[[@B20]\]. In these respects down regulation of keratin 8 by quercetin treatment of SW480 cells would be akin to the cells becoming more \"normal\", i.e. less tumorigenic. Further support for this contention derives from analysis using the cDNA macroarray technique of the differential gene expression in isolated human colorectal cancer and respective normal mucosa from two patients \[[@B21]\]. The tumors showed up-regulation of expression of type II cytoskeletal 8 keratin and other angiogenesis-related genes to over 5-fold the levels in normal mucosa. In the present study the down-regulation of type II cytoskeletal 8 keratin by quercetin may therefore demonstrate the decreased potential for angiogenesis and hence reduction of any tumor. Further support for the reduction in expression of type II cytoskeletal 8 keratin following quercetin treatment of SW480 colon cancer cells being reflective of decreased neoplasia is derived from a proteomic analysis of lung adenocarcinomas \[[@B22]\]. Cytoskeletal 8 keratin, in addition to other keratins, had reduced expression in normal lung samples compared with lung adenocarcinomas. Furthermore, several isoforms of cytoskeletal 8 keratin were identified. These had differing pI, but similar molecular weights, and apparently result from differing extents of posttranslational phosphorylation \[[@B22]\]. This supports our contention that unidentified protein 1 in the present study may be an isoform of type II cytoskeletal 8 keratin. A crucial role for this keratin in the malignant phenotype is suggested by studies on its increased expression by transfecting stratified epithelial cells with the cytoskeletal 8 keratin gene \[[@B23]\] and the epidermal consequences for transgenic mice expressing the human keratin 8 \[[@B24]\]. In each case neoplastic transformation of the cells to the malignant phenotype occurred. NADH dehydrogenase (ubiquinone) Fe-S protein 3 is a component of complex 1 of the mitochondrial electron transport chain. It is involved in transfer of electrons from NADH to ubiquinone in the respiratory chain. A similar component of complex 1 of the respiratory chain, NADH-ubiquinone oxidoreductase, has been determined from a proteomic analysis to be present in colon tumor tissue and the surrounding normal mucosa \[[@B19]\]. Interestingly, its abundance was higher in the tumor than in the normal mucosa. This suggests that the lower level of NADH dehydrogenase (ubiquinone) Fe-S protein 3 in our quercetin-treated colon cancer cells compared with its level in the untreated cells is linked to a decreased tumorigenicity of the cells following quercetin treatment. Furthermore, NADH dehydrogenase (ubiquinone) has also been determined by others to have a decreased abundance in HT-29 human colon cancer cells following exposure for 24 h to 150 μM quercetin \[[@B25]\]. This supports what we determined for NADH dehydrogenase (ubiquinone) Fe-S protein 3. The implication is that the consequent reduction in oxidative phosphorylation of substrates by quercetin is associated with decreased tumorigenicity. Following quercetin treatment, the only protein to be up-regulated belonged to the annexin family. In another proteomic study annexins I, III, IV and V have been determined to have a greater abundance in normal mucosa compared with neighboring tumor tissue \[[@B19]\]. Thus the rendering of the SW480 colon cancer cells less tumorigenic by treatment with quercetin could be reflected by the increased abundance of members of the annexin family. To strengthen this argument the level of annexin II, a protein that inhibits cell migration, was found to increase in HT-29 human colon cancer cells following treatment with quercetin for 24 h and 48 h \[[@B25]\]. Inhibiting the ability of the cells to migrate would contribute to explaining the anti-cancer activity of quercetin. In addition, annexin I, which promotes apoptotic cell engulfment, increased in HT-29 cells exposed to quercetin for 48 h. A role for apoptosis was also suggested when annexin IV, a key regulator of apoptosis, was found to increase in NCOL-1 human preneoplastic colonocytes following treatment with quercetin for 24 h \[[@B26]\]. Apoptosis, mediated by members of the annexin family, may therefore also underlie the anti-cancer activity of quercetin. Conclusion ========== Three proteins have been identified as potential molecular targets for the proposed action against colon cancer of quercetin the plant flavonoid. The application of proteomic techniques demonstrated the response of these proteins to quercetin treatment of colon cancer cells. Other research in the field supported the response of each of these proteins to quercetin. This serves to validate the previous work. Furthermore, the responses could be rationally described in terms of an anti-cancer action. This strengthens earlier findings of quercetin\'s protection against colon cancer using cell culture and animal models. A basis is thus provided for further research with quercetin and perhaps even advancement to studies with humans. An intriguing aspect to consider is whether quercetin could form the basis of directed drug design to yield drugs with greater efficacy against the identified molecular targets and thereby provide an effective treatment of colon cancer. List of abbreviations used ========================== NADH, nicotinamide adenine dinucleotide, reduced; MALDI-TOF, matrix-assisted laser desorption and ionization-time of flight; MS, mass spectrometry 2-D, two-dimensional; SDS, sodium dodecyl sulfate; DTT, dithiothreitol; IPG, immobilized pH gradient; CHAPS, 3-\[(3-cholamidopropyl) dimethylammonio\]-1-propane-sulfonate; TBP, tributylphosphine; BPB, bromophenol blue; DMSO, dimethylsulfoxide; NCBI, National Center for Biotechnology Information. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= MFM treated the cells, performed the two-dimensional electrophoresis and gel analysis, submitted protein spots for mass spectrometry, undertook the literature search on the identified proteins, drafted the manuscript, submitted the manuscript and completed all the necessary revisions to make it acceptable for publication. KK carried out trypsin digestion of the protein spots and undertook the mass spectrometry. RO provided oversight for the mass spectrometry. JLH and AG participated in conception and design of the study. All the authors read and approved the final manuscript. Acknowledgements ================ We express our gratitude to Elizabeth Buck and Joan G Fischer for culturing the SW480 cells and providing them to us. Funding was from the Agricultural Experiment Station, Hatch Project Number GEO00922.	PubMed Central	2024-06-05T03:55:54.108233	2005-3-4	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555539/", "journal": "Nutr J. 2005 Mar 4; 4:11", "authors": [ { "first": "Michael F", "last": "Mouat" }, { "first": "Kumar", "last": "Kolli" }, { "first": "Ronald", "last": "Orlando" }, { "first": "James L", "last": "Hargrove" }, { "first": "Arthur", "last": "Grider" } ] }
PMC555540	Background ========== Dacryocystorhinostomy (DCR) is the treatment of choice for patients with chronic stenosis and obstruction of the nasolacrimal duct. External dacryocystorhinostomy (EXT-DCR) was first described by Toti \[[@B1],[@B2]\] in 1904. The endonasal approach was first introduced in 1893 by Caldwell \[[@B3],[@B4]\], but it was inherently limited by poor visibility of endonasal anatomy during surgery. The introduction of high-resolution fiberoptic endoscopes in the late 1980s enabled adequate visualisation of the nasal cavities, and permitted minimally invasive surgery, under local anaesthesia, avoiding visible facial scarring\[[@B5],[@B6]\]. Endonasal dacryocystorhinostomy (ENL-DCR) can be performed either entirely surgically\[[@B7]\] or with the assistance of laser to create the fistula. Massaro, Gonnering and Haris \[[@B8],[@B9]\] were the first to describe the endonasal dacryocystorhinostomy (ENL-DCR), using Argon laser for the creation of the DCR fistula. Since then, carbon dioxide (CO~2~), holmium:Yag (Ho:Yag), neodymium:Yag (Nd:Yag), and potassium-titanyl-phosphate (KTP) laser systems have been employed in an attempt to identify the optimal delivery system that would achieve sufficient bone ablation with effective haemostasis\[[@B10]\]. Reported primary ENL-DCR success rates vary from 68% to 99% \[[@B5],[@B6],[@B11]\], depending on the type of laser, the size of the osteotomy and the use of antimetabolites, such as mitomycin C \[[@B12]\]. In this study, we evaluate the clinical outcome of 41 consecutive primary ENL-DCRs that were performed on 29 patients using the KTP laser over a period of one year. Methods ======= The records of all 47 ENL-DCR procedures that were performed in Freeman Hospital, Newcastle upon Tyne, United Kingdom, within a period of twelve months were retrospectively studied. The patients\' main symptom was moderate to severe epiphora. Obstruction of the nasolacrimal system distal to the lacrimal sac was diagnosed with nasolacrimal syringing. Radiographic imaging was not part of the routine preoperative evaluation. Otorhinolaryngological preoperative assessment included full endoscopic examination of nasal cavities, looking for evidence of mucosal disease including polyps particularly in the middle meati. Exclusion criteria for ENL-DCR were: noticeable lower lid laxity, previous lacrimal surgery, suspicion of malignancy and previous radiation therapy. Each patient with primary nasolacrimal duct obstruction was counselled as to the advantages and disadvantages of EXT- DCR versus ENL-DCR, together with estimated success rates of the two different types of DCR. The operations were performed by the same ophthalmologist (C.N.) and ENT surgeon (S.C.). The majority of patients had surgery under local anaesthesia on an outpatient basis, except for one who opted for general anaesthesia. For local anaesthesia, Amethocaine drops were instilled in the conjunctival sac, followed by injection of Xylocaine 2% with 1:200000 Adrenaline in the medial third of both eyelids and transcaruncularly to the lacrimal sac. Cophenylcaine spray and intranasal cocaine 4% paste was applied to achieve anaesthesia and haemostasis. Dilatation of the lower punctum was performed and a 20G vitreoretinal probe was inserted in the lower canaliculus and advanced into the nasolacrimal sac. The light was directly visualised endonasally with a 0° rigid nasal endoscope, and the laser energy was delivered, with full laser precautions, via a KTP laser probe guided by the light. The nasal mucosa and lacrimal bone were ablated and the ostium was enlarged anteriorly as necessary with a microronguer. Bicanalicular O\'Donoghue silicone tubes were inserted and secured with a Watzke sleeve. A course of topical Chloramphenicol drops was given for 1 week. Patients were examined 1 week postoperatively and then at 6 months for removal of tubes, or earlier, if discomfort was experienced. Mean follow up period was 16 months (range 12--24 months). Results ======= Forty- seven consecutive primary ENL-DCR operations with lacrimal intubation were performed from March 2001 to February 2002 on 35 patients with primary acquired nasolacrimal duct obstruction. Five cases with incomplete follow up and one which had to be converted to EXT-DCR due to very thick lacrimal bone were excluded from the study. Forty-one ENL-DCR procedures on 29 patients (7 males and 22 females) were included in the study. Mean age was 75 years (range 47--90, SD 13.7). 12 patients (41.4%) underwent simultaneous bilateral surgery and 17 (58.6%) had unilateral ENL-DCR. In total, 19 right sided (46.3%) and 22 left sided (53.7%) procedures were recorded (Table [1](#T1){ref-type="table"}). Pre-operatively, all patients were suffering from significant epiphora, which was affecting their quality of life. Mucocele was present in 6 cases (14.6%) and previous dacryocystitis in 4 (9.8%), while in another 3 cases (7.3%) these two conditions co-existed (Table [2](#T2){ref-type="table"}). Mild medial ectropion was noted in 5 cases (12.1%) and intranasal pathology (including sinus disease, deviated nasal septum, polypoidal medial turbinate and previous nasal fractures) in 7 cases (17.1%). Two patients (4.8%) had systemic sarcoidosis. During the operation the mean laser energy used was 400.2 joules (range 96--797, SD 188.5). Serious intra-operative complications did not occur, although in some patients\' records per-operative mild discomfort was documented. The removal of silicone tubes was scheduled at 6 months post-operatively, though in ten patients extubation was performed at 1--4 months due to discomfort. In two patients, mild nasal haemorrhage was noted on removal of tubes. The operation was defined as being successful if the patient was asymptomatic or if there was significant improvement of symptoms, not requiring any additional procedure. Success rate at 12 months postoperatively was 78.1%. No improvement of symptoms was noted in 8 patients (19.5%), while 1 patient reported worsening of epiphora (Table [3](#T3){ref-type="table"}). Pathology associated with failure (9 cases -- 21.9%) included: intranasal pathology (sinus disease, septum deviation, polypoidal medial turbinate and previous nasal fracture) in 5 cases (55.5%), mucocele in 3 cases (33.3%), and systemic sarcoidosis in 1 case (11.2%) (Table [4](#T4){ref-type="table"}). EXT-DCR or treatment of the nasal pathology was offered to all patients with persistent epiphora. Discussion ========== ENL-DCR is a well established surgical technique with some advantages compared to the conventional EXT-DCR. These include limitation of tissue injury to the discrete fistula site, avoidance of a skin incision, excellent haemostasis, the ability to perform a lacrimal bypass operation on an outpatient basis, quicker patient rehabilitation, decreased overall health care expense and patients\' preference \[[@B8],[@B13]\]. Main limitations of the technique are its steep learning curve, the higher equipment cost and its contraindication in cases of severe pre-existing nasal deformities or scarring and suspected lacrimal sac neoplasms \[[@B13]\]. ENL-DCR avoiding the use of laser is a well described procedure. Several surgical instruments have been employed to remove the bone overlying the lacrimal sac, including drills, osteotomes, curettes and rongeurs. Weidenbecher reported resolution or improvement of symptoms in 95% of the patients\[[@B6]\]. In another study of surgical ENL-DCR results, Sprekelsen achieved good results on 96% of the operations\[[@B5]\]. Both studies describe no major complications associated with the technique. Various types of laser (Argon, CO~2~, Ho:Yag, Nd:Yag), have been employed in an attempt to achieve better bone ablation and haemostasis. Massaro et al \[[@B8]\] and Christenbury \[[@B14]\] reported a 70% success rate using an argon blue laser, but they both encountered difficulties in creating an adequate osteotomy. A prospective randomised comparison of EXT-DCR and ENL-DCR with the CO2-Nd:Yag laser by Hartikainen et al \[[@B15]\] revealed far superior results of the external approach (91% success compared to 63% with the endonasal technique), admitting though that their ENL-DCR technique was possibly suboptimal. Szubin et al \[[@B10]\] achieved an impressive successs rate of 97% with the Ho:Yag laser. This laser seems to outperform the rest, delivering better haemostasis and ablation, but its cost is higher and it is not so diverse in its applications \[[@B10]\]. The KTP laser, already utilised by ENT surgeons in other procedures, offers excellent haemostasis but its ablating properties are relatively poor, thus requiring the use of a microrongeur if the underlying bone is thick \[[@B16]\]. Mirza et al \[[@B17]\] reported improvement of symptoms in 64% of patients by KTP laser ENL-DCR, rising to 82 % including revision procedures. Using the same type of laser, Reifler found 68% success rate in a retrospective study of 19 cases, with a longer follow up of 10--16 months \[[@B18]\]. Interestingly, though, he noted that the first 10 cases showed a success rate of only 50%, compared to 89% in the following 9 cases. This observation reflects the steep learning curve of this technique. Mickelson et al \[[@B19]\] reported a series of 19 patients with 100% success (follow up 5--25 months). Hofmann et al \[[@B2]\] performed ENL-DCR with KTP using miniendoscopes to visualise the exact site of obstruction, and achieved success rate of 83% at one year follow up. In our experience, the success rate of primary ENL-DCR using the KTP laser at 12 months was 78.1%. We defined success as complete resolution of epiphora or improvement of symptoms with no further procedure required, as this outcome carries the most significant implication on the patient\'s quality of life. The anatomic result was not evaluated at postoperative follow up, as the healed intranasal ostium size and patency do not always correlate with symptomatic relief. A significant number of patients have been reported to have symptoms in spite of a patent fistula (54% for EXT-DCR and 39% for ENL-DCR)\[[@B20]\], where in some cases, paradoxically, resolution of symptoms is achieved despite a negative fluorescein test \[[@B21]\]. As the aim of our study was to evaluate the results of primary procedures only, repeat procedures were excluded. The optimum duration of stent retention following DCR is controversial. In published series it varies from 4 weeks to 6 months, though there is some evidence that prolonged silicone intubation may increase the incidence of DCR failure by inciting a granulomatous reaction at the internal ostium, with subsequent stenosis \[[@B22]\]. All our patients underwent lacrimal intubation with silicone tubes, and in most cases the tubes were removed at 6 months postoperatively. Early extubation (1--4 months) was necessary in 10 cases due to discomfort, and the operation failed in 2 of them (20%). Thus, in our experience, early removal of silicone tubes was not associated with lower success rate. Reported pre-operative risk factors for ENL-DCR failure include pre-existing sinus disease, mucocele, nasal septum deviation, connective tissue diseases such as sarcoidosis, previous EXT-DCR, other nasal surgery, nasal fracture, and thickened lacrimal bone \[[@B23]\]. In our series, failure was associated mostly with intranasal pathology (sinus disease, septum deviation, polypoidal medial turbinate and previous nasal fracture), but also with mucocele and sarcoidosis (table [4](#T4){ref-type="table"}). Radiographic imaging, particularly CT DCG or CT of nose and sinuses could be of potential help in clarifying the extent of concomitant sinus and nasal disease and increase our success rate. However, as this would add to the cost and the complexity of the preoperative assessment in a busy clinical setting \[[@B24]\], it was not part of our routine evaluation. The common outcome in the failed cases is blockage of the ostium due to cicatrisation, adhesions between the ostium and the medial turbinate, synechiae between the ostium and the septum, or granuloma formation within the ostium \[[@B22]\]. Opinions differ about size and location, with some surgeons favouring smaller ostium size at the lower thinner part of the lacrimal bone \[[@B25]\], while others recommend larger size and removal of the thicker frontal process of the maxilla \[[@B6],[@B26]\]. The ideal technique is yet to be defined. It has been proposed that the thermal energy produced by the laser may lead to scarring and subsequent blockage of the ostium \[[@B10]\]. It has also been suggested that the adjunctive intra-operative application of Mitomycin C (MMC) can be considered in high risk cases or primary failures, as it appears to be safe and efficient in improving the patency rate \[[@B9]\]. ENL-DCR performed under local anaesthesia is reported to be generally well tolerated by the patients\[[@B27]\]. In our study, all operations were performed under local anaesthesia, except for one patient who opted for general anaesthesia. No significant discomfort was reported by any of our patients, which confirms the reported positive patients\' views. Reported complications associated with ENL-DCR include per-operative or post-operative haemorrhage, punctal erosion related to silicone intubation, silicone tubing prolapse, canalicular obstruction, orbital fat herniation, orbital and subcutaneous emphysema, conjunctival fistula formation, retrobulbar haemorrhage, and transient medial rectus paresis (23). In our series, no severe per-operative or post-operative complications were encountered. The only documented complications were discomfort caused by the silicone tubes in 10 patients requiring early extubation, and mild, transient nasal haemorrhage during removal of the silicone tubes in 2 patients. In our experience, the use of KTP laser in ENL-DCR under local anaesthesia with the adjunctive use of an osteotome is a safe and efficient technique, with good results. The particular advantages of this laser are its superior haemostatic properties and its diversity, which reduces the cost of the operation, as it is already employed in other procedures by the ENT surgeons. Significant complications are not common with this technique. In cases of failure, revision ENL-DCR or EXT- DCR can be performed. ENL-DCR with KTP laser is routinely performed under local anaesthesia, thus avoiding the risks of general anaesthesia usually required for EXT-DCR \[[@B24]\] The operative time is also shorter compared to the EXT- DCR \[[@B16]\]. Conclusion ========== ENL-DCR using KTP laser appears to be an efficient technique, with low complication rate and it is well tolerated by the patients. It still needs refinement in order to achieve the higher success rate of the EXT- DCR, which remains the gold standard method for the treatment of nasolacrimal duct obstruction. At present, we believe that patients should be involved in the decision on the type of operation, after comprehensive consultation on the advantages and disadvantages of each technique. Competing interests =================== The author(s) declare that they have no competing interests. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2415/5/2/prepub> Figures and Tables ================== ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of the study group ::: ------------------------------------ ------------------- Number of patients/procedures 29/41 Mean age (years) 75 Range of age 47--90 (mean, 75) Male:Female ratio 7:22 Laterality of surgery (right/left) 19/22 Simultaneous bilateral (patients) 12 ------------------------------------ ------------------- ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Pre-operative lacrimal drainage system abnormalities ::: ------------------------------- ----------- NLD obstruction with epiphora 41 (100%) Mucocele 6 (14.6%) Dacryocystitis 4 (9.7%) Mucocele and Dacryocystitis 3 (7.3%) ------------------------------- ----------- ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Success rate ::: --------------------------------------------------------- ------------ Asymptomatic 17 (41.5%) Significant improvement (no further procedure required) 15 (36.6%) Symptoms unchanged 8 (19.5%) Symptoms deteriorated 1 (2.4%) Total success 32 (78.1%) --------------------------------------------------------- ------------ ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Pathology associated with failure ::: ----------------------------------------------------------------------------------------------------------------- ----------- Intranasal pathology (sinus disease, septum deviation, polypoidal medial turbinate and previous nasal fracture) 5 (55.5%) Mucocele 3 (33.3%) Sarcoidosis 1 (11.2%) ----------------------------------------------------------------------------------------------------------------- ----------- :::	PubMed Central	2024-06-05T03:55:54.110255	2005-3-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555540/", "journal": "BMC Ophthalmol. 2005 Mar 3; 5:2", "authors": [ { "first": "Thomas", "last": "Ressiniotis" }, { "first": "Gerasimos M", "last": "Voros" }, { "first": "Vasilios T", "last": "Kostakis" }, { "first": "Sean", "last": "Carrie" }, { "first": "Christopher", "last": "Neoh" } ] }
PMC555541	Following publication of this work \[[@B1]\], the authors became aware that Local FDR has been originally defined by Efron & al. (2001) in a mixture model framework \[[@B2]\].	PubMed Central	2024-06-05T03:55:54.111843	2005-3-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555541/", "journal": "BMC Bioinformatics. 2005 Mar 3; 6:42", "authors": [ { "first": "J", "last": "Aubert" }, { "first": "A", "last": "Bar-Hen" }, { "first": "JJ", "last": "Daudin" }, { "first": "S", "last": "Robin" } ] }
PMC555542	Background ========== Invasive ductal carcinoma of the male breast comprises approximately 1% of all breast cancers. Invasive ductal carcinoma of the male breast is distinct from invasive ductal carcinoma of the female breast in both presentation and immunophenotype. Male breast cancer generally presents in older patients and at a more advanced stage than its female counterpart \[[@B1]-[@B3]\]. In contrast to female breast cancers, ductal carcinoma in situis quite rare in men \[[@B4],[@B5]\]. Male breast cancers are also predominantly of the invasive ductal adenocarcinoma, not otherwise specified (NOS) type. Invasive ductal carcinoma of the male breast, despite being a high-grade tumor is more likely to express estrogen receptor and/or progesterone receptor and is less likely to over-express P53 or Erb-B2 when compared to invasive ductal carcinoma in the female breast \[[@B6],[@B7]\]. The combination of a unique male hormonal environment, in addition to the unique immunophenotype, points to a distinct, non-p53-dependant, pathway of tumor progression in the male. Yet, despite these differences, it appears that the overall prognosis for male and female breast invasive ductal carcinomas are similar in age and stage-matched studies \[[@B1],[@B8]-[@B10]\]. Angiogenesis is the growth and proliferation of blood vessels from existing vasculature. This process is quiescent in normal tissues and becomes active in rapidly growing tissues -- including solid tumors. It has been shown that, in order to overcome tissue death by hypoxia, tumor growth beyond 1--2 mm^3^is dependant upon the formation of new vasculature \[[@B11]\]. Angiogenesis is, thus, an established step in solid tumor progression. This has been studied in many cancers including colorectal cancer \[[@B12]\] non-small cell lung cancer \[[@B13],[@B14]\], hepatocelullar cancer \[[@B15]\], melanoma \[[@B16]\] prostate cancer \[[@B17]\], breast cancer \[[@B18]-[@B24]\] and bladder carcinoma \[[@B25]\]. Most assessments of angiogenesis in female breast carcinoma have shown it to be of significant prognostic value \[[@B18]-[@B22]\]. However, not all studies in this field have observed such important clinical correlations to MVD \[[@B23],[@B24]\]. There are a variety of techniques used to evaluate angiogenesis and the variability between studies is probably related to the varying techniques employed in this process. Invasive ductal carcinoma of the male breast appears to be a unique and biologically different carcinoma \[[@B1]\]; it is not simply the appearance of female invasive ductal carcinoma in a male breast. Due to the rarity of the disease large cohorts are not readily available, and there is only a limited pool of published data exploring various facets of this important disease. In one study of 26 men with breast cancer, elevated MVD was associated with advanced stage of disease and poor outcome \[[@B26]\]. Another Japanese study confirms that angiogenesis is part of tumor progression in male breast cancer \[[@B27]\]. In an attempt to further characterize this rare tumor, the aim of the current study was to evaluate angiogenesis in invasive ductal carcinoma of the male breast by the assessment of microvascular density in tumor samples. Specifically, we investigated three questions: (1) do survival times and times to relapse differ between levels of MVD, demographic, and clinico-pathological variables; (2) do MVD measures differ between levels of demographic and clinico-pathological variables, and finally; (3) are different measures of MVD correlated within a section of tumor tissue? This study is an extension of our established work on immunophenotypic characterization of male breast carcinoma in Saskatchewan \[[@B6]\]. Patients and methods ==================== Patients -------- After obtaining appropriate ethics approval from the University of Saskatchewan Advisory Committee on Human Experimentation, all cases (n = 75) of invasive ductal male breast cancer diagnosed between 1975 and 1997 were selected from the records of the Saskatchewan Cancer Agency. Detailed chart review was performed for cases where paraffin-embedded tissue samples were available (n = 59). After the removal of all cases with inadequate tissue sample, tissue staining and chart data, there remained 47 cases. Clinical and pathological studies --------------------------------- Sections were cut from paraffin-embedded tissue samples. The sections were stained with hematoxylin and eosin (H & E). A detailed histopathological assessment was performed. Clinical features were recorded including age at diagnosis, date of birth, node status, tumor size, treatment method, date of relapse, and date of death. Age at diagnosis, tumor size, node status, disease-free survival and overall survival were the clinical variables of interest in this study. Age at diagnosis was determined from the patient chart. Tumor size and node status were determined from the pathology report. Overall survival (number of years patient survived since the diagnosis of invasive breast carcinoma) and disease-free survival (number of consecutive years the patient was alive without breast cancer or other cancer relapse related to the breast carcinoma since the date of diagnosis) were calculated from the information gathered in the chart review. In cases where multiple tissue blocks were available, all H & E sections were examined in order to select a representative tissue block with a large area of invasive tumor and satisfactory tissue integrity. Microvessel density determination --------------------------------- MVD determination was modeled after the method described by Kato et al., \[[@B18]\] and Weidner et al., \[[@B28]\]. Immunohistochemical staining was performed for CD31, CD34, CD105, VWF and VEGF. Staining was carried out on a representative section by the avidin-biotin-peroxidase (ABC) technique after antigen retrieval using appropriate positive and negative controls in all cases. The source and dilution for each antibody are presented in table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Source and dilution of antibodies used in this study ::: Antibody Clone Dilution Source Positive Control Negative -------------- ------------ -------------- ------------ ------------------------ ------------------------------------------------------------------------------------------------- CD31 JC70A 1/20 Dako Human Tonsil All markers used patient tissue stained in the absence of primary antibody as negative control. CD34 QBEnd10 1/20 Dako Human Tonsil CD105 4G11 1/25 Novacastra Human Tonsil VWF F8/36 1/40 Signet Human Tonsil VEGF Polyclonal 1/20 Zymed Human Colon Cancer CEA ::: Brown-staining areas, whether single endothelial cells or clusters of endothelial cells, regardless of the absence/presence of a lumen were counted as individual microvessels. Vessels that had a thick muscular layer were excluded from the count. Cases were evaluated in a random order. Two observers using a double-headed light microscope simultaneously performed all counts for CD31, CD34, VWF and VEGF. A single experienced observer assessed CD105. Observers were blinded to all clinical and pathological data. Average, central and highest microvessel counts (AMC, CMC, and HMC, respectively) were performed. Ten high power (200×) fields along the border between cancer nests and the stroma were evaluated for each section (figure [1](#F1){ref-type="fig"}). The average number of microvessels per high power field was determined and reported as AMC. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### *Average microvessel count -- VEGF.Ten high power (200×) fields along the border between cancer nests and the stroma were evaluated for each section. The average number of microvessels (arrows) per high power field was determined and reported as AMC. ::: ![](1477-7819-3-16-1) ::: After scanning at low power (40×), the central area of the tumor was estimated. From this area, six high power (200×) fields were evaluated for each section (figure [2](#F2){ref-type="fig"}). The average number of microvessels per high power field was determined and reported as CMC. For tumors with a central necrotic area, determination was completed using areas near the centre of the tumor that were viable (non-necrotic). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Central microvessel count -- VEGF.After scanning at low power (40×), the central area of the tumor was estimated. From this area, six high power (200×) fields were evaluated for each section. The average number of microvessels per high power field was determined and reported as CMC. ::: ![](1477-7819-3-16-2) ::: After scanning at low power (40×), three areas with the highest concentration of microvessels (vascular hot spots) were selected. Each area was evaluated with one high power (200×) field in such a way as to include the maximum number of microvessels (figure [3](#F3){ref-type="fig"}). The highest value obtained among the three fields was reported as HMC. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Highest microvessel count -- VEGF.After scanning at low power (40×), three areas with the highest concentration of microvessels (vascular hot spots) were selected. Each area was evaluated with one high power (200×) field in such a way as to include the maximum number of microvessels. The highest value obtained among the three fields was reported as HMC. ::: ![](1477-7819-3-16-3) ::: Statistical analysis -------------------- Analysis was completed using the Statistical Package for the Social Sciences (SPSS) version 11.0 on an IBM PC 300PL computer. All tests were two tailed with the level of statistical significance set at p \< 0.05. The demographic and clinico-pathological variables of interest included age at diagnosis (\<65 and ≥ 65 years), tumor size (T1 is ≤ 2 cm, T2 is \>2 cm but ≤ 5 cm, and T3 is \>5 cm) and node status (positive and negative). To compare the distribution of survival times and disease free survival times (time to relapse) we produced Kaplan-Meier curves and made statistical comparisons using the log-rank test between levels of demographic and clinico-pathological variables. In addition to this we dichotomized the MVD variables based on the median and repeated the Kaplan Meier with log-rank analysis to compare survival times and times to relapse between levels of MVD. For comparison of survival times, the outcome of interest was death while the remaining subjects (those surviving to the end of the study period) were censored. For comparison of time to relapse, the outcome of interest was relapse while the remaining subjects (those surviving to the end of the study or those who died before relapse) were censored. Levels of MVD were also compared with levels of demographic and clinico-pathological variables using the Mann Whitney test or Kruskal Wallis test when MVD was considered as a continuous variable and chi squared or Fisher\'s Exact test when MVD was considered as dichotomous variable. Finally, for each vascular marker (CD31, CD34, CD 105, VWF, and VEGF), correlation between the different measures of MVD (i.e. AMC with CMC, AMC with HMC, and CMC with HMC) was assessed using the Spearman\'s correlation coefficient. Correlations with a coefficient (ρ) of ≥ 0.80 were considered strong, moderate-strong correlations had coefficients that were \<0.80 but ≥ 0.50, moderate-weak correlations had coefficients that were \<0.50 but ≥ 0.30, weak correlations had coefficients that were \<0.30. Results ======= Age at diagnosis and clinicopathological characteristics -------------------------------------------------------- In this study of 47 cases of male breast cancer, the median age of diagnosis was 65.9 years with the youngest being 32 years and the oldest being 94 years. The frequency of male breast cancer cases by age is illustrated in figure [4](#F4){ref-type="fig"}. As seen in Table [2](#T2){ref-type="table"}, most of the patients had a tumor size of T1 to T2 and were node status negative. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Frequency of male breast cancer cases by age.This illustrates the age distribution of male breast cancer patients in this study. This is expressed as a percentage of the total number of patients. Note the predilection for older men. ::: ![](1477-7819-3-16-4) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Clinico-pathological characteristics and survival of the study population. The adjacent table is a summary of clinico-pathological data of interest in this study. ::: ------------------------------ ------------------ ------- Characteristics* No. of cases % Number of patients 47 100 Tumor size, T T1 (≤ 2 cm) 20 43 T2 (\>2 cm, ≤ 5 cm) 22 47 T3 (\>5 cm) 5 11 Node status N(-) 26 55 N(+) 21 45 Overall survival, years \<10 27 64 ≥ 10 15 36 Total evaluated 42 100 Relapse-free survival, years \<10 29 69 ≥ 10 13 31 Total evaluated 42 100 ------------------------------ ------------------ ------- ::: Treatment regimens ------------------ All patients underwent some form of surgical resection -- most frequently a modified radical mastectomy. In 31 out of 47 cases, surgical resection was followed by some form of adjuvant therapy (radiotherapy, chemotherapy, hormonal therapy (tamoxifen), or some combination of the aforementioned). Specifically, 6 patients received only radiotherapy, 7 patients received only hormonal therapy and 2 patients received only chemotherapy. For combined therapies, 6 patients received radiotherapy with hormonal therapy, 3 patients received radiotherapy with chemotherapy, 4 patients received hormonal therapy with chemotherapy and 3 patients received all three methods of adjuvant therapy. Patient outcome --------------- All cases reviewed in this study came from the records of the Saskatchewan Cancer Agency between 1975 and 1997. Thirty-three of 47 patients (70%) died in the time period considered. Of the remaining 14 patients (30%), 9 (64%) had been followed for 10 years or more and 5 (36%) patients had been followed for under 10 years. Seventeen patients (36%) had documented relapse. The average age at death for patients with relapse was 72 years while the average age at death for relapse-free patients was 78 years. Although 70% of patients did die in this study, thirty-two patients (68%) survived at least 5-years after the diagnosis of breast cancer. The Kaplan-Meier curves relating prognostic variables to death and relapse are illustrated in figure [5](#F5){ref-type="fig"}. There were significantly shorter survival times when the age of diagnosis was ≥ 65 years (p \< 0.001) and when tumor size was larger (p \< 0.01). However, there were no significant differences in the times to relapse by any of the clinical variables. In addition to this, there were no significant differences in survival times or times to relapse for any of the MVD markers when categorized by the median score (Table [3](#T3){ref-type="table"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### *Kaplan Meier curves for clinical variables with time to death (left column) and time to relapse (right column).This figure illustrates the percentage of patients with relapse-free survival across clinical groupings of age, node status and tumor size. ::: ![](1477-7819-3-16-5) ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Results from log rank test based on Kaplan Meier curves for microvascular density variables categorized at their median. The adjacent table reports the test statistic and p-value from the log rank test comparing differences in survival time and time to relapse between different levels of clinico-pathological variables. ::: Death* Relapse ------- ----------- ------------- ------ ------ VWF AMC 0.04 0.83 0.46 0.50 CMC 0.70 0.40 0.02 0.88 HMC 0.01 0.93 0.45 0.50 CD31 AMC 1.79 0.18 0.05 0.83 CMC 0.36 0.55 0.00 0.98 HMC 0.15 0.70 0.60 0.44 CD34 AMC 0.19 0.67 0.91 0.34 CMC 0.19 0.66 0.37 0.54 HMC 0.08 0.78 0.30 0.59 CD105 AMC 0.59 0.44 0.01 0.93 CMC 0.10 0.75 0.70 0.40 HMC 0.23 0.63 0.06 0.80 VEGF AMC 0.21 0.65 0.32 0.57 CMC 1.70 0.19 0.44 0.51 HMC 0.47 0.49 0.25 0.61 ::: Microvascular density and clinical variables -------------------------------------------- Regardless of whether MVD was considered as a continuous variable or as a categorical variable, there were no significant differences in MVD by demographic (age group) or clinico-pathological features (tumour size or node status) although some of the differences may be clinically important. Table [4](#T4){ref-type="table"} provides median values of MVD markers at different levels of clinico-pathological variables. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Microvascular density levels at various levels of clinico-pathological variables. The adjacent table reports median MVD levels at different levels of clinico-pathological variables. None of the comparisons are statistically significant ::: ----------- -------------------------------- -------------------------------- ------------------------ ------------------------ ------------------------ ------------------------------ ------------------------------ -------- ------ --------- ------ -------- ------ -------- Age group Tumor size Node status *[\<65 years]{.underline}* *[\<65 years]{.underline}* *[T1]{.underline}* *[T2]{.underline}* *[T3]{.underline}* *[Negative]{.underline}* *[Positive]{.underline}* VWF AMC 15.6 (5.7) 15.0 (5.8) 15.8 (5.5) 13.5 (5.3) 17.5 (27.9) 14.2 (5.2) 15.6 (5.2) CMC 53.7 (24.7) 47.2 (50.2) 44.8 (30.5) 50.3 (43.7) 67.5 (40.3) 48.5 (39.8) 50.0 (35.6) HMC 84.0 (50.3) 70.0 (48.5) 86.5 (48.6) 69.0 (58.5) 70.0 (41.8) 84.0 (53.5) 69.3 (44.0) CD31 AMC 5.9 (20.9) 14.5 (28.8) 6.8 (28.6) 11.4 (30.5) 15.4 (61.7) 7.2 (17.9) 10.4 (32.6) CMC 18.8 (41.4) 26.2 (28.6) 15.7 (38.0) 29.3 (28.9) 20.7 (81.0) 17.9 (33.7) 32.5 (36.5) HMC 36.5 (44.8) 38.0 (27.0) 28.0 (40.0) 43.6 (22.3) 30.0 (107.4) 35.9 (40.0) 43.0 (42.6) CD34 AMC 19.7 (9.6) 19.8 (10.5) 21.0 (9.0) 17.3 (11.5) 20.0 (81.8) 18.8 (8.3) 19.9 (11.4) CMC 52.0 (21.8) 41.3 (35.0) 43.3 (29.4) 52.0 (34.2) 48.5 (46.5) 47.2 (35.0) 50.7 (29.0) HMC 89.0 (50.5) 70.0 (77.0) 79.0 (54.3) 86.0 (83.1) 88.0 (79.2) 90.0 (63.3) 70.0 (60.9) CD105 AMC 2.7 (4.4) 4.4 (4.3) 2.7 (3.8) 4.4 (5.4) 5.4 (10.0) 3.0 (4.7) 4.0 (3.6) CMC 8.8 (14.3) 10.7 (13.2) 7.0 (13.8) 10.9 (14.9) 14.3 (31.7) 9.2 (14.5) 10.2 (10.6) HMC 29.0 (24.5) 32.0 (34.5) 28.5 (30.3) 30.5 (49.3) 39.0 (156.0) 26.5 (33.0) 31.0 (20.0) VEGF AMC 7.5 (20.0) 17.9 (25.1) 11.9 (25.5) 15.4 (26.8) 5.8 (64.9) 13.4 (21.8) 13.2 (29.2) CMC 42.5 (47.0) 39.4 (33.6) 47.5 (72.9) 40.8 (39.5) 36.3 (45.6) 41.1 (45.1) 42.3 (58.8) HMC 58.8 (103.1) 80.0 (87.6) 70.3 (113.6) 66.0 (79.1) 80.0 (55.9) 69.8 (86.2) 79.0 (92.8) ----------- -------------------------------- -------------------------------- ------------------------ ------------------------ ------------------------ ------------------------------ ------------------------------ -------- ------ --------- ------ -------- ------ -------- None of the differences between levels of age or clinico-pathological variables are statistically significant ::: Microvascular density within individual markers ----------------------------------------------- Measures of MVD (AMC, CMC and HMC) were compared within each marker. These correlations are illustrated in figure [6](#F6){ref-type="fig"}. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### *Microvascular density correlations within each marker.This figure illustrates all the relationships between the different methods of MVD measurement for each marker. ::: ![](1477-7819-3-16-6) ::: ### CD31 Significant correlations (p \< 0.01) were observed between all methods of measure (AMC correlates with CMC, CMC correlates with HMC, and HMC correlates with AMC) for CD31. The correlations between AMC and CMC, and AMC and HMC were moderate-strong (ρ = 0.76 and ρ = 0.60 respectively); the correlation between CMC and HMC was strong (ρ = 0.88). ### CD34 Significant correlations (p \< 0.01) were observed between all methods of measure (AMC correlates with CMC, CMC correlates with HMC, and HMC correlates with AMC) for CD34. All correlations were moderately-weak or moderate-strong (ρ = 0.45 for AMC and CMC, ρ = 0.41 for AMC and HMC, and ρ = 0.77 for CMC and HMC). ### CD105 Significant correlations (p \< 0.01) were observed between all methods of measure (AMC correlates with CMC, CMC correlates with HMC, and HMC correlates with AMC) for CD105. The correlations between AMC and CMC, and AMC and HMC were moderate-strong and moderate-weak (ρ = 0.62 and ρ = 0.49 respectively); the correlation between CMC and HMC was strong (ρ = 0.82). ### VWF A significant correlation (p \< 0.01) was observed between CMC and HMC for VWF. A trend correlation (p \< 0.10) was observed between AMC and HMC. Correlations were moderate-weak and weak (ρ = 0.47 and ρ = 0.25 respectively). There was no significant relationship between AMC and CMC for this marker. ### VEGF Significant correlations (p \< 0.01) were observed between AMC and CMC, and between CMC and HMC for VEGF. Correlations were moderate-weak and moderate-strong (ρ = 0.43 and ρ = 0.68 respectively). There was no significant relationship between AMC and HMC for this marker. Discussion ========== The markers ----------- VEGF also called vascular permeability factor (VPF) is an important angiogenic activator, for both physiological and pathological angiogenesis \[[@B29],[@B30]\], and it may be associated with inflammation. VEGF plays an essential role in embryonic vasculogenesis and angiogenesis \[[@B31],[@B32]\]. It has also been implicated in postnatal development of the glomerulus \[[@B33],[@B34]\] and endochondral bone \[[@B35],[@B36]\]. VEGF mRNA has been shown to be up-regulated in the majority of human tumors investigated \[[@B37]\], and carcinoma of the human breast is one of these. \[[@B38],[@B39]\]. In addition, VEGF has been implicated in psoriasis \[[@B40]\], brain edema \[[@B41]\], polycystic ovary syndrome \[[@B29]\], age-related macular degeneration (AMD) and other intraocular neovascular syndromes \[[@B42]-[@B44]\] The expression of VEGF is triggered by hypoxia. That is to say, low oxygen tension provokes VEGF mRNA expression \[[@B45]\]. An excellent review of CD105 and its involvement in angiogenesis has been written by Duff et al., \[[@B46]\]. CD105 (endoglin) is commonly expressed by angiogenic endothelial cells \[[@B46]-[@B48]\]. CD105 is an important pro-angiogenic factor. Transforming growth factor β exerts an inhibitory influence on cell proliferation, migration and microvessel formation. The suppressive effect of CD105 on transforming growth factor β, thus, contributes to angiogenesis \[[@B49]\]. It is, therefore, no surprise to observe elevated CD105 expression in various tumor endothelia \[[@B50]-[@B52]\], including breast cancer \[[@B53]\]. CD105 may be shed into the blood stream. The measure of serum endoglin appears to provide important prognostic information in cancer patients \[[@B54],[@B55]\]. CD31 is an important part of the endothelial intercellular junction \[[@B56]\] and it plays a crucial role in leukocyte migration through vascular endothelial intracellular junctions \[[@B57]-[@B59]\]. This molecule is at least partially responsible for the adhesion between leucocytes/endothelial cells, leucocytes/platelets, and endothelial cells/endothelial cells \[[@B57],[@B60]-[@B65]\]. This adhesion is likely the result of CD31-CD31 \[[@B66]\] interactions (homophilic interactions) although adhesion between CD31 and other components of the cell membrane has been demonstrated (heterophilic interactions) \[[@B61],[@B67]-[@B70]\]. CD31 also exhibits signal transduction; its dimerization appears to upregulate integrin function \[[@B71]\]. This molecule appears to be involved in thrombosis, angiogenesis, wound healing, and inflammation \[[@B61]\]. CD31 is known to be a co-signal transducer for macrophages, inducing respiratory burst. CD34 is a glycosylated type I transmembrane protein \[[@B72]\] which is expressed on hematopoietic stem cells, committed hematological progenitor cells \[[@B73]-[@B75]\], small vessel endothelial cells \[[@B76],[@B77]\], tumors of epithelial origin \[[@B78],[@B79]\] and a limited number of other cell populations including some haematological malignancies \[[@B72]\]. As specific ligands are still undefined, the precise role CD34 plays in early hematopoiesis remains uncertain. It is thought that differential splicing of sugar residues on CD34 may permit it to host a variety of ligands under different conditions \[[@B80]\]. Despite our meager understanding of this complex molecule there is evidence indicating that hematopoietic CD34 plays a role in modulating adhesion (this has been reviewed previously \[[@B72]\]). Factor VIII related antigen, or von Willebrand factor (VWF), is a plasma protein produced by endothelial cells \[[@B81],[@B82]\]. VWF is also present in platelets, as it is produced by their megakaryocytic precursor \[[@B83]\]. VWF is a multifunctional protein. It is known to mediate adhesion/aggregation of platelets in clot formation (reviewed in \[[@B84]\]). In addition to this, VWF acts as a chaperone for circulating factor VIII. About 1 -- 2% of VWF is bound by factor VIII \[[@B85]\]. This non-covalent bond prolongs the survival of factor VIII in the plasma. When the coagulation cascade is triggered, thrombin cleaves the complex, thereby freeing factor VIII to participate coagulation \[[@B86]\] (reviewed in \[[@B87]\]). Age at diagnosis ---------------- Male breast cancer is a disease of older men. The likelihood of this occurring in older men that is illustrated in this study is not surprising as this is the case for most studies of e breast cancer in males \[[@B88],[@B89]\]. As mortality from common conditions (e.g. cardiovascular disease) within this group improves due to advances in treatment/intervention and a larger proportion of the population enters this age group, it seems that the relative incidence of male breast cancer is likely to rise. Such is the finding in a recent meta-analysis of male breast carcinoma \[[@B1]\]. Survival -------- In this study, 70% of the reviewed patients died. Though this number may seem high, only half of those who died had documented relapse prior to the time of death. There is, however, an interesting difference between average age at death for relapsed and relapse-free patients, 72 years versus 78 years respectively. It appears that male breast cancer is contributing to mortality, but this study did not examine the effects of co-morbid conditions. The expected life remaining for a 65 year old male in Saskatchewan between 1995 and 1997 was 16.7 years (expected age approximately 82 years) \[[@B90]\]. Increased tumor size increases the likelihood of death for male breast cancer patients in this study (figure [5](#F5){ref-type="fig"}). One possible explanation for this relationship is as follows: a tumor\'s size may be a function of its rate of growth and time of growth; these characteristics seem likely to increase the opportunity for relapse and metastasis. Thus, we might expect large tumors to relapse more frequently than small ones, and therefore, also contribute to death. It appears that younger patients had a significantly better chance of not experiencing death (figures [5](#F5){ref-type="fig"}). This phenomenon is possibly related to improved response to treatment in younger patients; alternatively, this relationship may be demonstrating that younger patients are diagnosed with less advanced disease and vice versa. Evidence supports advanced age \[[@B88],[@B89]\] and tumor size \[[@B91]\] as important negative prognostic factors. This study was not able to clearly demonstrate statistically significant differences in survival for node status. In the available literature axillary node status is an important prognostic factor \[[@B91]-[@B94]\]. Microvascular density, though it was the primary focus of this study, did not demonstrate statistically significant association with survival, demographic or clinico-pathological features. However, we cannot discount the importance of angiogenesis in tumor progression. The lack of correlation in this study may have been influenced by the lack of statistical power, the methods used, the age of the tissue, advanced stage of disease at presentation and method of analysis. In most tumors studied, MVD has been identified as a prognostic factor and has had important correlations to clinical variables \[[@B12]-[@B16]\]. In most studies where angiogenesis has been evaluated in cancer of the female breast, MVD is an important prognostic factor \[[@B19]-[@B22]\]. In one study of male breast cancer using CD34 to highlight vessels, it was concluded that MVD was an important prognostic tool \[[@B26]\]. In an angiogenesis methodology study of 109 women with breast cancer by Kato et al., \[[@B18]\] it was found that CMC and HMC did not correlate to clinico-pathological variables other than peritumor vascular invasion. AMC was found to have prognostic value. The methods used to report microvessel density were modeled after this work by Kato et al, \[[@B18]\]. Despite a lack of strong evidence, in our study, to support angiogenesis as an independent prognostic factor, there is no evidence to disprove angiogenesis plays a critical role in tumor development. As angiogenesis remains a likely step in tumor progression, we must continue to recognize this process as a potential target for anti-tumor therapy. Microvascular density within each marker ---------------------------------------- There were some important correlations between the different methods of measure for MVD (AMC, CMC and HMC) within the various markers. CD31, CD34 and CD105 were the strongest in this regard with correlations that were very significant (p \< 0.01) and correlations that were usually moderate to strong. The correlations within VWF and VEGF were not all significant, and the relationship was moderate to weak. It could also be that VWF and VEGF are differentially expressed in male breast cancer tissue. This seems to be the case for VEGF. In fact, it was observed that VEGF had a propensity to be over-expressed in regions where there were invading lymphocytes. This may produce a patchy pattern of expression, which could have an important effect on microvessel counts. For the most part, this study saw strong correlations between the various microvessel count methods within the markers. Critics may suggest that evaluation of microvascular density for prognosis in tumors is flawed because, within a tumor, microvascular density is heterogeneous \[[@B24],[@B95]\]. However, the correlations observed in this study support the notion that tumor vasculature is predictable (but not ubiquitous or necessarily homogeneous) from the centre, periphery and vascular hotspot of a tumor. Notably, similar research in female invasive ductal carcinoma of the breast using VWF MVD assessment techniques also demonstrated correlation between central, peripheral and highest microvessel densities \[[@B18]\]. Microvessel determination, by the methods used in this study, is dependant on a predictable pattern of vasculature within a tumor. Such predictability allows for practical (in terms of time, money and ease of use) application of important clinical prognostic features of the markers. Further research to examine the relationship between these markers in cancer is wanting. Such information may prove important in improving the prognostic value of MVD determination. Conclusion ========== From this evaluation of angiogenesis in male breast cancer, we can draw the following conclusions: Microvascular density does not appear to be an independent prognostic factor in male breast cancer. Tumor vasculature (as measured by microvessel determination using antibodies to endothelial markers such as CD31, CD34, CD105) is strongly related throughout a tumor section (p \< 0.01). Other endothelial markers such as VWF and VEGF appear to have a moderate to weak relationship. Advanced age at diagnosis and increased tumor size increases the likelihood of death for men with breast cancer. Abbreviations ============= AMC Average microvessel count CD\# Cluster designation or cluster of differentiation (CD31, CD34, CD105) CMC Central microvessel count HMC Highest microvessel count MVD Microvessel density TNM Tumour nodes metastasis VEGF Vascular endothelial growth factor VWF Von Willebrand factor Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= EFwrote this manuscript, aided in collection and analysis of data and is the first author. JLprovided statistical analysis of the collected data. RK*conceived the design of this study, aided in data collection and remains the corresponding and senior author. All authors have read and approved this manuscript. Acknowledgements ================ This study was supported in part by a grant from the Royal University Hospital Foundation Fund. The authors also wish to extend their thanks to the Saskatchewan Cancer Agency for their assistance in obtaining the records requested for this study and to Todd Reichert and Mitch Hesson for all their help in the preparation of the figures for this manuscript.	PubMed Central	2024-06-05T03:55:54.112118	2005-3-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555542/", "journal": "World J Surg Oncol. 2005 Mar 2; 3:16", "authors": [ { "first": "Evan M", "last": "Frangou" }, { "first": "Joshua", "last": "Lawson" }, { "first": "Rani", "last": "Kanthan" } ] }
PMC555543	Background ========== Japanese quail is both a model animal in biology and a bird used for meat and egg production under a large variety of settings \[[@B1]\]. In the recent past, a special attention was given to the study of its plumage, and several major genes have been described \[[@B2]\]. Since the last compilation of plumage mutations of Japanese quail \[[@B2]\], new loci were described \[e.g. \[[@B3]\]\], linkage and epistasis relationships were explored \[[@B4],[@B5]\], and some genes were recently mapped \[[@B6]\]. This knowledge has already been put to use for running comparisons between chicken and quail based on plumage genetics \[[@B7],[@B8]\], and for tagging commercial quail lines with a visible plumage trait, like the \"fawn\" mutation \[[@B9]\], or with an auto-sexing mutation like the \"roux\" gene \[[@B10]\]. Interestingly, some of the mutations described in quail, like the sex-linked \"roux\" and the lethal \"yellow\" mutations still have no known homologues in the chicken. Moreover, the fact that some plumage colors, like \"lavender\", are common to several avian species \[[@B5]\] is an added incentive to enrich the panel of characterised Japanese quail mutations as potential tools for comparative studies among bird species. In the present work, a new feather structure phenotype (curly) and a new plumage color (rusty) were described, and their mode of inheritance and linkage were studied in two successive generations (F1 and F2) from an F0 made of eight quail with curly feathering and eight birds with rusty plumage. Growth of the F2 quail was also monitored and compared according to their phenotype for the two plumage mutations. Results and discussion ====================== Phenotypic description ---------------------- The quail line with the curly feathers was developed in 2001 from group mating 6 founding (G0) curly quail. Starting in G3, only quail for which the curly phenotype observed at 10 days of age was expressed most strongly were kept for breeding. No differential survival was observed in curly quail after hatching, but the hatching rate in the fixed line at G5 under pool mating was only 34%, mainly because some females did not get mated under this type of mating. In curly chicks, the calamus of the growing wing feathers are not independent from one another, but they are connected through the follicle walls which appear to be joined together. This phenomenon is best observed around 10 days of age and is associated with the curly growth (Figure [1](#F1){ref-type="fig"}). The expression of the trait is variable, however, and the penetrance of the curly mutation appears to be incomplete. The difference between normal and curly adults (Figure [2](#F2){ref-type="fig"}) is not as marked as for other feather structure mutations, like porcupine \[[@B11]\], for example. Following the gene nomenclature proposed for the chicken \[[@B12]\], the locus for this new mutation was named CU, and the symbols of the allele responsible for the curly mutation and of the normal allele at this locus were CU\C and CU\N, respectively. In all instances, inheritance of the trait was similar for both sexes, and heterozygotes CU\C/CU\N had normal plumage structure, indicating that the locus CU was autosomal and the mutation was recessive. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Wing of a 10 day Japanese quail with the curly phenotype.In curly quail, the calamus of the adjacent growing wing feathers are not independent from one another, but they are connected through the follicle walls which appear to be joined together. This phenomenon which lasts one to two weeks is best observed around 10 days of age. It hinders the normal growth of the feathers, thereby inducing the permanent curly structure of the feathers. ::: ![](1471-2156-6-14-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Japanese quail with the curly and the normal feather structures.The adult curly quail is on the left. Its plumage has an overall fluffier look when compared to the normal quail on the right, but the intensity of the curliness varies between curly quail, as the penetrance of this autosomal recessive mutation is not complete. ::: ![](1471-2156-6-14-2) ::: The quail line with the rusty plumage color was started in 2000 from a founding base (G0) made of a single rusty female bred to a wild-type male from another origin. It was followed by sib mating of the all wild-type G1 quail to produce G2 and G3 pedigreed progeny and selection of the few rusty G4 birds. Survival rate to sexual maturity of the rusty descendance of the early generations was poor, and reproductive performance was low (hatched/incubated = 31%) in the fixed line under pool mating at G6, because of the mating system but also of inbreeding derived from having a single rusty ancestor. The plumage of the mutant chicks was rusty, but the down underneath retained the usual wild-type dark-slaty color. A similar color pattern was maintained in rusty adults: their contour and flight feathers had dark barbs on the bottom and rusty colored barbs on the top third of their length which produced the overall rusty look (Figure [3](#F3){ref-type="fig"}). The effect of this mutation appears to be different from the dilution of the pigmentation \[[@B5]\] associated with the roux mutation which affects the whole feather and produces a paler color (Figure [4](#F4){ref-type="fig"}). The locus for this new mutation was named RU, and the symbols of the allele responsible for the rusty mutation and of the wild-type allele at this locus were RU\R and RU\N, respectively. In all instances, inheritance of the trait was similar for both sexes, and heterozygotes RU\R/RU\N had wild-type plumage color, indicating that the locus RU was autosomal and the mutation was recessive. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Japanese quail with the rusty plumage color.Quail with the rusty phenotype are homozygous for an autosomal recessive mutation. ::: ![](1471-2156-6-14-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Comparison of colors of feathers from Japanese quail.On the left, the contour feather of a wild-type Japanese quail shows the usual brownish colored tip with a transversal lighter stripe and slaty colored barbs at the bottom. On the center, the feather of a quail with the rusty phenotype has a different, rusty colored, tip, but keeps the same slaty colored barbs than the wild-type bird at the bottom. On the right, the feather of a roux quail (caused by a sex-linked recessive mutation) shows a diluted color, paler than rusty, over its whole length. ::: ![](1471-2156-6-14-4) ::: Linkage analysis ---------------- All 68 F1 quail were wild-type birds, confirming that both mutations should be recessive, and not sex-linked. Four phenotypes were obtained in the 531 F2 progeny: 326\"wild-type\", 95 \"rusty\", 79 \"curly\" and 31 \"rusty and curly\" (Figure [5](#F5){ref-type="fig"}). The high hatching rate (81%) obtained in the F2 confirms that the much lower value obtained in the two lines fixed for the rusty or the curly mutation might have resulted from inbreeding due to the small number of founders and from the pool mating system used rather than from detrimental reproductive effects directly associated with the two mutations. Observed and expected distributions under two different null hypotheses (A: independent segregation given complete penetrance of the curly mutation, and B: independent segregation given incomplete penetrance of the curly mutation) are shown with the corresponding values of χ^2^~s~(7.5 and 1.9, respectively) in Table [1](#T1){ref-type="table"}. None of the two hypotheses could be rejected because the probabilities for these values of χ^2^~s~were high enough (p \> 0.05 and p \> 0.1, respectively). The maximum likelihood estimation of the penetrance parameter (± SE) was 1-λ = 0.83 (± 0.07) which indicates that 17% of F2 curly quail might have been misclassified as wild-type birds. This result is consistent with the observation that the expression of the curly trait was variable, and it would account for the relative deficit of curly birds in the F2. Overall, however, the segregation of the F2 results fits a simple two-locus Mendelian inheritance of two autosomal recessive and independent mutations. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Japanese quail with the rusty plumage color and the curly feather structure.This phenotype was the least frequent of the four quail types produced in the F2 between the curly and the rusty lines. These quail are homozygous for the two recessive mutations. ::: ![](1471-2156-6-14-5) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Segregation of plumage color and feather structure in the F2 from rusty plumage and curly feather quail lines ::: Phenotype Observed (n = 531) Expected under independent segregation ----------------- -------------------- ---------------------------------------- ------------------------- Wild-type a = 326 9n/16 = 298.7 9n/16+3λn/16 = 315.6^¥^ Rusty b = 95 3n/16 = 99.6 3n/16+λn/16 = 105.2 Curly c = 79 3n/16 = 99.6 3n(1-λ)/16 = 82.7 Rusty and curly d = 31 n/16 = 33.2 n(1-λ)/16 = 27.6 Goodness of fit χ^2^~s~= 7.5^£^ χ^2^~s~= 1.9^\$^ ^¥^: calculated using 1-λ = 0.83 ^£^:0.10 \> p \> 0.05. ^\$^:0.5 \> p \> 0.1 ::: Growth ------ At hatching, none of the plumage mutations were associated with differences of body weight (Table [2](#T2){ref-type="table"}), but after two weeks quail with curly feathers had become significantly heavier than wild-type ones, and the difference reached 3.4% (p \< 0.001) of the average body weight at 35 days of age (Table [2](#T2){ref-type="table"}). On the opposite, quail with rusty plumage were 2.3 % lighter (p \< 0.05) than wild-type ones. The mean body weight at 5 weeks of age was 176.8, 170.5, 181.0 and 182.0 g, respectively for the \"wild-type\", \"rusty\", \"curly\" and \"rusty and curly\" quail. The fact that both \"curly\" and \"rusty and curly\" birds had a similar high body weight, whereas \"rusty\" quail were the smallest ones is an indication that the effect on growth of the \"curly\" mutation might be epistatic over that of the \"rusty\" mutation. The association of plumage color mutations with lower growth have been previously reported in albino \[[@B13]\] and roux \[[@B10]\] quail, with respectively 9 and 3% lower body weight at a similar age, but no mutation with a favorable effect had been found so far. A large scale experiment focused on the associated effects of the curly mutation on growth is needed, however, to confirm and extend the present results. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Analyses of variance of body weight of the F2 from rusty plumage and curly feather quail lines ::: Age At hatch 1 week 3 weeks 5 weeks -------------------------------------------- ----------- ------------ -------------- -------------- Sample size^1^ 348 348 346 347 Mean body weight (g) (SD) 8.1 (0.8) 27.6 (4.5) 105.7 (12.5) 176.7 (17.0) R^2^ 0.82 0.45 0.47 0.50 [Significance of main effects]{.underline} Hatch \\ ns \\\* \\\* Family \\\* \\\* \\\* \\\* Sex \\ ns ns \\\* Feather structure ns ns \\ \\\* Plumage color ns \\\* \\ \* [Contrast]{.underline} (g) \"curly\" -- \"wild-type\" -0.08^ns^ 0.8^ns^ 3.8\\ 6.1\\\* \"rusty\" -- \"wild-type\" 0.04^ns^ -2.1\\\* -3.6\\ -4.1\* ^1^: The quail sample was made up of 215 wild-type, 59 curly, 58 rusty, and 16 curly and rusty quail from 17 full-sib families and born in two successive hatches. \: p \< 0.05; \\: p \< 0.01; \\\: p \< 0.001; ^ns^: not significant. ::: Conclusion ========== The two new \"curly\" and \"rusty\" mutations will enrich the small number of plumage mutations already available in Japanese quail for studying the genetics and the biology of feathers, a field of research with many perspectives \[[@B14]\]. They may have also some interest from a zootechnical standpoint to tag commercial lines, and, if the positive effect of the \"curly\" mutation was confirmed, this gene might be worth introgressing in parental meat quail lines. Methods ======= Birds ----- The two mutations originated in the experimental quail population maintained and selected on behavioral traits at the INRA Station de recherches avicoles in Nouzilly, France. After two quail lines were established by fixing separately the curly and the rusty phenotypes, eight reciprocal single pair matings (three \"rusty × curly\" and five \"curly × rusty\") were set up with G5 rusty and G4 curly quail from the two pure lines to produce the F1. Then, 531 F2 birds were produced in three consecutive hatches from 17 single pair matings of F1 birds. Sib-mating was avoided, and the hatching rate across all pair matings and hatches was 81%. All chicks were pedigreed, and they were phenotyped for plumage color at hatching and for feather structure at 10 days of age. F2 quail from the first two hatches were raised in two group pens (one per hatch) with free access to ad libitum commercial feed and drinking water, and they were weighed weekly until 5 weeks of age. Genes ----- Gene nomenclature used in this paper followed recommendations published for chicken genes \[[@B12]\], with a two-part symbol: \"locus abbreviation\"\\"allele abbreviation/locus\", and \"locus abbreviation\N\" as the symbol for the wild-type allele. Statistical analyses -------------------- Analysis of the segregation in the F2 to test for linkage was carried out using maximum likelihood methodology and the χ^2^test \[[@B15]\]. Penetrance and its standard error were estimated as: 1-λ = (3c/(a+c)) + d(b+d) and SE = (9ac/(a+c)^3^) + bd/(b+d)^3^, derived for misclassification of phenotypes due to \"partial manifestation\" \[[@B16]\]. In the formulae, a, b, c and d are the numbers of observations in the four phenotypic classes described in Table [1](#T1){ref-type="table"}. Five-way analyses of variance of individual body weights (BW) between hatching and 5 weeks of age were carried out by the GLM procedure \[[@B17]\] for the 348 quail hatched alive in the first two hatches, using the following linear model: BW=(overall mean) + hatch + family + sex + (feather structure phenotype) + (plumage color phenotype) + error. The number of classes for the five main effects were respectively, 2, 17, 2, 2 and 2. Contrasts between least-squared means for each mutant phenotype and for the wild-type quail were estimated from the analyses of variance, using data adjusted for systematic effects of hatch, family and sex. Authors\' contributions ======================= FM coordinated the study and wrote the paper, DG and CM participated in the design of the study and carried out the the data collection. Acknowledgements ================ The authors wish to thank J.M. Faure (INRA SRA, Nouzilly, France) for kindly providing curly and rusty quail he had found in his experimental population.	PubMed Central	2024-06-05T03:55:54.116243	2005-3-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555543/", "journal": "BMC Genet. 2005 Mar 11; 6:14", "authors": [ { "first": "Francis", "last": "Minvielle" }, { "first": "David", "last": "Gourichon" }, { "first": "Chantal", "last": "Moussu" } ] }
PMC555544	Background ========== Chronic granulomatous disease (CGD) is a rare inherited disorder of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex of phagocytic cells resulting in failure to generate reactive oxidants and the absence of a respiratory burst \[[@B1]\]. The disease is characterized by recurrent or persistent intra-cellular bacterial and fungal infections. Approximately, the incidence of fungal infections in CGD patients has been reported up to 20% of infections \[[@B2]\]. Aspergillus sppare ubiquitous saprophytic fungi and are considered as the major causative fungal agent in these patients \[[@B2],[@B3]\]. The spectrum of infection caused by Aspergillusspecies varies from flu-like pneumonia to life-threatening invasive aspergillosis \[[@B4]\]. The most common form of the aspergillosis in CGD patients is Aspergilluspneumonia which can be accompanied by dissemination to the ribs, chest wall and soft tissues \[[@B1],[@B2]\]. Here, we describe a case of invasive aspergillosis in CGD patient with hepatic abscesses and osteomyelitis. Case Presentation ================= A 5-year-old male patient was admitted to Children Medical Center (CMC) with inflammation and swelling in his left mandible and wrist without a history of trauma. In the past, he had suffered from several episodes of pneumonia which started at the age of seven months. On admission, laboratory findings included erythrocyte sedimentation rate (ESR) 84 mm/h, WBC count 12100/mm3 (61% neutrophils, 39% lymphocytes), hemoglobin 11.3 gr/dl and thrombocyte 386000/mm3. As the CRP analysis displayed 20 mg/dl, cephalexin (150 mg/kg/day) was initiated. In his roentgenogram, osteolytic lesions in the distal metaphase of hand and maxillary bone were observed. Considering history of several infections and multifocal osteomyelitis, bone biopsy was performed and his immune system function was evaluated. In the bone biopsy, non-necrotizing granulomatoid lesions were seen. The induration of purified protein derivative reaction was 10 mm diameter. Besides, HIV, hepatitis B surface antigen (HBs), rheumatoid factor and brucella agglutination tests were all negative. The serum IgG level was 1650 mg/dl (normal: 441--1135 mg/dl). IgM and IgA were in high normal range at 250 and 175 mg/dl, respectively. Because no defect was found in his humeral and cellular immunity, the phagocytic cells function was tested with a nitroblue-tetrazolium (NBT) slide test. Based on his hematological and immunological tests (NBT = 0), CGD was considered as underlying disease in this case. Regarding his NBT test, antibiotic therapy was changed from cephalexin to co-trimoxazole (20 mg/kg/day, iv) plus (along with) interferon-γ (50 microgram/m^2^every other day). After two weeks of treatment, the patient\'s condition improved and he was discharged with prescription of both cephalexin (100 mg/kg/day) and co-trimoxazole (10 mg/kg/day) to be taken orally as prophylaxis. The patient was readmitted to our center after eight months with a tender mass in his right upper quadrant (RUQ) (Fig [1](#F1){ref-type="fig"}). On admission, his major complaint was severe dyspnea, a persistent cough and also chest and abdominal pain in epigastric area which was started 10 days ago. He was placed on antibiotic therapy including cephalexin (100 mg/kg/day). A computerized tomography (CT) scan of the chest and abdomen was performed which revealed the hypodense area in liver (Fig [2](#F2){ref-type="fig"}). Adjacent to this opacity, involvement of lower right ribs and reaction to soft tissue were also observed, indicating ribs osteomyelitis. After sonography guided drainage of the above-mentioned hepatic abscess, a sample was sent to the Mycology Department in Tehran University of Medical Sciences. The microscopic examination of clarified specimen with KOH 10% indicated the branched, septated and dichotomous mycelia (Fig. [3](#F3){ref-type="fig"}). The remaining specimen was also cultured on Brain Heart Infusion agar (BHI), Sabouraud\'s dextrose agar (S) and Sabouraud\'s containing 0.005% chloramphenicol (Sc). The S and Sc culture media were incubated at 25°C and BHI at 37°C. The colonies grew rapidly, attaining the diameter of 5 cm within 5 days and their color was bluish green. Cellophane tape preparations and slide cultures demonstrated septated, branched and hyaline hyphae with rough-walled conidiophores and radiated conidial heads. Based on these microscopic and macroscopic findings, Aspergillus fumigatuswas determined as causative agent in this case. Deoxycholate Amphotericin B (1 mg/kg/day, iv), interferon-γ (50 microgeram/m^2^every other day, sc) and rifampicin (10 mg/kg/day) were administered with diagnosis of invasive aspergillosis. The only adverse event observed during treatment was hypokalemia, which was adjusted by administration of potassium chloride 15%. One month after initiation of antifungal therapy, his follow-up CT scan of the abdominal and thoracal region demonstrated relative resolution of hepatic abscess. After four weeks of intravenous treatment, the patient\'s clinical condition improved. He was discharged upon his parents\' responsibility while continuing taking rifampicin (10 mg/kg/day) for two more weeks as a treatment in addition to co-trimoxazole (5 mg/kg/day) and itraconazole (4 mg/kg/day) as long term prophylaxis. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Subcutaneous swelling and granuloma formation in right upper quadrant. ::: ![](1476-0711-4-4-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Computerized tomography showed a hypodense area in right lobe of liver with peripheral enhancement ::: ![](1476-0711-4-4-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### KOH 10% preparation of hepatic abscess showing dichotomous septated hyaline hyphae. ::: ![](1476-0711-4-4-3) ::: Discussion ========== CGD is a rare inherited immune disorder whose prevalence is estimated to be about 1/1,100,000 -- 1/1,300,000 individuals worldwide \[[@B1]\]. Similar to the presented case, the most common form of CGD is X-linked recessive that consists of about two thirds of cases and the rest are autosomal recessive \[[@B5]\]. In the absence of minimal oxidative metabolism in CGD which can be ascertained easily using nitro blue tetrazolium (NBT) slide test, other immune mechanisms are triggered \[[@B6]\]. The relative evaluated immunoglobulin levels in the above-mentioned case might be due to persistent antigenic stimulation and it is a common phenomenon in all chronic infections. This defect is characterized by recurrent or persistent infections due to catalase-positive fungal and bacterial agents despite aggressive antibiotic therapy \[[@B1],[@B6]\]. The incidence of aspergillosis in these patients has been reported to be 78% of all fungal infections \[[@B2]\]. Among Aspergillus spp, Aspergillus fumigatusis considered to be the predominant cause of invasive aspergillosis in CGD patients \[[@B1],[@B7]\]. Pulmonary aspergillosis has been reported in CGD patients infected with Aspergillus fumigatus. As shown in this case, Aspergillusmight spread from lungs to the bones of thoracic wall and cause osteomyelitis \[[@B7]-[@B9]\]. Although Aspergillus fumigatusis considered to be the most isolated species, Aspergillus nidulansosteomyelitis is reported to have a higher incidence and more mortality rate in these patients \[[@B7],[@B9]\]. The treatment of infections in CGD patients is not easy. Since the underlying immunodeficiency is the most important factor with respect to the outcome of treatment, these patients should be treated either with immunomudulative agents such as recombinant INF-γ or with stimulating factors \[[@B10]\]. Recently, on the basis of cytochrome b (558) expression and NADPH oxidase activity, three different sub-type of X-linked chronic granulomatous disease were described \[[@B11]\]. Therefore, therapeutic response to INF-γ in this case and other X-linked CGD patients might be elucidated. Besides, similar to other systemic fungal infections, antifungal drugs such as amphotericin B should be added to therapeutic regimen of CGD patients with established invasive aspergillosis. Our patient responded to the above-mentioned therapeutic protocol and was discharged with long term anti-microbial and immunomudulatory prophylactic treatment as well as anti fungal drug \[[@B12]\] to enhance the quality of life and lessen the risk of re-infection. Acknowledgements ================ Written consent was obtained from the patient or their relative for publication of the study.	PubMed Central	2024-06-05T03:55:54.118373	2005-3-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555544/", "journal": "Ann Clin Microbiol Antimicrob. 2005 Mar 3; 4:4", "authors": [ { "first": "Setareh", "last": "Mamishi" }, { "first": "Kamiar", "last": "Zomorodian" }, { "first": "Farshid", "last": "Saadat" }, { "first": "Mohsen", "last": "Gerami-Shoar" }, { "first": "Bita", "last": "Tarazooie" }, { "first": "Seyad Ahmad", "last": "Siadati" } ] }
PMC555545	Introduction ============ The World Health Organization (WHO) estimates that chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide. In the year 2000, it was estimated that 2.74 million people died from COPD worldwide \[[@B1]\]. COPD is defined by the presence of irreversible or partially irreversible airway obstruction in patients with chronic bronchitis or emphysema \[[@B2],[@B3]\]. The disease is characterized by recurrent (1--4 per year) acute exacerbations of chronic bronchitis (AECB), defined by a subjective increase from baseline of one or more symptoms including shortness of breath, cough, sputum production, and sputum purulence \[[@B4]\]. The precipitating factors for AECB have been extensively researched and determined to be heterogeneous with complex aetiology \[[@B5]-[@B10]\]. Results from a number of placebo-controlled clinical investigations have demonstrated that antibacterial agents are of significant clinical benefit in the treatment of AECB, particularly for those patients with at least two of the three cardinal symptoms of AECB (worsening dyspnoea, increased sputum volume, and increased sputum purulence) and/or severe airway obstruction \[[@B11]-[@B13]\]. Other clinical trials measuring non-traditional endpoints have shown that antibiotic therapy reduces the time to symptom resolution and has long-term benefits including greater intervals between episodes of exacerbation \[[@B14],[@B15]\]. Consequently, antibiotic therapy is considered an important component in the medical management of patients with AECB. Bacteria can be isolated from 40--60% of sputum samples of patients experiencing AECB \[[@B16]\]. The three predominant bacterial species isolated are non-typeable Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae. Other less frequently isolated potential pathogens are Gram-negative enterobacteria, Haemophilus parainfluenzae, Staphylococcus aureus, and Pseudomonas aeruginosa. Gram negative enterobacteria and Pseudomonas aeruginosaare more frequently isolated in patients with severe underlying disease \[[@B10]\]. Viral infections are present in approximately 30% of exacerbations, Mycoplasma pneumoniaein 1--10%, and Chlamydophila pneumoniaein 4--5% (serologically identified) \[[@B6]-[@B10]\]. Amoxycillin, ampicillin, sulfamethoxazole-trimethoprim (trimethoprim-sulphamethoxazole), tetracyclines, and erythromycin are considered first-line antimicrobial therapy for AECB \[[@B17]\]. The clinical utility of these agents is, however, being hampered by the increasing global spread of pathogens with resistance to one or more of these agents. Up to 40% of H. influenzaeisolates and more than 90% of M. catarrhalisisolates produce β-lactamase and this limits the value of penicillins and some other β-lactams \[[@B18]\]. Furthermore, resistance to penicillin and macrolides has spread rapidly among isolates of S. pneumoniae\[[@B19]\]. Other agents used include extended spectrum cephalosporins, amoxycillin/clavulanate, azithromycin, clarithromycin, and levofloxacin. Telithromycin is the first ketolide available for clinical use. Derivatives of erythromycin-A, the ketolides, like the macrolides, exert their antimicrobial action by binding to the bacterial ribosome. Although both macrolides and ketolides bind strongly to a region of domain V in the 23S rRNA of the ribosome, telithromycin has additional strong binding to a region in domain II to which the macrolides bind weakly \[[@B20]\]. Ketolides are also poor substrates for the efflux pump (mefA) responsible for macrolide resistance in S. pneumoniae\[[@B21]\]. Consequently, telithromycin has been found to have potent activity against macrolide resistant S. pneumoniaewith methylase, efflux or ribosomal mutations as the mechanisms of resistance \[[@B22],[@B23]\]. There is a need for alterative therapeutic options for the treatment of AECB and surveillance data are needed to help determine the suitability of new agents. The PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) study is an international, longitudinal, antibacterial resistance surveillance study, which was initiated in 1999 to monitor the spread of resistance among respiratory tract pathogens worldwide. Here we analyze the in vitroantimicrobial activity of bacterial isolates obtained from patients clinically diagnosed with AECB in 3 consecutive years of the PROTEKT study. Using these data, and previously published clinical data, the potential role of telithromycin in the treatment of AECB will be discussed. Materials and Methods ===================== Patients and bacterial isolates ------------------------------- Details of the study design, including the selection of patients and the methodology for the identification of isolates and their storage in the PROTEKT study has been described previously \[[@B24]\]. Isolates in this study were obtained from patients diagnosed with AECB from in 85 centres in 29 countries (Table [1](#T1){ref-type="table"}). To be included in this analysis, an isolate was deemed pathogenic in AECB by clinical and laboratory findings. Isolates were only acceptable if the patient was ≥ 30 years old and the specimen was obtained from blood, bronchoalveolar lavage (BAL), or sputum. Isolates from patients diagnosed with AECB obtained from other sites (e.g., ear, throat, nasopharynx) and isolates obtained from patients \<30 years of age were excluded from this analysis because AECB is more likely to be present in patients ≥ 30 years of age and the responsible bacterial pathogen is more likely to be correctly isolated from the blood, BAL, or sputum. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Geographical distribution of isolates from AECB patients used in this study ::: Area Countries Centres Isolates --------------- ----------- --------- ---------- North America 2 4 319 South America 6 14 427 Europe 13 41 1847 Australasia 6 19 437 South Africa 1 6 13 Totals 29 85 3043 ::: In Year 1 (1999--2000), each centre had a quota of 60 isolates of S. pneumoniae, 40 H. influenzae, 15 H. parainfluenzae, 20 M. catarrhalis, 25 Streptococcus pyogenesand 20 S. aureusto collect. In years 2 (2000--2001) and 3 (2001--2002), H. parainfluenzaewere not collected and 15 extra isolates of S. pneumoniaewere collected instead. Antimicrobial testing --------------------- The comparator agents used were four β-lactams; penicillin (for S. pneumoniaeand S. aureus), ampicillin (for H. influenzae, H. parainfluenzaeand M. catarrhalis), amoxycillin/clavulanate, and cefuroxime, three macrolides/azalides; erythromycin, clarithromycin, and azithromycin, the folate synthesis inhibitor; trimethoprim-sulphamethoxazole, the tetracycline; tetracycline and a fluoroquinolone, levofloxacin. Minimum inhibitory concentrations (MIC) of each antibacterial were determined using the National Committee for Clinical and Laboratory Standards (NCCLS) broth microdilution methodology and lyophilised microtitre plates (Sensititre, Trek Diagnostics) at a central laboratory (GR Micro Ltd., London, UK) \[[@B26]\]. NCCLS breakpoints \[[@B25],[@B26]\] were used to interpret the MIC data and to determine susceptibility status. The NCCLS breakpoints for telithromycin for S. pneumoniaeand for S. aureusare ≤ 1 mg/l is susceptible, 2 mg/l is intermediate, and ≥ 4 mg/l is resistant, and for H. influenzae≤ 4 mg/l is susceptible, 8 mg/l is intermediate, and ≥ 16 mg/l is resistant \[[@B27]\]. Results ======= A total of 3043 bacterial pathogens were isolated from patients in 29 countries around the world, with by far the largest number of specimens (1841, 60.5%) coming from Europe (Table [1](#T1){ref-type="table"}). Percentage of isolates by country were as follows: Argentina 8.0%, Australia 1.1%, Austria 0.6%, Brazil 4.0%, Canada 9.1%, China 1.7%, Colombia 0.1%, Ecuador 0.6%, Eire 0.03%, France 3.4%, Germany 14.3%, Hungary 1.2%, Indonesia 0.03%, Italy 18.9%, Japan 10.0%, Mexico 1.3%, Poland 10.2%, Portugal 2.8%, Russia 0.2%, South Africa 0.4%, South Korea 0.9%, Spain 5.3%, Sweden 0.4%, Switzerland 0.6%, Taiwan 0.7%, Turkey 0.3%, United Kingdom 2.4%, United States 1.4%, Venezuela 0.1%. Of these isolates identified as causative pathogens for bacterial AECB, S. pneumoniaeand H. influenzaeformed the majority (1075 and 1037 respectively), followed by M. catarrhalis(536) (Table [2](#T2){ref-type="table"}). Patients were predominantly male (63.5%), with 47.5% of patients belonging to the (30--64) year age group and 52.5% in the \>64 year old age group. No difference in the distribution of pathogens by age group was observed (data not shown). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Distribution of specimen types by species for the 3043 bacterial pathogens described in this study ::: Specimen S. pneumoniae H. influenzae M. catarrhalis S. aureus H. parainfluenzae^1^ Total\[n (%)\] -------------------- ----------------- ----------------- ------------------ ------------- ------------------------ -------------------- Sputum 832 895 492 219 43 2481 (81.5) BAL^2^ 144 135 44 66 17 406 (13.4) Blood 99 7 0 50 0 156 (5.1) Total\[n (%)\] 1075 (35.3) 1037 (34.1) 536 (17.6) 335 (11.0) 60 (2.0) 3043 (100) ^1^Only isolated in the first year of the study ^2^Bronchoalveolar lavage ::: Table [3](#T3){ref-type="table"} shows the range of MIC values, the MIC~50~and MIC~90~of the various agents against the five species. Where breakpoints were available the percentage of isolates to the various agents is also included. Telithromycin had similar or better in vitrosusceptibility than the comparator agents against all of these species. Activity against S. pneumoniaewas particularly good, with telithromycin being the most active agent; 99.9% of isolates were classified as susceptible and the MIC~90~(0.12 mg/L) was substantially lower than all other compounds tested. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### In vitroactivity of antibacterial agents against 3043 bacterial pathogens isolated from patients with AECB and % susceptibilities to antibacterial agents. ::: Organism Antibacterial MIC mg/l % susceptible --------------------------- -------------------------------- -------------- ------------------- ------- ------ ------ ------ S. pneumoniaeN = 1075 Telithromycin 0.004--2 0.015 0.12 99.9 Azithromycin 0.03-\>64 0.12 \>64 71.2 Clarithromycin 0.015-\>32 0.03 \>32 71 Erythromycin 0.03-\>64 0.06 \>64 71 Penicillin 0.008--8 0.03 2 75.3 Amox/clavulanate^1^ 0.015--8 0.03 2 96.1 Cefuroxime 0.015--16 0.03 2 82.2 Trimethoprim-sulphamethoxazole 0.12--32 0.25 8 62 Tetracycline 0.12--32 0.25 32 69.6 Levofloxacin 0.5-\>32 1 1 98.9 H. influenzaeN = 1037 Telithromycin 0.002--16 1 2 99.6 Azithromycin 0.06--32 1 2 99.7 Clarithromycin 0.25-\>64 8 16 82.4 Erythromycin 0.25-\>64 4 8 -^2^ Ampicillin 0.12--32 0.25 16 87.3 Amox/clavulanate^1^ 0.12--4 0.5 1 100 Cefuroxime 0.12--16 1 2 99.5 Trimethoprim-sulphamethoxazole 0.03--32 0.06 4 80.7 Tetracycline 0.12--32 0.5 1 97.4 Levofloxacin 0.008--8 0.015 0.015 99.8 M. catarrhalisN = 536 Telithromycin 0.004--0.5 0.06 0.12 Azithromycin 0.06--0.25 0.06 0.06 Clarithromycin 0.25--0.5 0.25 0.25 Erythromycin 0.25--1 0.25 0.25 Ampicillin 0.12--32 8 16 Amox/clavulanate^1^ 0.12--0.5 0.12 0.25 Cefuroxime 0.12--16 1 2 Trimethoprim-sulphamethoxazole 0.06--4 0.25 0.5 Tetracycline 0.12--32 0.25 0.5 Levofloxacin 0.008--0.06 0.03 0.03 S. aureusN = 335 Telithromycin 0.03-\>32 0.06 \>32 85.1 98.9 24.2 Azithromycin 0.12-\>64 1 \>64 70.4 84.2 9.7 Clarithromycin 0.03-\>32 0.25 \>32 70.4 84.2 9.7 Erythromycin 0.12-\>64 0.25 \>64 70.4 84.6 9.7 Penicillin 0.008--8 4 8 23.6 28.9 0 Amox/clavulanate^1^ 0.06--8 0.5 8 83.1 100 0 Cefuroxime 0.12--16 1 16 81.2 100 0 Trimethoprim-sulphamethoxazole 0.12--32 0.12 0.25 94.9 97.4 83.9 Tetracycline 0.12--32 0.5 32 84.8 92.7 50 Levofloxacin 0.5--64 0.5 8 76.7 81.7 6.5 H. parainfluenzaeN = 60 Telithromycin 0.06--4 1 2 100 Azithromycin 0.06--2 0.5 1 100 Clarithromycin 0.25--16 4 8 93.3 Erythromycin 0.5--8 2 4 -^2^ Ampicillin 0.12--32 0.25 1 90 Amox/clavulanate^1^ 0.12--2 0.5 1 100 Cefuroxime 0.12--4 0.25 0.5 100 Trimethoprim-sulphamethoxazole 0.03--32 0.03 1 88.3 Tetracycline 0.12--16 0.5 4 88.3 Levofloxacin 0.008--8 0.015 0.06 98.4 ^1^Amox/clavulanate = Amoxycillin/clavulanate ^2^No CLSI interpretive criteria for erythromycin and Haemophilusspp. ::: One hundred and three (9.6%) S. pneumoniaeisolates (from 51 and 53 patients in the 30--64 and \>64 year old age groups respectively)) were resistant to both penicillin (MIC ≥ 2 mg/L) and erythromycin (MIC ≥ 1 mg/L) and this was reflected in resistance to amoxycillin, cefuroxime, clarithromycin and azithromycin also (Table [4](#T4){ref-type="table"}). These isolates were found in 35 centres in 16 countries. Sixty of these resistant isolates were also resistant to both trimethoprim-sulphamethoxazole and tetracycline. Both telithromycin and levofloxacin had good activity against these isolates, 99% susceptibility to telithromycin and 98.1% to levofloxacin. The MIC~50~and MIC~90~values for telithromycin in this population were 0.06 mg/L and 0.5 mg/L, respectively. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Antibacterial activity against 103 Streptococcus pneumoniaeisolates with combined macrolide and penicillin resistance ::: Antibacterial % susceptible % intermediate % resistant -------------------------------- ------------------- -------------------- ----------------- Telithromycin 99.0 1.0 0.0 Azithromycin 0 0 100 Clarithromycin 0 0 100 Erythromycin 0 0 100 Penicillin 0 0 100 Amoxycillin 0 0 100 Amoxycillin-clavulanate 72.8 13.6 13.6 Cefuroxime 1.0 1.0 98.0 Trimethoprim-sulphamethoxazole 19.4 16.5 64.1 Tetracycline 11.7 0.0 88.3 Levofloxacin 98.1 0.0 1.9 ::: Over 99% of H. influenzaeisolates were susceptible to amoxycillin-clavulanate, cefuroxime, telithromycin, azithromycin, and levofloxacin. Tetracycline also had good activity with 97.4% of isolates susceptible. Only 11.7% of H. influenzaeisolates produced β-lactamase. There were only 60 isolates of H. parainfluenzaeand 100% of these were susceptible to four of the eight compounds tested, telithromycin, amoxycillin/clavulanate, cefuroxime and azithromycin. Trimethoprim-sulphamethoxazole and tetracycline were the least active compounds. In terms of MICs, levofloxacin, azithromycin and telithromycin were the most potent compounds against M. catarrhaliswith MIC~90~values of 0.03 mg/l, 0.06 mg/l and 0.12 mg/l respectively. There are currently no interpretative NCCLS guidelines available for M. catarrhalisto allow classification into susceptible or resistant categories. The total number of isolates of S. aureus was 335 and of these only 62 were resistant to methicillin (MRSA). Trimethoprim-sulphamethoxazole was the most active compound overall, with 94.9% of all isolates being susceptible. Telithromycin and tetracycline were the next most active with 85.1% and 84.8% of all isolates susceptible. Telithromycin was the most active compound against the MSSA isolates, with 98.9% being susceptible. The susceptibility of MSSA to tetracycline and trimethoprim-sulphamethoxazole was 92.7% and 97.4% susceptible respectively. These three compounds were the only ones to have activity against the MRSA isolates (trimethoprim-sulphamethoxazole 83.9%, tetracycline 50% and telithromycin 24.2%). Less than 10% of the MRSA isolates were susceptible to the remainder of the compounds. Discussion ========== The primary cause of COPD is exposure to tobacco smoke, the major risk factor being cigarette smoking. The demography of the disease in this study and others reflects this, as the majority of patients in this analysis were male and half were elderly (\>64 yrs of age) (2). S. pneumoniaeis most frequently isolated in the least severe cases of AECB, whereas H. influenzaeis more commonly isolated from moderate to severe cases, with P. aeruginosaoccurring in severe hospitalised cases \[[@B28]\]. Telithromycin does not have good activity against Pseudomonasspp. (GR Micro Limited, data on file, internal report number 141-02-99) and hence may not be an appropriate empirical therapeutic option for AECB patients with severe underlying disease who are hospitalized for an acute exacerbation. Whether the isolation of a pathogen during AECB represents an infection responsible for the exacerbation has been debated for many years \[[@B29]-[@B31]\]. Bacteria have been isolated almost as frequently from patients with stable COPD as those with an AECB, and clinical trials of antibiotic therapy in AECB show contradictory and sometimes unconvincing results \[[@B30]\]. The presence of bacteria in the lower airways is, however, regarded as abnormal since these airways are sterile in healthy adults, and it has been hypothesized that the presence of bacteria in stable COPD represents a low-grade smouldering infection. In addition, a recent study has shown that infection with different strains of pathogens that are new to the patient is associated with development of exacerbation \[[@B32],[@B33]\]. Amoxycillin-clavulanate, azithromycin, and levofloxacin have been shown to be effective in the treatment of AECB, however, there is concern regarding their long-term usefulness, because of the development of resistance to these agents among the causative pathogens \[[@B34],[@B35]\]. Telithromycin has a more focused spectrum of activity than the β-lactams and the fluoroquinolones; it is specifically targeted against pathogens causing community-acquired respiratory disease, including those most commonly associated with AECB. In addition, it is active against penicillin- and macrolide-resistant strains of S. pneumoniaeand hence offers a viable potential option for the empiric treatment of AECB in non-hospitalised patients \[[@B36]\]. The data in this study demonstrate that telithromycin has high in vitroactivity against the commonest bacterial pathogens causing AECB. These data also show that telithromycin has the highest overall activity against bacterial isolates from patients with AECB, regardless of species. Almost 10% of S. pneumoniaeisolated were resistant to penicillin, macrolides, and at least one of the other antibiotics tested, with only telithromycin and levofloxacin retaining high activity against these isolates (99.0% and 98.1%, respectively). The validity of this finding is strengthened as the isolates were obtained from a large number of patients over a wide geographical distribution. Although atypical pathogens were not examined in the PROTEKT study, telithromycin has been shown to have superior activity in vitroagainst Chlamydophila pneumoniaeto the other macrolides with the exception of clarithromycin and has similar activity to the fluoroquinolones \[[@B37]\]. In guinea pig models, telithromycin had better activity than erythromycin against Legionella pneumophilainfections \[[@B38]\]. In vitro, the activity of telithromycin against L. pneumophilawas similar to levofloxacin but better than erythromycin \[[@B38]\]. β-lactams and cephalosporins have no activity against Mycoplasma pneumoniaeas this species lacks a typical bacterial cell wall, the site of activity for these drugs. Telithromycin has been found to have higher activity than doxycycline and levofloxacin against M. pneumoniae\[[@B39]\]. As the atypical pathogens can represent up to 10% of infections associated with AECB, the efficacy of telithromycin against these pathogens could be a consideration in the selection of empiric therapy for AECB. Telithromycin has been shown to penetrate into respiratory tissues well \[[@B40]\]. The concentration of telithromycin in alveolar macrophages and epithelial lining fluid exceeds that of plasma markedly and remains at therapeutic levels for 24 hours after dosing. Bactericidal levels are also maintained in plasma. A good post-antibiotic effect has also been observed \[[@B41]\]. Telithromycin causes only moderate ecological disturbance to oral and intestinal flora comparable to that associated with clarithromycin and it does not significantly increase the development of resistance in the normal flora, although the MIC of oral streptococci can be slightly raised \[[@B42]\]. Telithromycin can be administered once a day for AECB. Clinical studies have demonstrated that 800 mg administered once daily for 5 days was as effective and well tolerated as a 10-day course of amoxycillin/clavulanate (500/125 mg 3 times daily for 10 days), cefuroxime axetil (500 mg twice daily for 10 days) or clarithromycin (500 mg twice daily for 10 days) \[[@B43]\]. Other clinical studies have also confirmed the safety and tolerability of telithromycin 800 mg administered for 5 -- 10 days \[[@B44]\]. Once a day dosing schedules and shorter courses may promote patient adherence to therapy, and this in turn could delay the development of resistance. Although this study provides valuable information on the overall antimicrobial profile of bacteria causing AECB, care should be taken when interpreting data related to specific demographics. The prevalence of species could not be calculated in this study as a major limitation, inherent to most surveillance studies, is the requirement for collecting centres to fulfil a specified quota of isolates over a defined time period (1 year). If, for instance, a centre managed to fulfil the quota for S. pneumoniaeisolates from patients with community-acquired pneumonia, it could then only send H. influenzaefrom patients with AECB to fulfil the quota for this organism. In addition, atypical pathogens were not sampled and they can represent up to 10% of the causative pathogens \[[@B28]\]. In summary, the data presented here demonstrate that telithromycin has good in vitroactivity against H. influenzae, S. pneumoniae, and M. catarrhalis, respiratory pathogens commonly isolated in AECB. It is as active as or more active than agents that are currently used in this clinical setting. Additionally, although not shown here, telithromycin has better in vitroactivity against atypical pathogens than other agents; an important advantage in this clinical setting as these pathogens may represent 10% of AECB associated infections. The development of resistance will always be a threat to the usefulness of antibacterial compounds, however surveillance studies such as PROTEKT allow the rapid detection and characterization of resistance mechanisms and highlight the need for and examine the in vitroefficacy of newer antibacterial agents. Providing careful surveillance for the development of resistance is maintained, telithromycin currently offers a useful agent in the treatment of AECB. Acknowledgements ================ We are grateful to our colleagues worldwide for the supply of bacterial isolates as part of the PROTEKT study and the GR Micro PROTEKT team who performed the MIC testing. Aventis is acknowledged for their financial support of the PROTEKT study. The data and discussion in the manuscript were presented in part at the European Respiratory Society Congress held in Glasgow in 2004 (Abstract P3902).	PubMed Central	2024-06-05T03:55:54.119396	2005-3-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555545/", "journal": "Ann Clin Microbiol Antimicrob. 2005 Mar 8; 4:5", "authors": [ { "first": "Sanjay", "last": "Sethi" }, { "first": "Antonio", "last": "Anzueto" }, { "first": "David J", "last": "Farrell" } ] }
PMC555546	Atherosclerosis is a generalized disease that causes lesions in large- and medium-sized elastic and muscular arteries. As lesions progress, arterial walls are remodeled, a process through which the size of the arterial lumen is preserved. Because of this, the disease is clinically asymptomatic during its earlier stages and may go unnoticed for decades as the risk for its clinical manifestation as acute vascular disease grows \[[@B1],[@B2]\]. Epidemiological studies and intervention trials based on the incidence of acute vascular disease endpoints require years of follow-up, the participation of large populations, or both. As a consequence, such studies consume considerable time and financial resources \[[@B3]\]. The use of surrogate markers for atherosclerosis extent and progression is widespread. Currently, the most established of these is based on carotid intima-media thickness (IMT) as measured by B-mode ultrasound. It is a natural extension to consider these measures as surrogate markers for cardiovascular disease clinical endpoints \[[@B4],[@B5]\]. If this extension is valid, the time, expense, and participant burden in understanding and developing treatments to reduce the risk of clinical endpoints can be reduced. To be rigorous, this definition must be based on accepted definitions and/or set of criteria for surrogacy. This document examines the evidence that carotid IMT, a marker for atherosclerosis, meets two prominent set of criteria for defining surrogate outcomes. Defintions of surrogate markers =============================== Both clinical and statistical criteria for surrogacy have been proposed. Clinical Criteria for Surrogacy ------------------------------- Boissel, et al. lay out criteria that markers must meet to be considered as valid surrogates for clinical endpoints \[[@B6]\]. We group these into three domains. B1:([Efficiency]{.underline}) The surrogate marker should be relatively easy to evaluate, preferably by non-invasive means, and more readily available than the gold standard. The time course of the surrogate should precede that of the endpoints so that disease and/or disease progression may be established more quickly via the surrogate. Clinical trials based on surrogates should require fewer resources, less participant burden, and a shorter time frame. B2:([Linkage]{.underline}) The quantitative and qualitative relationship between the surrogate marker and the clinical endpoint should be established based on epidemiological and clinical studies. The nature of this relationship may be understood in terms of its pathophysiology or in terms of an expression of joint risk. B3:([Congruency]{.underline}) The surrogate should produce parallel estimates of risk and benefit as endpoints. Individuals with and without vascular disease should exhibit differences in surrogate marker measurements. In intervention studies, anticipated clinical benefits should be deducible from the observed changes in the surrogate marker. Statistical Criteria for Surrogacy ---------------------------------- Prentice views surrogacy as a statistical property and defines it with mathematical expressions \[[@B7],[@B8]\]. Four criteria are required for S to serve as a surrogate for endpoint T with respect to intervention Z. P1:The intervention should affect the distribution of T. P2:The intervention should affect the distribution of S. P3:The distribution of T should be dependent on S. P4:Endpoint T should be conditionally independent of Z given S, i.e. S should fully account for the impact of Z on T. This definition may be specific to a particular setting and cohort; a marker may meet the criteria for surrogacy for one intervention, but fail criteria for others. The criteria for surrogacy are based on explicit models, and may also be dependent on covariates and additional explanatory factors being collected and incorporated into these models. Establishing Surrogacy ---------------------- These clinical and statistical definitions require different approaches to establish surrogacy, neither of which is clear-cut. To meet the criteria outlined by Boissel, et al. \[[@B6]\], experience and data from clinical trials are required to demonstrate efficiency and congruence, and data from bench and cohort studies are required to establish plausible linkage. Arguments for surrogacy address whether these data are sufficiently compelling. To meet the criteria outlined by Prentice \[[@B7]\], decisions must be made on the parametric model describing the relationship between intervention and outcomes. The plausibility for surrogacy is argued by the ability of the surrogate marker, once incorporated in this model, to account (induce conditional independence) for this relationship using experimental data (and by P1 is limited to interventions that affect outcomes). Since statistical relationships cannot be established with certainty, arguments are required that the empirical evidence for conditional independence provided by data are sufficiently compelling to adopt the hypothesis of conditional independence required by the Prentice criteria. B-mode ultrasound imt ===================== B-mode ultrasound imaging technology has evolved to the extent that the walls of superficial arteries can be imaged non-invasively, in real-time, and with high resolution. Unlike angiography or \'luminology\', ultrasound imaging can visualize the arterial wall at every stage of atherosclerosis, from \'normal\' arterial wall to complete arterial occlusion. Arterial wall thickness can therefore be measured as a continuous variable from childhood to old age, in patients and healthy controls \[[@B9]\]. Studies that have evaluated the origin of the lumen-intima and the media-adventitia ultrasound interfaces in relation to carotid and femoral far-wall arterial histology have demonstrated that the distance between these interfaces reflects the intima-media complex. Consequently, this distance is referred to as IMT \[[@B10],[@B11]\]. IMT has been widely used in both observational studies and intervention studies. Surrogacy of carotid imt with respect to statins ================================================ We are interested in examining the potential of carotid IMT to serve as a surrogate marker for cardiovascular events, in particular cardiovascular mortality, myocardial infarctions, and clinical stroke. The clinical and statistical arguments for surrogacy are contextual, i.e. are based on specific relationships and mechanisms. It is unreasonable to make open-ended claims that a carotid IMT is a surrogate for these endpoints for all interventions and all cohorts, a point that has not been emphasized sufficiently. Our specific focus is to examine surrogacy in clinical trials of HMG-CoA reductase inhibitors (statins). Empirical evidence is largely drawn from statin clinical trials conducted on cohorts of adults at elevated risk for cardiovascular endpoints. Our choice of statins is based, in part, on the many published trials available for these agents. We acknowledge that it is quite possible that IMT may be a valid surrogate for cardiovascular events with respect to statins (i.e. accounting for the effects of these agents on cardiovascular events), but may not be a valid surrogate for other agents (e.g. diuretics or postmenopausal hormone therapy) or endpoints. We used Medline searches to identify seven placebo-controlled clinical trials of statins that report both IMT outcomes and cardiovascular events (see Table [1](#T1){ref-type="table"}): the Asympotomatic Carotid Artery Progression Study (ACAPS), the Kuopio Atherosclerosis Prevention Study (KAPS), the Pravastatin, Lipids, and Atherosclerosis in the Carotid Arteries Study (PLAC-2), the Carotid Atherosclerosis Italian Ultrasound Study (CAIUS), the Regression Growth Evaluation Statin Study (REGRESS), the Beta-Blocker Cholesterol Lowering Asymptomatic Plaque Study (BCAPS), and the Fukuoka Atherosclerosis Trial (FAST) \[[@B12]-[@B18]\]. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Clinical trials involving HMG-CoA reductase inhibitors and reporting both carotid IMT and cardiovascular event outcomes. ::: Clinical Trial (N\) Statin Relative Impact on IMT Progression of Primary Outcome (mm/yr): Mean \[95% CI\] (reported p-value) Relative Impact on Reported Cardiovascular Endpoints: Odds Ratio \[95% CI\] ------------------------ ------------- --------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------- ---------------------- ACAPS^(25)^(N = 919) Lovastatin -0.015 \[-0.023, -0.007\] (p = 0.001) CVD Death, MI, Stroke 0.34 \[0.12, 0.69\] KAPS^(26)^(N = 447) Pravastatin -0.014 \[-0.022, -0.006\] (p = 0.005) CVD Death, MI, Stroke 0.57 \[0.22, 1.47\] PLAC-II^(47)^(N = 151) Pravastatin -0.009 \[-0.031, 0.013\] (p = 0.44) Clinical Coronary Events 0.37 \[0.11, 1.24\] CAIUS^(48)^(N = 305) Pravastatin -0.014 \[-0.021, -0.005\] (p = 0.0007) CVD Death, MI 1.02 \[0.14, 7.33\] REGRESS^(28)^(N = 255) Pravastatin -0.030 \[-0.056, -0.004\] (p = 0.002) Clinical Events 0.51 \[0.24, 1.07\] BCAPS^(49)^(N = 793) Fluvastatin -0.008 \[-0.013, -0.003\] (p = 0.002) CVD Death, MI, Stroke 0.64 \[-0.24, 1.66\] FAST^(50)^(N = 164) Pravastatin Significant Benefit (p \< 0.001) CVD Death, MI 0.32 \[0.10, 1.06\] Pooled Estimate -0.012 \[-0.016, -0.007\]\\* 0.48 \[0.30, 0.78\] \Arms used in meta-analysis; \\*Excludes FAST ::: Do IMT measurements meet clinical criteria for surrogate markers of cardiovascular disease events? -------------------------------------------------------------------------------------------------- The three criteria described by Boissel, et al. \[[@B6]\] for surrogate markers relate to efficiency, linkage, and congruency and will be described in turn. ### B1: Efficiency Carotid IMT has been widely used in clinical trials. Reliable protocols have been established for its measurement and it is arguably more sensitive to the effects of interventions than cardiovascular disease events. Six of seven clinical trials in Table [1](#T1){ref-type="table"} reported a significant beneficial impact of statins on IMT progression with respect to their primary IMT outcome measure; the seventh trial, PLAC-II found no significant impact on its primary IMT outcome measure, but reported a significant impact on a secondary IMT measure. In six of seven trials, there were beneficial trends with respect to reported cardiovascular disease endpoints; however only for one trial (ACAPS) did this trend reach nominal statistical significance. Thus, while IMT measures were sufficiently sensitive so that benefit could be established within trials of this size, the general benefit with respect to cardiovascular events could not be generally established. ### B2: Linkage The strong association between carotid IMT and cardiovascular events has been demonstrated repeatedly. For example, the Cardiovascular Health Study, found it to be the risk factor most strongly associated with incident cardiovascular events \[[@B19]\]. In the Rotterdam Study, Del Sol, et al. found that a single carotid IMT measurement was of the same importance as a battery of commonly used risk factors in the prediction of CHD and CVD \[[@B20]\]. The Atherosclerosis Risk in Communities (ARIC) study found that carotid IMT of 1 mm or more was associated with two to five times the increased hazard of CHD and four to eight times the increased hazard of stroke \[[@B21],[@B22]\]. Using a nested case-control approach and a mean duration of follow-up of 2.7 years, the Rotterdam Study found that per standard deviation increase (0.16 mm) in IMT, the odds ratio for stroke was 1.41 and for myocardial infarction was 1.43 \[[@B23]\]. Atherosclerosis is a manifestation of the pathophysiology underlying cardiovascular disease. The links between carotid IMT and atherosclerosis are well-established and IMT measures, as markers of atherosclerosis, have contributed greatly to the understanding of atherosclerosis progression \[[@B24],[@B25]\]. These measures have characterized the role of many risk factors for atherosclerosis and currently serve the basis for several studies examining its genetics. The mechanisms by which atherosclerosis is causally related to cardiovascular events are also well-established. ### B3: Congruency IMT (continuous) and events (categorical) represent different measurement scales, thus it is difficult to argue they are influenced by statin therapy to quantitatively similar degrees. We drew evidence that the impacts are qualitatively similar using a meta-analysis of the clinical trials listed in Table [1](#T1){ref-type="table"} and developed pooled estimates of the relative impact of HMG-CoA reductase inhibitor (statin) therapy on IMT progression and on the odds ratio of cardiovascular endpoints \[[@B26]\]. Because standard errors for IMT changes were not reported for the FAST trial, it was excluded from this analysis. Across the trials, statin therapy was associated with an average decrease of IMT progression of 0.012 mm/yr with 95% confidence interval \[-0.016, -0.007\]. This pooled estimate confirms with greater precision the results from the individual trials. More importantly, the meta-analysis yields a significant odds ratio of 0.48 \[0.30, 0.78\] for the reduction cardiovascular events associated with statin therapy. Thus, a meta-analysis across a number of trials demonstrates a benefit with respect to cardiovascular disease events that is congruent with the benefits established by individual IMT trials. Do IMT measurements meet the criteria of statistical criteria for surrogate markers of cardiovascular disease events? --------------------------------------------------------------------------------------------------------------------- The four criteria of Prentice \[[@B7]\] are as follows. ### P1: Impact of Interventions on Endpoint There is convincing evidence, some of which is summarized in the meta-analysis described above, that statin therapy reduces the risk of cardiovascular events, to the extent that this is now an indication for their use. ### P2: Impact of Interventions on Carotid IMT As noted above, this association is supported by the results of our meta-analysis (Table [1](#T1){ref-type="table"}) and elsewhere (e.g. \[[@B4]\]). ### P3: Link Between Carotid IMT and Cardiovascular Events The considerable evidence of this association has been discussed above. ### P4: Conditional Independence Between Statin Therapy and Cardiovascular Events Given Carotid IMT We know of no published literature that examines this conditional independence for statin therapies. Such a study is difficult to mount as it requires both sufficient power to demonstrate the relative impact on IMT progression of an intervention and sufficient size and follow-up time after this demonstration to assess the ability of measured IMT progression to account for subsequent risk. The only published account to examine the conditional independence of cardiovascular events given carotid IMT is for colestipol-niacin therapy in the Cholesterol Lowering Atherosclerosis Study (CLAS) clinical trial \[[@B27]\]. The 2-year CLAS trial demonstrated that colestipol-niacin therapy reduced IMT progression \[[@B28]\]; the trial cohort was surveyed an average of 8.8 years after the conclusion of CLAS to tally post-trial incidence of coronary events (nonfatal MI, coronary death, and coronary artery revascularization). These investigators found that while treatment assignment, by itself, was significantly related to occurrence of these events (relative risk 0.41; p = 0.01), when on-study IMT progression was included as a covariate, this relationship evaporated (relative risk 1.1; p \> 0.2). Friedman, et al. use the term proportion of treatment effect captured (PTE) to describe how well a surrogate marker meets criterion P4 \[[@B29]\]; at face value, the findings from CLAS produce an estimate that PTE exceeds 1. In our meta-analysis, when IMT progression is included as a covariate in regression models linking cardiovascular disease events to statin treatment, the relative odds ratio is mediated from 0.48 (as tabulated below) to 0.64 and is no longer statistically significant (p = 0.13). This suggests that changes in IMT may account for some, but not all, of the effect of statins on cardiovascular events (i.e. a PTE of 0.3). Several issues complicate this argument, however. Even if a surrogate successfully meets Prentice\'s criteria for surrogacy within individual trials, because designs, cohorts, and endpoints vary it is to be expected that a surrogate would only account for some, not all, of treatment effects in regression models across trials. Secondly, like many markers, IMT is subject to measurement error and that is not insubstantial. This measurement error, if uncorrected, may lead to marked underestimates of relationships \[[@B30]\]: measured IMT progression may appear to account for less of the relationship between interventions and events than true progression. These issues obscure the validation of surrogacy from meta-analyses based on published summary statistics. We can only conclude that IMT progression may account for at least some of the treatment effects attributable to statin therapy, but that it is difficult to quantitate the degree of this relationship and that full surrogacy cannot be ruled out. Summary ======= We have examined, in a structured and rigorous manner, the evidence that carotid IMT progression may serve as a surrogate for cardiovascular disease endpoints in statin trials. Each of the criteria for surrogacy described by Boissel, et al. appears to be met. The first three of Prentice\'s criteria are met, and the fourth is met by the one published study for which it can be evaluated (although not for statin therapy). Meta-analyses of statin trials provide support for Boissel\'s criteria and the first three of Prentice\'s criteria, and are not inconsistent for Prentice\'s fourth criterion. It is possible that these arguments may generalize to other agents whose mechanisms are similar to statins, however additional analyses, based on criteria for surrogate outcomes, would be required to make this extension. Competing interests =================== MAE received an honorarium from Sankyo Pharma, Inc for a meeting during which ideas for this manuscript were developed. He is an occasional consultant to other companies concerning the design of clinical trials involving carotid ultrasonography. DHO serves on data safety and monitoring boards for Pfizer and Astra/Zeneca and serves as consultant to Sankyo Pharma and to Merck. JGT received an honorarium from Sankyo Pharma, Inc for a portion of this work. TO has no competing interests. GE has received an honorarium and consulting fees from AstraZeneca Pharmaceuticals for assistance in the planning and implementation ofa clinical trial involving carotid ultrasonography and statin therapy. He also serves as an occasional consultant to other companies on the design and conduct of trials involving carotid ultrasonography in which statins may be included as background therapies, but are not part of the experimental intervention. HM is an occasional consultant to Sankyo Pharma, including attending the meeting during which ideas for this manuscript were developed. He very occasionally consults with other Pharma companies like MSD, Essex, and Lilly. He is a consultant to Boston Scientific for interventional cardiology and intravascular ultrasound and is also a regular teacher in carotid stenting for the Guidant and Cordis companies. Authors\' contributions ======================= MAE, DHO, and HM conceived and drafted this manuscript. MAE, JGT, and TM organized and conducted its meta-analysis. GE provided oversight to analyses and contributed to interpretation of results. All authors read and approved the final manuscript. Acknowledgements ================ This paper was developed with support from Sankyo Pharma GmbH Prinzenallee 7, 40549 Duesseldorf, Germany.	PubMed Central	2024-06-05T03:55:54.122631	2005-3-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555546/", "journal": "Curr Control Trials Cardiovasc Med. 2005 Mar 10; 6(1):3", "authors": [ { "first": "Mark A", "last": "Espeland" }, { "first": "Daniel H", "last": "O'Leary" }, { "first": "James G", "last": "Terry" }, { "first": "Timothy", "last": "Morgan" }, { "first": "Greg", "last": "Evans" }, { "first": "Harald", "last": "Mudra" } ] }
PMC555547	Background ========== We are now witnessing a post-genomic era of cancer research. Thousands of papers are devoted to discovering molecular mechanisms of this extremely complicated program, the latter term being understood as a prescribed sequence of events with an inevitable result. In almost all studies cancer is considered as a given entity with no attempts made, to my knowledge, to search into its evolutionary origin. However, cancer almost certainly fulfills some evolutionary tasks. First, although cancer is usually mentioned as a representative of a large group of age-related diseases, it principally differs from all the others. Cardiovascular diseases, diabetes, Alzheimer\'s disease, and many other diseases are manifestations of the \"loss-of-function\" phenotype due to degeneration and/or death of corresponding cells. Cancer, on the contrary, is an active \"gain-of-function\" process. Cancer cells acquire numerous new functions, including the unique abilities to adapt to a changing environment and to dodge the blows of the body\'s protective means, as well as a striking capacity to recruit surrounding normal tissues. The tumor transforms its natural enemies (normal cells) into unnatural allies, being able to grow only having such a paradoxical support \[[@B1]-[@B15]\]. The functional relations between normal and cancer cells are so diverse and the tumor structure is so complex and hierarchical that a growing tumor is sometimes viewed as a special developing \"organ\" \[[@B2],[@B16]\]. Such an \"organ\" must have serious evolutionary grounds to have evolved. Second, cancer is an inevitable attribute of the animal world. It hits every species despite the fact that there are huge differences among them in the intracellular machinery, organization of signal pathways and, accordingly, in anticancer defense (or, transformation resistance, see below). In no case, however, is the anticancer defense of a species high enough to exclude completely this illness, despite presumably high cellular plasticity in this respect. This fact would indicate some evolutionary advantages of possessing such a trait. It seems likely that anticancer defense of a species is counterbalanced by opposing forces acting on the population level. In other words, cancer incidence among a species is presumably maintained at certain favorable level, which is coupled in each particular case with an evolutionary adaptation of the intracellular machinery. In conclusion, evolutionary grounds for cancer seem to be evident; it is the explanation of its expediency which presents difficulties. A clue to this problem would be the fact that cancer cell possesses, apart from the well known necessary and sufficient capabilities \[[@B17]\], a killer function directed against the host. Strikingly, this evident capability that seems to be a key property of the tumor cell does not attract the attention it deserves. It is believed to be a self-evident consequence of the other traits that constitute the malignant phenotype, although the available evidence would rather suggest that killer function is a distinct capability. Taking into account this overlooked property, one can view cancer as a suicidal act of an individual, since the inevitable outcome of this illness is demise (if treatment is absent or delayed). By analogy with apoptosis, which has evolved as altruistic suicidal act of a damaged cell \[[@B18]\] that otherwise would threaten the genetic stability of the cell population, cancer might be viewed as an individual suicidal act that brings some benefits to the population. Presentation of the hypothesis ============================== Killer function is a key capability of the cancer cell ------------------------------------------------------ Carcinogenesis is a multistage process of accumulation of gene defects that determine the characteristic traits of the cancer cell: self-sufficiency in growth signals, insensitivity to anti-growth and pro-apoptosis signals, limitless replicative potential, sustained angiogenesis, tissue invasion, and metastasis \[[@B17]\]. These acquired capabilities determine the malignant phenotype of a cell but do not explain the clinical manifestations of cancer. Indeed, it seems astonishing that the human body, which consists of ca. 10^14^cells, can not endure a relatively small fraction of cancer cells (0.01 -- 0.1% of the total), this burden often being incompatible with life. Rarely, the immediate cause of death is evident (brain compression, bleeding, perforation of the intestine), but in general it remains obscure. This suggests a deadly influence of cancer upon the body through some unknown mechanisms. Although each of many different forms of cancer has clinical peculiarities, the overall course of the illness and the final result are always the same. So, the notion that the tumor cell, regardless of origin, possesses a special killer function is a statement of an obvious fact. What is surprising in this regard is that no room has been allocated to it in the existing paradigm of carcinogenesis. Paraneoplastic syndromes present evidence that tumors may affect normal tissues remote from the primary site. These syndromes are extremely diverse and affect almost all organs and tissues \[[@B19]\]. The most frequent clinical manifestations are cachexia, anorexia, nausea, neuropathy, retinopathy, general sickness, and malfunctions of many body systems. A long time (16 to 20 months) before cancer is diagnosed, some patients show body weight loss, which is indicative that even at early stages tumors may have a generalized effect upon the body, which increases progressively with tumor growth \[[@B20]\]. Since, however, pronounced cachexia (a loss of more than 5% body weight) occurs in about a third of patients and becomes the cause of death in only 20% of the cases \[[@B21]\], it is clear that tumor cells have other, yet unrecognized means of killing the body. It remains unknown whether the paraneoplastic syndromes are direct manifestations of the killer function itself or they are mere side effects of tumor growth, such as autoimmune diseases. It is noteworthy that cancer is not always accompanied by paraneoplastic syndromes \[[@B22]\], yet its killer function never fails. On the other hand, the most effective treatment of paraneoplastic syndromes is specific cancer therapy, while the reverse approach, a symptomatic treatment targeted at particular manifestations of tumor growth, rarely gives positive results and never offers a radical cure. This fact suggests that most of the mentioned effects, justifying their name of [para]{.underline}neoplastic, take place not within the killer function pathway but somewhere aside. Because of this, the term killer function will be used here, without going into its mechanism, as indicating the obvious capability of cancer cells to kill the body. The killer capability is crucial to the achievement of the final goal, body demise, whereas all the other, promoting proliferation and spread of killer cells are de factoonly accessory. It seems to be a distinct capability of cancer cells, rather than a derivative of other capabilities, such as uncontrolled proliferation and metastasizing. It is unlikely that active proliferation can by itself exert such a deleterious effect since dozens of billions of cells divide daily in the human body, which is many-fold greater than the proliferation pool of the biggest tumor. Even the metastases, these relatively small foci of ectopic proliferation, can not account by themselves for the inevitable demise. The killer function is also a universal property of the cancer cell since without treatment the lethal outcome is inevitable no matter what is the type of tumor, its ability to develop metastases, recur, induce cachexia, or affect biochemical indices. Finally, this property is specific to the cancer cell, as in normal cell physiology there are no examples of such activity. The killer function seems radically different from all other acquired capabilities in that it apparently gives no selective advantage to the cancer cell. On the contrary, for the latter, as a part of the body, acquiring such a function is the same as committing suicide. This changes radically the understanding of the role of cancer cells: they can be regarded not as selfish \"cheats\" \[[@B23]\], which propagate at the expense of all others, but rather as altruists which sacrifice themselves and the whole body for the sake of some higher (apparently population) benefits, as suggested in the recent hypothesis of phenoptosis \[[@B24]\]. Mutations as death program trigger ---------------------------------- As cancer cells do not acquire selective advantages during realization of their killer function, it seems unlikely that the latter is created each time de novoin the same way as the other properties are, namely in the way of numerous step-by-step cycles of mutation-selection. Rather, the cells possess a built-in and ready-for-use program of deadly events, which, just like apoptosis, is launched under certain conditions and then functions automatically. In such a case, mutations of cancer-related genes are not only transformation steps, as generally considered, but also a trigger countdown mechanism that activates the death program directed against the \"host\". This dual activity leads to appearance of an expanding clone of killer cells progressively strengthening their effect upon the organism (Fig. [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Step-wise accumulation of specific gene defects.Mutations (red triangles) trigger the built-in cell killer function (red asterisks). m(0), normal cells; m(1), m(2), m(3), m(n), mutant cells with 1, 2, 3, and ndefects in cancer-related genes, respectively. (a), (b), (c), (n), selection \"sieves\" which determine the stages of transformation. The trigger \"count-down\" is shown on the left. Double-headed arrows indicate that transformation and trigger steps are amenable to species- and tissue-specific variations (see text). ::: ![](1475-2867-5-5-1) ::: A specific feature of the genetic defects underlying the tumor phenotype is that they do not decrease cell viability (basic cellular functions, in contrast to specialized, are even enhanced). In a certain sense, these are the defects of not the cell per sebut rather of the cell/organism \"interface\" that mediates subordination of cell functions to the common interests. Under normal conditions, numerous signal pathways tie the cell to the tissue homeostasis mechanism, whereas their defects are nothing but a step-wise liberation of the cell from its \"fetters\". The number of liberating mutations necessary for cell transformation to occur ranges from 4 to 12 \[[@B25],[@B26]\], this value being a quantitative measure of the cell \"transformation resistance\". If one can compare the cancer cell with an explosion device aimed at destroying the body, then mutagenesis serves there the function of a trigger countdown clock with 4--12 intermediate positions (Fig. [1](#F1){ref-type="fig"}). Peto paradox ------------ Cell transformation is an extremely rare event because it requires the coincidence in a single cell of several very improbable events \[[@B26]-[@B28]\]. However, the life-span risk of human cancer is high (ca. 20%) because of a huge number of cells in the body (ca. 10^14^) and large longevity. Indeed, no matter how unlikely is the event by itself it has a real chance to occur under such conditions. If so, one might suggest that animals with a small body weight and short life-span (e.g., rodents) should not suffer from cancer at all, while big animals (whales) should get cancer in their mothers\' wombs. Reality, however, does not follow this theory\'s predictions (the well known Peto paradox \[[@B29]\]). All animals regardless of body weight and longevity suffer from cancer, but, on the other hand, cancer incidence does not threaten the species existence. The Peto paradox is explained by various transformation resistance of cells from different species \[[@B23],[@B30]\]. This may be due to (i) different efficiency of DNA repair systems, (ii) difference among species in the degree of selective advantages acquired by the cell from similar mutations \[[@B30]\], (iii) different setting up of the trigger (e.g., more signal pathways have to be damaged to transform human than mouse fibroblasts \[[@B31]\]). The trigger is probably most reliable (i.e., has a greatest number of intermediate positions) in whale cells and least reliable in mouse cells. The Peto paradox can apparently be applied to body tissues as well \[[@B23]\]. Indeed, cancer develops in all human tissues, which differ greatly in the number of cells as well as their proliferation activity. Just as in the case of interspecies variability, it can be assumed that cells of different tissues have different transformation resistance \[[@B32],[@B33]\]. The presented examples suggest that the transformation resistance of cells from different tissues and species underwent evolutionary adaptation to the selective pressure exerted by tumor growth \[[@B23]\]. Cancer is a local manifestation of generalized mutagenesis ---------------------------------------------------------- Most patients develop only one tumor, which presents carcinogenesis as allegedly a local process. Experiments with exposure of animal skin to chemical carcinogens as well as cases of occupational cancer, which demonstrate clearly the link between site of exposure and tumor localization, support this notion. Without questioning the significance of such observations, they seem to have an exceptional and limited character (see below). In fact, mutagenesis is intimately related to metabolism and is therefore omnipresent: every day in each cell many thousands of DNA lesions occur due to replication and repair errors, spontaneous depurination, methylcytosine deamination, reactive oxygen species attacks, and telomere shortening. This list should perhaps be extended to include the mutagenic effect of apoptosis resulting from uptake by phagocytosis of DNA from dead cells \[[@B34]\]. Because of imperfect repair of DNA damage, the mutation rate is estimated to vary in the range of 10^-4^--10^-8^per gene per cell division \[[@B26],[@B27]\]; mutations occur in all tissues and increase with age \[[@B10],[@B27],[@B35],[@B36]\]. Calculations show, for instance, that by the age of 65 over 10^5^mutations accumulate in the normal stem cell of human colonic crypt \[[@B28]\]. When a tumor nodule appears in the body, it seems to be only the tip of an iceberg, maturing in the body for decades and consisting of a multitude of damaged cells at different stages of transformation. This assumption is supported by the clinical experience showing that overt symptoms of the disease are always preceded by precancerous lesions, such as hyperplasia, metaplasia, and dysplasia. This idea finds further development in the concept of \"field cancerization\", i.e., large (more than 7 cm in diameter) and surrounding the tumor \"patches\" of damaged cells, recognized on the basis of mutations in TP53, but remaining undetectable by routine diagnostic techniques \[[@B37]\]. Similar conclusions can be drawn from a notion of mutation as a random and rare event and carcinogenesis as accumulation of genetic defects. Mutagenesis can be described as the process both extensive (measured by number of affected cells) and intensive (measured by number of mutations per individual cell). Evidently, these parameters are positively correlated with each other: the wider the damaged zone, the deeper the damage of individual cells. The reverse is also true: the deeper the damage of the individual, most \"advanced\" cells, the wider the lesion area (this means that the very fact of a tumor appearance is, in general, the sign of a significant mutagenic lesion). In other words, appearance of the cell having a complete set of mutations (i.e., cancer cell) is accompanied by formation of a large pool of precancerous cells. The simple model of accumulation of mutant cells (i.e., cells with 1--4 mutations, the latter being the arbitrary transformation threshold) in an exponentially growing cell population is shown in Fig. [2](#F2){ref-type="fig"}. With time mutant cells inevitably appear \[[@B28]\], since mutagenic load is increasing and the repair systems become less efficient \[[@B35]\]. The first to appear and start to grow is a layer of cells with one defect, then a layer of cells with two defects, and so on. Each subsequent cell layer grows quicker than the preceding one because each new mutation confers a selective advantage to affected cells \[[@B38]\]. Additional momentum is conferred to the entire process by acquisition of chromosomal instability \[[@B39]\] or a mutator phenotype \[[@B26]\]. At each stage of carcinogenesis, transition from quantity (of damaged cells) to quality (a cell with a new mutation) takes place, the latter having a chance to appear only from a large enough pool of its predecessors. On the whole, maturation of a tumor looks like a \"pyramid\" growing until a completely transformed cell appears at its top. The latter gives rise to the overt tumor. The overall process may be symbolized by a \"mushroom\", in which the \"stem\" and \"cap\" are the latent and overt stages, respectively (Fig. [2](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Maturation of cancer \"mushroom\" in exponentially growing tissue.k, cycles of cell exponential growth; u, 4 × 10^-5^. a -- e, stages of carcinogenesis. m(0), normal cells; m(1), m(2), m(3), and m(4), mutant cells with 1, 2, 3, and 4 mutations in cancer-related genes, respectively. Cell numbers in each layer are indicated on the right (see text). ::: ![](1475-2867-5-5-2) ::: It follows from the model of multistage carcinogenesis \[[@B23]\] that in an exponentially growing tissue the number of mutant cells is approximated by the formula ![](1475-2867-5-5-i1.gif) where ![](1475-2867-5-5-i2.gif) is the mean number of cells with mmutations after kcell generations and uis the mutation rate. If we assume that the body is composed of 3.5 × 10^13^cells (i.e., 2^45^, k= 45), and uis 4 × 10^-5^(for a single gene uis 4 × 10^-7^, thus if there are one hundred cancer-related genes ubecomes 4 × 10^-5^\[[@B40]\]), then at the moment of appearance in the body of the cell with 4 mutations (a tumor founder), there will be \~3.9 × 10^3^cells with 3 mutations, \~1.4 × 10^7^cells with 2 mutations, and \~3.1 × 10^10^cells with 1 mutation (Fig. [2d](#F2){ref-type="fig"}). In other words, in the human body one cell from every thousand (0.1%) will be mutated at least in one of the cancer-related genes. In reality, mutation frequency should be much higher because cell death occurs during developmental growth so that replenishing cell divisions must increase kvalue significantly. This calculation, though very approximate, agrees with experimental data \[[@B27]\] and can give an idea of the magnitude of the mutation frequency (see also \[[@B28]\]). They might indicate that cancer appears as mutation frequency reaches a certain security threshold. On the other hand, this calculation showing that the human body is flooded with mutant cells prior to tumor appearance points to the validity of chemoprevention as an essential approach to controlling cancer \[[@B41]\]. Cancer as a mechanism of negative selection of mutant alleles ------------------------------------------------------------- As discussed above, the size of the fraction of pre-malignant cells is dependent on trigger tuning -- the more mutations needed for a malignant transformation, the bigger the pool of damaged cells. Hence, the first completely transformed cell plays de factoa triple role: (i) a [sensor]{.underline} for general mutagenesis (since its \"trigger clock\" counts down in accordance with accumulation of mutant cells in the body), (ii) an [executioner]{.underline}that unleashes a built-in death program after mutagenesis exceeds some threshold level, and (iii) a [founder]{.underline} of a clone of killer cells. Any stem cell in an organism can apparently play such a triple role, thus ensuring reliability of the mechanism. Evolutionary conservation of the death program prompts us to look for an explanation for its appearance. It may consist of the counter-selection of genetically defective individuals. As a matter of fact, if an arising tumor manifests significant whole body mutagenesis, then germ cells are most probably not an exception: a similarity of spontaneous germinal and somatic cell mutation rates was shown for human cells in vitro\[[@B42]\]. High levels of somatic mutation seem to be a direct reflection of the germ line mutation rate selected over evolutionary time \[[@B35]\]. Due to its prolonged solitary existence and relative lack of protective, repair and self-destruction mechanisms, sperm cells may be even more susceptible to genetic damage than somatic cells \[[@B27],[@B43],[@B44]\]. Besides, some gene mutations are paradoxically enriched because they confer a selective advantage to the spermatogonial cells in which they arise \[[@B45],[@B46]\]. In conclusion, germ cells are apparently not protected from mutagenesis more reliably than somatic cells are. If so, one can hypothesize that the killer program (i.e., cancer) is unleashed in a somatic cell when its vital (\"cancer-related\") genes are damaged to such an extent that testifies to intolerably high mutation frequency in body tissues and, most importantly, in germ cells (\"in mutant soma a mutant semen\" principle). This may endanger the genetic stability of the population. Therefore, if at the cellular level cancer cells look like aggressive \"cheats\" \[[@B23]\], at the body level the process may be an altruistic suicide to remove mutant alleles from the genetic pool. While in the case of sporadic tumors the notion that cancer is a local manifestation of generalized mutagenesis is only a more or less validated assumption, it is a truism in the case of hereditary tumors \[[@B47]\]. Carcinogenesis in such an individual has significant \"odds\" compared to wild-type individuals \[[@B11]\] because all his cells are mutant (the trigger, in other words, has been moved one position ahead from the very outset). The association between the evident threat of germinal mutations to the population, on one hand, and their extremely high cancer risk, on the other, seems not to be by chance. The life-span risk of getting cancer for persons with germinal mutations in the suppressor genes reaches 50--80%, and their tumors, often multiple, appear at a reproductive age \[[@B11]\]. Owing to an extremely strong selection pressure, the alleles that predispose to cancer are very rare (ca. 1:1000 or less), suggesting that the mechanism is efficient. The most convincing argument for the hypothesis that malignant tumors have an altruistic function comes from hereditary forms of cancer in which the association \"mutant semen in a mutant soma\" is most evident. The altruism here is that the carriers of mutant alleles die at a reproductive age. It is germinal mutations arising in a population with a certain frequency that could have been the driving force for cancer evolution. It is evident that the greater the number of pre-cancerous stages, the more strongly the individual is protected against cancer. A computational model of cancer progression was elaborated recently to show that the appearance of an extra stage and the additional buffering, which arises as a result, reduce the impact of any single hereditary mutation and therefore allow the accumulation of more nonlethal mutations in the population \[[@B48]\]. Because natural selection cannot purge mutations that are mostly hidden by robust pathways, mutations will continue to accumulate until their consequences become sufficiently deleterious that they are balanced by natural selection. An additional protection from cancer by extra stages thereby leads to the evolution of partially decreased cancer mortality and significantly increased genetic variability in the population as a whole \[[@B49]\]. This point of view is in accordance with the assumption that if such purging mechanism as cancer was absent, deleterious mutations would be widespread in the population. There are many germinal mutations with phenotypic expression but just a few of them (affecting a small group of \~50 genes \[[@B50],[@B51]\]) are linked with hereditary cancer. Probably, only mutations in key genes that present the greatest threat to the population are prevented by this mechanism from being spread. Many other germinal mutations are unable to pass the \"sieve\" of embryonic development because they induce early abortions \[[@B27]\]. There are a number of barriers to prevent spreading of mutant alleles in a population, and cancer seems to be only one of them. A favorable trait is retained only if it manifests itself during the reproductive period. So, the fact that cancer is predominantly a disease of the elderly would seem to be at conflict with a supposition of its evolutionary significance. This is perhaps an apparent conflict: the incidence of cancer among the young may be relatively low exactly because of the efficient selection against the adverse alleles that constantly appear in the population and exist as an inevitable background. As regards the high incidence of cancer at the old age, which seems to have no evolutionary significance, this can be explained from the viewpoint of the evolutionary theory of antagonistic pleiotropy \[[@B52]\]. A genetic program that has played a positive role in youth continues to be active in the older age, despite its possible counter-productive effects, simply \"from force of inertia\" because no correction mechanisms are available \[[@B53]\]. This, together with an increased mutation and weaker repair, results in the exponential growth of cancer incidence in old age \[[@B10],[@B35]\]. Germinal mutations in functionally important genes are a strong stimulus for development of a mechanism to prevent their spread in the population. Apart from these highly penetrant rare alleles with a strong hereditary predisposition to cancer, which are merely eradicated from the population, there are many alleles that determine a weak predisposition \[[@B49]\] (their cumulative effect can be very significant \[[@B11],[@B54],[@B55]\]). The frequency of such alleles in a population seems to be negatively correlated with the cancer risk they contribute. There is another apparent contradiction between the thesis that tumor appearance manifests generalized mutagenesis and the numerous cases when a tumor is clearly linked with a local exposure -- inflammation, bacterial infection, or UV-irradiation. The occupational cancer, on one hand, and experimentally induced tumors in animals, on the other hand, makes this association unquestionable. This linkage is probably a side effect of the evolutionary mechanism described here, in which the tumor cell plays a dual role, the mutagenesis sensor and the death program executor. In such a mechanism, a local fluctuation can unleash a process even when the overall mutation level is low (similarly, a sensor designed to respond to whole system temperature is activated by a local heating). In carcinogenesis, the hypothetical sensor/executor functions under the conditions of continuous interference and noise, which are generated by numerous external and internal locally acting mutagens. From this, many false actions ensue. If one takes the hypothesis that cancer is a means of protection, one must admit that in humans this mechanism is hyperactive and operates beyond the originally set objectives. Both the high cancer incidence in old age and the multitude of cancer cases resulting from local exposure to carcinogens are examples of this hyperactivity that obscure the true evolutionary nature of the phenomenon. Evolution hypotheses that attempt at explaining the appearance of cooperation and altruistic behavior are based on the ideas of kin or group selection \[[@B56],[@B57]\]. Their weakness is sometimes seen as an inequality between gross individual losses and relatively small population benefits, thereby questioning the validity of the proposed mechanisms. However, in the case of germinal mutations affecting important genes, the threat of genetic imbalance in the population is perhaps so high (see \[[@B48]\]) that it justifies the individual losses due to such a protection means as cancer. Here again one can perceive an analogy with cell suicide, which is likewise hyperactive in \"forestalling\" the potentially hazardous consequences of genetic defects (a weakening of such a preventive defense is undesirable \[[@B58]\]). Maintaining DNA integrity is one of the main priorities of living organisms. Depending on the extent of DNA damage, three outcomes are possible: (i) small damage induces repair which restores the initial state; (ii) strong damage launches apoptosis thus preventing cell-to-cell transfer of damaged DNA; (iii) accumulating lesions, when apoptosis is impossible, trigger cancer thus preventing individual-to-individual transfer of damaged alleles of vitally important genes. From this point of view, apoptosis is not a protection means against cancer as generally believed, but rather they are the first and second lines of defense against genome instability, respectively. Testing the hypothesis ====================== A striking discovery was made recently that a tumor can survive, propagate, and spread in the body only through the unnatural help coming from normal tissues \[[@B6],[@B13],[@B59]\]. One more step further in elucidation of tumor-host relationships is yet to be made, namely, a discovery of a mechanism of killer function. Although it is this feature that imparts so much significance to malignant growth, the current paradigm of carcinogenesis does not envisage the killer function as some special property; as a result, this function does not attract the attention it deserves. Meanwhile, there are possibilities to unveil the mechanism of tumor malignancy at present. The modern DNA array technology is capable of revealing gene expression profiles responsible for killer function. This can be done by comparison of (i) malignant tumors having different expression of this trait and (ii) benign and malignant tumors. Besides, identification of genes responsible for killer function is to be supplemented, using the same technology, with serial analyses of expression profiles of various organs and tissues of tumor-bearing animals at various stages of tumor progression. This can help to identify those specific targets within the body that are most strongly affected by the tumor growth. Implications of the hypothesis ============================== Recognition of killer function as a crucial capability of cancer cells suggests not only new avenues of cancer research (see above), but also principally new therapeutic strategy. Achievement of better understanding of mechanisms of body death may help to pinpoint new targets for therapy, such as some factors presumably emitted by cancer cells (unusual cytokines, for example), that exert the deadly effect. These factors are likely to be cancer-specific, so their elimination would not entail severe side effects. The present day cancer therapy is based, without much success, on the imperative to \"exterminate the evil\" (i.e., the cancer cells). The essence of an alternative strategy which may turn out to be more effective is not to kill the cancer cells, but to neutralize them. Competing interests =================== The author(s) declare that they have no competing interests. Acknowledgements ================ I thank G. Sardanashvilli for help in mathematics and V. Gurtsevitch and B. Kopnin for helpful discussions. This work was supported in part by the Russian Foundation for Basic Research (grant 04-04-48094).	PubMed Central	2024-06-05T03:55:54.124868	2005-3-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555547/", "journal": "Cancer Cell Int. 2005 Mar 2; 5:5", "authors": [ { "first": "Anatoly V", "last": "Lichtenstein" } ] }
PMC555548	Background ========== The successful trials of pyrethroid insecticide-treated nets for malaria control in various endemic settings has led to the Roll Back Malaria initiative adopting the approach as one of the cornerstones of its malaria control programmes \[[@B1]-[@B3]\]. However, the increasing prevalence of insecticide resistance in Anopheles gambiae, the major vector of malaria in sub-Saharan Africa, threatens to compromise the successful use of insecticide-treated materials \[[@B4]\]. Resistance to pyrethroid insecticides was first seen in An. gambiae sensu strictoin West Africa \[[@B5]\] and has subsequently been detected in East Africa \[[@B6]\]. Whilst much of the observed resistance is thought to have been selected for by the use of pesticides in agriculture \[[@B7]\], there is already some evidence in East Africa that the introduction of treated bednets has selected for reduced susceptibility to permethrin \[[@B6]\]. One allele commonly associated with resistance to permethrin is the knock-down resistance or kdrallele. This allele encodes a modified voltage-gated sodium channel that has reduced sensitivity to DDT and pyrethroids. Molecular studies identified a single point mutation in the kdrallele that causes an amino acid substitution in domain II of the protein \[[@B8]\]. Two different mutations have been found in An. gambiae; the first causes a leucine to phenylalanine amino acid change and has been found in several West African countries \[[@B8]-[@B11]\], whilst the second found mainly in East African populations causes a leucine to serine substitution at the same amino acid position \[[@B6],[@B12]\]. The importance of these mutations to the control of Anophelesmosquitoes is not yet fully understood. However, monitoring its frequency, as a rapid indicator of the development of resistance, should be an integral component of insecticide resistance management programmes. The most commonly used method for identifying the kdrmutations involves a multiplexed PCR technique. Single Nucleotide Polymorphism (SNP) detection is problematic with simple PCR approaches, requiring the use of highly toxic reagents \[[@B13]\] or prohibitively expensive equipment. Many of these approaches are difficult to transfer to field laboratories where the ability to monitor gene frequencies is most acutely needed. The technique detailed here, adapted from one originally designed by W.C. Black IV requires only a thermal cycler and provides an easily interpretable, colorimetric genotyping system. No toxic reagents are involved. While this system has been specifically designed to assay kdrresistance allele frequencies in An. gambiae, it is broadly applicable where target-site insensitivity is an important mechanism of resistance to insecticides and to chemotherapeutics. Methods ======= Mosquito strains and bioassays ------------------------------ Specimens were obtained from laboratory colonies of RSP (a homozygous line for the East African kdrmutation), Kisumu (a susceptible line from Kenya, established in 1953), and Odumasi (a partially resistant line, not yet fixed for the West African kdrmutation). Adult females were stored at -20°C before extraction. Field caught specimens were collected using resting catches from Asembo in western Kenya in May 2004, and by pyrethrum spray collections in Odumasi, Ghana in June 2003. Samples were dried over silica gel for later analysis. PCR --- All PCR reactions were performed in ABI GeneAmp^®^PCR system 2700 or MJ Research PTC-200 DNA Engine thermal cyclers. Primers Agd1 and Agd2 \[[@B8]\] were used to amplify a 293 bp fragment from domain II of the voltage-sensitive sodium channel protein sequence (EMBL \#Y13592). PCR was carried out with the DNA of 1/80^th^or 1/160^th^of a single mosquito in a 25 μl volume with a final concentration of 1x Buffer, 2.0 mM MgCl~2~, 0.2 mM dNTP\'s (Sigma dNTP-100), 0.3 μM each primer (Qiagen), Taq DNA polymerase 0.034 U/μl (Qiagen 201203). Reaction conditions were 94°C for 4 min, 25 cycles of 94°C for 25 sec, 56°C for 20 sec, 72°C for 8 sec; and a final extension step of 72°C for 10 min (modified from \[[@B12]\]). Artificial heterozygote controls were created using DNA from two homozygous samples. DNA from a single mosquito was extracted using the Livak method, \[[@B14]\] or the Ballinger Crabtree method \[[@B15]\] and resuspended in 100 μl or 200 μl of ddH~2~0. Species identification was carried out on all specimens using a PCR method \[[@B16]\]and specimens were characterized for kdrstatus using PCR methods \[[@B8],[@B12]\]. PCR products were visualized under UV light on 1.5% agarose, 0.5x TBE gels stained with ethidium bromide. Hot Ligation ------------ 3 μl of PCR product from the above reaction was used in a hot ligation with Detector and Reporter oligonucleotides (MWG Biotech) (Table [1](#T1){ref-type="table"}). Aliquots were made for each oligo pair containing 1 μM detector and 1 μM reporter in ddH~2~0. A 20 μl reaction volume containing 1x Buffer, 50 nM detector and reporter mix and 0.05 U/μl Ampligase^®^(Cambio A32250) was set up for each oligo pair. Four reactions were set up for each PCR sample to test for the East and West resistant alleles and the susceptible allele (two different oligo pairs must be used to test for the susceptible allele in these assays, as the potential oligo binding site differs by one base pair). The reaction conditions were 95°C for 5 min, 25 cycles of 94°C for 1 min, 58°C for West African kdrdetection or 60°C for East African kdrdetection for 2 min; with a final hold at 4°C. Ligated products were kept at 4°C in the dark and used as soon as possible for SNP analysis. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Oligonucleotide sequences used in the Hot Ligation ::: ------------------------------------------------------------------------------------------------------------------------- Description Oligo Name bp Position^a^ Oligo sequence 5\' -- 3\' Modifications ---------------------------- ---------------- -------------------- ------------------------------- ---------------------- Suspt. East kdrdetector Kdr104L-DTe 311-15i ATTTGCATTACTTACGACTA 5\' Biotin Resist. East kdrdetector Kdr104S-DTe 311-15i ATTTGCATTACTTACGACTG 5\' Biotin East kdrreporter Kdr104-RTe 291--310 AATTTCCTATCACTACAGTG 5\' Phosphorylation\ 3\' Fluorescein Suspt. West kdrdetector Kdr104L-DTw 312-16i AATTTGCATTACTTACGACT 5\' Biotin Resist. West kdrdetector Kdr104F-DTw 312-16i AATTTGCATTACTTACGACA 5\' Biotin West kdrreporter Kdr104-RTw 292--311 AAATTTCCTATCACTACAGT 5\' Phosphorylation\ 3\' Fluorescein ------------------------------------------------------------------------------------------------------------------------- ^a^Using sequence from Martinez-Torres et al., as reference; iintron 2 position. ::: SNP Detection ------------- 96-well plates (VWR 402 200 402) were prepared using 100 μl of 5 μg/ml streptavidin (Sigma S4762) per well. The plate was left to dry overnight and then washed 4 times in 250 μl of 1 x PBS with 0.1% v/v Tween 20. Buffer was removed by tapping the plate upside down and 200 μl of blocking solution (1x PBS, 0.1%v/v Tween 20, 2%w/v BSA) added for 1 hour. Four more washes of 250 μl with PBS were carried out before plates were covered with a plastic seal and stored at 4°C for up to one week. 20 μl of TNE (10 mM Tris-HCl pH7.5, 1 mM EDTA pH 8.0, 0.2 M NaCl) was added to the hot ligation reaction and then all 40 μl was placed in a well of the streptavidin plate and allowed to incubate at room temperature for 30 min in the dark. The ligation reaction was carefully removed with a multichannel pipette and the plate washed twice in 250 μl of freshly prepared wash buffer 1 (10 mM NaOH, 0.05%v/v Tween 20) and then twice in 250 μl of wash buffer 2 (0.1 M Tris-HCl pH7.5, 0.15 M NaCl, 0.05%v/v Tween 20). 40 μl of 75 mU/ml HSP-conjugated antifluorescein Ab (Roche 1 426 346) solution in 1% w/v BSA solution was placed in each well and incubated at room temperature for 30 min. The plate was then washed three times in 250 μl of wash buffer 2. All buffer traces were removed by tapping the plate upside down on a paper towel and 100 μl of room-temperature TMB solution (Roche BM Blue Pod Substrate 1 484 281) added. At least 5 min were allowed for the colour to develop before plates were scored. Plates were read at 680 nm in a Molecular Devices Versa Max plate reader to provide a quantitative method of scoring which could be compared to the visual method of scoring to check reliability. Results and Discussion ====================== A schematic of the HOLA approach is given in Figure [1](#F1){ref-type="fig"} and a photograph of the HOLA 96-well plate is shown in Figure [2](#F2){ref-type="fig"}. Susceptible individuals score positively for both the East and West African susceptibility tests although a somewhat weaker reaction may be seen in East African susceptible individuals for the West Susceptibility test. Resistant individuals show a positive colour change only for their specific kdrallele. Heterozygotes are easily distinguishable. The protocol presented here for kdrdetection is reliable and gives unambiguous results (Table [1](#T1){ref-type="table"}). Visual and colorimetric scoring results were always comparable (data not shown). A double-blind trial was carried out on 12 wild-caught specimens of An. gambiaefrom East Africa compared to the commonly used PCR multiplex approach. The genotype was unambiguously determined by the HOLA technique, whereas the PCR results were more difficult to interpret and often required a repeat reaction (Table [2](#T2){ref-type="table"}). There was one discrepancy between the two approaches which was not resolved after repeated analyses (Specimen Kenya 3, Table [2](#T2){ref-type="table"}). It is believed that the HOLA method gave the correct result since three HOLA repetitions were carried out on the sample which all scored the specimen as heterozygous. Contamination may be excluded as a cause of this discrepancy as HOLA reactions were performed before and after the PCR tests. Furthermore the kdrallele is rare in the Kenyan population \[[@B17]\] and so would be much more likely to occur more frequently in a heterozygous rather than homozygous state. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Schematic of Hot Oligonucleotide Ligation Assay for West African Allele ::: ![](1475-2875-4-16-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Photograph of HOLA plate, including DNA extraction method and expected results.Abbreviations: SS, homozygous susceptible. RR, homozygous resistant. RS, heterozygous. ^a^Livak \[14\] extraction method ^b^Ballinger-Crabtree \[15\] extraction method ^c^Artificially created heterozygote ::: ![](1475-2875-4-16-2) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Double blind trial of HOLA approach versus conventional PCR ::: Specimen HOLA PCR 1^e^ PCR 2^e^ -------------- ---------- -------------- -------------- NK5^a^ SS SS SS NK6 SS X SS NK7 RR RR RR NK8 SS SS SS Kenya 1^b^ SS X SS Kenya 2 SS X SS Kenya 3 RS X RR Thyolo 7^c^ SS SS SS Thyolo 33 SS SS SS Thyolo 34 SS X SS Thyolo 64 SS X SS Thyolo 75 SS X SS RSP^d^ RR RR RR ^a^Specimens labelled NK collected by Pie Muller, Ben Oloo, and Nadine Randle from Asembo, Kenya on 05/2004, DNA extracted by Ballinger-Crabtree method \[15\] on 09/2004. ^b^Specimens labelled Kenya collected by Pie Muller, Ben Oloo, and Nadine Randle from Asembo Bay, Kenya on 05/2004 DNA extracted by Livak method \[14\] on 08/2004. ^c^Specimens labelled Thyolo collected by Philimon Tambala and Bill Hawley from Thyolo, Malawi on 01/1995, DNA extracted by Ballinger-Crabtree method \[15\] on 09/1997. ^d^Specimen from RSP colony. Abbreviations: SS, homozygous susceptible. RR, homozygous resistant. RS, heterozygous. ^e^Conditions for the PCR reactions were identical. ::: The HOLA method allows for over 40 samples to be screened on a single microtitre plate. As shown in Figure [2](#F2){ref-type="fig"}, the method works for a variety of DNA extraction techniques, on fresh and stored material. Although costs per reaction are slightly higher than for the traditional multiplex PCR, the greater reliability ensures that repeat reactions are unlikely to be required, reducing costs in the long term. In addition, since this technique dispenses with the need for gel electrophoresis apparatus there is a lower initial equipment outlay, greater comparative safety and greater ease of this technique, making the method ideal for field laboratories. Conclusion ========== The HOLA method allows fresh and stored An. gambiaemosquitoes to be characterized for the East and West African kdrmutations. Homozygotes and heterozygotes can be easily distinguished using low cost equipment and a simple methodology which makes this technique suitable for use in resource-poor countries. In our hands the method is more reliable than the current multiplex PCR approach, less ambiguous and may be more sensitive for the detection of heterozygotes. List of Abbreviations used ========================== DNA -- Deoxyribonucleic acid. ELISA -- Enzyme-linked immunosorbent assay HOLA -- Heated oligonucleotide ligation assay. Kdr-- Knock down resistance. PCR -- Polymerase chain reaction. SNP -- Single nucleotide polymorphism. Authors\' contributions ======================= AL developed the HOLA method for the kdrmutation and drafted the manuscript. HR conceived of the study and participated in its design. NPR carried out the multiplex PCR. PJM and EDW helped draft the manuscript. WCB developed the HOLA technique. MJD participated in the design of the study and substantially helped draft the manuscript. Acknowledgements ================ We thank Alexander Egyir-Yawson, Pie Muller, Ben Oloo, Bill Hawley, and Philimon Tambala for providing wild caught specimens. Funding for this work came from NIH grant U01 AI58542.	PubMed Central	2024-06-05T03:55:54.127823	2005-3-14	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555548/", "journal": "Malar J. 2005 Mar 14; 4:16", "authors": [ { "first": "Amy", "last": "Lynd" }, { "first": "Hilary", "last": "Ranson" }, { "first": "P J", "last": "McCall" }, { "first": "Nadine P", "last": "Randle" }, { "first": "William C", "last": "Black" }, { "first": "Edward D", "last": "Walker" }, { "first": "Martin J", "last": "Donnelly" } ] }
PMC555549	Background ========== Sequence-specific hybridization of a long single-stranded labeled DNA or RNA target molecule to shorter oligonucleotide probes is the basis of the gene expression microarray experiment. In this type of microarray experiment, gene specific probemolecules are either synthesized in situ or are printed to the microarray slide, and are either non-specifically cross-linked to the surface or are attached specifically using a method such as poly-Lysine linkers. Targetmolecules (most often fluorescently labeled cDNA molecules, although cRNA and aRNA are used in some protocols) hybridize transiently to the probe oligomers until they form stable double helices with their specific probes. At some point, the rate of on and off reactions reach equilibrium, and the concentration of the target in the sample solution can be calculated. Transcript abundance is assessed by the relative intensity of signal from each spot on the array. This interpretation of array data relies on the assumption that each hybridization reaction goes to completion within the timeframe of the experiment and that the behavior of all pairs of intended reaction partners in the experiment is somewhat uniform. There are three major types of DNA microarrays, which differ in the approach used for probe design: Affymetrix type microarrays \[[@B1]\], which assay each transcript with a distributed set of 25-mer oligonucleotides, full length cDNA microarrays, in which long cDNA molecules of lengths up to several hundred bases are crosslinked to the slide surface to probe their complement \[[@B2]\], and synthetic long-oligomer probe microarrays, which usually assay each transcript only once. The latter class of microarrays encompasses a variety of commercial and custom platforms, and there has yet to emerge a consensus on an optimal probe length for particular experimental designs. Oligo lengths ranging from 35 to 70 nucleotides have been shown to perform well under different conditions \[[@B3]-[@B5]\], though recent studies have shown that oligomers of up to 150 nucleotides may be desirable for assessing transcript abundance \[[@B6]\]. In general, the use of synthetic oligomers has been shown to result in improved data quality \[[@B7],[@B8]\] relative to cDNA arrays, and 70mers have been shown to detect target with a sensitivity similar to that of full length cDNA probes \[[@B9]\]. Short probes have been promoted because they facilitate finding unique sequence matches while forming fewer, and less stable, hairpin structures and because they display more uniform hybridization behavior overall. However, the need for sensitivity and detection of transcripts in low copy number drives the use of long-oligonucleotide arrays. In this study, we have modeled the accessibility of transcripts to hybridization with 70mer oligonucleotides. A number of oligonucleotide design software packages have been published in recent years, each having design strengths in one of a number of criteria \[[@B10]-[@B14]\]. Several factors are considered by almost all microarray design software packages: in particular, the sequence specificity of the probe-target interface and the overall balance of GC content across the array. Unique regions of the target sequence are identified using sequence comparison methods; the unique regions become the search space for probe selection based on other criteria. The number of probes per sequence and location of the probe in the sequence also restrict sequence availability. A relatively uniform melting profile generally is achieved simply by selecting for probes with similar GC content and uniform or close-to-uniform length, although some design methods explicitly compute the duplex melting temperature for each candidate probe-target pair and filter unique probes to find those which match a specified range of melting temperatures. Another biophysical criterion that is sometimes applied is the elimination of probes having the ability to form stable intramolecular structures under the conditions of the experiment. This is usually done by eliminating regions of self-complementarity, although at least one design program \[[@B13]\] does explicitly compute the melting temperature of the most stable structure to form in the probe molecule and uses that information to filter out stable secondary structures in the probe. Few of the available array design packages explicitly consider the possible structures of the transcript-derived molecules in the sample solution and their impact on whether the microarray will provide an effective assay, although the OligoDesign web server \[[@B14]\] does compute this information for use in design of locked nucleic acid probes. It has been shown that a hairpin of as little as six bases in an oligonucleotide can require a 600-fold excess of the complementary strand to displace the hairpin even partially \[[@B15]\]. Since the target molecules are generally longer than the probe and may be of a different chemistry, it is not sufficient to conclude that their behavior will mirror that of the complementary probe. Prediction of secondary structure in a sample transcript using a standard nucleic acid secondary structure prediction algorithm (Mfold) demonstrates that while longer-range interactions are reduced at high temperatures, stable local structures persist in the transcript even at high salt concentration and high temperature (Figure [1](#F1){ref-type="fig"}). Because unimolecular reactions within the target can occur on a much shorter timescale than the diffusion-mediated, bimolecular, duplex hybridization reaction, competition for binding by intramolecular structures is expected to block the specific probe annealing sites on the target sequence in some cases and result in misinterpretation of the signal obtained from the assay if these effects are not taken into account. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Secondary structure in a sample transcript.Circular diagrams of structure in a sample transcript (moeB homolog designated BR0004) from Brucella suis. Circular diagrams show hydrogen bonds between individual nucleotides, color-coded according to single-strandedness -- the fraction of structures in which that bond is not present. Black bonds indicate 0% single-strandedness; red bonds indicate 100% single-strandedness. ::: ![](1471-2164-6-31-1) ::: In order to estimate the prevalence of stable secondary structure in long target molecules, and thus the impact such structures might have on the analysis of microarray data, we have modeled secondary structure formation in mRNA transcripts of the intracellular pathogen Brucella suis. We have assessed the stability of structures formed in the transcript and the accessibility of the binding sites of optimal probes generated using commonly applied design criteria. Because random shearing of the full-length target molecule is used in some protocols, we have also modeled the effects of shearing to an average length on the prevalence of secondary structure in selected targets. Results ======= Extent and stability of target secondary structure -------------------------------------------------- Our modeling results obtained for the genome-wide set of intact single-stranded DNA or RNA targets demonstrate that stable secondary structures are widespread in target mixtures from Brucella suis(Figure [2](#F2){ref-type="fig"}) and in randomly chosen transcripts from the genomes of E. coliand L. lactis. Figure [2](#F2){ref-type="fig"} shows the ΔG of formation for the most stable predicted secondary structure of the full-length transcript, as a function of reaction temperature. The major energy components of the Mfold ΔG are hydrogen bond energy and base pair stacking energy. These can be assumed to have a roughly linear relationship with transcript length. In order to make energies from different-length transcripts comparable, energies were normalized by computing a per-residue folding ΔG for each transcript and then multiplying that value by the global mean target length, for all transcripts considered from all organisms, of 851 bp. Average ΔG of secondary structure formation decreases with increasing temperature, but even at 65°C, the average ΔG of secondary structure formation for a full-length transcript is -98.2 kcal/mol (-27.9 kcal/mol when modeled as cDNA), meaning that the transcript is quite stable in that structure and a considerable energy input will be required to displace or melt the remaining structure. The trend in ΔG of secondary structure formation from the high-GC genome of B. suisto the low-GC genome of L. lactisis a decrease in overall stability. The average normalized ΔG of secondary structure formation for transcripts selected from the GC-balanced genome (E. coli) is near 70% of the average for Brucella, while the average ΔG for transcripts from the GC-poor genome (L. lactis) are even lower (30% at 52°C). However, even in the most GC-poor genome, stable target secondary structure in the single-stranded target is widespread. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### *Stability of transcript secondary structure in Brucella suis.**Average free energy change on global secondary structure formation for Brucella suistargets, modeled as DNA or RNA. ΔG values are normalized to global mean target length. ::: ![](1471-2164-6-31-2) ::: Our results demonstrate that a significant fraction of nucleotide sites in the average target mixture, whether single stranded DNA or RNA, will be found in stable secondary structure under the hybridization conditions used in oligonucleotide microarray experiments, and will be relatively inaccessible for intermolecular interactions. Figure [3](#F3){ref-type="fig"} shows the percentage of nucleotides that are in a double-helical state in at least 50% of the secondary structure conformations predicted by Mfold, at various reaction temperatures. The measure of accessibility used is the fraction of structures in which a nucleotide is found in a single-stranded conformation, when all optimal and suboptimal structures predicted are considered. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Fractional accessibility of nucleotides in the target.Fraction of the complete transcript classified as inaccessible due to the presence of stable structure in \>50% of predicted conformations. Data shown are for 37, 42, 52 and 65°C simulations in Brucella suis. ::: ![](1471-2164-6-31-3) ::: Extent and stability of target secondary structure -------------------------------------------------- Figure [4](#F4){ref-type="fig"} is a plot of the average ΔG of structure formation when shearing of the target molecule is simulated by dividing the target into overlapping 200, 100, and 50mer fragments. Shearing the target into smaller fragments destabilizes secondary structure, especially at very short fragment lengths. However, shearing does not eliminate occlusion of nucleotides by secondary structure, even in the shortest fragments examined. When a DNA target is modeled at 52°C, for example, the double stranded fraction decreases by only about 30% -- from 41% to 29% -- when the target is simulated as sheared into 50mer fragments. However, in hybridization experiments involving low copy number targets and longer oligos, creating extremely short target fragments may reduce or eliminate the signal on the chip, because the target can not be sheared specifically to present an unbroken hybridization site for the probe, and so some fragments will be created that match the probe only partially. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Stability of secondary structure in sheared fragments.Free energy change on secondary structure formation for the ureG-1 RNA transcript from Brucella suis. The transcript is modeled as sheared into fragments of length 200 nt, 100 nt or 50 nt; fragments are chosen starting at every 10th residue. ::: ![](1471-2164-6-31-4) ::: Interference of secondary structure with the hybridization site --------------------------------------------------------------- Figure [5](#F5){ref-type="fig"} shows the average percentage of nucleotides within a probe binding region in the target that are inaccessible, when different fractional accessibility cutoffs are used to classify the sites. Even when a relatively demanding criterion -- double-strandedness in over 75% of optimal and suboptimal structures -- is used to classify a nucleotide as inaccessible, an average of 21 ± 13% of nucleotides in the probe binding region are found in stable secondary structures at 65°C. Figure [6](#F6){ref-type="fig"} shows a representative transcript and the challenge it presents to hybridization when modeled as full-length cDNA and fragments of various lengths. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Accessibility of the probe binding site.Fraction of the average probe binding site in the Brucellagenomic array that is found to be inaccessible at 37°, 42°, 52° and 65°C, for DNA or RNA target. Inaccessible sites are defined here using three different cutoffs for the fraction of structures in which the site is base-paired: 25%, 50%, and 75%. ::: ![](1471-2164-6-31-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Structure in a binding site -- full length target and sheared fragments.The position of a 70mer oligonucleotide probe (green) binding site (red dots) within a full-length optimal transcript structure, as well as examples of stable structure in 200mer and 100mer fragments which overlap the probe binding site. Corresponding ΔG values for these fragments modeled at 42° and 52°C are shown in Table 1. ::: ![](1471-2164-6-31-6) ::: Discussion ========== Lack of bioinformatics tools that incorporate experimentally validated biophysical properties of nucleic acids as a criterion for synthetic oligomer probe design is a major challenge for do-it-yourself microarray designers. One biophysical characteristic, which we predict will reduce the binding efficiency of microarray probes to their targets, is the propensity of long single-stranded DNA or RNA molecules to form stable secondary structure. 3-D structures such as hairpins and stacked regions have the potential to pre-empt target nucleotides, thus blocking regions of the target molecules from hybridizing to their intended probes. Prediction and thermodynamic analysis of secondary structure at a range of temperatures in full length target sequences, as well as in subsequences formed by in silicoshearing, revealed the likely presence of stable secondary structures in both full-length target and sheared target mixtures. These structures do not convert completely to random coil with either increasing hybridization temperature, more extensive shearing, or both. These secondary structures may therefore compete with the intended target for effective probe annealing in a microarray experiment, resulting in a misinterpretation of the amount of target present in the sample. Applying target secondary structure as a criterion in array design ------------------------------------------------------------------ Based on the results of this in silicoexperiment, secondary structure prediction in the target is being used to develop a new criterion for oligonucleotide probe design. Our results from this modeling experiment demonstrate that the implicit assumption used until now -- that eliminating probe secondary structure by avoiding self-complementarity eliminates target secondary structure as well -- is valid only when the target and probe are of the same length. Use of target secondary structure as an explicit criterion will allow for masking or preferentially avoiding the regions of the target sequence in which base pairs are directly involved in secondary structure formation, to eliminate these regions from the sequence for the purpose of the search for the optimal probe. In this study we have assigned accessibility scores to sites in the target sequence based only on the fraction of predicted structures within 5% of the energy optimum, in which a residue is found in a single-stranded conformation. While this measure is not too computationally intensive to compute, and can be applied to genome-scale problems using readily available software (Mfold), it is not the most physically rigorous definition of accessibility. By equally weighting each possible structure in the ensemble of optimal and suboptimal structures that a molecule can form, it is possible that secondary structure at some positions in the molecule is overcounted; bonds which form only in rare conformations are considered equal to bonds which are present in the lowest-energy structure. The program Sfold \[[@B16]-[@B18]\] assigns accessibility based on an ensemble-weighted average of secondary structure. The program RNAfold\[[@B19]\], part of the Vienna RNA package, implements McCaskill\'s partition function approach\[[@B20]\] to arrive at pairing probabilities for each pair of bases in the sequence, from which a summary per-base accessibility can be derived. These methods are more rigorous than MFold and we expected they might produce somewhat different results, although it has also been shown that predicted binding states from MFold optimal structures perform almost as well as SFold and RNAFold predictions when applied to molecules of known 3D structure \[[@B16]\]. When we compared MFold-based accessibility predictions for an individual transcript to those generated by SFold and RNAFold, we found that the difference in average predicted accessibility over an entire transcript is small. We computed accessibility for the transcript of human 1CAM-1, which has been mapped experimentally to determine its accessibility \[[@B21]\]. The average fractional accessibility derived from MFold results is about 3--4% greater than that predicted by RNAFold or SFold. Therefore use of this fractional accessibility measure will not impose an unnecessary constraint on the design process relative to other predictive approaches. The accessibility profiles calculated for ICAM-1 using each method are shown in Fig. [7](#F7){ref-type="fig"}. In each section of the figure, antipeak locations (having lower pairing probability and therefore likely to be more accessible) can be compared to the extendable sites detected by Allawi et al \[[@B21]\], which are indicated by green dots at the bottom of the plot. In each prediction, there are a number of apparently correct predictions and obvious errors, and it is not clear which method is yielding the best results at the residue level. A systematic, competitive test of these predictions against solution accessibility data gathered on various experimental platforms is called for, although available data sets for validation are still rare. In the absence of such validation, the MFold accessibility predictions are sufficient to predict the scope of the secondary structure problem in a genome-based array design, even if some details of the prediction are not correct. An experimental approach will eventually be required to determine which approach best represents the conditions of the microarray experiment. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Accessibility prediction using three common methods.Pairing probabilities computed using RNAFold (top), MFold (middle) and SFold (bottom) for the human ICAM-1 transcript. Extendable sites detected by Allawi et al \[21\] ::: ![](1471-2164-6-31-7) ::: Loop length and other considerations ------------------------------------ In this study, we focused specifically on the DNA/RNA base pairs that are actively involved in hydrogen bond formation. We realize that other accessibility considerations will have to be added to the scoring scheme in practice. The structure of a long single stranded DNA or RNA molecule can contain many nucleotides that, while not part of a double-helical stem, remain inaccessible to hybridization due to their location inside small loops within the target secondary structure. A loop is a somewhat constrained structure as well, and the length at which it presents accessible sequence that favors hybridization has been shown to be on the order of 10 nucleotides and longer \[[@B22]\], while nucleotides found in shorter loops may be classifiable as inaccessible. However, there is a need for quantitative hybridization experiments that would elucidate how loops and loop-like structures in tethered long-oligo probe and target molecules affect the performance of assays, and we have chosen not to formulate a system for scoring the accessibility of single-stranded loop structures or weighting this criterion relative to the double-strandedness criterion until we have carried out some of these experiments. Development of a target secondary structure criterion for oligonucleotide array design is expected to impose restrictions on the probe selection beyond the sequence similarity and melting temperature criteria that are currently used, especially in cases where short probe length restricts the annealing temperature used in the hybridization protocol to 22--37°. In the B. suisexample, use of a low annealing temperature, e.g. 42°C which is the temperature used in some published 70-mer array experiments \[[@B9]\], would result in only about 30% of the average transcript being accessible for intermolecular hybridization, not counting \'free\' bases found in short loops in secondary structures. There will be greater design latitude for experiments carried out at higher hybridization temperatures. Recommended hybridization temperatures for long synthetic oligomer arrays may prove to be closer to 65°C, when only 50% of a typical RNA transcript or 30% of the corresponding cDNA molecule remain inaccessible. To shear or not to shear ------------------------ We have shown here that while shearing reduces overall ΔG of secondary structure formation for individual molecules in the target solution, shearing does not in itself eliminate formation of secondary structure in single-stranded DNA or RNA. The question of whether shearing should be used for long oligomer arrays is still an open one. While some signal may be gained by reducing the stability of secondary structure in the target molecule, random shearing by its nature creates a mixture of targets that may have substantially different affinities. For instance, in a 300 nt transcript that is targeted by a 70mer oligonucleotide, there is nearly a one in four chance that a random break in the sequence will occur within the target site for which the probe is designed. Short fragments may present a substantially different binding site, and therefore have a different binding affinity, than the full-length transcript that is considered when the probe is designed. This is illustrated in Figure 8d, where binding of a 50mer sheared fragment to a 70mer probe leaves a dangling end in the probe. A break very close to one end or the other of the target site may create a target that still binds to the probe, though with reduced affinity; a break closer to the middle of the target site may produce fragments that bind partially to the probe, competing for binding with perfect matches. The utility of experimentally validated biophysical criteria ------------------------------------------------------------ In other experimental contexts where hybridization is critical to success, the impact of secondary structure in single stranded polynucleotides on results has been recognized and is now being systematically studied (18--21). Intramolecular folding of mRNAs is so extensive that only 5--10% of most transcripts is accessible to binding of complementary nucleic acids; however the modeling of long molecules has not proven to give very accurate binding predictions \[[@B23]-[@B25]\]. In fact, array-based screens have been utilized to empirically select oligonucleotides that bind effectively to transcripts for siRNA experiments \[[@B23],[@B26]\]. Several studies have demonstrated that, at 37°C and 0 mM Mg2+ oligonucleotides of length \>20 yield good binding/RNAseH digestion at low concentrations relative to shorter oligonucleotides (30 nM vs 300 nM compared) and found that microarray binding was a good predictor of siRNA activity despite the 3\' tethering and 1M NaCl used in array experiments vs siRNA experiments \[[@B26]\]. Systematic \"scanning\" of mRNA sequences with libraries of short oligos \[[@B27]\] has also been shown to be successful in locating sites for siRNA targeting; however, such methods are likely to become extremely expensive if applied to the large number of targets in a microarray design. We have begun to develop an experimental approach to this problem, in which structure predictions like those used in this study are experimentally evaluated to determine whether the structures we can predict using existing modeling approaches will detectably affect signal in the microarray context. Conclusion ========== The results of the current study suggest a significant role for target secondary structure in hybridization to oligonucleotide arrays, which will warrant further investigation. Oligonucleotide probe binding sites in a significant fraction of transcripts are found in double-stranded conformations even in cases where self-complementarity was avoided during the probe design process. We find that at 52°C, for example, approximately 57% of probes designed for Brucellahad binding sites in the target which were predicted to contain a stretch of unpaired bases of at least 14 nt in length; at 65°C, that fraction increased to 93%. Based on these findings we would expect that at 52°C only 57% of our probes would encounter optimal conditions for hybridization and therefore would demonstrate the expected behavior in the experiment, where intensity is expected to scale with target concentration. We predict that the remaining probes, which have shorter, or no, accessible sequences, will exhibit modified binding behavior, and we plan to conduct experiments to characterize this behavior. We have shown conclusively that avoiding self-complementarity in the probe when designing an oligonucleotide array is insufficient to eliminate secondary structure from the binding site in the target. By combining the procedure for systematic computational assessment of transcript accessibility described in this study with selective experimental validation of the impact of predicted accessibility on hybridization, we will develop a useful criterion for avoiding troublesome secondary structure when designing microarray targets. Methods ======= Prediction and thermodynamic analysis of secondary structure was performed for all protein-coding gene transcripts predicted from 3264 CDSs in the Brucella suis1330 genome. Brucella suishas a relatively high (57%) genomic GC content. Brucella suiswas chosen for this experiment because our collaborators have previously acquired a custom synthetic oligomer microarray for this organism, developed using standard oligo array design software, and we have access to both target sequences and to a set of unique probe sequences that define the interaction sites for which expression results have been obtained by the laboratory. In order to determine whether Brucella sequences form atypical structures we randomly picked and analyzed 50 gene coding sequences from a compositionally balanced genome (Escherichia coli), and 50 from the GC-poor genome of the nonpathogenic AT-rich gram-positive bacterium Lactococcus lactis(35% genomic GC content). The Brucella suisgenes ranged in length from 90 to 4,803 bp, with an average transcript length of 851 bp. The E. coligenes ranged in length from 140 to 2,660 bp, with an average transcript length of 792 bp. The range of GC content in the genes chosen was 37% to 57% with an average value of 50%, which is reasonably representative of the E. coligenome. The L. lactisgenes chosen ranged in length from 140 to 2,730 bp, with an average transcript length of 765 bp., and ranged in GC content range from 30% to 42% with an average value of 35%. Microarray design ----------------- 70-mer probes for each Brucella suistarget were previously designed (Stephen Boyle, personal communication) using ArrayOligoSelector (pick70) \[[@B10]\]. ArrayOligoSelector uses sequence uniqueness, self-complementarity, and sequence complexity as criteria but does not explicitly evaluate ΔG of secondary structure formation for the probe. 72% of the probes designed using this method were found to contain secondary structures with melting temperatures greater than 65°C, and 10% contained secondary structures with melting temperatures greater than 80°C. The Brucella probes defined the interaction sites within the target transcripts for which structural accessibility was evaluated. Secondary structure prediction ------------------------------ Probe and transcript secondary structure were predicted using the Mfold 3.1 software package \[[@B28],[@B29]\]. Mfold identifies the optimal folding of a nucleic acid sequence by energy minimization and can identify suboptimal foldings within a specified energy increment of the optimum as an approach to modeling the ensemble of possible structures that a single-stranded nucleotide molecule can assume. We modeled secondary structure in the single-stranded target, modeling the target both as DNA and as RNA, at a range of temperatures which is inclusive of hybridization temperatures commonly used in microarray protocols: 37°C, 42°C, 52°C and 65°C. The modeling conditions were chosen within the allowed settings of Mfold to approximate a microarray experiment: solution conditions of 1.0 M sodium concentration and no magnesium ion were used. The free energy increment for computing suboptimal foldings, ΔΔG, was set to 5% of the computed minimum free energy. The default values of the window parameters, which control the number of structures automatically computed by Mfold 3.1, were chosen based on the sequence length. Free energy changes on formation of secondary structure were extracted from the Mfold output. Accessibility calculation ------------------------- Accessibility in folded single-stranded DNA or RNA has recently begun to be addressed in a few experimental studies, mainly with the goal of targeting appropriate sites for RNAi. Because the structure of single-stranded nucleotide molecules is much more dynamic than that of proteins, with each molecule likely to exist in an ensemble of structures, and because the 3D structure of these molecules is rarely known, there is not yet a consensus representational standard of per-residue accessibility for single-stranded nucleic acids. Ding et al. \[[@B17],[@B18]\] implement probability of single-strandedness, when the weighted ensemble of likely structures is taken into account, as an accessibility criterion. However, use of their Sfold server, with batch jobs limited to 3500 bases, is not currently practical for a genome-scale survey of accessibility. Another approach to accessibility prediction is McCaskill\'s partition function approach \[[@B20]\] which can be used to compute base pair probabilities and summary pairing probability for any base. This approach is implemented in RNAFold \[[@B19]\], a component of the Vienna RNA package. In this study, we chose to use the less physically rigorous approximation of probability of single strandedness as a simple fraction of predicted optimal and suboptimal structures in which a residue is found to be part of a single stranded structure, as computed by Mfold. Accessibility scores derived from MFold predictions have been used in limited studies of RNA structure focused on hammerhead ribozymes\[[@B30]\], antisense and siRNA targeting \[[@B22],[@B31]\] and have been shown to be predictive in cases where some experimental measure of accessibility has been made\[[@B32]\]. While MFold-derived accessibility scores may not be completely optimal, they have been used with reasonable success to predict accessibility in the siRNA targeting context, and so we use MFold here. Shearing simulation ------------------- Random shearing of the target mixture is an approach that is often offered as a solution for the problem of target secondary structure. The actual content of a sheared mixture of DNA or RNA fragments is complex. Shearing breaks the molecule not in predictable locations, but in random locations that give rise to a distribution of fragments around an average fragment length. In order to simulate the effects of different degrees of shearing on structure formation and stability in a transcript, we picked fragments of 200, 100, or 50 bases in length, choosing the start position via a sliding window of 10 bases. Secondary structure prediction for all fragments derived from every transcript in the B. suis genome is computationally intensive and produces an extremely large amount of output. Since our initial goal was to determine how much the method would affect the number and type of secondary structures probes would be expected to bind the shearing simulation was performed for fragments derived from the 300 bp Ure-1A gene of B. suis. Secondary structure and thermodynamics were computed for each of these fragments individually. Authors\' contributions ======================= VGR participated in the design of the study, carried out the simulations and analysis, and drafted the manuscript. JWW participated in the design of the study and helped to draft the manuscript. CJG conceived of the study, participated in its design, coordinated the research and analysis, and drafted the manuscript. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Stability of a sample transcript -- full length target and sheared fragments Folding ΔG of target transcript and fragment molecules shown in Figure 8, at hybridization temperatures commonly used for long oligomer arrays. ::: Molecule* ^2^G, kcal/mole --------------------------------------- --------------------- ---------- -------- ---------- 70-mer Probe \- 6.8 N/A \- 4.2 N/A Full Length Target \- 85.9 \- 188.4 -56.6 \- 140.2 200-mer sheared Target \- 25.5 \- 58.6 -15.9 \- 41.6 100-mer sheared Target -14.2 \- 25.7 -9.6 -18.0 50-mer sheared Target (not shown) \- 6.1 -10.5 \- 4.2 -7.3 ::: Acknowledgements ================ The authors would like to acknowledge the Fralin Biotechnology Center for research startup support (CJG) and the Department of Biology for graduate assistantship support (VGR).	PubMed Central	2024-06-05T03:55:54.129413	2005-3-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555549/", "journal": "BMC Genomics. 2005 Mar 8; 6:31", "authors": [ { "first": "Vladyslava G", "last": "Ratushna" }, { "first": "Jennifer W", "last": "Weller" }, { "first": "Cynthia J", "last": "Gibas" } ] }
PMC555550	Background ========== Chronic ethanol exposure has been shown to cause damage to the adult and developing nervous system \[[@B1],[@B2]\]. For example, in vivochronic ethanol has been shown to cause aberrant sprouting of hippocampal neurites in developing rats \[[@B3]\], increase the length of dendrites in cerebellar Purkinje neurons \[[@B4]\], the size of synaptic terminals of cerebellar granule cells \[[@B5]\], and the number of dendritic spines on hippocampal dentate granule neurons in adult rats \[[@B6]\]. Furthermore, in vitroethanol enhances neurite outgrowth in cultured rat cerebellar neurons \[[@B7]\]. Contrary to the enhancement of neurite outgrowth, other studies have shown that chronic ethanol exposure inhibits the growth of dendrites in CA1 hippocampal neurons and cerebellar Purkinje cells in vivoand inhibits chick spinal cord neurite formation in vitro\[[@B8],[@B9]\]. However, the mechanisms underlying this alteration of dendrite formation induced by ethanol exposure remain unknown. PC12 cells have been used as a cell culture model system to study the underlying mechanisms of ethanol\'s alteration of neurite outgrowth \[[@B10],[@B11]\]. PC12 cells are a rat chromaffin cell line that differentiate into neuronal-like cells in the presence of Nerve Growth Factor (NGF) \[[@B12]\]. Using these cells, chronic ethanol has been shown to enhance NGF-induced neurite outgrowth \[[@B10],[@B11]\]. Thus, PC12 cells have proven to be a valuable system for studying the mechanisms underlying ethanol-induced enhancement of neurite outgrowth. Nerve growth factor-induced neurite outgrowth in PC12 cells involves an induction of microtubule assembly \[[@B13],[@B14]\]. Microtubules are formed from α and β tubulin proteins, which form head to tail protofilaments \[[@B15]\]. Studies have shown that Protein Kinase C (PKC) activation enhances the polymerization of tubulin to form microtubules \[[@B16]-[@B19]\]. Schultz et al. \[[@B20]\] have also demonstrated that microtubules containing phosphorylated tubulin are more stable than those containing unphosphorylated tubulin, although it remains unclear whether tubulin phosphorylation is the cause or the result of microtubule stabilization. PKC also modulates the activity of several microtubule associated proteins, including those involved in microtubule polymerization and vesicle transport \[[@B21]-[@B24]\]. Specific isoforms of PKC have also been implicated in mediating NGF-induced neurite outgrowth. Using both antisense oligonucleotides and specific inhibitors of PKC delta, Corbit et al. \[[@B25]\] have demonstrated that this isoform of PKC is required for NGF-induced neurite outgrowth. Other studies have found that in PC12 cells which over-express PKC epsilon, there is an enhancement of NGF-induced neurite outgrowth, while PC12 cells which over-express a dominant negative inhibitor of PKC epsilon show an inhibition of neurite outgrowth \[[@B26],[@B27]\]. Thus, both the epsilon and delta isoforms of PKC have been implicated in modulation of NGF-induced neurite outgrowth. It is possible that the enhancement of NGF-induced neurite outgrowth produced by chronic ethanol may be due to ethanol\'s known alterations of PKC signaling. Chronic ethanol has multiple effects on PKC, including altered PKC subcellular localization following chronic exposure \[[@B28],[@B29]\]. Messing et al. \[[@B30]\] have shown that chronic ethanol exposure actually increases total cellular content of PKC delta and epsilon (membrane associated and cytosolic) in PC12 cells. However, other studies have shown that membrane-associated PKC activity is down-regulated following chronic ethanol exposure \[[@B31]\]. Thus, while total cellular content of PKC may increase with chronic ethanol exposure, membrane-associated PKC may be down-regulated. Interestingly, Hundle et al. \[[@B32]\] have shown, using PC12 cells which over-express an inhibitory fragment of either delta or epsilon PKC, that PKC epsilon is required for ethanol\'s enhancement of neurite outgrowth. In this study, we examined the effect of chronic ethanol exposure on the neuronal microtubule cytoskeleton using PC12 cells as a model system. Here we show that chronic ethanol exposure increases microtubule content, while decreasing free-tubulin content. Thus, it appears that ethanol enhances microtubule polymerization in PC12 cells. We also investigated the role of microtubule polymerization in mediating ethanol\'s effects on neurite outgrowth using PC12 cells which over-express an inhibitory fragment of either delta or epsilon PKC. Importantly, it is the PKC epsilon isoform which is required for ethanol\'s enhancement of neurite outgrowth. Here, we found that the PKC delta isoform, but not the PKC epsilon isoform, is required for the enhancement of microtubule polymerization following treatment with chronic ethanol. Thus, it appears that neurite outgrowth does not correlate with enhanced microtubule polymerization in PC12 cells. Results ======= Chronic ethanol exposure increases microtubule content in PC12 cells -------------------------------------------------------------------- For the following studies, we used 100 mM ethanol for four days as a chronic exposure; a dose and duration used by previous researchers to demonstrate ethanol\'s enhancement of neurite outgrowth \[[@B10]\]. This is a concentration of ethanol which can easily be achieved by chronic alcoholics (0.46 g/dl). Figure [1](#F1){ref-type="fig"} demonstrates that following a 96 hour exposure to 100 mM ethanol, there was approximately a 13% increase in polymerized microtubules compared to control cells (t~9~= 5.2; p \< 0.001; n = 10). Similarly, there was a significant decrease (about 15%) in free-tubulin concentration (t~9~= 5.7; p \< 0.001; n = 10) following 96 hours of ethanol exposure. There was no effect on total tubulin expression following four days of chronic ethanol exposure (t~9~= 0.034; data not shown). Chronic ethanol exposure in PKC dominant-negative PC12 cells ------------------------------------------------------------ We next used PC12 cells which over-express the first variable domain of PKC epsilon or delta, which acts as an isozyme specific inhibitor of PKC epsilon or delta \[[@B32]\], respectively, to investigate the role of PKC in ethanol\'s enhancement of microtubule polymerization (Figure [2](#F2){ref-type="fig"}). Interestingly, in the cells which express the inhibitor of PKC epsilon (DNE cells), we found that chronic ethanol exposure significantly increased microtubule content (t~5~= 7.15; p \< 0.001; n = 6) and decreased tubulin content (t~5~= 3.4; p \< 0.01; n = 6) (similar to control PC12 cells). In the cells which express the inhibitor of PKC delta (DND cells), there was no significant effect on microtubule (t~5~= 0.02; n = 6) or tubulin (t~5~= 0.44; n = 6) content. Ethanol had no effect on total tubulin content in either the DNE (t~5~= 0.2) or DND cells (t~5~= 0.013). There was no difference between the control vector transfected PC12 cells and wild-type PC12 cells, thus these groups were combined and expressed in Figure [1](#F1){ref-type="fig"}. Importantly, for both the DNE and DND experiments, these experiments were replicated in two different sub-clones of transfected PC12 cells. In other words, we had two strains of dominant-negative PKC epsilon cells (DNE1 and DNE4) and two strains of dominant-negative PKC cells (DND21 and DND24). Results were similar between the two epsilon lines and between the two delta lines. Discussion ========== Cells maintain a balance between free tubulin in the cytoplasm and tubulin which is polymerized into microtubules of the cytoskeleton. In this study, we find that chronic ethanol exposure increases microtubule content while decreasing free tubulin content in PC12 cells. Ethanol appears to be enhancing polymerization of tubulin into microtubules. While there was no increase in total tubulin within the cells, there was a change in the proportion of tubulin in the polymerized (microtubule) versus non-polymerized state. We initially hypothesized that the increase in microtubule content was due to ethanol\'s enhancement of neurite outgrowth. Presumably, neurite outgrowth is not due to simple stability of microtubules, but to an increase in dynamic microtubule growth. Microtubules are particularly abundant along the axons of nerve cells and multiple labs have show that chronic ethanol exposure increases NGF-induced neurite outgrowth in PC12 cells \[[@B10],[@B11]\]. Therefore, since the cells that were exposed to ethanol have increased neurite outgrowth, this could be reflected as an increase in microtubles. However, it is not clear that there would necessarily be a concomitant decrease in free tubulin content or what effect of ethanol may have on microtubule stability, per se. Our data from experiments utilizing PC12 cells which express dominant negative inhibitors of PKC do not seem to support the idea that the increased microtubule content reflects increased neurite outgrowth. Hundle et al. \[[@B32]\] have demonstrated the specificity of these inhibitory fragments by measuring PMA-induced translocation of PKC epsilon and delta to the particulate fraction of the cells using Western blotting with isoform specific antibodies. They show (see Figure [2](#F2){ref-type="fig"} of Hundle et al. \[[@B32]\]) that PKC epsilon, but not PKC delta, translocation was specifically inhibited in the cells expressing the epsilon inhibitory fragment. Further, PKC delta, but not PKC epsilon, was specifically inhibited in the cells expressing the delta inhibitory fragment. Thus demonstrating that the delta and epsilon inhibitory fragments selectively inhibit PMA-induced translocation of their corresponding PKC isozymes. Hundle et al \[[@B32]\] has shown, using these same cells, that chronic ethanol exposure enhances NGF-induced neurite outgrowth in control cells and cells expressing a dominant-negative inhibitor of PKC delta (DND cells) but not in cells expressing the inhibitor of the epsilon isoform of PKC (DNE cells). While it was not a goal of the current study to measure neurite outgrowth, daily observation of the cells was that neurite outgrowth was most dramatic in wild-type cells, slightly less so in the DND cells, and very limited in DNE cells (C. Reiter-Funk, personal observation). The work of Hundle et al \[[@B32]\] suggests that PKC epsilon but not PKC delta is involved in the effect of ethanol on neurite outgrowth. Therefore, we would have predicted that ethanol would cause an increase in microtubule content in DND cells but not in the DNE cells which do not have increased neurite outgrowth. However, we found that chronic ethanol enhances polymerization of tubulin into microtubules in dominant-negative PKC epsilon cells but not dominant-negative delta cells. Thus it appears that the delta isoform is involved in ethanol\'s enhancement of microtubule polymerization. Further, our data suggest that neurite outgrowth does not correlate with enhanced microtubule polymerization in PC12 cells. Interestingly, PKC delta has previously been shown to be involved in NGF-induced neurite outgrowth in PC12 cells \[[@B25]\]. It should be noted however, that our experimental paradigm varied slightly from that of Hundle et al \[[@B32]\]. For example, we allowed our cells to differentiate for 4 days prior to beginning ethanol. Therefore, apparent discrepancies of our findings with those of Hundle et al \[[@B32]\] could be related to these differences. Based on our data, we speculate that ethanol\'s enhancement of microtubule polymerization may involve phosphorylation of tubulin by PKC. However, it should be noted that we have, thus far, not directly measured phosphorylation of tubulin. Studies have shown that chronic ethanol increases expression of delta and epsilon PKC in PC12 cells \[[@B30]\] and PKC activation enhances tubulin polymerization into microtubules \[[@B16]-[@B19]\]. Alternatively, chronic ethanol could be acting to alter important microtubule associated proteins. Many of these proteins, including Microtubule-associated Proteins (MAPS) are important for promoting microtubule assembly \[[@B34],[@B35]\] and it has been shown that PKC phosphorylation mediates the assembly-promoting activity of these proteins \[[@B21],[@B22],[@B36],[@B37]\]. Further studies are required to determine the mechanism of ethanol\'s enhancement of microtubule formation and the apparent role of delta PKC. Conclusion ========== Our studies demonstrate that chronic ethanol alters the relative ratio of free versus microtubule-associated tubulin content in PC12 cells, resulting in an increase in microtubule content and a corresponding decrease in free tubulin. This alteration was found to occur in wild type cells, as well as those expressing a dominant-negative inhibitor of epsilon PKC but not in cells expressing a dominant-negative inhibitor of delta PKC. These ethanol-induced changes could be important during activity-dependent remodeling of synapses or developmental growth of axons and dendrites which may lead to cognitive dysfunction. Methods ======= Materials --------- Wild type PC12 cells were a gift from Nicholas Pantazis, Ph.D (University of Iowa). PC12 cell lines expressing dominant negative inhibitors of delta or epsilon PKC were a gift from Robert Messing, M.D (University of California, San Francisco). RPMI, Dulbecco\'s Modified Eagle Medium, and PenStrep were purchased from Gibco, NGF from R&D Systems, horse and fetal bovine serum was purchased from Hyclone, and laminin was purchased from Invitrogen. The microtubule assay kit was purchased from Cytoskeleton, Inc (\#BK038). Cell Culture ------------ PC12 cells were cultured in RPMI medium supplemented with 10% horse serum, 5% fetal bovine serum, and 1% PenStrep. Medium for the dominant-negative PC12 cells also included G418 (250 μg/ml) for selection purposes. The dominant-negative lines are cells that have been transfected to express isozyme specific dominant-negative inhibitors of delta or epsilon PKC and have been used to show specific PKC isozyme involvement in multiple ethanol effects \[[@B32],[@B33]\]. These cell lines stably express the fragments δV1 or εV1, which are derived from the first variable domains of delta PKC or epsilon PKC, respectively. There was also a cell line transfected with vector alone, which served as a control. Dr. Messing has shown that these fragments can function as isozyme specific inhibitors \[[@B32],[@B33]\]. The cells were maintained in an incubator at 37°C in 5% CO~2~. For all experiments, PC12 cells were plated into six-well, laminin-coated (5 μg/ml) plates at a density of 270,000 cells/well. The cells were differentiated into neuronal-like cells with Nerve Growth Factor (25 ng/ml) for four days prior to addition of any chronic drug exposure. For chronic exposures, ethanol (100 mM) was added to the culture media (along with NGF) for 96 hours and plates (control and ethanol) were wrapped in parafilm to prevent ethanol evaporation. Media were changed every other day. Microtubule assay ----------------- Measurement of microtubule and free-tubulin contents in cells are well-established assays. The materials are available as a commercially kit (Cytoskeleton, Inc). Microtubules are very sensitive to changes in temperature; therefore, all equipment and buffers were warmed to 37°C before use (unless otherwise indicated). To prevent free tubulin from polymerizing onto existing microtubules during the assay, lysis buffer was added at a ratio of 10 volumes of buffer to 1 volume of cell pellet. Cells were homogenized via syringe trituration and incubated for 10 minutes in lysis buffer (contents listed below). 10 μL of cell homogenates were saved for protein measurement using the Bradford Assay. Homogenized cells were then centrifuged at 100,000 × g for 30 minutes to separate microtubules from free-tubulin. The polymerized microtubules settle in the pellet, while the free-tubulin remains in the supernatant. Following centrifugation, the supernatant (free-tubulin) was removed and frozen until Western blot analysis. The pellet was resuspended in ice-cold water containing CaCl~2~(200 μM) and incubated for one hour. CaCl~2~acts to enhance microtubule depolymerization. Thus, the microtubules remaining in the pellet were depolymerized to free-tubulin. The samples were then centrifuged at 14,000 × g (4°C) for 10 minutes. The supernatant (containing free-tubulin representing the original microtubules) was collected and frozen. Tubulin concentrations in both fractions were measured using Western blotting as described below. Buffer contents --------------- [Lysis buffer]{.underline}: LMS1 solution containing GTP (100 mM) + ATP (100 mM) + protease inhibitor cocktail (10 μM) + Okadaic Acid (100 nM). [LMS1]{.underline}: PIPES (100 mM) containing MgCl~2~(5 mM) + EGTA (1 mM) + glycerol (30%)+ Nonidet P40 (0.1%) + Triton X-100 (0.1%) + Tween 20 (0.1%) + beta-mercapto-ethanol (0.1%) + Antifoam (0.001%). [Protease inhibitor cocktail]{.underline}: Pepstatin (1 μg/ml) + Leupeptin (1 μg/ml) + benzamidine (10 μg/ml) + tosyl arginine methyl ester (500 μg/ml). Measurement of tubulin concentrations ------------------------------------- Western blot analysis was used to determine tubulin content. Samples for Western blot were boiled with electrophoresis sample buffer (containing 25% glycerol, 5% beta-mercaptoethanol, 2% SDS, and 0.01% bromophenol blue in 62.5 mM Tris-HCl, pH 6.8). Equal amounts of protein (25 μg) were separated by SDS-PAGE using a 12% polyacrylamide gel. Molecular weight standards were also loaded. The separated proteins were transferred to nitrocellulose membranes using an electroblotting apparatus. The membrane was blocked \[Tris-Buffered Saline (TBS) containing 4% nonfat dry milk and 0.05% Tween-20\] for 20 minutes at room temperature. Membranes were then incubated in primary antibody (500 ng/ml; monoclonal anti-beta Tubulin; Cytoskeleton, Inc) overnight at 4°C. Following incubation in the primary antibody, membranes were washed with TBS/0.05% Tween-20 and incubated in secondary antibody (1:1000 dilution; HRP conjugated anti-mouse antibody; Santa Cruz). Membranes were again washed and tubulin bands were visualized by Enhanced Chemiluminescence (ECL) using standard luminol reagents captured using a BioRad Gel Doc 1000. The computer analysis software Molecular Analyst (version 2.1; BioRad) was used to quantify tubulin concentrations (relative density using the Volume Analysis setting with equal sized rectangular boxes set around individual bands). All blots were very clean and no filtering was needed. Data expression and statistical analysis ---------------------------------------- Tubulin concentrations in both the free-tubulin and microtubule fractions were expressed as a percent control relative density and presented as mean (+/-SEM). The data represent multiple replicates of the same experiment (n values listed in figure legends). Student\'s t-tests were used to make comparisons between control and ethanol-treated PC12 cells. Each individual band analyzed was an independent extract from the same experiment, with the experiment being replicated 5 times. Each replication of the experiment was composed of 2 separate wells of cells for each treatment condition. For each of the 5 replications, the 2 control bands were averaged and each sample, including the controls, from that replication normalized to the average. The data was combined after all 5 replications. Authors\' contributions ======================= CKR-F conceived and performed the experiments and participated in writing the manuscript. DPD participated in the design of the study and writing of the manuscript. Both authors read and approved the final manuscript. Acknowledgements ================ The authors wish to thank Drs. Nicholas Pantazis and Wei-Jung Chen for helpful comments on the manuscript. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Chronic ethanol exposure increases microtubule content in PC12 cells. Chronic ethanol exposure (100 mM for 96 hours) significantly decreases free tubulin content (A) and significantly increases microtubule-associated tubulin content (B) in PC12 cells. The graphs in A and B represent the combined results of five independent replications (n = 10) of the same experiment (Three of these experiments are from wild type PC12 cells; two are from control vector transfected PC12 cells. We found no significant differences between these two cells types). Data are the mean +/- SEM. Asterisks represent significant differences from corresponding control (p \< 0.001). Figure C is a representative Western Blot from one replication. ::: ![](1471-2202-6-16-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Chronic ethanol exposure in PKC dominant-negative PC12 cells. Ethanol (100 mM for 96 hours) has no effect on microtubule or tubulin content in dominant-negative delta PC12 cells (A). Ethanol significantly increased microtubule content and decreased tubulin content in dominant-negative epsilon cells (B). The graphs represent the combined data from three independent replications of the same experiment (n = 6). Data are presented as the mean +/- SEM. Asterisks represent significant differences from corresponding control (p \< 0.005). ::: ![](1471-2202-6-16-2) :::	PubMed Central	2024-06-05T03:55:54.132426	2005-3-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555550/", "journal": "BMC Neurosci. 2005 Mar 11; 6:16", "authors": [ { "first": "Cindy K", "last": "Reiter-Funk" }, { "first": "Douglas P", "last": "Dohrman" } ] }
PMC555551	Introduction ============ Chronic fatigue syndrome (CFS) is characterized by persistent debilitating fatigue often accompanied by a complex of other symptoms (e.g., impaired memory, sore throat, post-exertional fatigue, aching and stiffness in muscles) lasting at least six months that is unresolved with rest or medication \[[@B1],[@B2]\]. A primary component of the case definition of CFS is the inability of patients to maintain their own pre-illness level of activity. Using both self-report as well as accelerometers, previous research on activity levels in this population suggests that individuals with CFS have physical activity levels that are 15% to 40% reduced from those of otherwise healthy sedentary individuals \[[@B3]-[@B6]\]. These data suggest that most CFS patients are on the lowest end of the activity spectrum. It is widely accepted that a sedentary lifestyle may greatly increase the risk of development of cardiovascular disease and Type II diabetes, as well as contributing to bone loss, and an age-related loss of function in a person\'s ability to perform daily activities \[[@B7]-[@B9]\]. Recent recommendations by the Surgeon General suggest that accumulating thirty minutes of moderate intensity physical activity per day could provide positive health benefits \[[@B10]\]. Individuals are encouraged to increase their daily physical activity not only by traditional, structured exercise programs, but also by increasing the amount of unstructured physical activity they perform each day (i.e. the amount they walk each day). The CFS population, with its low daily activity, could derive many positive health benefits from increasing its daily activity, and given their extremely sedentary nature the needed exercise stimulus could easily be met by increasing the amount of walking each subject performs daily. Few randomized controlled trials designed to assess the efficacy of exercise training have been conducted in the CFS population. Two studies used graded exercise performed several days-a-week over a period of 12--26 weeks and both reported an improvement in fatigue-related symptoms and aerobic capacity \[[@B11],[@B12]\]. Additionally, a single subject performed graded aerobic exercise as well as strength training and reported moderate improvements in fatigue-related and other CFS symptoms \[[@B13]\]. Results from these studies suggest that exercise, in addition to providing positive health outcomes, could also provide beneficial effects to CFS symptomology. Although exercise has been shown to be beneficial, it is unknown to what extent a structured, formal exercise program may alter daily activity in CFS patients. It is common for CFS patients to report exacerbation of their symptoms when too much physical activity is undertaken. CFS patients may have to \"rest\" more often and/or decrease other types of physical activity in order to compensate for their periods of exercise. The purpose of this study was to assess normal daily physical activity levels in CFS patients using accelerometers over a two-week period and compare these values to a group of healthy sedentary individuals. Additionally, the CFS patients were asked to increase their daily physical activity approximately 30% over a four week period by walking a prescribed amount each day while maintaining their non-exercise daily physical activity. Daily ratings of mood, fatigue, and muscle pain intensity were assessed and compared between the groups as well as in response to increased daily activity in the CFS patients. Methods ======= Participants ------------ All experimental procedures were approved by the Institutional Review Board at the University of Georgia, and informed consent was obtained from each participant. All participants were recruited from the general community and either responded to a newspaper ad, responded to a flyer placed around campus, or were referred to the study by their physician. Seventeen CFS and twenty-one controls responded and were screened as possible study participants. A physician\'s diagnosis of CFS was required for inclusion. Additionally, CFS patients were required to confirm a self-report of decreased physical activity compared to pre-CFS levels, and self-reported inability to sustain high levels of physical activity without a subsequent exacerbation of CFS symptoms for study inclusion. CFS participants with a self-reported history of depression or other psychiatric illness were excluded. Control participants were chosen to be similar in age, height, and weight to the CFS patients and were defined as sedentary by self-report of one bout of regular exercise per week or less. The most sedentary participants (those with desk jobs, etc.) were given first priority for study inclusion. Control participants were also apparently healthy and reported no illnesses or disease conditions. Medications were monitored in all participants. The CFS participants were found to be taking many medications, both prescription and over-the-counter. Analgesics such as Vioxx, Celebrex, Advil, and Aleve were common. Study Design ------------ Initially, participants received instructions for wearing the activity monitors and for completing a daily activity log. A \"pre\" score from the 30-item Profile of Mood States short form questionnaire (POMS) was obtained (The Educational and Industrial Testing Service, San Diego, CA). Participants proceeded to wear the monitors for two weeks during which time they were instructed to maintain normal daily activity. After the two weeks, data were collected from the activity monitors, and the monitors were reset. The participants then wore the monitors for an additional four weeks. The CFS patients were asked to increase their daily physical activity (30% above baseline) during this four week period by walking a prescribed amount each day in order to approximate the daily physical activity of a healthy sedentary person. This was based upon averaging the findings of others that suggested CFS patients had activity levels that were 15% to 40% reduced from healthy but sedentary individuals \[[@B3],[@B4],[@B6]\]. CFS patients were given neutral instructions as to whether or not increasing their daily physical activity would alter their mood and fatigue symptoms. Control participants maintained their normal activity for a six week period. Daily activity logs were completed each day. Participants recorded all daily activities, time spent in each activity, as well as time periods when the monitor was not worn (e.g., bathing). Participants also completed a series of questions documenting their daily mood, perceptions of fatigue and muscle pain intensity, and the duration of time fatigue and muscle pain were experienced each day. Objective Measurement of Physical Activity ------------------------------------------ Daily physical activity was assessed by a CSA accelerometer (Computer Science Associates Inc., Fort Walton Beach, FL). To ensure accurate measurements, the procedure recommended by CSA was followed -- the monitors were positioned over the subject\'s anterior superior iliac spine with the belt fitting snuggly so as to limit extraneous monitor movement. Participants were asked to wear the monitors at all times of the day, including sleep. Two minute epochs were used. Data were retrieved from the monitors using a specially designed docking module that input data into a computer. Recommended percent increases in daily activity were calculated based on each subject\'s average daily counts during their two-week baseline activity period. Counts are arbitrary units assigned to movements detected by the accelerometer. Counts are assigned based upon the magnitude of a change in velocity during a given time period. The number of counts needed to raise daily activity approximately 30% was calculated. Participants were then asked to walk on a treadmill at what they considered to be a comfortable walking pace. Walking speed was recorded, and used to calculate the recommended daily walking time. The approximate number of counts per minute for various walking speeds was assessed prior to the onset of the study (unpublished observations). Additionally, a pedometer was also used to aid participants in achieving the desired daily activity increase. Participants were given an approximate number of steps to take each day during their walk based upon their prescribed walking pace and time. Steps per minute for various walking speeds were assessed in a similar manner as counts per minute prior to the study (unpublished observations). Self-report of Daily Activity and Feelings ------------------------------------------ Data concerning daily activity, mood, fatigue, and muscle pain were obtained from each subject via daily self-report. Participants were asked to report all daily activities and time spent engaged in each. Time periods when the activity monitor was not worn were also reported. A series of five questions concerning daily mood, fatigue intensity, and muscle pain intensity were also answered. Participants ranked, using a 10 cm (0 to 100 mm) visual analog scale their general overall daily mood for that day (with 0 being their best possible overall mood and 100 being their worst possible overall mood), the intensity of their fatigue that day (with 0 being no fatigue and 100 being the highest intensity fatigue imaginable), and the intensity of their muscle pain that day (with 0 being no pain and 100 being the worst imaginable pain). A similar visual analog scale has been used previously in CFS patients to rate daily fatigue \[[@B13]\]. Additionally, participants reported the amount of time each day they experienced fatigue as well as muscle pain. Once each week participants completed a Profile of Mood States (POMS) short form questionnaire consisting of 30 questions (The Educational and Industrial Testing Service, San Diego, CA) in which participants reported how they had been feeling during the prior seven days. These forms were scored for both fatigue and vigor ratings. Statistical analysis -------------------- Independent samples t-tests were conducted to compare for differences in subject characteristics between the groups. A repeated measures ANOVA was conducted to determine differences between activity level and mood in CFS and control participants. When a significant interaction was observed a one-way repeated measure ANOVA was performed to analyze simple effects with planned comparisons performed to analyze differences in treatment means. All values are reported means ± SD. Analyses were conducted and significance was assumed at an α level of 0.05. Results ======= Participants ------------ No participants (CFS or controls) reported adverse events associated with the increased activity program. Data were obtained from six CFS patients as well as seven sedentary control participants. Additionally, two other CFS patients began the testing protocol but were removed from the study at their own request. One was removed on doctor\'s recommendation due to an injury (unrelated to the study) and the second was removed due to a change in residence. The physical characteristics of all participants are presented in Table [1](#T1){ref-type="table"}. Mean age, height, and weight, were not different between the CFS patients and the sedentary controls. Five of the six CFS patients also had a physician\'s diagnosis of fibromyalgia. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Participants characteristics for six CFS and seven sedentary control participants, values are mean ± SD. ::: Age (Years) Height (cm) Weight (kg) ------------- ----------------- ----------------- ----------------- CFS 43 ± 4.6 164 ± 7.3 73 ± 21.2 Control 43 ± 6.5 167 ± 7.0 70 ± 16.7 ::: Daily Physical Activity ----------------------- Individual as well as mean group daily activity counts are presented in Table [2](#T2){ref-type="table"}. All 24-hour periods in which the monitor was not worn for at least 23 hours were removed from the analysis. No trends were observed in daily activity counts in either subject group across all monitoring periods. Day-to-day counts were also relatively stable within a given activity period. Based on this, activity levels are presented as average daily counts during a given activity period. During baseline activity, CFS participants demonstrated 39% lower daily activity counts compared to controls (P= 0.017). All six of the CFS participants were successful in increasing their daily physical activity. Their daily activity counts increased 28%, on average, during the four-week training period (P\< 0.001). However, it should be noted that 4 of the 6 CFS participants did not reach the prescribed 30% increase in daily activity. Interestingly, following their activity increase the CFS participants had activity levels that were still 24% reduced from those of the control group (P= 0.08). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Average daily activity counts for CFS and control participants. Data are mean ± SD. ::: Subject Average Daily Activity(Coun\'ts × 10^3^) %Difference ------------- ---------------------------------------------- ------------------ --------- CFS Baseline Increase 1 88.3 ± 24.1 143.4 ± 42.5 \+ 62.3 2 126.7 ± 19.5 179.1 ± 44.9 \+ 41.4 3 199.8 ± 38.7 226.7 ± 57.5 \+ 13.5 4 167.4 ± 32.8 197.4 ± 32.5 \+ 17.9 5 234.2 ± 37.2 263.8 ± 31.2 \+ 12.6 6 158.3 ± 32.9 193.5 ± 33.0 \+ 22.2 Mean 162.5 ± 51.7 200.6 ± 41.2 Control Baseline 1 Baseline 2 1 415.1 ± 79.4 360.7 ± 67.3 \- 13.1 2 284.1 ± 87.1 289.5 ± 81.3 \+ 1.9 3 150.6 ± 61.6 146.2 ± 48.7 \- 3.0 4 254.9 ± 60.8 281.9 ± 76.5 \+ 10.6 5 223.7 ± 73.5 190.9 ± 68.8 \- 14.7 6 263.7 ± 60.7 233.9 ± 58.1 \- 11.3 7 278.1 ± 106.2 274.0 ± 113.9 \- 1.5 Mean 267.2 ± 79.5 253.9 ± 70.0 ::: Self-Report Mood/Feeling Ratings -------------------------------- Figures [1](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"}, and [3](#F3){ref-type="fig"} contain daily ratings of overall mood, fatigue intensity, and muscle pain intensity averaged over two week periods. Days where missing data were found (i.e. ratings were not completed), were checked against the activity monitor data to ascertain if the participants simply forgot to fill out the form or if some problem was present that could prevent them from filling out the ratings. Out of 692 possible days, missing data were found for only 19 days. Activity monitor data appeared normal on all 19 of these days. This suggests that the missing data were likely the consequence of participants forgetting to fill out the form rather than due to any adverse medical event. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Overall mood ratings. Each time point represents an average of the scores from the previous two weeks. \"0\" represents the best possible mood and \"100\" represents the worst mood imaginable. For CFS participants, the two week time point is from baseline activity and the four and six week time points are from increased activity. For control participants, all time points are from baseline activity. \* Significant group × time interaction (P= 0.016). Values are mean ± SD. ::: ![](1476-5918-4-3-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Ratings of fatigue intensity with \"0\" being a complete lack of fatigue and \"100\" being the highest intensity fatigue imaginable. Each time point represents an average of the daily scores from the previous two weeks. For CFS participants, the two week time point is from two weeks of baseline activity and the four and six week time points are from increased activity. For the control participants all time points are from baseline activity. CFS participants did not change significantly over time with increased activity. \# Significant difference between CFS and control participants (P\< 0.001). Values are mean ± SD. ::: ![](1476-5918-4-3-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Ratings of muscle pain intensity with \"0\" being a complete lack of muscle pain and \"100\" being the worst muscle pain imaginable. Each time point represents an average of the daily scores from the previous two weeks. For CFS participants, the two week time point is from two weeks of baseline activity and the four and six week time points are from increased activity. For the control participants all time points are from baseline activity. \* Significant group by time interaction (P= 0.030). Values are mean ± SD. ::: ![](1476-5918-4-3-3) ::: Figure [1](#F1){ref-type="fig"} demonstrates a significant group-by-time interaction between the CFS and control participants (P= 0.016, Eta^2^= 0.311) in overall mood. CFS participants reported a worsening of overall mood over time compared to controls. Neither an interaction nor a time main effect was observed in ratings of fatigue intensity. A significant group difference was observed between the CFS and control participants (Fig. [2](#F2){ref-type="fig"}, P\< 0.001, Eta^2^= 0.892). Although not statistically significant, ratings of fatigue intensity in the CFS group did increase from 58.2 ± 8.5 to 67.0 ± 17.5 (indicating a worsening of symptoms). A significant group-by-time interaction (P= 0.03, Eta^2^= 0.295) was seen between the CFS and control participants in their ratings of muscle pain intensity (Fig. [3](#F3){ref-type="fig"}). As their daily activity was increased, the CFS participants reported higher intensity muscle pain compared to controls. The amount of time spent with fatigue each day as well as the amount of time spent with muscle pain each day was also reported by both groups. During baseline activity, the CFS participants reported experiencing a significantly greater amount of time spent with fatigue per day compared to the control participants (930 ± 397 min/day. vs. 43 ± 73 min/day; P\< 0.001). Additionally, the CFS participants also reported experiencing a significantly greater amount of time spent with muscle pain each day (552 ± 505 min./day vs. 9 ± 22 min./day; P= 0.011). A significant time main effect was found for time spent with fatigue each day (P= 0.047, Eta^2^= 0.243). A significant difference was found between baseline activity, 451 ± 528 min/day, compared to the final two weeks of increased activity 521 ± 566 min/day (P= 0.048, Eta^2^= 0.287). The CFS participants also demonstrated a non-significant increase in time spent with muscle pain each day during baseline activity, the first two weeks of increased activity and the final two weeks of increased activity (554 ± 507 min/day vs. 642 ± 546 min/day vs. 713 ± 557 min/day). Control participants demonstrated no change over time in time spent each day with fatigue or muscle pain. Figures [4](#F4){ref-type="fig"}[5](#F5){ref-type="fig"} shows the mean weekly scores on the POMS fatigue and vigor scale for the CFS and control participants. A large and significant difference was observed between the CFS participants and the control participants with respect to their fatigue scores (P\< 0.001, Eta^2^= 0.916). No change was observed in the fatigue scores of the CFS participants as their daily physical activity was increased. The control participants also demonstrated no change over time. Similarly, a significant difference was also observed between the CFS and control groups when vigor scores were compared (P= 0.036, Eta^2^= 0.343). No change was observed in the vigor scores over time or in response to increased activity in either group. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Mean POMS short form ratings (taken once a week) scored for fatigue. Higher scores represent greater fatigue. For CFS participants scores were obtained prior to beginning the study (pre), at the end of each week of baseline activity (1--2) and after each of four weeks of increased activity (3--6). For control participants scores were obtained prior to beginning the study and at the end of each of six weeks of baseline activity (1--6). \* Significant difference between the CFS and control participants at all time points (P\< 0.001). Scores did not change over time in either group. Values are mean ± SD. ::: ![](1476-5918-4-3-4) ::: Discussion ========== A primary finding of this study was that individuals with CFS can increase their daily physical activity 28% on average over a four week period. Average daily activity counts, measured by accelerometer, increased in all six of the CFS participants who participated in the study. The magnitude of the observed increases in daily activity ranged from approximately 13% to 60%. These results are consistent with previous studies that have shown that CFS patients can sustain training programs lasting 12 to 26 weeks \[[@B11],[@B12]\]. The major difference between this study and previous training studies was that our goal was to increase total daily physical activity opposed to participating in an aerobic training program several days per week. Participation in a traditional program, even if the subject is compliant, does not insure that the subject increased total daily physical activity. With a traditional training program, it is conceivable that the CFS patients could \"rest\" between training sessions and consequently not experience a net gain in daily activity. This study demonstrates that with encouragement CFS patients can not only exercise daily, but also sustain enough of their non-exercise daily activities to result in sustained increases in daily physical activity over a period of four weeks. Another interesting aspect of this study was the fact that even after increasing their daily activity 28%, our CFS participants were still approximately 25% less active than our sedentary control participants. We attempted a 30% increase in daily physical activity with the hope of bringing the daily activity levels of our CFS participants up to those of sedentary individuals. This was based upon the findings of others that suggested CFS patients had activity levels that were 15% to 40% reduced from healthy but sedentary individuals \[[@B3],[@B4],[@B6]\]. Our results suggest that previous estimates of daily activity in this population may have been overestimated. However, given our small sample size it is possible that our CFS participants were more inactive than those examined in previous studies. It is also possible that our sedentary control group may not have been as inactive as those used in other studies, even though great care was taken to insure the sedentary nature of the group. While additional accelerometer data on the daily physical activity of sedentary women may exist, comparison of data between studies may prove difficult. Variables such as epoch period, monitor location, monitor type (brand), and calibration may all contribute differences in observed daily physical activity. For that reason, we feel it best to limit our comparison to only those subjects in our own study. To our knowledge, this study was one of the first to obtain daily ratings of fatigue intensity from a group of CFS participants under normal daily physical activity conditions as well as during periods of increased daily physical activity. Consistent with their diagnosis, CFS patients reported much greater daily ratings of fatigue intensity, time spent each day with fatigue, as well as fatigue recalled during the prior week compared to healthy sedentary participants. This large difference was expected based on the diagnosis and demonstrated the usefulness of ratings of this type to confirm fatigue symptoms. We found that overall mood, muscle pain intensity, and time spent each day with fatigue worsened following increased activity in our CFS participants compared to controls. Additionally, daily ratings of fatigue intensity (VAS) increased moderately over time while weekly fatigue (POMS) remained stable but elevated in the CFS patients. These observations are contrary to previous studies of increased exercise in this population where exercise training has been shown to reduce fatigue-related symptoms \[[@B11],[@B12]\]. These data demonstrate that, at least in our CFS participants, increases in daily physical activity had no beneficial effects on self-rated fatigue over the measured four week time period. It is not clear why our study did not find increased daily physical activity to reduce symptom severity, as reported in previous studies \[[@B11]-[@B13]\]. One possible explanation is that our fatigue ratings provided a more accurate and discriminatory measure of our participants\' fatigue symptoms. By providing them with a 0 to 100 visual analog scale to rate fatigue as well as asking them to assess their symptoms each day we may have obtained a more thorough and accurate picture of their fatigue symptoms than those obtained in previous studies using different rating scales. This is an important distinction between the present study and prior related studies. In our assessment of fatigue, we included daily fatigue intensity and duration. Previous studies have employed measures of fatigue-related symptoms that incorporated more symptoms than simply fatigue intensity or duration. For example, Wearden et al. \[[@B12]\] measured fatigue using the Chalder Fatigue Scale which includes items beyond the scope of fatigue intensity or duration such as sleepiness and \"slips of the tongue\". Additionally, the reliability of the factor structure of the Chalder Sale has been questioned in the CFS population \[[@B14]\]. Presentation of exercise as a possible treatment for CFS symptoms could have played a role in the improvements in symptom severity observed with exercise training \[[@B11]-[@B13]\]. Cognitive behavior therapy has been shown to be beneficial in CFS patients \[[@B15]\], and if CFS participants were made to believe that exercise would be beneficial to them then the observed improvements in other studies could represent some form of a placebo effect. Exact instructions given in previous studies were not reported. Care was taken in this study to present the increase in daily physical activity as a neutral intervention. A third explanation of our findings is that there may have been something inherent in our exercise protocol that prevented fatigue symptoms from improving. Our method of activity increase, self-paced walking, was no more strenuous than the exercise used in other studies \[[@B11]-[@B13]\]. However, the participants were asked to walk every day, as opposed to 2--4 times per week as in other studies, and to attempt to maintain all non-exercise daily activities \[[@B11]-[@B13]\]. It is possible the marked increase in activity each day over several weeks had a cumulative effect. By not providing \"rest\" days, it is possible that the CFS patients were approaching their daily \"activity limit.\" Conversely, while using larger training volumes, previous training studies in this population may not have observed evidence of an \"activity limit\" due to the fact \"rest\" days were provided and that 24-hour physical activity levels were not assessed. This hypothesis is based on self-reports that CFS is associated with an inability to sustain normal daily activity levels without a subsequent worsening of symptoms. If the prescribed increase in daily physical activity caused the CFS patients to approach their tolerable activity limit, this could result in a worsening of their fatigue symptoms. Our data suggests that the CFS participants were able to sustain an increase in daily activity over the course of the study. However, it is worth noting that four of our six CFS patients did not reach the goal of a 30% increase in daily activity. Whether this simply represents non compliance is unclear. However, the CFS patients with the lowest baseline daily activity were able to sustain the greatest increase while the patients with the highest baseline activity experienced the smallest increase. This perhaps points toward an activity limit in the CFS patients. An \"activity limit\' hypothesis might be consistent with the athletic overtraining syndrome in which athletes report heightened feelings of fatigue and loss of energy \[[@B16]-[@B19]\]. In addition to fatigue and energy loss, other features of overtraining such as immune dysfunction are similar to those observed in CFS \[[@B1],[@B2],[@B20]\]. Our prescribed exercise was less intense and occurred over a shorter time period than that of previous studies \[[@B11]-[@B13]\]. It is possible that more intense exercise over a longer time period is needed to see improvements in fatigue symptoms, and that we simply did not exercise our participants enough to see any improvements. This seems unlikely to us. Although not objectively measured, our CFS participants did subjectively report that maintaining the increased daily activity was difficult for them and they expressed doubts about the possibility of a further increase their daily activity levels. Five of our six CFS participants also reported having fibromyalgia. Fibromyalgia is a related syndrome with a primary symptom of muscle pain and tenderness \[[@B21]\]. Interestingly, our CFS-FM participants reported no reduction in the intensity of their daily muscle pain as their daily physical activity increased. Similar to their ratings of fatigue intensity, muscle pain showed a trend toward worsening as activity was increased. Exercise programs have been shown to have modest benefits with respect to muscle pain in FM patients \[[@B22],[@B23]\], and it is possible that our muscle pain results should be interpreted in a similar manner to our fatigue results. It is important to note that our small sample size is a key limitation in our study. The study is under powered to detect differences between our groups. Most of the measured variables had small to moderate effect sizes (Eta^2^values from 0.101 to 0.377), suggesting that more participants would be needed to detect differences between the subject groups. Consequently, additional studies will be needed to confirm our results. In conclusion, this study found that individuals with CFS were able to increase their daily physical activity by approximately 28% for four weeks without serious health complications. At baseline activity our CFS participants exhibited significantly lower daily activity than sedentary controls. This reduced level of daily activity was larger than previously reported values\[[@B3],[@B4],[@B6]\]. Large differences were seen between CFS and controls in several fatigue ratings as well as ratings of muscle pain (consistent with an additional diagnosis of FM). Ratings of overall mood, muscle pain, and time spent each day with fatigue worsened as daily activity increased, and ratings of fatigue intensity did not improve. These findings are in contrast to those of prior exercise studies in this population, which have suggested exercise as a possible clinical treatment for CFS. It is possible that our CFS participants were approaching their daily \"activity limit\" and this prevented improvements in fatigue symptoms. Future studies are needed to understand the complex interaction between daily physical activity and fatigue symptoms, and to determine if a daily \"activity limit\" can be quantified in CFS patients. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Mean POMS short form ratings (taken once a week) scored for vigor. Higher scores represent greater vigor. For CFS participants scores were obtained prior to beginning the study (pre), at the end of each week of baseline activity (1--2) and after each of four weeks of increased activity (3--6). For control participants scores were obtained prior to beginning the study and at the end of each of six weeks of baseline activity (1--6). \* Significant difference between CFS and control participants (P\< 0.036). Values are mean ± SD. ::: ![](1476-5918-4-3-5) ::: Acknowledgements ================ We wish to thank the research volunteers for their participation. We would also like to thank Chris Elder, Matt Reifenberger, and Manning Sabatier for their aid in data collection, and to Dane Cook for helpful comments of the manuscript.. This project was supported by National Institute of Health Grant HL65179.	PubMed Central	2024-06-05T03:55:54.134697	2005-3-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555551/", "journal": "Dyn Med. 2005 Mar 3; 4:3", "authors": [ { "first": "Christopher D", "last": "Black" }, { "first": "Patrick J", "last": "O'Connor" }, { "first": "Kevin K", "last": "McCully" } ] }
PMC555552	Background ========== Creatine kinase (CK) isoenzymes catalyse the reversible transfer of the phosphate group of phosphocreatine (PCr) to ADP, to yield ATP and creatine (Cr). The CK/PCr/Cr system is present primarily in tissues with high and fluctuating energy demands such as brain, heart and skeletal muscle, and serves as a temporal and spatial \"energy buffer\" that helps to maintain a high intracellular phosphorylation potential in situations of increased metabolic demand (for reviews, see \[[@B1],[@B2]\]). In mammals, Cr can be taken up by the intestine from the food, or can be synthesized de novo. The liver is the main site of Cr production in the body (see \[[@B2]\]). After its synthesis, Cr is transported through the blood and is taken up by Cr-containing tissues via a specific Cr transporter. Whereas the importance of the liver in Cr biosynthesis is undisputed, some confusion still exists on the CK activity and PCr content in this organ. The majority of findings suggest no or minute levels of CK and PCr in liver tissue and, in particular, in hepatocytes (e.g., \[[@B3]-[@B6]\]). Other studies that used more sensitive experimental approaches provided evidence for low levels of PCr and CK, specifically localized in sinusoidal endothelial cells (\[[@B7]-[@B9]\]; see also \[[@B10]\]). Finally, in a few cases, more extreme findings were made: unusually high levels of CK activity were measured in liver tissue by Shatton et al. \[[@B11]\], Goullé et al. \[[@B12]\], and Wali & Makinde \[[@B13]\]. The majority of studies indicated that the low levels of CK activity in liver are due solely to the brain-type cytosolic CK (BB-CK) isoenzyme. On the other hand, besides BB-CK which was suggested to be present in endothelial and Kupffer cells, Vaubourdolle et al. \[[@B14]\] also provided evidence for the presence of the muscle-type cytosolic (MM-CK) isoenzyme in Ito cells, and for mitochondrial CK (Mi-CK) in hepatocytes. Similarly, Kanemitsu et al. \[[@B15]\] purified Mi-CK from normal human liver, which would imply significant amounts of this isoenzyme in liver tissue. Finally, increases in serum CK activity were frequently observed in cases of severe liver disease, with the most obvious source of CK being the pathological liver tissue itself \[[@B16]-[@B18]\]. In a number of studies reporting significant levels of CK activity in liver, interference by adenylate kinase (AdK) isoenzymes in the CK activity assays \[[@B19]-[@B21]\] is very likely (e.g., \[[@B13]\]), or can at least not be excluded, thus questioning the validity of these studies. Another possible cause for inconsistent findings might be compensatory up-regulation of CK expression in pathological liver tissue. Two lines of evidence that favour this hypothesis are: (i) partially hepatectomized rat liver was reported to show an increase in BB-CK activity (see \[[@B22]\]); and (ii) overexpression of CK isoenzymes in the liver of transgenic mice was shown to stabilize energy metabolism under low-oxygen stress and after a metabolic challenge \[[@B23],[@B24]\], to accelerate regeneration of liver mass following major hepatectomy \[[@B10],[@B25]\], and to increase endotoxin tolerance \[[@B5],[@B26]\]. Because of these conflicting data, the goal of the present study was to analyse in detail the CK and AdK activities in pathological liver tissue of patients undergoing orthotopic liver transplantation. Methods ======= Liver samples ------------- The present project was approved by the ethics commission of the University of Innsbruck. In total, 25 liver samples were analysed. Twenty-three samples were obtained from 18 explanted organs of liver transplant recipients, one sample was obtained at autopsy (no. 1), and the last sample was from a normal rat liver. According to pathomorphological criteria, the 25 samples can be divided into 5 groups: (1) Nine samples of cirrhotic liver tissue (nos. 5, 7, 11, 13, 17--19, 23, 24: 4 due to hepatitis B or C virus infection, 3 due to primary or secondary biliary cirrhosis, 1 due to chronic alcohol abuse, and 1 due to vena hepatica occlusion); (2) six samples of neoplastic tissue (nos. 4, 9, 14, 15, 20, 21: 3 cholangiocellular carcinomas, 2 primary hepatocellular carcinomas, and 1 liver metastasis of a malignant melanoma); (3) three samples of necrotizing liver tissue due to acute or subacute organ rejection (nos. 2, 6, 22); (4) five samples of macroscopically normal liver parenchymal tissue (nos. 3, 8, 10, 16, 25) surrounding focal liver pathologies (i.e., 2 primary HCCs \[samples 4 and 9\]; metastasis of malignant melanoma \[sample 15\]; vena hepatica occlusion \[sample 24\]); (5) two samples originating from a normal rat liver (no. 12) and from a patient with steatosis hepatis (no. 1). Preparation of homogenate, cytosolic and mitochondrial fractions of human and rat liver --------------------------------------------------------------------------------------- All steps were performed on ice or at 4°C. Approximately 5 g of liver tissue was homogenized in 45 ml buffer A (250 mM sucrose, 5 mM HEPES, 0.5 mM EGTA, pH 7.4). The homogenate was subjected to centrifugation for 5 min at 800 g. The pellet was discarded, and the supernatant centrifuged for 4 min at 5,100 g (centrifugation C2). The supernatant of C2 was further clarified by centrifugation for 12 min at 12,300 g, thus yielding the cytosolic fraction. The pellet of C2 was resuspended in 10 ml buffer A, followed by centrifugation for 2 min at 12,300 g (C3). After resuspension of the C3 pellet in 10 ml buffer A and centrifugation for a further 10 min at 12,300 g, the sediment was resuspended in 4 ml buffer A, thus yielding the mitochondrial fraction. One-ml aliquots of the different fractions were immediately frozen in liquid nitrogen and stored at -80°C until analysis. Measurements of CK and AdK activity ----------------------------------- For CK and AdK activity measurements, the following assay medium was used: 110 mM imidazole, pH 6.7, 20.5 mM glucose, 11 mM Mg-acetate, 2.05 mM EDTA·Na~2~, 2.1 mM ADP, 2.1 mM NADP, 21 mM N-acetylcysteine, 9 U/ml of hexokinase, and 5.8 U/ml of glucose-6-phosphate dehydrogenase (both from Sigma). Enzymatic activity was measured at 25°C as an increase in NADPH absorbance at 340 nm. For AdK, three separate measurements were made for each sample in the same assay medium. For CK measurements, 5.1 mM AMP was added to the assay medium to inhibit AdK activity. For each sample, three measurements with 10.3 mM PCr and three measurements without PCr (blank measuring residual AdK activity after inhibition with AMP) were made, and the CK activity was calculated as the difference of the respective means. All values in this paper represent specific activities per mg of homogenate, cytosolic or mitochondrial protein. Protein amounts were measured according to the method of Bradford \[[@B27]\] with bovine serum albumin as standard. Cellulose polyacetate electrophoresis (CPE) ------------------------------------------- CPE was performed at room temperature for 90 min at a constant voltage of 150 V, but otherwise as described previously \[[@B28]\]. CK and AdK isoenzyme bands were visualized at 37°C with an overlay gel technique in a reaction protocol similar to the one described above for the measurement of enzymatic activity. NADPH was reacted with nitrobluetetrazolium in the presence of phenazine methosulfate to yield formazan. For visualization of CK bands, AMP was added to the overlay gel to inhibit AdK activity. Since AMP alone may not be sufficient to inhibit all AdK activity \[[@B21]\], two identical cellulose polyacetate strips were run; one was developed with PCr in the overlay gel, whereas for the other, PCr was omitted from the overlay gel (blank). Results ======= CK and AdK activities were measured in total homogenate (Fig. [1](#F1){ref-type="fig"}), cytosolic and mitochondrial fractions (data not shown) obtained by differential centrifugation from 25 normal and pathological liver samples. Highest CK activities were observed in the two primary HCCs analysed (liver samples no. 4 and 9), with specific CK activities in the homogenate of 0.36 and 0.21 U·(mg protein)^-1^, respectively. In most other liver samples, the specific CK activities in the homogenate were below 0.05 U·(mg protein)^-1^. Whereas enzymatic activity measurements revealed low, but consistent CK activity in many of the cytosolic fractions, no CK activity was detected in the mitochondrial fractions, except for HCC sample no. 9 with a specific CK activity of approx. 0.1 U·(mg protein)^-1^(due to limited sample size, subcellular fractionation was not feasible for HCC sample no. 4). These findings were corroborated qualitatively by isoenzyme electrophoresis on cellulose polyacetate strips. Visualization of the different CK isoenzymes by an overlay gel technique revealed that the brain-type cytosolic BB-CK isoenzyme was present in all liver samples. Conversely, bands for the dimeric and octameric forms of Mi-CK were only observed in the two primary HCC samples (nos. 4 and 9; Fig. [2](#F2){ref-type="fig"}). The CK/AdK activity ratio in the homogenate was 1.4 and 2.6 for the two primary HCCs (liver samples no. 4 and 9, respectively), 0.5 for liver sample no. 5 (secondary biliary cirrhosis), and \< 0.2 for all other liver samples. Similar findings were made for the cytosolic and mitochondrial fractions, with CK/AdK activity ratios of \< 0.3 for the cytosolic fraction and \< 0.05 for the mitochondrial fraction. For HCC sample no. 9, however, these ratios were significantly higher: 4.8 (cytosolic fraction) and 0.33 (mitochondrial fraction). Discussion ========== CK is an enzyme still widely analysed in clinical diagnostics. Although a wealth of CK measurements have been reported in the scientific literature, there still exist inconsistency and incomplete knowledge on such an apparently simple question as the CK (isoenzyme) content of mammalian liver in both health and disease. In the present study, we detected the presence of BB-CK in all liver samples analysed by using CK activity measurements and cellulose polyacetate electrophoresis. However, in the normal and most pathological liver samples that we analysed, the specific CK activity was very low (\< 0.05 U·\[mg protein\]^-1^), levels which are comparable with or lower than data reported for rat and human liver \[[@B29],[@B30]\], but much lower than the specific CK activities in skeletal muscle, heart and brain (2--37 U·\[mg protein\]^-1^; \[[@B29],[@B31]-[@B33]\]). We additionally observed that (i) the specific AdK activities in these samples were consistently higher than the specific CK activities (on average, \> 10-fold), (ii) both activity measurements and cellulose polyacetate electrophoresis revealed similar specific AdK activities in the cytosolic and mitochondrial fractions (although from different AdK isoenzymes; data not shown), and (iii) mitochondrial respiration, in the presence of ATP, could be fully stimulated by AMP, but not by creatine (data not shown). This last observation favours the interpretation that in normal hepatocytes, CK isoenzymes are not expressed, and that the AdK isoenzyme system plays a function in high-energy phosphate buffering and transport, which is similar to the role of CK in brain, skeletal muscle and heart. Although histochemical data are missing, the results obtained here are most consistent with a localization of small amounts of BB-CK in sinusoidal endothelial cells \[[@B14]\]. Interestingly, we observed a strong induction of both BB-CK and Mi-CK expression in two samples of primary HCC. Despite CK/AdK activity ratios in vitroof 1.4--2.6, the specific CK activities were still relatively low (0.21--0.36 U·\[mg protein\]^-1^). Therefore, in the absence of histochemical data, it cannot be concluded with certainty whether the increased levels of CK are due to increased vascularization of the tumour (possibly associated with a higher proportion of CK-containing endothelial cells), or to induction of CK expression in the malignant cells. Induction of CK expression has been observed previously in many types of tumours (see \[[@B2]\]) and may reflect an adaptation of the tumour tissue to the increased energetic demands. Evidence for induction of CK in liver tumours mostly comes from hepatoma cells grown in tissue culture \[[@B34],[@B35]\] or, indirectly, from increased amounts of circulating BB-CK and Mi-CK in the blood of patients with liver tumours \[[@B36]\]. On the other hand, analysis of the tumour tissue itself, both by classical biochemical methods and by microarray technology, provided inconsistent results. Some authors reported induction of CK expression in liver tumours (\[[@B37]-[@B39]\]; and, in part, \[[@B40]\]; M. Sakamoto and S. H. Yim, personal communication), others repression \[[@B41]\], and still others observed no statistically significant differences between normal and malignant liver tissue \[[@B11],[@B30]\]. This may be a reflection of the diverse clinicopathological and biological phenotypes of HCC, with different underlying molecular defects. A key player in the picture might be the p53 tumour suppressor gene. Mutations in p53 are quite prevalent in HCC, especially in tumours with low cellular differentiation \[[@B42],[@B43]\]. On the other hand, p53 was shown to control BB-CK expression: transrepression as observed for wild-type p53 is prevented by different mutations in the p53 gene \[[@B44]\]. Therefore, it is tempting to speculate that induction of BB-CK in HCCs is caused directly or indirectly by mutations in p53. Expression of CK in HCC may have therapeutic implications, which is all the more important given (i) the limited responsiveness of HCC to currently available therapeutic approaches and, thus, (ii) the poor prognosis associated with this disease. Cr analogues (cyclocreatine and β-guanidinopropionic acid) and also Cr itself were previously shown to have antitumour activity, both in cell culture and in in vivomodels (\[[@B45],[@B46]\]; see also \[[@B2]\]). The responsiveness of tumour cells to growth inhibition by cyclocreatine seems to be correlated with their specific CK activity; cell lines with a specific CK activity of \> 0.10 U·(mg protein)^-1^were generally sensitive to the drug. As for the liver, β-guanidinopropionic acid and creatine slowed the growth of AS30-D ascites tumour cells in culture (chemically induced rat hepatoma; \[[@B34]\]). Similarly, cyclocreatine revealed antitumour effects in a rat model of chemically induced hepatocarcinogenesis \[[@B47]\]. Conclusion ========== The present findings shed light on some old enigmas and open up fascinating avenues for future research. Our findings do not support significant expression of CK in normal liver and most liver pathologies, but rather indicate that many of the previous misconceptions in this field can be explained by interference from AdK isoenzymes. On the other hand, given the need for improved understanding of the molecular pathogenesis of HCC, and for improved therapies and cures, the induction of CK expression in HCC described here calls for a more in-depth analysis of the interplay between p53 mutations, HCC, CK expression, and the growth-inhibitory effects of cyclocreatine in HCC. List of abbreviations ===================== AdK, adenylate kinase; BB-CK, brain-type cytosolic CK isoenzyme; CK, creatine kinase; Cr, creatine; HCC, hepatocellular carcinoma; Mi-CK, mitochondrial CK; MM-CK, muscle-type cytosolic CK isoenzyme; PCr, phosphocreatine. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= GM and RM covered the medical part of this study. GM, FNG and MW performed the biochemical experiments. MW drafted the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-230X/5/9/prepub> Acknowledgements ================ Ursula Walterscheid-Müller, Erich Gnaiger and all colleagues at the Department of General and Transplant Surgery of the University Hospital of Innsbruck are gratefully acknowledged for stimulating discussions and support, John Perkins for critical comments on the manuscript, and Michiie Sakamoto and Sunhee Yim for providing unpublished information. This work was supported by the Swiss National Science Foundation (fellowship No. 823A-037106), the Austrian Science Foundation (Lise Meitner fellowship No. M00198-MED), and the \"Ciba-Geigy-Jubiläums-Stiftung\". Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### CK and AdK activities in pathological and normal liver tissue.Specific CK (A) and AdK activities (B) in total homogenate fractions were determined as described under \"Methods\". The liver samples were classified into five groups: group 1 -- cirrhotic liver tissue (nos. 5, 7, 11, 13, 17--19, 23, 24); group 2 -- neoplastic tissues (nos. 4, 9, 14, 15, 20, 21; 2 primary hepatocellular carcinomas \[nos. 4 and 9\]; 3 cholangiocellular carcinomas \[nos. 14, 20 and 21\]; 1 liver metastasis of a malignant melanoma \[no. 15\]); group 3 -- samples of necrotizing liver tissue due to acute or subacute organ rejection (nos. 2, 6, 22); group 4 -- samples of macroscopically normal liver parenchymal tissue (nos. 3, 8, 10, 16, 25) surrounding focal liver pathologies (2 primary HCCs \[nos. 4 and 9\]; metastasis of malignant melanoma \[no. 15\]; vena hepatica occlusion \[no. 24\]); group 5 -- samples originating from a normal rat liver (no. 12) and from a patient with steatosis hepatis (no. 1). No AdK activity measurements are available for liver sample no. 1. ::: ![](1471-230X-5-9-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### CK isoenzymes in HCC.Subcellular fractions of HCC liver samples no. 4 (A, B) and 9 (C, D) were analysed by cellulose polyacetate electrophoresis as described under \"Methods\". H = total homogenate; C = cytosolic fraction; M = mitochondrial fraction; Ct = control containing BB-CK and Mi-CK; Mi-CKd = dimeric Mi-CK; Mi-CKo = octameric Mi-CK; O = place of sample application. The cellulose polyacetate strips were exposed to an overlay gel containing PCr (A, C = CK activity staining), or lacking PCr (B, D = corresponding negative controls). Whereas Mi-CKd and Mi-CKo were well separated in (A), they were less so in (C). ::: ![](1471-230X-5-9-2) :::	PubMed Central	2024-06-05T03:55:54.137484	2005-3-5	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555552/", "journal": "BMC Gastroenterol. 2005 Mar 5; 5:9", "authors": [ { "first": "Georg", "last": "Meffert" }, { "first": "Frank N", "last": "Gellerich" }, { "first": "Raimund", "last": "Margreiter" }, { "first": "Markus", "last": "Wyss" } ] }
PMC555553	Background ========== Colorectal cancer is a widespread fatal malignancy in the West \[[@B1]\] and its incidence in Japan is increasing \[[@B2]\]. Epidemiological studies have shown that regular doses of nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with lower rates of colorectal polyps, colorectal cancer, and colorectal cancer-associated mortality. Waddell and Loughry initially reported that sulindac decreased the number of polyps in patients with familial adenomatous polyposis (FAP) \[[@B3]\]. The largest study of aspirin and colon cancer was undertaken by Thun et al. in 1991 \[[@B4]\]. They found that colon cancer mortality decreased among those who used aspirin, and that the protective effect was increased by increasing doses. NSAIDs inhibit prostaglandin production mainly by inhibiting cyclooxygenase (COX) that is expressed as COX-1 and COX-2 isoforms. Cox is a key enzyme, which converts arachidonic acid to prostaglandins. COX-1 is constitutively expressed in most tissues and is thought to be responsible for homeostatic functions. On the other hand, COX-2 expression is induced by various factors including cytokines, growth factors, and tumor promoters \[[@B5],[@B6]\]. Many studies have indicated that COX-2 expression is associated with both the carcinogenesis and growth of colon cancer. COX-2 is overexpressed in 85% of human colorectal cancers \[[@B7]\]. Oshima et al. disclosed that COX-2 gene knockouts and a COX-2 inhibitor decreased the number of polyps of apcΔ716 knockout mice \[[@B8]\]. In addition, several studies have shown that the selective inhibition of COX-2 reduces colorectal tumorigenesis in various models of carcinogenesis. Reddy et al. found that the COX-2 inhibitor, SC-58635 (celecoxib), significantly suppressed colonic aberrant crypt foci and crypt multiplicity in rats \[[@B9]\]. This supports the hypothesis that COX-2 inhibitors are chemopreventive. Kawamori et al. showed that celecoxib inhibited both the incidence and multiplicity of colon tumors in a rat model \[[@B10]\], and another study showed that COX-2 expression in colon cancer cells increased metastatic potential \[[@B11]\]. We reported that increased COX-2 levels in human colorectal carcinomas correlated with both the size and invasiveness of tumors \[[@B12]\]. Tomozawa et al. demonstrated a correlation between COX-2 overexpression and the recurrence of colorectal cancer \[[@B13]\]. It is well known that colorectal metastasis occurs primarily in the liver and secondarily in the lung \[[@B14]\]. A laparotomy is necessary for making experimental model of liver metastasis in rats, but it is also a stressful event. On the other hand, it is less invasive to make lung metastasis by injection of tumor cells into the tail vein of rats. Thus, we considered that the experimental model of pulmonary metastasis was appropriate for the evaluation of the real effect of JTE-522, a selective COX-2 inhibitor, on hematogenous metastasis of colon cancer in rats. The aim of this study was to examine the effect of JTE-522 on hematogenous metastasis of colon cancer in a rat model. Methods ======= Preparation of JTE-522 ---------------------- The selective COX-2 inhibitor, JTE-522 \[[@B15]\], was suspended in 0.5% carboxymethyl cellulose sodium salt (Wako Life Science Reagents, Osaka, Japan). Animals ------- Twenty-four 4-week-old male F344/DuCrj rats, weighing 50 to 70 g purchased from SLC (Shizuoka, Japan) were acclimatized for one week before use in this study. The rats were maintained in an environment with a controlled temperature (22 to 24°C), humidity (40 to 50%), and lighting (12 hours light-dark cycle) with free access to tap water and standard rodent chow (CE-2, Nihon Clea, Tokyo, Japan). Tumors ------ The RIKEN CELL BANK donated the rat colon cancer cell line, RCN-9, which was established by Inoue et al. after inbred F344/DuCrj rats were subcutaneously injected with neutralized 1, 2- dimethylhydrazine (DMH) \[[@B16]\]. The tumor cells were stored at -80°C. After thawing, tumor cells were washed in phosphate-buffered saline (PBS) and resuspended in RPMI-1640 (Sigma Chemical Co., St. Louis, MO) medium containing 10% heat-inactivated fetal bovine serum (FBS; Sigma) and 0.05% penicillin-streptomycin (Sigma). After repeated subculture, cell viability was assessed by Trypan blue dye exclusion and the cells were counted using a hemocytometer. Induction of lung metastases ---------------------------- F344 male rats were anesthetized with ether and then a tumor suspension containing 5 × 10^6^RCN-9 cells in 0.4 ml of PBS was injected into the tail vein. Pulmonary metastases of colon cancer in rats -------------------------------------------- Twenty-four rats were divided into four groups. Group 1 (control) was not given JTE-522. The other three groups all orally received daily doses of 3, 10 and 30 mg/kg JTE-522 (groups 2, 3 and 4, respectively) by gavage from the day before RCN-9 injection until the end of the study. The rats were sacrificed 24 days after injection and the lungs were removed and weighed. After 3-day formalin fixation, lungs were embedded in paraffin. Paraffin-embedded sections were cut into 3-μm serial sections, which stained with hematoxylin and eosin to evaluate the pharmacological effects of JTE-522 on pulmonary metastases. The number and the largest diameter of metastatic tumors were examined microscopically in the largest sagittal cross section. The largest metastatic tumor was selected after measuring the diameter of 10 tumors in each slide. Immunohistochemistry -------------------- Slides were immunohistochemically examined using the Universal Immuno-enzyme Polymer (UIP). Sections of 3 μm thick were cut from formalin-fixed and paraffin-embedded blocks of rat tissues. The slides were deparaffinized in xylene, rehydrated through a graded series of ethanols and washed with distilled deionized water. Antigen was retrieved by incubating the sections in 10 mmol/L citrate buffer (pH 6.0) in an autoclave (120°C) for 5 minutes. Thereafter, slides were cooled to room temperature and then washed with distilled deionized water. To quench endogenous peroxidase activity, slides were immersed in 3% H~2~O~2~in methanol for 20 minutes. After a short rinse in phosphate-buffered saline (PBS), non-specific binding was blocked by incubating the sections in 10% normal goat serum for 10 minutes at room temperature. Thereafter, the slides were incubated with primary antibodies directed against COX-2 (rabbit polyclonal, Cayman Chemical, Ann Arbor, MI; dilution 1:250) for 2 hours or vascular endothelial growth factor (VEGF) (rabbit polyclonal, VEGF A-20: sc-152; Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:200) for 1 hour at room temperature. The slides were washed in PBS (3 × 5 minutes) and incubated with secondary antibody (N-Histofine^®^Simple Stain MAX PO, Nichirei, Tokyo, Japan) for 30 minutes at room temperature. After washing in PBS, color was developed using 3, 3\'-diaminobenzidine tetrahydrochloride (Sigma) in 50 mmol Tris-HCl (pH 7.5) for 5 minutes and counterstaining with 1% Mayer\'s hematoxylin. Sections known to show intense immunostaining for COX-2 and VEGF were included in each experiment, having been incubated with either primary antibody or PBS as positive and negative controls, respectively. The positive controls were always obviously stained and negative controls were never stained. Evaluation of immunostaining ---------------------------- The specimens immunostained for COX-2 and VEGF were independently scored by two blinded investigators. Both the distribution (the percentage of positive cells) and the intensity of staining were assessed in a semiquantitative fashion. Sections were scored, according to the method of Yukawa et al\[[@B17]\]. Both the number of positive cells and the intensity of staining were evaluated as follows. The number of positive cells: none, 0; focal (one third of cells stained), 1; multifocal (two third of cells stained), 2; and diffuse (most cells stained), 3. The intensity of staining: none, 0; mild (between 0 and 2), 1; and strong (clearly identified by × 40 magnification), 2. The score for distribution and intensity were added and graded as follows: 0, 1, and 2: negative; and 3, 4, and 5: positive. Statistical analysis -------------------- The effects of JTE-522 on pulmonary metastasis were analyzed by the Kruskal-Wallis test and are expressed as means ± standard deviation. A correlation between COX-2 and VEGF expression was analyzed by Spearman\'s rank test. In all instances, statistical significance was established at p \< 0.05. Results ======= Pulmonary metastases -------------------- Each group consisted of 6 rats. JTE-522 significantly and dose-dependently decreased the weight of the lungs (p = 0.0001, Figure [1A](#F1){ref-type="fig"}). Cross sections contained many metastatic tumors that were not visible on the surface (Figures [2](#F2){ref-type="fig"}). The size of the metastatic tumors was significantly and dose-dependently decreased (p = 0.0002, Figure [1B](#F1){ref-type="fig"}), although the number of metastases did not differ (Figure [1C](#F1){ref-type="fig"}). JTE-522 appeared to have no significant adverse effects. Immunohistochemistry -------------------- Immunohistochemical staining showed that both COX-2 and VEGF were expressed at high levels in pulmonary metastatic tumors of all groups (Figures [3](#F3){ref-type="fig"}). The large airways and vascular structures were stained for both proteins, as were tumor cells in all groups. However, there was no correlation between COX-2 score and VEGF score (p = 0.60, Figure [4](#F4){ref-type="fig"}). The degree of COX-2 and VEGF immunohistochemical staining in tumor cells did not significantly differ among groups (Table [1](#T1){ref-type="table"}). Discussion ========== JTE-522 dose-dependently reduced the weight of the lung with metastases and the size of metastatic tumors. These observations are consistent with those of other studies of selective COX-2 inhibitors. In the present study, however, the number of metastatic lesions determined microscopically was not reduced by JTE-522, which conflicts with the earlier findings. Masferrer et al. described the inhibition of tumor growth and lung metastasis of Lewis Lung carcinoma in mice by continuous dietary celecoxib (160--3200 ppm) supplied from the date of implantation \[[@B18]\]. Celecoxib at a dose of 160 ppm did not affect the number and size of lung metastases, but doses between 480 and 3200 ppm reduced the number of metastatic nodules by \>50%. Histopathological analysis revealed that celecoxib dose-dependently reduced the size of the metastatic lung tumors. Masferrer et al. evaluated the pharmacological effects of celecoxib on lung metastasis by counting the metastatic tumors using a stereomicroscope and by histochemical analyses of consecutive lung sections. Tomozawa et al. investigated the effect of JTE-522 on lung metastasis of colon cancer in mice \[[@B19]\]. Their study showed that JTE-522 significantly reduced the number of metastatic nodules and lung weight. They macroscopically counted the metastatic tumors on the lung surface. Our study using cross sections disclosed that many nodules were not visible on the surface and could not be seen by the naked eye. These observations may indicate that JTE-522 blocks the growth of metastatic tumors, but does not inhibit the process of metastasis. On the other hand, a recent study in vitro, using cancer cell lines preincubated with celecoxib, suggests that COX-2 is involved in adhesion of cancer cells to vascular endothelium \[[@B20]\]. If the cancer cells had been pretreated with JTE-522 for an appropriate period in the present study, it might reduce the number of the lung metastases. Here, we immunohistochemically stained COX-2 and VEGF because some reports indicate that prostaglandins regulate VEGF expression \[[@B21],[@B22]\] and that COX-2 inhibitors directly affect angiogenesis \[[@B23]\]. Other recent studies have confirmed the notion that tumor growth and metastasis are dependent on angiogenesis and require the development of new vessels \[[@B24]-[@B26]\], and that COX-2 is related to tumor angiogenesis. COX-2 modulates the production of angiogenic factors derived from colon cancer cells, while COX-1 regulates angiogenesis in endothelial cells \[[@B27]\]. COX-2 is also related to tumor angiogenesis in human colorectal cancer. Cianchi et al. identified a significant correlation between COX-2 and VEGF, the latter of which might be one of the most important mediators of the COX-2 angiogenic pathway \[[@B28]\]. Immunohistochemical staining in the present study revealed parallel COX-2 and VEGF expression that was mainly localized in the cytoplasm of tumor cells, epithelial cells of the airways, and vascular structures. These findings may indicate that COX-2 is involved in the production of VEGF. Furthermore, the present study supports our previous report that JTE-522 interferes with the growth of metastatic tumors of colon cancer, due to inhibition of neovascularization \[[@B29]\]. Our results showed that high levels of COX-2 were expressed in the groups administered with JTE-522 as well as in controls. This is consistent with the fact that JTE-522 suppresses not the production, but the activity of COX-2 in prostaglandin biosynthesis \[[@B15]\]. VEGF was also highly expressed in all groups. One of the reasons may be hypoxia induced by multiple pulmonary metastases. Christou et al. reported that hypoxic animals have significantly higher VEGF concentrations compared with normoxic controls \[[@B30]\]. On the other hand, these findings may indicate that VEGF is produced via a pathway, which does not include COX-2, as well as through a COX-2 pathway. We reported that COX-2 is related to human colorectal tumorigenesis. COX-2 mRNA expression is obviously increased in sporadic adenomas compared with normal colorectal mucosa and is significantly greater in adenomas with larger diameters \[[@B31]\]. Additionally, COX-2 expression and degree of dysplasia are significantly associated in adenomas \[[@B32]\]. COX-2 levels in human colorectal carcinomas are significantly higher in larger tumors and in those that invade more deeply \[[@B12]\]. We therefore postulated that COX-2 might correlate with metastasis as well as primary colorectal tumor, because hematogenous metastasis is the key for prognostic factor in colorectal cancer. The advances in the treatment for metastasis would improve the prognosis of the patients with colorectal cancer. The present study found that JTE-522 dose-dependently suppressed metastatic tumor growth, although the function to prevent cancer cells from metastasizing might not be so strong. Our recent report has revealed that administration of rofecoxib, a selective COX-2 inhibitor, decreased the number and size of rectal polyps in familial adenomatous polyposis patients, whereas the relapse of rectal polyps was seen 3 months after completion of the treatment \[[@B33]\]. This phenomenon might indicate that COX-2 inhibitors make colorectal polyps invisible, although atypical cells still exist microscopically. This is consistent with the result of the present study. Recent reports have shown the inhibitory effect of rofecoxib on liver metastases. Yao et al. demonstrated that rofecoxib decreased the growth and metastatic potential of colorectal cancer in mice through multiple mechanisms \[[@B34]\]. Fenwick et al. reported that refecoxib negatively regulated angiogenesis in human colorectal cancer liver metastases \[[@B35]\]. It is considered that COX-2 inhibitors can be used as therapeutic agents for hematogenous metastases of colorectal cancer. Conclusion ========== In conclusion, JTE-522, a selective COX-2 inhibitor, dose-dependently inhibited the growth of pulmonary metastasis from colorectal cancer in a rat model. Selective COX-2 inhibitors may constitute useful therapeutic agents that can interfere with the growth of hematogenous metastasis, as well as the tumorigenesis of colorectal cancer. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= HK carried out the animal experiment and drafted the manuscript. HK and TH performed immunostaining and its evaluation. HU performed the statistical analysis. ME and KS participated in the design of the study and its 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/1471-2407/5/26/prepub> Acknowledgements ================ Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. JTE-522 was donated by Japan Tobacco Inc (Tokyo, Japan). We thank Yoko Takagi and Labile T. Soumaoro for their technical support. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Graphs of pulmonary metastases of rats.(A) Lung weight of control and JTE-522 treated rats injected with RCN-9 (group 1, 10.70 ± 0.57 g; group 2, 9.14 ± 0.62 g; group 3, 5.61 ± 0.76 g; group 4, 3.52 ± 0.81 g). JTE-522 significantly and dose-dependently decreased the lung weight of each group (p = 0.0001). (B) Maximal diameter of metastatic tumors in control and JTE-522 treated rats injected with RCN-9 (group 1, 5067 ± 499 μm; group 2, 4233 ± 244 μm; group 3, 2583 ± 211 μm; group 4; 1990 ± 96 μm). JTE-522 significantly and dose-dependently decreased size of metastases (p = 0.0002). (C) The number of metastatic tumors in control and JTE-522 treated rats injected with RCN-9 (group 1, 203 ± 26; group 2, 197 ± 26; group 3, 204 ± 18; group 4, 165 ± 33). Microscopy shows no significant differences in the number of metastases (p = 0.13) ::: ![](1471-2407-5-26-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Removed lungs and the largest cross sections.(A) Representative lungs of rats injected with RCN-9. Removed lungs on the left were those of group 4 (JTE-522: 30 mg/kg/day); and those on the right were group 1 (control). Surface of the lungs of group 1 has more metastatic tumors than that of group 4. (B) The largest cross-sections of group 1 (control) and group 4 (JTE-522: 30 mg/kg/day), which were stained with hematoxylin and eosin. The upper slide shows the largest cross-section of group 1, and the lower one shows that of group 4. Many metastatic tumors are not visible on the surface. ::: ![](1471-2407-5-26-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Immunohistochemical staining for COX-2 and VEGF.A, B, and C show immunohistochemical staining for COX-2 in lungs of rats injected with RCN-9. A, group 1 (control group); B and C, group 4 (JTE-522; 30 mg/kg/day). Tumor cells in all groups expressed COX-2. Degree of staining between groups 1 and 4 did not differ. Vascular structures (black arrow, B) and large airways (black arrow, C) in the rat lung were stained for COX-2. White arrow in C shows tumor cells stained for COX-2. Original magnification, A and B, ×400, C, ×200. D, E, and F show immunohistochemical staining for VEGF in lungs of rats injected with RCN-9. D and E, group 1 (control); F, group 2 (JTE-522; 3 mg/kg/day). Tumor cells of all groups expressed VEGF. Degrees of staining between groups 1 and 2 did not differ (D and F). Vascular structures (black arrow, E) and large airways (white arrow, F) were stained for VEGF in rat lung. Black arrow in F shows tumor cells stained for VEGF. Original magnification, ×400. ::: ![](1471-2407-5-26-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Immunohistochemical score of COX-2 and VEGF.Relation between the immunohistochemical score of COX-2 and that of VEGF is shown. The score was defined as positive, if it was 3 or more. The number of each score (COX-2 vs VEGF) was as follows: 3 vs 3, 1; 4 vs 4, 8; 4 vs 5, 4; 5 vs 4, 8; and 5 vs 5, 3. Both COX-2 and VEGF were positive in all groups, however, there was no correlation between COX-2 and VEGF scores (p = 0.60, Spearman\'s rank test). ::: ![](1471-2407-5-26-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Immunohistochemical staining score of COX-2 and VEGF. ::: Group COX-2 Expression Score VEGF Expression Score --------- ------------------------ ----------------------- Group 1 4.5 ± 0.55 4.0 ± 0.51 Group 2 4.5 ± 0.55 4.0 ± 0.41 Group 3 4.5 ± 0.55 4.0 ± 0.41 Group 4 4.0 ± 0.75 4.0 ± 0.82 p value NS NS NS: not significant :::	PubMed Central	2024-06-05T03:55:54.139118	2005-3-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555553/", "journal": "BMC Cancer. 2005 Mar 3; 5:26", "authors": [ { "first": "Hirotoshi", "last": "Kobayashi" }, { "first": "Hiroyuki", "last": "Uetake" }, { "first": "Tetsuro", "last": "Higuchi" }, { "first": "Masayuki", "last": "Enomoto" }, { "first": "Kenichi", "last": "Sugihara" } ] }
PMC555554	Background ========== SSIs are among the most common hospital acquired infections comprising 14--16 percent of inpatient infections\[[@B1]\]. SSI is a dangerous condition, a heavy burden on the patient and social health system\[[@B2]\]. Such infections lengthens bed stay for an average of seven days. Potential sources of infection are the patient (especially contamination by alimentary tract bacteria), hospital environment, food, other patients, staff, infected surgical instruments, dressings, and even drugs and injections \[[@B3]\]. The incedence of SSIs with regard to abdominal surgical sites and operating conditions is as follows: Clean wounds (1.5--3.7%); clean-contaminated wounds (3--4%); contaminated wounds (8.5%); dirty-infected wounds (28--40%); in laparoscopy (10%) umbilical hernia (2--5%); in the cancer of the colon without taking antimicrobial drugs (30--60%); or with antibiotic and proper intestine wash (10%); in colostomy (above 50%); in colon perforation (20%); in stomach cancer and surgery (20%); in hernionite (50%); in adult appendectomy (10--20%); in children\'s appendicitis (2--5%); in aged appendicitis and in pregnant women (10--50%); and in AIDS victims (above 50%); in liver abscess (20%); in hydatid cyst (2--5%); in acute and chronic cholecystectomy without stones (10%); in acute septic cholangitis (10--20%); in laparoscopic cholecystectomy (2--5%); and in splenectomy (2--5%) \[[@B4]\]. SSI is identified with redness, inflammation, heat, pain, a temperature of 38°C, and septic drainage from the surgical site during the 30 days following operation\[[@B4]\]. Several factors are to be taken into account for SSI, some of which are as follows: While we could not find any significant correlation between sex and SSI rate, age proved to be an important factor; the rate of wound infection for 15 to 24-year-old patients was only10% but increased significantly for those over 65 years of age. The extent of SSI was doubled for obese patients. The duration of surgical operation also proved to be a significant factor: only 3% of operations lasting 30 minutes or less led to infection, while for operations lasting more than 6 hours this rate leapt to 18%. SSI rate increased with longer durations of preoperative bed stay, but preoperative showers with a disinfecting soap such as chlorhexidine or Betadine decreased the cutaneous bacterial load. According to Kruise, the rate of infection was reduced to 1.3% among patients who showered with disinfecting soap containing hexachlorophene. In those who showered with ordinary soap the corresponding figure was 2.1%, and for patients who did not shower at all it increased to 2.3%. However, another study on 5536 subjects showed no decrease in SSI in patients who showered with chlorhexidine preoperatively; the rate was 4% \[[@B4]\]. Among other factors that delay wound healing or increase the infection rate are cigarette smoking, which increases the postoperative infection rate 5-fold, and the use of steroids, which delays wound healing and increases the infection rate by 9 percentage points from 7% to 16%. Aseptic surgical techniques are claimed to decrease the infection rate, though not to zero \[[@B3]\]. However, the administration of prophylactic antibiotics 30 to 60 minutes before surgery, decreases the incidence of SSI \[[@B4]-[@B10]\]. Obviously, remote infections increase SSI. Other contributing factors are the type of surgery and secondary infections. The object of this study was to assess SSI, and the incedence of the factors contributing to such infections at Imam Khomeini Hospital in Iran. Methods ======= In this study, 802 patients in a teaching hospital during the 15 months from April 2002 to July 2003 underwent abdominal surgery and were studied for SSI and the factors affecting it. Initially, 884 patients were recruited for the study but 82 were excluded on the basis of the following criteria: deficient medical records; patients operated at a different hospital and subsequently transferred to Imam\'s Hospital: or patients deceased during the operation or within the following 30 days. The dependent variable in this study was abdominal surgical site infection, defined as redness, swelling, pain, temperature above 38°C, during the 30 days after operation. The independent variables were: age, sex, site operated, body mass index, time of shaving the site of incision, administration of prophylactic antibiotics, type of surgical operation, duration of operation, duration of preoperative bed stay; preoperative shower, type of shaving, and accompanying conditions. The data were collected through a 20-item questionnaire. The stages for data collection and information completion were as follows: identification of patients; preoperative interview; postoperative interview; record completion; weekly examinations and telephone follow-ups for 30 days following operation; and pre-discharge examinations. The collected data were analyzed by the SPSS 10 package, using Student\'s t-test for continuous variables and chi-square test.for categorical variables. To the best of our ability, this study was conducted with due attention to research ethics.however, problems met in the follow-ups the patient\'s discharge imposed limitations on the study Results ======= Of the 802 patients studied who had undergone abdominal surgery 139 (17.4%) suffered from SSI as defined in Table [1](#T1){ref-type="table"}. No infections were observed in the other 663 cases (82.7%). So far as wound type was concerned, we found clean wounds in 109 cases (13.6%); clean-contaminated wounds in 214 cases (26.7%); contaminated wounds in 307 cases (45.8%); and dirty infected wounds in 112 cases (14%). While 255 cases (28.1%) did not shower before the operation, the other 577 patients (71.9%) did. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Population Distribution based on the type of operation and SSI Incidence. ::: Type of operation Frequency Percent SSI incidence Percent ----------------------- --------------- ------------- ------------------- ------------- splenectomy 24 3 \-- \-- cholecystectomy 272 33.9 25 9.2 umbilical hernia 35 4.4 5 14.3 appendicitis 132 16.5 19 14.4 stomach cancer 58 7.2 23 39.7 Excision biopsy 15 1.9 \-- \-- laparotomy 142 17.7 25 17.6 cystectomy 9 1.1 4 44.4 colon cancer 24 3 14 58.3 colostomy 15 1.9 10 66.7 laparoscopy 18 2.2 \-- \-- abdominal mass 10 1.2 2 20 ileostomy 10 1.2 5 50 Intestinal adhesion 5 0.6 4 80 others 33 4.2 4 12.1 total 802 100 140 17.4 ::: The body mass index for 73 patients (9.1%) was above 30, indicating obesity. Over half the patients (403 cases) suffered from accompanying conditions such as diabetes, high arterial blood pressure, kidney or liver failure, malignancy, febrile condition, cardiac disorders, thyroid disorders, blood disease, chronic obstructive pulmonary disease, convulsion, hyperlipidemia, or immunological disorders; or had previously undergone surgical operations. The rest were free of accompanying conditions (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### SSI Distribution based on the factors involved. ::: Variable Classification SSI (+) SSI (-) total P. Val. --------------------------- ------------------------------- ------------- ------------- ----------- ------------- ----- ----- ------------- Age Age groups under 25 4 3.7 103 96.3 107 100 \<0.001 Age groups 25--65 102 18.1 462 81.9 564 100 Age groups above 65 33 25.2 98 74.8 131 100 Sex Female 61 15.1 344 84.9 405 100 \<0.093 male 78 19.6 319 80.4 397 100 Type Of Wounds Clean wounds 5 4.6 104 95.4 109 100 \<0.001 Clean contaminated wounds 9 4.2 205 95.8 214 100 Contaminated wounds 115 31.3 252 68.7 367 100 Dirty -- infected wounds 10 8.9 102 91.1 112 100 Type Of Operation Urgent 29 14.9 166 85.1 195 100 \<0.001 elective 110 18.1 497 81.9 607 100 Operation Duration Below 1.5 hours 9 5.4 157 94.6 166 100 \<0.001 1.5 -- 4 hours 121 19.5 501 80.5 622 100 Above 4 hours 9 64.3 5 35.7 14 100 preoperative bed stay Emergency 24 11.7 181 88.3 205 100 \<0.018 1 -- 15 days 100 18.6 439 81.4 539 100 More than 15 days 15 25.9 43 74.1 58 100 Shaving Time One hour before operation 29 14.5 171 85.5 200 100 \<0.001 12 hours before operation 110 18.3 492 81.7 602 100 showering Before op. Taken 80 13.9 497 86.1 577 100 1.00 none 59 26.2 166 76.8 255 100 Body mass index Under 20 10 17.2 48 82.8 58 100 0.692 Ranging 20.1 -- 25 73 16 383 84 456 100 Ranging 25.1 -- 30 42 19.5 173 80.5 215 100 Above 30.1 14 19.2 59 80.8 73 100 ::: We found no significant correlations between SSI incidence and sex or preoperative shower. However, correlations with duration of operation, duration of preoperative bed stay, electivity of surgery, lengthening of preoperative shaving time, increasing age, wound infection, site of surgery, type of incision, accompanying disorders, and type of prophylactic antibiotic administered before operation were all significant at p \< 0.001. Although differences in SSI rates were not significantly related to BMI, a trend was apparent: SSI rate was higher with low and high BMI. The following prophylactic antibiotics were used: ampicillin, gentamicine, cephalothin, metronidazole, ceftriaxone and cefazoline (table [3](#T3){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### SSI Distribution based on the type of prophylactic antibiotic administered. ::: variables SSI cefazolin ceftriaxone Metronidazole cephalothin Gentimicine Ampicillin --------------- ----------- ------------- --------------- ------------- ------------- ------------ ----- ------ ----- ----- ----- ------ ----- ----- ----- ------ ----- ------ ----- ------ ---- ----- ----- ------ A NA A NA A NA A NA A NA A NA A N \% N \% N \% N \% N \% N \% N \% N \% N \% N \% N \% N \% positive 42 12.1 97 21.3 73 24.5 66 13.1 79 24 60 12.7 19 17 120 17.4 70 30.4 69 12.1 5 50 134 16.9 negative 305 87.9 358 78.7 225 75.5 438 86.9 250 76 413 87.3 93 83 570 82.6 160 69.6 503 87.9 5 50 658 83.1 total 347 100 455 100 298 100 504 100 329 100 473 100 112 100 690 100 230 100 572 100 10 100 792 100 P-Value P = 0.001 P \< 0.001 P \< 0.001 P = 1.000 P \< 0.001 P = 0.018 A = applied NA = not applied ::: Discussion ========== For the 802 participants in this study, the SSI reported was 17.4%, which is well above the 14--16% reported in other studies \[[@B1]\]. There were particularly high values in cases of umbilical hernia (14.3% compared with previously reported 5%) and stomach cancer (39.7%; in previous studies 20%) \[[@B4]\]. Increasing age is correlated with greater likelihood of certain chronic conditions, malnutrition and a fall in the body immunological efficiency, causing more extensive SSI \[[@B4]\]. The present findings supported this argument (p = 0.001). SSI is not correlated with sex \[[@B5]\], in agreement with previous findings (p = 0.093). The literature shows that SSI increases with obesity, one reason being a decrease in blood circulation in fat tissues \[[@B7]\]. Malnutrition is another factor predisposing to SSI \[[@B5]\]. In this study we considered a BMI of above 30 obese and that of below 20 as malnutrition, found no significant correlations between the two ranges and SSI extensity (p = 0.692). But, the previously reported correlation between SSI and pre-operation bed stay (p = 0.018) \[[@B4],[@B5]\] was supported by this study. This is one of the factors to be taken into account. Thus by reducing pre-operation bed stay we may decrease SSI. The findings of this study also proved the risk of SSI to be less in elective surgeries than those referred to emergency departments as cases of acute abdomen, which could result from lack of readiness for operation on the patient\'s side. Here we should reduce risk factors by preparing the patient for the urgent operation as much as possible. The findings supported the literature by showing that administration of prophylactic antibiotic half an hour before the operation would bring about the best results and the lowest SSI \[[@B10]\]. This was proved for all antibiotics (p = 0.001) with the exception of cephalothin with (p = 1), which requires a lot more research. The literature shows that with the duration of above 2 hours, the risk of SSI increases \[[@B4]\]. The average time in this study was 2.24 hours, which must be reduced to below 2 hours although the nature of surgical operations is not always the same. The time of shaving when it approaches the operation and if done by clippers, reduces the SSI risk. In this study the two times: one hour before surgical operations and 12 hours before that were contrasted which supported previous findings with p = 0.001. This is one area where we can lower the risk by approximating the time of shaving as much as possible to that of operation. Other such factors quoted in the literature as the conditions of the operating theatre, personal hygiene, acompanying diseases, immunological disorders, smoking, techniques of surgery, the surgeon\'s expertise, duration of surgical scrub, preoperative skin preparation, poor hemostasis, failure to obliterate dead space, tissue trauma, and inadequate sterilization of instruments, which were not included in this study might be considered as confounding factors. Conclusion ========== Considering the relatively higher rate of SSI in this study (17.4% compared with the 14% quoted in the literature), especially in such cases as stomach cancers and umbilical hernia, where the rate is considerablly higher, we should carefully reconsider the whole operation procedure. In general, we should do our best to reduce the average operation duration to less than 2 hours and the average preoperative bed stay to less than 4 days. Thus, the present average of 11.2 days for the total bed stay would be reduced to less than 11 days. The time of shaving should approximate the operation time as much as possible. Finally, when the administration of prophylactic antibiotics is required, cefazoline is recommended to substitute cephalothin. Razavy: main researcher (design, analysis, report) Ibrahimpour: (implementation, analysis) Sabouri Kashani: (report, edit) Jafarian: (counsult) Competing interests =================== The author(s) declare that they have no competing interests. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2482/5/2/prepub> Acknowledgements ================ Grateful thanks are due to the professors at surgery departments, their personnel, and the staff at medical records department. We should also express our gratitude to the statistics advisors at the dean\'s office for research at the school of medicine. Finally, we should thank Ms. Bakhshandeh for typing the final version.	PubMed Central	2024-06-05T03:55:54.141020	2005-2-27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555554/", "journal": "BMC Surg. 2005 Feb 27; 5:2", "authors": [ { "first": "Seyd Mansour", "last": "Razavi" }, { "first": "Mohammad", "last": "Ibrahimpoor" }, { "first": "Ahmad", "last": "Sabouri Kashani" }, { "first": "Ali", "last": "Jafarian" } ] }
PMC555555	Background ========== Three-dimensional (3D) reconstruction of virus particles like SARS (Severe Acute Respiratory Syndrome) and HSV (Herpes Simplex Virus) using electron microscopy yields crucial information for understanding the assembly and infectivity mechanism. The structural determination begins with acquisition of projection images in an electron-microscope. A major part of data processing is aimed at determining the direction of projection for each particle image (2D projection of virus) so that a 3D reconstruction can be computed. The first step in a virus reconstruction is the detection and selection of the individual particle images from a large area of an electron micrograph. There are different criteria to determine the particle orientation. One criterion is based on the computational search of the common lines in the computed Fourier Transforms of individual or multiple particle images \[[@B1]\]. An improvement of the Fourier Common Line algorithm \[[@B2]\] has been proposed, but a significant amount of the low contrast particle images are still discarded, partly because of the impossibility of obtaining a reliable estimate of their orientations. Another criterion for the particle orientation estimate is to find the correlation match between the raw images with many projections from a 3D model \[[@B3]\]. Regardless of the criterion used, finding the orientation determination for a particle image such as that in Fig. [1a](#F1){ref-type="fig"} is difficult because of its extremely low contrast. One approach is to take two consecutive pictures of the same particles one close-to-focus (Fig. [1-a](#F1){ref-type="fig"}) and another farther from focus with a higher contrast (Fig. [1-c](#F1){ref-type="fig"}) from which the initial orientations are easily determined \[[@B4],[@B5]\]. The initial orientations are then assigned to the corresponding particles in the close-to-focus images for structural refinement (henceforth, called focal pair method). In a high resolution structure determination, one would require over 6000 particles of data for 8.5 Å resolutions \[[@B6]\]. If a focal pair is required, one would need over 12,000 particles and hence it is a labor-intensive process of data recording, digitization and archiving. In this paper, we propose a method for determining the initial orientations of the particles from low contrast (close-to-focus) images without necessity for a second set of highly defocused images. In this technique, we use the wavelet transformation in a multi-resolution analysis \[[@B7],[@B8]\] to enhance the contrast of the image and the hierarchical weighted projection matching to accelerate the processing. The wavelet-transformed images have the same size as the original images. Wavelet decomposition separates the low-resolution information, called \"approximation\", from the high resolution information, called \"details\". This method computationally generates an image equivalent to the far-from-focus picture taken by the microscope and separates images containing details and noise. The technique proposed here is a model-based approach in wavelet space, which we call Hierarchical Wavelet Projection Matching (HWPM). Results ======= A data set of 600 HSV-1 capsid particle images in different orientations was used to test the HWPM method. The defocus range of herpes particles was chosen to be close to focus between 1.7 μm and 0.4 μm. An initial model of about a 40 Å resolution \[[@B4]\] was used to generate projections uniformly covering the asymmetric triangle of the icosahedrally symmetric HSV-1 capsid particle \[[@B4],[@B5]\]. A grid sampling of 0.5° in each direction of the asymmetric triangle of icosahedral particles was used. The number of projections obtained with this grid was relatively high (2616 projections). First, the 2616 projections were grouped into 200 classes, each class containing about 13 projections. A match of the particle into the best 3 of the 200 classes was obtained using the wavelet correlation coefficient (wccf) criterion. Next, the particle was compared to the 39 projections of the best three classes, and the correct orientation was that of the projection giving the highest wccf. The hierarchical implementation wavelet projection matching reduced the time at least by a factor of 10 compared with the classical projection matching method. In the example of 600 particles, by using HWPM it took approximately 3 hours to determine the orientations, instead the 33 hours it took with the classical matching algorithm. Both algorithms were running on the SGI Origin-2000 supercomputer using 10 processors. At this point, each particle had been assigned the orientation of the closest projection. A quality factor was assigned to each orientation, which was the wavelet correlation coefficient. Particles having high wccf coefficients were selected for reconstruction of a first 3D model of the virus. Refinement of initial orientations obtained by HWPM was realized by the same iterative refinement process used in focal pair method \[[@B5]\]. This refinement process uses both local and global refinement. Local-refinement refines orientations against a set of projections from the 3D density map. In global refinement, all the raw particle orientations are refined against each other, without using projections from the 3D model. A potential merit of global refinement is the absence of possible bias arising from the 3D model. In order to assess the accuracy of the orientations obtained with the HWPM. A comparison with the focal pair method (Fig. [5.a](#F5){ref-type="fig"}), which is currently the most appropriate method for low contrast virus images was accomplished. The following steps were executed. First the initial orientations of the far-from focus particles were determined by using the cross-common line method between real particles and a set of projections obtained from the low resolution model. Next, a global refinement process was realized in order to determine the initial orientation. The same software as in \[[@B4]\] was used with the same initial parameters. The parameters used in this software were the minimum radius and maximum radius limiting the resolution and the sampling step size of 4.67Å/pixel. The minimum valid radius ensured that the minimal radius was computationally accurate when the two common lines angles were close and also to avoid the biasing of the orientations of particles by the very low frequency components. This parameter choice for herpes at the above sampling step was 5 pixels. The maximum radius was chosen to limit the maximum resolution expected from the reconstruction, here in the initial orientation the maximum radius corresponding to a resolution about 40 Å. Then an assignment of the particle orientations from the far to focus to the close to focus is realized. Next, an iterative refinement process to the close-to-focus data was accomplished as described in \[[@B5]\]. A 3D reconstruction using the best 300 HSV-1 particle orientations was performed for each method. Fig. [5.a](#F5){ref-type="fig"} and Fig. [5.b](#F5){ref-type="fig"} show surfaces density contour, displayed at one standard deviation above the mean density \[[@B9]\], obtained respectively from focal pair method and HWPM method. Both structures show a similar visual resemblance. In order to assess the reliability of the 3D density maps and the quality of particles orientations obtained from each method, the Fourier Shell Correlation (FSC) criterion, which is the most robust criterion \[[@B10],[@B11]\], was employed. The FSC was calculated between 2 independent reconstructions from the same set of orientations for each method. The effective resolution assessment of the 3D structure obtained from each method is estimated at FSC correlation value of 0.5, which correspond to 45° phase difference. Fig. [6](#F6){ref-type="fig"} shows three different plots. The green curve shows a resolution of 32 Å of the reconstruction using the best 300 particles with orientations obtained from the focal pair method. The blue curve shows a resolution of 24 Å of the reconstruction using 300 particles with orientations obtained from the HWPM method. This result shows that the resolution of the structure obtained from the HWPM is higher than the one using the orientations from the focal pair method. Therefore, the orientations obtained from HWPM method are more accurate. Furthermore, HWPM method uses only one set of close-to-focus data instead of the two sets used by the focal pair method. The purple curve shows a resolution of about 14.5 Å of the reconstruction using 500 particles with orientations assigned by HWPM. The red curve plots twice the expected FSC for Gaussian noise. A less stringent criterion to assess the resolution as the intersection between the FSC curve and the curve plotting the 2 times expected Gaussian noise. HWPM was tested on a P22 empty shell capsid which was circular and whose shell is very thin (\~40 Angstrom). Twenty micrographs of the P22 empty shell capsid with defocus range \[0.5 to 2 μm\] were used for testing purpose. The total number of particles is 1340, each image has a size of 300 × 300 pixels, and the dimension of each pixel is 2.8 Å. Concerning the initial orientations determinations using HWPM method, an initial model of around 20 Å resolutions was used to generate projections which uniformly covered the asymmetric triangle of the icosahedrally symmetric model. A grid sampling of 2° in each direction of the asymmetric triangle of icosahedral symmetry was used to obtain an initial orientation, targeting a structure of 30 Å. The number of projections obtained with this grid was about 200 projections. A match of the particle with the projections was obtained by using the wccf criterion. The correct orientation was selected as the one of the projection giving the highest wccf. The better half of the orientations projections (650) according to wccf criterion was chosen for final reconstruction. The initial orientations for the same set of data were determined using the Improved Common Line (ICL) method, with the same input parameters for the software described in \[[@B2]\]. ICL use one single micrograph and does not use focal pair technique. The best half of the particles orientations (650) was chosen, according to the phase residual criterion, in the 3D reconstruction of the P22. Fig. [7](#F7){ref-type="fig"} shows three surface views of the P22 empty shell capsid. The Top image shows the original surface \[[@B12],[@B13]\]. The lower right image shows the surface obtained by HWPM, which shows a very similar view to the original structure. The resolution assessment of the structure, by Fourier shell correlation criterion, gives a resolution of 14.5 Å. The lower left surface shows the result obtained by ICL method. The surface view of the reconstruction obtained from the ICL of the P22 empty shell capsid is different from the original P22 capsid. Fig. [7](#F7){ref-type="fig"} proves the inaccuracy of some of the initial orientations obtained from the ICL method for such a smooth virus. Discussion ========== During the last thirty years the common lines methods were a great method to resolve icoshedral particles up to 7--8 Å \[[@B6]\]. Recently, a method using polar transformation and projection matching were used for the purpose of orientation determination \[[@B3]\], but this last method is not suitable for the high resolution of large virus because the resulting transformed images, could be double the size of the original image. The proposed method combines the projection matching of wavelet denoising for an initial determination of particle orientation, with the common lines method for refinement to a higher resolution. It is clear that HWPM method works only if the initial low resolution model of the particle is already known. This method is very interesting if we need to add more particles to an existing intermediate resolution reconstruction in order to increase the resolution. Particles having high resolution information are very noisy \[[@B9],[@B14]\]. The best that we can get using the ICL method is less than 40 % of good orientations, for defocus values between 1.9 μm and 1.2 μm, for the P22 capsid \[[@B2]\]. Usually, very high resolutions use defocus values which go much lower than 1.2 μm as in the HSV data, or the current P22 data which goes to 0.5 μm. The 40% rate of correct orientations would certainly become smaller if we used data at closer defocus. The study accomplished on high resolution for HSV reconstruction showed that using a close-to-focus single micrograph with CL method was not effective, because a small number of orientations were found to be correct \[[@B9]\], for this reason a focal pair method was used for 8.5 Å structure\[[@B9]\]. At high resolution reconstructions, the number of particles needed increases drastically, and the data with a signal-to-noise ratio valid up to the targeted resolution, tend to be very noisy. For an 8.5 Å structure of HSV-1 it took about 6000 particles for a final reconstruction. For a 6.5 Å structure resolution, the estimated value was about 50,000 particles using the same electron microscope \[[@B14]\]. To further increase the resolution of the HSV virus to 6.5 Å or higher (4 Å), the focal pair method would be impracticable. The focal pair method, for intermediate resolution up to (8 Å) for big viruses like HSV, works well for orientations determinations. The number of particles selected for the final reconstruction about 40% of the original number of particles (taking into account the far-focus and close-focus micrographs). It is necessary to emphasize that results from both methods are very similar in terms of visual resemblance. But, there are two advantages of HWPM over the focal pair method. First, focal pair method uses as much as double the data used for the HWPM. Second, the quality of the density maps shows that HWPM gives a better resolution for the same number of particles (figure [6](#F6){ref-type="fig"}). This proves a better accuracy of orientations determinations obtained by the HWPM. One of the more obvious advantages of the HWPM for orientation accuracy appears in two examples of real reconstructions. The first is for the P22 capsid, the ICL method does not give a good initial orientation, and the refinement of the orientations does not help to converge toward the right orientations. The probable reasons why the ICL method did not work properly for the P22 capsid are: first the P22 capsid has a smooth surface (the thickness of the shell is about 40 Å); second most of the data are very close-to-focus with defocus range of 0.5 μm to 1.3 μm. The data was noisy and had a very low contrast. The ICL method was able to give 40% of good orientations for the defocus range between 1.9 and 1.2 μm, here the data was closer to focus, which reduced the percentage of good orientations to less then 22%. The application of the HWPM to the P22 empty shell capsid gave the expected structure (Fig. [7](#F7){ref-type="fig"}). The wavelet denoising in the HWPM not only helped in reducing the noise and enhancing the contrast of the particles, but also used the entire information from the image (instead of using several lines) which is enhanced accuracy for highly noisy particles. Another example of real data reconstruction is the VP5-VP19C recombinant. After long investigation using CL and ICL algorithms, the classical projection matching scheme was also tested in order to determine the orientations, but unfortunately all those methods failed. The wavelet filtering and matching was used during the classification step of the recombinant particle VP5-VP19C \[[@B15],[@B16]\], which significantly improved the quality of the class averages \[[@B16]-[@B18]\] and enabled the determination of the structure of that particle. A study \[[@B16]\] shows the superiority of the wavelet projection matching over the Gaussian filtered projection matching. The third examples for low PH sindbis: Three years of investigation using CL and ICL methods failed to obtain the correct density map of the low PH sindbis capsid which is subject to conformational changes and an alteration of the symmetry. Recently the proposed method (HWPM) was tested on low PH sindbis and the correct structure was finally observed and analyzed \[[@B19]\]. Wavelet multi-resolution analysis and processing improves particle detections \[[@B8]\], classification \[[@B15],[@B16]\], and orientation determination on a variety of electron microscopy images which are highly noisy and have an extremely low contrast. This prove that wavelet techniques are adequate in the 3 main steps of 3D virus reconstruction and in the classification step of single particle reconstruction \[[@B16],[@B17]\]. Conclusion ========== This paper describes the development and implementation of a new method for orientation determination for low contrast images of virus particles. This method is based on wavelet filtering, which enhances the contrast of the particles and reduces the noise, and on weighted projection matching in wavelet space. A hierarchical implementation of this method increases the speed of orientation determination. Results show that, HWPM have been able to determine accurately more than 85% of the orientations of low-contrast particles. Compared to the focal pair method (for orientation determination from low contrast data) the HWPM reduced the amount of data required in a reconstruction by at least 50 %. In addition the accuracy of the orientations obtained by the proposed method is higher than those obtained by focal pair method \[[@B9]\]. This improved accuracy is shown clearly by the resolution assessment in Fig. [6](#F6){ref-type="fig"}. The estimated number of particles needed for a 6.5 Å reconstruction of the HSV-1 capsid was about 50,000 \[[@B14]\]. By using the HWPM method, only half as much data was necessary. The proposed method could save 2 to 3 man-years invested in acquiring images from the microscope and data processing. Another advantage of this method is the ability to give accurate orientations for some particles having conformational changes or alteration of symmetry as seen for VP5-VP19C recombinant and recently with the low PH sindbis capsid. Methods ======= Choice of wavelet Base ---------------------- The choice of wavelet filter bases depends on the signal. Signals coming from different sources have different characteristics. For audio, speech, image and video signals the best choices of wavelet bases are known. The best choice for electron microscopic images is not clear. The problem is to represent typical signals with a small number of convenient computable functions. An investigation to choose the best wavelet bases for electron microscopic images was performed here. During this study, simulated and real electron microscopy images were used. The majority of the wavelets basis existing in Matlab-5 software \[[@B20]-[@B24]\] was tested. The criterion used to determine the best wavelet base was the one which optimizes the signal to noise ratio in a broad spectrum of spatial frequencies. The bi-orthogonal wavelets basis \[[@B25]-[@B27]\] especially the 3.5 basis in Matlab-5 yielded the best average signal to noise ratio in the range of the spatial frequency (1/100 - 1/8 Å^-1^) relevant to data analysis. Wavelet Projection Matching (WPM) Principle ------------------------------------------- The principle of the wavelet decomposition is to transform the original raw particle image into several components: one low-resolution component called \"approximation\" \[[@B21]\], which is mainly used in this method, and the other components called \"details\" (Fig. [2](#F2){ref-type="fig"}). The approximation component is obtained after applying a bi-orthogonal low-pass wavelet filter in each direction (horizontal and vertical) followed by a sub-sampling of each image by a factor of 2 for each dimension. The details are obtained with the application of a low-pass filter in one direction and a high-pass filter in the other, or a high-pass filter in both directions. The noise is mainly present in the detail components. A higher level of decomposition is obtained by repeating the same filtering operations on the approximation. The wavelet correlation coefficient between two wavelet-transformed images, for a given level, is : ![](1472-6807-5-5-i1.gif) Where W~1~to Wp are weights given for each components of the wavelet correlation, p is the number of components of wavelet decomposition. A~1~, A~2~are the approximations. ⊗ denote the correlation between two components images. D~1i~, D~2i~are the details (Fig. [2](#F2){ref-type="fig"}). This implementation starts first by a wavelet filtering which is performed by thresholding \[[@B21],[@B28],[@B29]\] of the details components in order to reduce the noise effects in the correlation matching. Higher weight is given to the approximation component to further reduce the noise effect in the decision. The weights given in this implementation are 0.75 for the approximation and 0.25 for the details. Orientation determination with Hierarchical WPM (HWPM) ------------------------------------------------------ Initial orientation determination is based on model-based projection matching approach \[[@B3]\]. The level of wavelet decomposition depends on the dimension of the virus and the sampling rate. For herpes simplex virus type-1 (HSV-1) B-capsid, which has a diameter of 1250 Å with a sampling of 2.1 Å/pixel, a level two of wavelet decomposition (Fig. [2](#F2){ref-type="fig"}) is appropriate for the initial orientation estimate, because of the contrast enhancement and the consideration of computational speed. The method starts by generating the wavelet decomposition at level two for each projection and raw image. In order to have accurate orientation estimation a small angular grid (figure [3](#F3){ref-type="fig"}) to generate projections from the initial model is needed, and this results in a large number of projections. The classical projection matching, which consists of comparing the wavelet-transformed raw images with every projection, is very slow even when using multiple processors on a parallel computer. In order to significantly increase the speed of processing, a hierarchical implementation is performed. This consists of grouping projections into classes of similar orientations \[[@B30]\]. Fig. [3](#F3){ref-type="fig"} shows the classification scheme applied for the icosahedral viruses, only an asymmetric triangle representing the possible orientations for icosahedrally symmetric object \[[@B4]\] is considered. The choice of the number of classes is optimized to give the best tradeoff between speed and accuracy. The classification gives a uniform distribution of projections into the classes. The next step is to compare each wavelet-transformed raw image with the closest projection to the center of each class, and then rank the classes in terms of wccf (Fig. [4](#F4){ref-type="fig"}). The final step is to compare the raw image with all the projections of the three classes given the highest wccf coefficients. Next, the orientation of the projection yielding the highest wccf will be assigned to the raw image as the correct orientation. The software is written in C++ (a parallel version of the software has been written to run on the SGI Origin-2000 supercomputer). Acknowledgements ================ I thank Wah Chiu for helpful and interesting discussions. Frazer Rixon, Joanita Jakana, Zhang Zhixian for providing the HSV-1 and P22 capsid data and Kelechi Ogbueh for the English revision of the manuscript. This work was supported by the National Institutes of Health (P41RR002250, R01AI38469) and Human Frontier Science Program. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Different defocus images with their spatial frequency resolution.Figure 1 -a shows a typical image taken at 0.8 μm defocus with JEOL 400 kV electron cryo-microscope with a LaB6 gun. This image has low contrast but contains a detectable signal above noise at a relatively high spatial frequency out to 1/7 Å^-1^(b). The image contrast can be enhanced by setting the defocus value to a larger value (i.e. 2.6 μm as shown in c). However, such an image contains much lower resolution data as shown in (d), the detectable signal is present out to spatial frequency (1/12Å^-1^). In order to obtain a high-resolution reconstruction, we would use images taken at condition similar to that of (a). ::: ![](1472-6807-5-5-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Wavelet decomposition of a virus at level 2.Level 2 decomposition of wavelet uses bi-orthogonal base. This transformation is applied on a projection of phi 13 model downloaded form the Proteins data bank and then reconstructed to 10 Å. ::: ![](1472-6807-5-5-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Classification scheme of projections into classes.Classification of projections using (2 of the 3) Euler angles for viruses having Icosahedral symmetry like the herpes virus. Only an asymmetric triangle is needed in the case of an Icosahedrally symmetric virus instead of the whole plan in the case of an asymmetric object. Each rectangle represents a class of projections. The closest projection to the center of the rectangle, represents the center of the class. This figure represents the classification of 2600 projections into 300 classes. ::: ![](1472-6807-5-5-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Schematic diagram of the HWPM method.Schematic diagram of the HWPM method: The first step is the wavelet decomposition of each of the particles, and projections and then classification of projections into 200 classes. The final step is to determine the correlation by using the centers of classes and then using all members of the best 3 classes to get the best orientation estimates. ::: ![](1472-6807-5-5-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Comparison between 3D reconstruction obtained from both methods.Reconstruction of the B-capsid of the herpes simplex virus type-1: in (a) the initial orientations are determined from the second picture and then refined using the first picture. In (b) Initial orientation is determined from the wavelet-projection matching algorithm with one refinement iteration using the common line program. Both iso-surfaces are displayed at 1 standard deviation above the mean. ::: ![](1472-6807-5-5-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### FSC between 2 independent reconstruction for each method.Green and blue lines show the correlation as function of the spatial frequency using 300 particles from the Focal pair method and HWPM respectively. Using the 0.5 correlation point criterion to assess the resolution of the 3D structure, the dotted line gives a resolution of 32 Å and thin line gives a resolution of 24 Å. The purple line shows a reconstruction using 500 particles where the orientation is obtained from the HWPM method the estimated resolution using 0.5 correlation point shows 14.5Å. The red line shows the 2 times expected FSC for Gaussian noise. ::: ![](1472-6807-5-5-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Surface visualization of P22 empty shell capsid.For surface visualization of P22 empty shell capsid, three views were taken at the 5 fold direction and surfaces were displayed at 1.5 standard deviations above the mean. Top image is the original surface from Zhang \[13\], the lower left image is the reconstruction obtained from ICL, the lower right image is the reconstruction obtained from the proposed method. ::: ![](1472-6807-5-5-7) :::	PubMed Central	2024-06-05T03:55:54.144740	2005-3-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555555/", "journal": "BMC Struct Biol. 2005 Mar 2; 5:5", "authors": [ { "first": "Ali Samir", "last": "Saad" } ] }
PMC555556	Background ========== Osteoporosis is defined as a systemic skeletal disease characterized by low bone mass and micro-architectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture \[[@B1],[@B2]\]. This definition indicates that measurement of bone mineral density (BMD) is a central component to diagnosis of the disease \[[@B3]\]. \'T score\' is a statistical definition which indicates the difference between patient\'s BMD and mean bone density of normal population in the age of 20 -- 30 (reference population) \[[@B3]\]. This value shows the difference in terms of standard deviations. According to the World Health Organization (WHO) classification system, T scores under the value of -2.5 are considered as osteoporosis and between -1 and -2.5 as osteopenia. These figures are usually calculated separately for two different sites of lumbar spine and total hip. Discordance in diagnosis of osteoporosis is defined as presence of different categories of T scores (osteoporosis, osteopenia, and normal) in two skeletal sites of an individual patient \[[@B4]\]. This phenomenon has been divided into two groups: major and minor \[[@B5]\]. Minor discordance happens when the different diagnostic classes are adjacent; i.e., patient is diagnosed as osteoporotic in one site and osteopenic in the other site, or, osteopenic in one site and normal in the other site. If the diagnosis is osteoporosis in one site and the other site is in the normal range, the discordance falls into the major class. Actually, one of the reasons for measuring BMD in several sites is the presence of discordance, which can affect the diagnosis and therapeutic plan in an individual person. Various studies have analyzed the prevalence and impact of T-score discordance on different aspects of management of osteoporosis \[[@B5]-[@B9]\]. However, most of these studies did not evaluate risk factors for this phenomenon. Given this background and concerning the need for the estimation of the impact of this phenomenon in our country, we aimed to evaluate the presence and risk factors for T-score discordance in a large sample of Iranian population. Methods ======= Participants in this study were 4229 persons who underwent bone densitometry in outpatient clinic of Endocrinology & Metabolism Research Center in Tehran from 2000 to 2003. A considerable proportion of these cases were healthy post-menopausal women referred by clinicians for densitometric evaluations. All study participants signed the informed consent for any scientific approach to their medical registered data. Our Institutional Review Board approved this study. A standardized questionnaire was filled before densitometry for all participants. Demographic data (including age and sex) as well as other known or suspicious risk factors for osteoporosis (including menopause, age at menopause, age at menarche, history of osteoporotic fractures, drugs, and smoking) were collected. All participants had their standing height measured using a stadiometer to the nearest 0.5 cm. Weight was measured on a standard weighting scale with a precision of 0.5 kg. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared. All the BMD measurements were done for diagnostic purposes and none of the participants were on the treatment with bone active agents (hormone replacement therapy was not considered a bone active agent). BMD was measured at the lumbar spine and total hip with dual X-ray absorptiometry (DXA) using a Lunar DPXMD densitometer (Lunar 7164, GE, Madison, WI) by a trained operator according to the manufacturer\'s instruction. The instrument was calibrated weekly by using appropriate phantoms. Precision error for BMD measurements was 1--1.5% in the lumbar and 2--3% in the femoral regions. The device normative data of US population for spine BMD and NHANES III study for femur BMD were used as reference values. All the data gained from densitometry and questionnaires were entered into a comprehensive relational database. The participants with incomplete data were excluded from the study. To compare presence of various risk factors in participants with and without T-score discordance, chi-square test and independent sample t-test were used firstly. Potential risk factors were entered to a multivariate binary logistic regression analysis and the resulted odds ratios with 95% confidence intervals were reported. Pvalues less than 0.05 were taken to indicate statistical significance. Statistical analyses were performed using Stata Statistical Package, version 8.0 (Stata Corporation, College Station, Tx). Results ======= In sum, 4188 persons were enrolled in the study. Characteristics of all participants are summarized in Table [1](#T1){ref-type="table"}. The main reasons of referral for BMD measurement were menopause in 49%, old age in 16%, glucocorticoid use in 9%, history of low energy fractures in 1.5%, and other reasons (such as metabolic disorders, rheumatoid arthritis, positive family history, leanness, and transplantation) in 4.5% of participants. In 20% of participants, no major risk factor was identified as the referral reason. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of the study population\* ::: Male participants (n = 340) Female participants (n = 3848) -------------------------------------- --------------------------------- ------------------------------------ Age (years) 49.7 (16.3) 53.8 (11.2) Weight (kilograms) 68.5 (13.1) 67.1 (11.9) Height (centimeters) 168.5 (7.7) 156.1 (6.1) Body Mass Index (kg/cm^2^) 24.1 (4.2) 27.6 (4.7) History of osteoporotic fracture 8 (2.4) 47(1.2) Smoking 35 (10.3) 94 (2.4) Corticosteroid use 89 (26.2) 298 (7.7) Hormone Replacement Therapy 231 (6.0) Age at menarche (years) 13.6 (1.5) Menopause 2137 (55.5) Age at menopause (years) 47.2 (5.8) Femoral T score -0.93 (1.24) -1.43 (1.18) Lumbar T score -1.40 (1.48) -1.45 (1.54) \* Numbers are presented as mean (standard deviation in parenthesis) for numerical variables and frequency (percentage in parenthesis) for categorical variables. ::: Totally, 518 participants were diagnosed in osteoporotic range in hip area and 1036 participants in the lumbar area. T-score classifications are presented in Table [2](#T2){ref-type="table"}. Major discordance was observed in BMD results of 115 (2.7%) participants. Minor discordance was observed in 1631 (38.9%) participants and T-score categories of two measurement sites in other 2442 (58.3%) participants were not different. Distribution and pattern of this variable in different genders is depicted in Table [3](#T3){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Classification of T scores according to WHO criteria in different sites\* ::: Lumbar spine Total hip --------------------------------- ------------------ --------------- ---------------------------- ------- ------ ---------------------------- No. % 95% Confidence Intervals No. % 95% Confidence Intervals Osteoporosis (T = -2.5) 1036 24.7 23.4--26.0 518 12.4 11.4--13.4 Osteopenia (-2.5 \< T = -1) 1605 38.3 36.8--39.8 1592 38.0 36.5--39.5 Normal (T \> -1) 1547 36.9 35.5--38.4 2078 49.6 48.1--51.1 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Distribution of diagnostic discordances according to WHO criteria in different genders\* ::: Male participants (n = 340) Female participants (n = 3848) Total (n = 4188) ------------------------------------- ----------------------------- -------------------------------- ------------------ Major T-score Discordance 7 (2.1) 108 (2.8) 115 (2.7) Hip Osteoporosis, Normal Lumbar 5 16 21 Hip Normal, Lumbar Osteoporosis 2 92 94 Minor T-score Discordance 117 (34.4) 1514 (39.3) 1631 (38.9) Hip Osteoporosis, Lumbar Osteopenia 10 99 109 Hip Osteopenia, Lumbar Osteoporosis 39 515 554 Hip Osteopenia, Normal Lumbar 35 220 255 Hip Normal, Lumbar Osteopenia 33 680 713 T-score Concordance 216 (63.5) 2226 (57.8) 2442 (58.3) Hip and Lumbar Osteoporosis 50 338 388 Hip and Lumbar Osteopenia 93 690 783 Hip and Lumbar Normal 73 1198 1271 \* Numbers are presented as frequency (percentage in parenthesis). ::: T-score discordance was more prevalent in women than men (42.2% versus 36.5%, P = 0.042). The mean age of participants with discordance (54.8 years) was higher than the other group (52.5 years, P \< 0.001). In 3848 female participants, the number of post-menopausal women with diagnostic discordances (951 of 2027) was significantly higher than pre-menopausal participants with discordance (671 of 1821; P \< 0.001). In multivariate analysis (Table [4](#T4){ref-type="table"}), two genders lost their difference in occurrence of discordance. Effects of age and menopause were established with their significant odds ratios. Participants with late menopause (age at menopause \> 50) were more likely to show T-score discordances. Obesity defined as BMI over 30 was recognized as a risk factor for major discordance and smoking as a protective factor against minor discordance. Hormone replacement therapy was a significant protector against both. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Results of multivariate logistic regression analysis for risk factors of major and minor discordance getting T-score concordance at lumbar and femoral sites as the reference ::: Variables Minor Discordance Major Discordance -------------------------------------- ----------------------- ----------------------- Gender (female) 1.09 (0.85 -- 1.4) 1.02 (0.45 -- 2.3) Age decade 1.2 (1.1 -- 1.3)\* 1.5 (1.2 -- 1.9)\* Age group (\>65 years) 1.2 (1.01 -- 1.6)\* 1.4 (0.70 -- 2.7) Corticosteroid use 0.89 (0.73 -- 1.1) 0.71 (0.37 -- 1.3) Body Mass Index (\>30 kg/cm^2^) 1.01 (0.87 -- 1.2) 1.7 (1.2 -- 2.6)\* History of osteoporotic fracture 1.1 (0.59 -- 2.0) 1.3 (0.29 -- 5.5) Smoking 0.66 (0.45 -- 0.97)\* 0.49 (0.12 -- 2.1) Menopause 1.3 (1.1 -- 1.5)\* 1.7 (1.01 -- 2.7)\* Hormone Replacement Therapy 0.37 (0.16 -- 0.82)\* 0.54 (0.36 -- 0.82)\* Age at menarche (\> 13 years) 1.1 (0.90 -- 1.3) 0.82 (0.50 -- 1.3) Age at menopause (\> 50 years) 1.4 (1.1 -- 1.7)\* 2.0 (1.2 -- 3.4)\* \* indicates significant odds ratio. Numbers are presented as odds ratio (95% confidence intervals in parentheses). ::: Discussion ========== This study reveals that, using WHO criteria for definition of osteoporosis and osteopenia, a significant fraction of patients (41.7% in this study) would show T-score discordance between hip and spine sites. Most of these discordances (38.9%) are from minor category, presenting difference on only one class, and could be due to minor variation in BMD techniques or some minor physiologic dissimilarity. Minor discordance generally does not influence the overall prognosis of patients; however, in the case of patients with one site normal and the other osteopenic, follow up of patients with hip osteopenia seems reasonable \[[@B7]\]. The multivariate analysis we have implemented to the data could aid clinicians and diagnosticians to approach patients with different characteristics. According to our results, BMD measurement in both sites is necessary at least for older patients and post-menopausal women especially those with delay in menopause. Hormone replacement therapy, however, could decrease the diagnostic discordance and patients receiving estrogen and progesterone are more likely to have similar results in DXA scans of lumbar and femoral areas. This could be the result of drug effects on the BMD of lumber area \[[@B10]\]. Generally, five different causes have been proposed for occurrence of discordance \[[@B5]\]. Physiologic discordance is related to the skeleton\'s natural adaptive reaction to normal external and internal factors and forces. An example of this type of discordance is the difference observed between the dominant and non-dominant total hip. Pathophysiologic discordance is seen secondary to a disease. Common examples include vertebral osteophytosis, vertebral end plate and facet sclerosis, osteochondrosis, and aortic calcification. Anatomic discordance is owing to differences in the composition of bone envelopes tested. An example is the difference in T-scores found for the PA lumbar spine and the supine lateral lumbar spine in the same patient. Artifactual discordance occurs when dense synthetic substances (such as metal from zipper, coin, clip, etc) are within the field of region of interest of the test. And finally, technical discordance occurs when the technician improperly positions the patient for the test or the hardware or software used to acquire the test data is out of order. Major discordance was observed in 2.7% of our participants, which is in agreement with the results of similar studies. In both major and minor discordances, lower BMD for lumbar spine was more prevalent. This could be due to several reasons. The difference between velocities of bone loss in different parts of human body could be the main reason \[[@B11]\]. Trabecular bones (typical of lumbar area) are known to have a more rapid rate of deprivation in early post-menopausal state in comparison to cortical bone (typical of proximal femur) \[[@B12]\]. Moreover, most of the etiologies of the secondary osteoporosis (such as glucocorticoid excess, hyperthyroidism, malabsorption, liver disease, rheumatoid arthritis, and medications) first affect spinal column \[[@B13]\]. This will lead to higher prevalence of lumbar osteoporosis. In addition, weight bearing is a known cause of physiologic dissimilarity, which can cause rise in bone density especially in the hip and femur regions \[[@B14]\]. This mechanism could be the reason of more major T-score discordances observed by increment of BMI in this study. In 30% of our participants, the lumbar T-score was higher than hip T-score and this culminated in poorer hip diagnoses in 9.2% of participants. This phenomenon could be regarded as \'inverse discordance\' and several factors may be involved in its occurrence. One of these reasons is the prevalent vitamin D deficiency in our participants. A recent nationwide study with random sampling from five major cities in Iran reported a high prevalence (about 80%) for vitamin D deficiency in Iranian population \[[@B15]\]. Other studies have confirmed this finding \[[@B16],[@B17]\]. Basic studies have revealed that decrease in serum concentrations of vitamin D by means of raising serum parathyroid hormone (PTH) would induce reduction in density of cortical bones and may have a supportive role for density of trabecular bones \[[@B18]\]. The other reason for \'inverse discordance\' could be due to other diseases such as minor compression fractures in lumbar area, joint sclerosis, and aortic calcification \[[@B19],[@B20]\]. These ailments can induce errors in the estimation of lumbar BMD and falsely higher values. The observation of \'inverse discordance\' could not be regarded as a direct influence of more significant bone loss in femoral region. A known phenomenon named \'birth cohort effect\' can play a role \[[@B21]\]. This indicates that, in the particular section the data have been gathered, a specific observed finding could not be interpreted for the effects of age and time passing. In this study, the reason for lower femoral BMD can be insufficient bone gain during puberty in this area. Latest findings indicates that peak bone mass of Iranian population are about 5% lower than that of western population \[[@B22],[@B23]\]. Decreased bone density in hip region could lead to start of bone loss from lower amounts in older ages and post-menopausal states. This can lead to femoral osteoporosis without significant decrease in lumbar BMD. This study, as every other cross-sectional study, has a number of limitations. We could not rule out the possibility of referral bias for this study. As the study was performed in a referral center affiliated to a teaching hospital, the assumption of similarity of study population to exact community is not reasonable and we could not generalize the results to the Iranian population. The other limitation is the choice of multivariate analysis used in this study. With the current analysis, prediction of the presence or absence of T-score discordances is possible. However, prediction of the situation of one site according to results of the other site or choosing one site to measure BMD need further evaluations and analyses which was behind the scope of this study. Future studies using more powerful statistical analyses with larger sample sizes are needed to establish these imperative questions. The importance of existing discordance on the prognosis and fracture risk of patients needs further prognostic studies with long follow-up designs. The high prevalence of T-score discordance could induce some problems for the physicians in decision-making regarding these patients. In general, high prevalence of discordance in this study and similar studies suggests some defects in the cut-off values for definition of osteoporosis and osteopenia proposed with the WHO \[[@B5]\]. To eliminate this problem, further studies to re-calculate ranges for definition of these diagnoses (considering diagnostic and therapeutic necessities) seem to be needed. Conclusion ========== In summary, this study indicates that about 40% of participants evaluated for bone density changes in a referral center may show diagnostic discordance, majority of them from minor class. This phenomenon should be regarded as a real and prevalent finding and physicians should become familiar with this topic. Clinicians should look for possible cause or causes of this occurrence and develop a particular strategy approaching to these patients. Competing interests =================== This study was supported by a grant from Endocrinology & Metabolism Research Center of Tehran University of Medical Sciences. Authors\' contributions ======================= In advance, suggestion of the design of the study was from AS. Data extraction and initial analysis were done by NKT and AH. AM performed additional analyses and wrote the first draft of the paper. AS and BL both had helpful and valuable comments in revising 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/1472-6823/5/3/prepub>	PubMed Central	2024-06-05T03:55:54.147249	2005-3-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555556/", "journal": "BMC Endocr Disord. 2005 Mar 11; 5:3", "authors": [ { "first": "Alireza", "last": "Moayyeri" }, { "first": "Akbar", "last": "Soltani" }, { "first": "Nasibeh Khaleghnejad", "last": "Tabari" }, { "first": "Mohsen", "last": "Sadatsafavi" }, { "first": "Arash", "last": "Hossein-neghad" }, { "first": "Bagher", "last": "Larijani" } ] }
PMC555557	Background ========== Alzheimer\'s disease is a progressive neurodegenerative disorder characterized by intra-cellular abnormally phosphorylated tau protein and extra-cellular beta amyloid plaques. It has been suggested that inflammation may be a key player in the pathophysiology of AD as evidenced by epidemiological studies which have revealed that the long term use of non-steroidal anti-inflammatory drugs reduces the risk of developing AD \[[@B1]-[@B3]\]. Transgenic mouse models of Alzheimer\'s disease that over-express β-amyloid (Aβ) exhibit significant cerebrovascular inflammation and microgliosis around areas of plaque deposition \[[@B4]-[@B7]\]. Chronic administration of ibuprofen can reduce plaque pathology and brain Aβ levels in these animal models of AD \[[@B8],[@B9]\]. There are numerous reports of increased levels of cytokines in the brains of Alzheimer\'s disease patients, and in transgenic mouse models of Alzheimer\'s disease \[[@B10]-[@B12]\]. However, all these reports have focused on a small number of cytokines within the same sample. It is not clear which cytokines are key in promoting and maintaining the inflammatory environment in the AD brain. Furthermore, it is unclear which Aβ species (1--40, 1--42, soluble or insoluble) are most closely related to cytokine levels. Multiplex technology enables the simultaneous quantification of many cytokines within a single sample. By examining different mouse models of AD using multiplex technology, it is possible to more clearly characterize the particular cytokines which maintain the inflammatory environment and to relate them to particular forms of Aβ (1--40, 1--42, soluble or insoluble). There is considerable debate over which length of Aβ and which conformations are most potently toxic. Recently, specific oligomeric forms have been shown to be most toxic to neurons. These soluble species of Aβ differ from the higher-molecular-weight aggregated insoluble forms that are found precipitated in the AD patient and mouse brain. This study sought to determine whether soluble or insoluble Aβ fractions were most closely related to cytokine levels. Materials and methods ===================== Organotypic brain slice cultures -------------------------------- Mouse brain slice cultures were prepared as previously described \[[@B29]\]. Briefly, 15-month-old PS1 (M146L), TgAPPsw (K670M / N671L), PS1/APPsw and wildtype littermates were humanely euthanized and the brains extracted under sterile conditions. One-mm-thick brain slices were sectioned from co-ordinates 1 to -4 from bregma using a mouse brain slicer. Sections were cultured in neurobasal medium with 5% B27 supplement (Gibco-Invitrogen, CA) and Penicillin-Streptomycin-Fungizone mixture (Cambrex Corp., NJ). After 40 hours, media was collected for quantification of cytokine levels. Multi-plex cytokine array analysis was performed using the Bio-plex protein multi-array system, which utilizes Luminex-based technology \[[@B13]\]. For the current experiments, a mouse 12-plex assay was used according to the recommendations of the manufacturer (BioRad, CA). Measurement of Aβ levels in brain slices ---------------------------------------- Brain slices were washed with PBS (BioSource, CA), and 300 μl of lysis buffer was added. Lysis buffer consisted of mammalian protein extraction reagent (Pierce-Endogen, IL) with 1X protease inhibitor cocktail XI (Calbiochem, CA), 100 μM Sodium Orthovanadate, and 1 μM Phenylmethylsulfonyl Fluoride (PMSF) (Sigma-Aldrich, MO). The resulting mixture was sonicated using a sonic dismembrator (Fisher Scientific, PA) Protein content in each slice was determined using the bicinchoninic acid (BCA) protein reagent kit (Pierce-Endogen, IL), as per the manufacturers protocol. Insoluble Aβ was extracted using 70% formic acid as previously published \[[@B14]\]. Aβ content in brain slices was determined using human Aβ 1--40 and Aβ 1--42 ELISA detection kits (Biosource, CA), as per the manufacturers protocol. Statistical analyses -------------------- For statistical analyses, ANOVA and t-tests were performed where appropriate using SPSS for Windows release 10.1. Hierarchical cluster analysis of Aβ-cytokine data from brain slices were performed with the R program <http://cran.r-project.org/>. A correlation matrix was constructed using the raw data and subsequently converted to a distance matrix by subtracting each element in the correlation matrix from 1. The distance matrix was used as the dissimilarity matrix for building an hierarchical cluster using the averaging method. The resulting dendrogram consists of closely related members under the same node. The farther one needs to traverse across the tree to reach another member, the higher the dissimilarity represented. The distance from the base in the y-axis represents dissimilarity or 1-r, where r is the correlation co-efficient. Results ======= Cytokine production by organotypic brain slice cultures ------------------------------------------------------- Cytokine production was evaluated by multi-plex cytokine array analysis using the cell culture supernatant of organotypic brain slice cultures from control, PS1 (Presenilin 1 mutant heterozygotes), TgAPPsw, and TgPS1/APPsw mice at 15 months of age. We chose non-transgenic littermates as controls for the TgAPPsw mice and the PS1 animals as controls for the PS1/APPsw mice as the PS1 animals were the littermates of the PS1/APPsw mice. There were no significant differences in cytokine production between control slices and PS1 slices showing that PS1 over-expression does not directly induce inflammatory events. Compared to control slices, production of IL-1α, TNF-α, GM-CSF and IL-6 was increased in TgAPPsw slices (figs. [1](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"}). Compared to TgAPPsw slices, PS1/APPsw brain slices produced significantly more IL-12p40, IL-1β, IL-1α, TNF-α, GM-CSF and IL-6. Across control, TgAPPsw, and PS1/APP transgenic brain slices, there was a graduated increase in IL-1α, TNF-α, GM-CSF and IL-6. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Cytokine production by brain slices from transgenic mouse models of AD at 15 months of age.Freshly harvested brain slices were incubated in neurobasal medium with B27 supplement. Media was collected after 24 hours, and cytokine levels measured. Mean concentrations (N = 15) +/- standard error are expressed in picograms per milligram of protein. P \< 0.05 was considered statistically significant. ::: ![](1742-2094-2-9-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Cytokine production by brain slices from transgenic mouse models of AD at 15 months of age.Freshly harvested brain slices were incubated in neurobasal medium with B27 supplement. Media was collected after 24 hours, and cytokine levels measured. Mean concentrations (N = 15) +/- standard error are expressed in picograms per milligram of protein. P \< 0.05 was considered statistically significant. ::: ![](1742-2094-2-9-2) ::: Correlation between Aβ level and cytokine production by transgenic mouse brain slices ------------------------------------------------------------------------------------- Quantification of amyloid levels in brain mouse slices revealed that PS1/APPsw mice produce significantly more total Aβ as compared to TgAPPsw mice at the same age, and levels of insoluble and soluble Aβ (both 1--40 and 1--42) correlated well with each other (Table [1](#T1){ref-type="table"}). Analysis of the ratio of soluble:insoluble Aβ revealed a wide range of values across the TgAPPsw and PS1/APPsw mouse brain slices, with a 15.3-fold variance for Aβ 1--40 and a 5.4-fold variance for Aβ 1--42 (for Aβ 1--40, comparison of soluble:insoluble ratios revealed an average difference of 3.9 fold, and an average 1.7-fold difference for Aβ 1--42). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Quantification of Aβ levels in TgAPPsw and PS1/APPsw mouse brain slices. Data expressed as picograms/mg protein, mean ± S.E.M. for 13 determinations. ::: TgAPPsw PS1/APPsw ------------------- -------------------- --------------------- Soluble Aβ1--40 331.15 ± 35.36 4957.79 ± 322.30 Soluble Aβ1--42 68.11 ± 6.82 1644.29 ± 90.30 Insoluble Aβ1--40 67619.38 ± 7089.61 4095442 ± 409212.3 Insoluble Aβ1--42 6837.22 ± 2741.70 286463.3 ± 31395.63 ::: Although all the cytokines that changed in the transgenic brain slices were correlated with increases in Aβ levels, some showed a closer relationship than others to Aβ levels (Figs. [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, and [5](#F5){ref-type="fig"}). A table of r-correlation values is given in [Additional file 1](#S1){ref-type="supplementary-material"}. It is important to note that the dendrograms depict the closeness of a correlation between a particular cytokine and Aβ levels, and that all the members in the dendrograms are in fact highly correlated with Aβ levels (1% significance was considered as r \>= 0.496, and 5% significance was considered as r \>= 0.388). IL-4 and IL-5 were not produced in detectable amounts, were therefore omitted from the dendrograms. Of all the cytokines, IL-12p40 showed the strongest correlation with levels of both Aβ1--40 and 42 (soluble or insoluble). IL-1α and IL-1β were also highly correlated with Aβ1--40 and 42 (soluble or insoluble). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Dendrogram correlations of Aβ1--40 and Aβ1--42-cytokine relationships.Closely related members appear under the same node. The farther one needs to travel across the tree to reach another member, the greater the dissimilarity. ::: ![](1742-2094-2-9-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Dendrogram correlations of Total Aβ (Aβ1--40+Aβ1--42)-cytokine relationships.Closely related members appear under the same node. Total Aβ levels were calculated by adding soluble and formic acid extracted Aβ. The farther one needs to travel across the tree to reach another member, the greater the dissimilarity. ::: ![](1742-2094-2-9-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Dendrogram correlations of (Aβ1--42:40 ratio)-cytokine relationships.Total Aβ1--42:40 ratio\'s were calculated for both soluble and formic acid extracted Aβ. Closely related members appear under the same node. The farther one needs to travel across the tree to reach another member, the greater the dissimilarity. ::: ![](1742-2094-2-9-5) ::: Discussion ========== Levels of both peripheral and local CNS cytokines are elevated in AD patients, indicating that there is cellular activation occurring in response to inflammatory stimuli \[[@B15]-[@B20]\]. However, there is still considerable debate over exactly what is triggering this inflammation. Studies using mouse models of AD have shown that ibuprofen is effective in reducing plaque pathology and also in improving behavioral deficits characteristic of these transgenic models \[[@B8],[@B21]\]. The transgenic mouse models used to study AD exhibit some of the pathological features seen in the AD patient brain and show an increased production of inflammatory markers such as COX-2, PGE~2~and also increased levels of the pro-inflammatory cytokines IFN-γ and IL-12, TNF-α, IL-1α, IL-1β and IL-6 \[[@B12],[@B22]\]. Pathological analysis of tissue from AD patients and from mouse models of AD shows that there is extensive astrocytic and microglial activation around areas of Aβ plaque deposition \[[@B6],[@B7]\]. In addition, the chronic use of non-steroidal anti-inflammatory drugs (NSAIDs) has been associated with a reduced risk of developing AD \[[@B23],[@B24]\], suggesting that inflammation is an important contributor to the pathophysiology of AD. One aim of this study was to create a cytokine expression profile for organotypic brain slice cultures from transgenic mouse models of Alzheimer\'s disease, and to further relate this increase to the level of Aβ present in the brain. Another purpose of our study was to determine whether inflammatory events may be correlated with the accumulation of particular forms of Aβ; either soluble or insoluble. In the current study, we used the organotypic brain slice culture model to assess multiple cytokine production in the culture medium surrounding brain slices from transgenic mice that are engineered to over-produce Aβ. Cytokine production from 15-month-old control, PS1, TgAPPsw and PS1/APPsw mouse brain slices was assessed using the Bioplex cytokine multi-array system. Cytokine levels were not significantly elevated in PS1 brain slices compared to control slices, indicating that the PS1 (M146L) mutation does not have a significant impact on cytokine production. No significant change in the production of IL-4 and IL-10 was observed in the brains of these transgenic mice compared to their respective controls, indicating the absence of an anti-inflammatory response. All of the cytokines that were increased in the TgAPPsw brain slices (IL-1α, TNF-α, GM-CSF and IL-6) were further increased in the PS1/APP brain slices. This suggests that the presence of these inflammatory molecules is related to the amount of β-amyloid protein present, in agreement with a pro-inflammatory effect of Aβ \[[@B25]-[@B29]\]. A recent report has also shown increases in IL-1β, IL-6 and TNFα in-vivo after intra-cerebral administration of fibrillar Aβ into rat brain \[[@B30]\]. In order to further understand the correlation between the amount of Aβ and cytokine levels in the brains of transgenic mice, levels of both soluble and insoluble (formic acid-extracted) Aβ1--40 and 1--42 were quantified in the same slices from which cytokine production was measured, allowing a direct correlation of Aβ-cytokine levels. Levels of soluble and insoluble Aβ1--40 correlated well with each other, and the same was observed for Aβ1--42. As expected, quantification of Aβ levels generally revealed significantly higher amyloid levels in the PS1/APPsw mouse brain slices compared to TgAPPsw (for soluble Aβ, approximately 15 fold more Aβ1--40, and 20 fold more 1--42) but there was considerable slice-to-slice variation in soluble and insoluble Aβ levels within and between genotypes. The TgAPPsw and PS1/APPsw mice express equal levels of the APPsw molecule, but the PS1/APPsw model produces greater levels of Aβ and develops plaques at an earlier age (10 weeks) \[[@B31]-[@B33]\]. This increased deposition of Aβ in the PS1/APPsw mouse is due to a PS1 mutation, resulting in increased production of Aβ1--42 \[[@B34]-[@B36]\]. The Aβ data in the current report found a significant range of values for soluble:insoluble Aβ ratios between brain slices. This broad spread of values allowed correlation with equally wide ranges of cytokine production. This approach of examining Aβ-cytokine correlations within the same slices in the same aged animals eliminated the confounding factor of age related changes in cytokine production. Both Aβ1--40 and 1--42 correlated closely with all the cytokines that changed in the brain slices, but the correlation was particularly striking with IL-12p40. IL-12 is a hetero-dimeric cytokine which can comprise two subunits; IL-12p40 and IL-12p35. It is produced mainly by monocytes and macrophages and is a crucial factor in directing the T-cell response to infection, by inducing a Th1-type cytokine response. Our data agrees with that of previous reports showing that IL-12p40 is strongly up-regulated in-vitro (in response to an inflammatory stimulus) and in-vivo in the cerebral cortex of TgAPPsw mice \[[@B12],[@B37],[@B38]\]. IL-1, which was increased in the transgenic brain slices, is a major immune-response molecule functioning in the periphery and brain. The family comprises three related proteins (IL-1α, IL-1β and IL-1 receptor antagonist (IL-1ra)). IL-1α and IL-1β are two different isoforms of IL-1 that have similar affinities for their receptor IL-1R, and therefore have similar activities. Both are capable of inducing inflammatory cascades in-vivo and in-vitro, and it has been shown that they are capable of up-regulating expression of astrocyte-derived S100B and APP \[[@B39],[@B40]\]. It has been shown that IL-1β can promote β-secretase cleavage of APP in human astrocytes and thereby increase production of Aβ1--40 and 1--42 \[[@B41],[@B42]\]. It is also known that accumulation of plaques and the formation of neurofibrillary tangles are correlated with increased IL-1 levels in the AD brain \[[@B43]-[@B45]\]. Certain polymorphisms of IL-1A (the gene for IL-1α) are associated with late onset AD, although there is controversy as to whether all IL-1 gene polymorphisms represent risk factors for AD \[[@B46]-[@B50]\]. Microglia, in particular, have been shown to locally up regulate IL-1α at both the protein and mRNA level when inflamed, a situation that occurs in chronic disease states such as AD \[[@B51]\]. Both IL-1α and IL-1β can enhance the translation of APP mRNA in human astrocytes \[[@B52]\]; an up-regulation of IL-1α/β production in-vivo could therefore increase Aβ production, and an inflammatory cycle with increased Aβ levels may further increase IL-1α/β production. The Aβ 1--42:40 ratio is also of considerable interest in relation to cytokine levels and although there are currently no studies correlating Aβ 1--42:40 ratio with cytokine levels in-vivo, certain reports have suggested that cytokines can modulate Aβ production \[[@B53]-[@B55]\]. PS1 mutations are known to cause a shift in the production of Aβ species, favoring the production of Aβ1--42 over 1--40 and causing an increase in the Aβ1--42:40 ratio \[[@B56]\]. Since TNF-α correlated better with the level of Aβ1--42 than with that of Aβ 1--40, and correlated particularly well with the Aβ1--42:40 ratio in our study, TNF-α levels may be partly determined by this ratio. Higher levels of Aβ1--42 can promote the formation of toxic oligomers \[[@B57]-[@B59]\], and it therefore seems possible that the increased level of Aβ oligomers in PS1/APP mice (compared to APPsw) and the level of oligomeric forms present in the brains of our transgenic mice may be related to the amount of TNF-α being produced. It is important to consider the nature of the exact form of Aβ that may be most responsible for the inflammatory events seen in AD brains. Aβ can exist in various forms (monomeric, dimeric, oligomeric and fibrillar), but it is not yet clear which of these forms are most potent in inducing inflammatory cellular responses \[[@B57],[@B60],[@B61]\]. This is of interest because the oligomeric forms of Aβ which are thought to be the most toxic are produced more readily by Aβ1--42 (for review see \[[@B62]\]). Future studies will assess the relative proportions of monomers/dimers, oligomers or fibrils occurring in these mice brains and their relationship with the cytokine increases observed. List of abbreviations ===================== AD: Alzheimer\'s disease APP: Amyloid precursor protein APPsw: Amyloid precursor protein Swedish mutation PS1: Presenilin 1 Aβ: Beta-amyloid Tg: Transgenic TNF: Tumor necrosis factor IL-x: Interleukin-x IL-1ra: Interleukin-1 receptor antagonist GM-CSF: Granulocyte macrophage colony stimulating factor PBS: Phosphate buffered saline COX-2: Cyclo-oxygenase-2 PGE2: Prostaglandin E2 IFN: Interferon NSAID: Non-steroidal anti-inflammatory drug Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= NP carried out the in-vitro brain slice assays, processed brain tissues, performed the Bio-plex assay, ELISAs and drafted the manuscript. DP conceived the design of the study, carried out Bio-plex assays, performed statistical analyses and aided in manuscript preparation. VM analyzed data and constructed dendrograms. AQ aided in ELISA and Bio-plex assays and collected mouse brain tissues. FC oversees management of the mouse colonies. MM aided in manuscript preparation and gave critical analysis of the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Correlation table of levels of different β-amyloid species with cytokines in transgenic mouse models of Alzheimer\'s disease. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ The authors would like to thank Bob and Diane Roskamp for their generous support.	PubMed Central	2024-06-05T03:55:54.149228	2005-3-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555557/", "journal": "J Neuroinflammation. 2005 Mar 11; 2:9", "authors": [ { "first": "Nikunj S", "last": "Patel" }, { "first": "Daniel", "last": "Paris" }, { "first": "Venkatarajan", "last": "Mathura" }, { "first": "Amita N", "last": "Quadros" }, { "first": "Fiona C", "last": "Crawford" }, { "first": "Michael J", "last": "Mullan" } ] }
PMC555558	Background ========== Computational protein annotation is a major goal of bioinformatics and annotation methods are widely used. A wide variety of annotation methods exist, many of which rely on some kind of scoring. Typically, when testing whether a protein should be given a certain annotation, a score threshold is set, and proteins that score higher than the threshold are given the annotation. Obviously, some annotation mistakes may occur. Such mistakes can be divided into false positives (FPs) and false negatives (FNs). FPs (or false hits) are annotations that were mistakenly assigned to a protein (type I error). FNs (or misses) are annotations that should have been assigned to a protein but were not (type II error). Adjustment of score thresholds allows tradeoff between these two types of mistakes. FPs annotations are considered to be of graver consequence than FNs. This is partially due to the fact that introduction of a false positive annotation into a protein database may cause other proteins to become incorrectly annotated on the basis of sequence similarity \[[@B1],[@B2]\]. A systematic evaluation of the source of false annotations that already contaminated current databases was reported \[[@B3]\]. Several automatic systems such as PEDANT \[[@B4]\] and GeneQuiz \[[@B5]\] were introduced with the goal of matching the performance of human experts. Still, over interpretation, FN errors, typographic mistakes and the domain-based transitivity pitfall \[[@B6]\] limit the use of such fully automatic systems for inferring protein function. Due to the importance of minimizing the amount of false annotations and maintaining highly reliable protein databases, three methods are generally used to avoid false annotations. The first method is manual validation of the annotation of each protein, which creates a serious bottleneck in the addition of new proteins and annotations to the database. The second method is using high score thresholds, thus lowering the rate of FPs but also increasing the rate of FNs (resulting in a loss of sensitivity). The third method is requirement for hits from different detection methods, eliminating advantages that are unique to some methods. Thus it would be beneficial to develop means by which FP annotations could be detected automatically, allowing both high throughput and high sensitivity. Here we present such a method that uses clustering of protein functional groups to separate true positives (TPs) from FPs automatically. Our method is based on the following notions: (a) protein annotations represent biological properties; (b) protein functional groups share specific combinations of biological properties, essentially constituting \"property clusters\"; (c) if two proteins have very different combinations of annotations, they are unlikely to share a single functional annotation and therefore there is a high chance that one of them was given that annotation incorrectly. These notions are not obvious, but were shown to correctly indicate false annotations in some individual cases tested manually using the graphical annotation-analysis tool of PANDORA \[[@B7]\]. We aim to generalize these sporadic observations and to address the feasibility of automating the detection of FP. Using these ideas, the method attempts to separate a group of proteins into \"property clusters\", by introducing a measure that quantifies the similarity between the annotation combinations of two proteins. According to our basic notions, these clusters are likely to be in accordance with false and true hits. We tested our method on the PROSITE protein signature database \[[@B8]\]. The database consists of 1189 protein signatures (essentially annotations) that were assigned to a protein database. PROSITE annotation of proteins is manually validated, stating for each protein hit whether the annotation is a TP or a FP. Out of this set of 1,189 signatures, we chose a subset of all signatures that have both true and false hits, and this served as our test set. Altogether 327 such signatures were collected and tested. For each of the signatures, the method examined the set of proteins that were assigned the signature. We called the separation successful only if at any step of the clustering process [all]{.underline} the TPs were clustered together without any FPs. We applied a stringent scoring, where a partial success is considered failure. Furthermore, we constructed a random FP simulation test in order to provide a more extensive test. In this test, all 5,551 InterPro \[[@B9]\] annotations were considered. For each InterPro annotation we selected the set of proteins in SwissProt \[[@B10]\] that were assigned that annotation, and added to that set random proteins, simulating proteins that were assigned the annotation by mistake (FPs). For each annotation we repeated the test 15 times: 5 times with 1 random protein, 5 times with 5 random proteins and 5 times with 10 random proteins. This artificial contamination of the annotation source strives to simulate mistaken annotations that may occur under some automation annotation inference schemes. Results ======= Property-based clustering ------------------------- We begin by describing the method of property-based clustering. Given a set P of all proteins that were given a certain annotation, and that there are both FPs and TPs in P, we would like to separate the set into disjoint subsets, so that one of the subsets will include all TPs and no FPs (leaving one or more subsets containing the FPs). Annotation-based clustering is used to detect these subsets. We define an annotation as a binary property assigned to a protein (each protein may or may not have a \"hit\"). At the first stage, annotations from GO (Gene Ontology) \[[@B11]\], InterPro (entries) and SwissProt (keywords) are gathered for all proteins in P. The clustering works in the following way: between each two proteins we define a similarity score that tries to quantify how much do the two proteins have in common from a biological perspective. The score between two proteins p~1~and p~2~is defined as: ![](1471-2105-6-46-i1.gif) where A~1~and A~2~are the set of annotations of proteins p~1~and p~2~respectively, iis the current annotation, and f(i) is the frequency of iin the database. This score uses the following logic: if two proteins share an annotation, they are biologically similar in some manner. The more annotations these proteins share, the more cause we have to believe that they are similar biologically. However, two proteins sharing an annotation like \"Enzyme\" (that appears 45,991 times in our database) should receive a worse similarity score than two proteins that share a much uncommon annotation like \"Heat Shock Protein\" (that appears only 832 times). This is taken into account by using log(f(i)). Obviously, one could think of different scoring schemes that would quantify this differently. For a specific example of how the score is calculated see Table [1](#T1){ref-type="table"}. The similarity score is calculated between every two proteins in P. Next, we define the similarity score between two clusters as the arithmetic average of scores of all inter-cluster protein pairs: ![](1471-2105-6-46-i2.gif) where C~1~and C~2~are clusters of proteins. Starting with clusters of 1 protein each, the method begins by an initial one-step clustering which merges all clusters that have the exact same combination of annotations. Following this the primary clustering commences: At each clustering step the two clusters that have the highest similarity score are merged. At each step the contents of the clusters are evaluated, and if all TP proteins appear in one cluster without any FPs, we say that the clustering process successfully separated the TPs from the FPs. Note that we do not require all the FPs to be grouped into one cluster, due to the fact that they cannot be expected to share biological similarity amongst themselves. PROSITE test ------------ Out of 327 sets of proteins that share a PROSITE signature, the method showed successful separation (as defined previously) in 227 sets, i.e. 69% of the cases. The average size of the protein sets was 156.1 and the median 76. Altogether 58,254 proteins were used for this test. The average and median FP rates (FP rate is defined as: FP/(TP+FP)) of the sets were 0.12 and 0.07 respectively. These general statistics about the test set indicate that the sets were large enough and had a high enough amount of TPs and FPs so that the chance of random success would be minimal. In order to demonstrate the method\'s performance in this test, we provide the following example of testing a single protein set. The set presented here is the set of all 37 proteins that matched the PROSITE \"Serum albumin family\" signature. Each protein in the set contains an average of 18.2 annotations (obviously not all are relevant). First, the score between every pair of proteins is calculated, based on their mutual annotations. Next, the proteins undergo a preliminary clustering step in which all proteins that have the exact same combination of annotations are merged into clusters. Following this, the proteins are clustered together based on their mutual similarity score. Finally, once the clustering has finished we examine the tree to see if the true positives were separated from the false positives. In the given example, there are 5 proteins that were incorrectly assigned the PROSITE annotation (FPs), and in Figure [1](#F1){ref-type="fig"} we see that they are indeed separated from the TP proteins. Random FP simulation test ------------------------- 5,551 sets of proteins were tested 15 times each and showed successful separation in 74% of the cases. Altogether 99,076 proteins were used for this test. This can be subdivided into 78% success for the sets that had 1 random protein added, 74% success for the sets that had 5 random proteins added and 68% for the sets that had 10 random proteins added. The average set size was 78 proteins. The drop in the performance by increasing the level of FPs is due to the fact that there is a higher chance that one of the randomly selected proteins will be biologically similar to the TPs. Since we consider only cases in which all FPs are detected, then there would be a higher chance of failure as the number of randomly-generated FPs increases. While the simulation of FP errors randomly provides endless amounts of test sets, which is a clear advantage over the limited test sets provided by a real database such as PROSITE, the simulation has its own limitations. The hidden assumption made by this approach is that the FP hits are independent of each other. This assumption is not necessarily true: for example, if annotation is done by means of sequence similarity, false hits may be more likely to be biologically similar to each other (e.g. belong to the same family). In fact, in many cases in the PROSITE test we find that the correct separation separates the TP proteins into one cluster and the FP into one or two clusters, suggesting that the FPs share some degree of biological similarity (see \"Determination of the correct halting step\"). This difference in the way that FP annotations are generated may also account for the difference in success rates between the PROSITE test set and the simulated test set. The way FP annotations are introduced into databases is impossible to model, but the combined success of the method on both a real database test set and on an extensive simulated test set seems promising. A further issue which concerns the simulation method is determining the amount of FPs to add to each set. Here we chose to add 1, 5 or 10 proteins to each set. This does not necessarily reflect the amount of FPs in real databases. Understandably, each database\'s average FP rate depends on its specific characteristics. However, the PROSITE database\'s average FP rate of 0.12 (median of 0.08) might give an indication as to what a typical rate is. In comparison, the average FP rate for our random simulation set was 0.11 (median of 0.07), which suggests that our choice was reasonable. Determination of the correct halting step ----------------------------------------- We call a clustering process successful if it managed at any step to separate the false annotations. However, this step must be somehow determined automatically. There are two approaches to this: one is to use an intrinsic parameter of the clustering process that would indicate where the correct halting step is located; the other is selecting a predetermined step of the process. We chose the similarity score at each merging step as an intrinsic process parameter. When plotting the score against the progression of the clustering (Figure [2](#F2){ref-type="fig"}), a knee shape in the plot would indicate a point of stability (biological similarity), suggesting it as a potential halting step. Analysis of the second derivative of this plot allows finding these knee-shaped stability points automatically. Using this method, 56% percent of the correct halting steps in the PROSITE test were correctly predicted. A different approach was to always choose the last step or the last two steps as the correct halting step. This resulted in 45% and 65% correct prediction, respectively. Furthermore, the union of the correct predictions made by both approaches indicates that together they correctly predict the halting step in 79% of the PROSITE test cases. Discussion ========== Prediction of success --------------------- Interestingly, we found that with certain sets the method tended to be more successful than with other sets, probably indicating that these sets are more coherent biologically. This might suggest exploring an approach in which for each annotation one could predict the level of success provided by this method. Furthermore, we used the InterPro categorization of annotations into types in order to check success in specific annotation types. InterPro divides its annotations into different categories, such as \"domain\", \"repeat\" and \"family\". Understandably, \"family\" type annotations had a \~30% higher success rate than the other annotation types, primarily due to the fact that the \"family\" annotations often represent protein sets that are biologically coherent whereas other types such as \"repeat\" or \"domain\" annotations are biologically diverse. This result would be expected by a method that performs a clustering based on biological similarity. This indicates that this approach should be aimed primarily at functional family annotations. However, functional families can be defined at different resolutions: an alcohol dehydrogenase belongs to the enzyme family, the dehydrogenase family and the alcohol dehydrogenase family. The test sets of the PROSITE and InterPro databases mainly represent mid-level and low-level annotations, with a typical size of tens or a few hundreds of proteins (see the statistics given previously). In order to further our understanding of the resolution in which this method is successful, we divided the protein groups into size categories and studied the relative amount of success in every size category. Figure [3](#F3){ref-type="fig"} shows that as the group size increases, the rate of success decreases. Assuming larger sets represent the higher level annotations of InterPro, this suggests that when the annotations are more general (\"higher\" in the biological functional hierarchy) they have less in common biologically. Therefore, we would not expect the method to succeed on very general terms such as \"enzyme\". Sporadic tests of several high level GO annotations suggest that this is indeed the case (data not shown). Annotation source interdependency --------------------------------- Because multiple annotation sources were used, concerns arose regarding interdependencies amongst them. For example, InterPro is highly dependent on PROSITE, so proteins that have a PROSITE annotation will very likely be assigned an InterPro annotation as well automatically. In order to minimize this effect, we did not allow the algorithm to use the InterPro annotations that matched the PROSITE annotation which was being tested. Furthermore, in order to increase reliability of the random FP simulation test, all known PROSITE FPs were removed from InterPro prior to the test. Still, there is some concern that the results are partially biased due to annotation source interdependencies. Furthermore, it is difficult to determine whether these dependencies represent true biologically dependent properties, or simply a duplication of the same property in different sources. Keeping this difficulty in mind, our results which show different levels of success for different types of annotations (see \"Prediction of success\") indicate that the success of the method is more likely due to biological dependency rather than artificial duplication. Sufficient annotation --------------------- It should be stressed that the clustering process is based on sufficient annotation. Therefore, it may be difficult to apply this method to proteins that are poorly annotated. Still, these cases should be relatively rare: Nearly 77% of the \~1,600,000 proteins in TrEMBL \[[@B10]\] have at least one annotation by InterPro, and when considering several annotation sources there are on average \~10 annotations per SwissProt protein. Note that the amount and richness of annotation is constantly increasing at a fast rate. Furthermore, the ability to detect false annotations automatically may allow an increase in the sensitivity of current methods, thereby allowing more extensive annotation of proteins. It is worthwhile noting that amongst the 58,254 proteins used in these sets there were 3,587 (6%) proteins annotated by SwissProt as \"hypothetical proteins\". 18% of the sets that were successfully separated contained such hypothetical proteins, with an average of 8% hypothetical proteins for each such set. These results suggest that the method is capable of handling to some extent hypothetical proteins of unknown function. Another helpful approach to the problem of insufficient annotation could be the introduction of quantitative protein properties that are easily determined and show some correlation with function (i.e. the protein length, its Isoelectric point, etc.) into this method. Preliminary testing showed some positive correlation between protein length and Isoelectric point with function in certain cases (not shown). Conclusion ========== Introduction of FP annotations into protein databases can be harmful. It has been shown that once a mistaken annotation is introduced into a database, it often transfers to other proteins that are sequentially similar causing a propagation of false annotation \[[@B1]\]. Due to the importance of keeping high-quality databases, either the proteins are manually checked one by one or the annotation detection sensitivity is reduced in order to minimize FPs. The error rate and the limited sensitivity of assigning structural annotations using PSI-BLAST \[[@B12]\] or SAM-T98 \[[@B13]\] and methodologies based on HMMs and SVMs had been reported \[[@B14]\]. Naturally the process of manual validation of the annotation of protein databases is extremely time-consuming and in many cases is subjective to the expert view. Automatic detection of false annotations greatly facilitates the task of manual validation of annotation, and allows using lower thresholds when trying to detect protein signatures, therefore allowing higher method sensitivity. Based on the notion that protein functional groups share specific combinations of annotations, we have introduced a method that by separating a set of proteins into biological \"property clusters\" shows successful separation of incorrectly annotated proteins from correctly annotated proteins. We test the method both with a manually validated test set and with a randomly constructed test set, and in both cases show a high degree of success. These results suggest that this tendency of certain annotations to appear in groups may be used as a basis of automatic methods that detect FPs. Naturally, different computer learning methods can be used to take advantage of these interdependencies of biological properties (for example see \[[@B15]\]). Methods ======= Sources ------- We created a database that includes all proteins from SwissProt 40.28 (114,033 proteins) \[[@B10]\]. The database also included annotation of these proteins by GO\[[@B11]\], SwissProt and InterPro \[[@B9]\]. GO terms represent a wide range of biological terms concerning molecular function, cellular localization and biological processes, and span various degrees of specificity: from very general terms to very specific ones. GO terms are assigned to proteins both manually and automatically. InterPro annotations are assigned automatically by sequence and typically represent functional families and domains of no more than a few hundred protein members. SwissProt keywords are assigned manually and cover various biological subjects. Annotation source and the number of annotation for each (in parenthesis) are: SwissProt version 40.28 (865 keywords), InterPro version 5.2 (5,551 entries), GO as of July 2002 (5,229 terms), PROSITE version 17.5 (1,189 signatures). Authors\' contributions ======================= NK and ML conceived of the study. NK designed the method. NK implemented and developed the method. NK designed the tests and analyzed the results. NK and ML wrote the manuscript. Acknowledgements ================ A preliminary version of this work has been presented in ISMB2004 by NK, under the name \"False Annotations of Proteins: Automatic Detection via Keyword-Based Clustering\". It can be accessed at: <http://www.iscb.org/ismbeccb2004/accepted_papers.html>. We thank Menachem Fromer for his support and useful suggestions. We thank the ProtoNet team for their constant support. This work is partially supported by The Sudarsky Center for Computational Biology and the EU framework VI BioSapiens NoE consortium. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Biological clustering example.The figure shows a dendrogram describing the clustering of 37 proteins that matched the PROSITE \"Serum Albumin Family\" signature. The clustering advances from right to left along the axis that shows the similarity score at each point of the process. The vertical axis shows 16 initial clusters of proteins that the clustering starts with after the initial clustering stage. The initial clusters are numbered 1--16 and in parentheses show the number of proteins in them. Clusters 1--3 contain 5 Vitamin D Binding proteins (TPs). Clusters 4--13 contain 24 Albumin proteins (TPs). Cluster 14 contains 3 Afamin proteins (TPs). Clusters 15 and 16 contain the 5 FPs. The colors indicate the correct separation of this set into TPs and FPs. ::: ![](1471-2105-6-46-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Similarity score plot.The figure shows the similarity score (solid line) plotted versus the progression of the clustering process for a sample protein set that was tested. The protein set includes 606 proteins that were annotated as \"Rhodopsin-like GPCR superfamily\". The score decreases from left to right as the clusters are merged, indicating decreasing biological similarity. The vertical dashed line indicates the correct halting step. Note that the correct halting step is located where there is a distinct knee in the graph, indicating a point of stability in the process. ::: ![](1471-2105-6-46-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Relative success and failure in group size categories.The figure shows the relative success and failure of the clustering method in different categories of group sizes (the group size of an annotations is the number of proteins that have the annotation). All tested sets were grouped into 30 categories according to the amount of proteins in them, from 0 to 1500 proteins (shown on the horizontal axis). Each category shows the relative amount of success (purple) and failure (blue) of the method in each of the categories. It is apparent that relative success decreases as the group sizes increase. ::: ![](1471-2105-6-46-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Similarity score calculation ::: Source Annotation CD63\_RABIT^a^ CD68\_HUMAN^a^ Frequency^b^ -ln(freq) --------------- ------------------------------------------------------- -------------------- -------------------- ------------------------------------- --------------- SwissProt Antigen 1 1 0.007130 4.9435114 SwissProt Lysosome 1 1 0.001929 6.2506136 SwissProt Glycoprotein 1 1 0.094727 2.3567562 SwissProt Transmembrane 1 1 0.159770 1.8340200 SwissProt Alternative splicing 0 1 0.029281 \- SwissProt Signal 0 1 0.123850 \- SwissProt Repeat 0 1 0.078968 \- InterPro Serum albumin family 1 0 0.000342 \- InterPro CD9/CD37/CD63 antigen 1 0 0.000666 \- InterPro Lysosome-associated membrane glycoprotein (lamp)/CD68 0 1 0.000123 \- GO Membrane 1 1 0.210869 1.5565182 GO Lysosome 1 1 0.002043 6.1932038 GO Vacuole 1 1 0.002184 6.1267895 GO Lytic vacuole 1 1 0.002043 6.1932038 GO Cell 1 1 0.440206 0.8205125 GO Integral membrane protein 1 1 0.160874 1.8271338 GO Cytoplasm 1 1 0.186569 1.6789541 GO Intracellular 1 1 0.307578 1.1790266 [Similarity Score:]{.underline} 40.960244 The table shows a calculation of the similarity score between two SwissProt proteins: Rabbit CD63 antigen (CD63\_RABIT) and Human Microsialin precursor (CD68\_HUMAN). The similarity score is the summation of -ln(freq) on all annotations that are shared by both proteins. ^a^-- 1 or 0 indicate if the given protein has or does not have the annotation respectively. ^b^-- The frequency is the portion of proteins in the database that have the annotation. :::	PubMed Central	2024-06-05T03:55:54.150921	2005-3-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555558/", "journal": "BMC Bioinformatics. 2005 Mar 8; 6:46", "authors": [ { "first": "Noam", "last": "Kaplan" }, { "first": "Michal", "last": "Linial" } ] }
PMC555559	Background ========== It is well documented that women who experience a full-term pregnancy early in their lives have a significantly reduced risk for developing breast cancer \[[@B1],[@B2]\]. The mechanisms by which pregnancy affects maternal breast cancer incidences are not fully understood. Previous studies suggest that the protective effect of pregnancy can be mimicked with a defined regimen of estrogen and progesterone combination (E/P) in rodent models \[[@B3],[@B4]\]. However, the effects of pregnancy levels of E/P in human and their underlying mechanisms have not been investigated. Retinoids are a promising class of chemopreventive agents against breast cancer because of their antiproliferative and proapoptotic properties \[[@B5],[@B6]\]. Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are nuclear transcription factors that modulate the biological effects of retinoids. Most retinoid forms, including 9-cis-retinoic acid (9-cis RA) and all-trans-retinoic acid (ATRA), activate RAR family members, whereas RXR family members are activated by 9-cis RA. N-(4-hydroxyphenyl) retinamide (HPR), a synthetic derivative of ATRA, may weakly interact with retinoid receptors \[[@B7]\]. The 76N TERT cells were derived from a reduction mammoplasty specimen \[[@B8],[@B9]\]. They are normal human mammary epithelial cells immortalized by plasmids containing hTERT, the human catalytic subunit of the reverse transcriptase protein of telomerase \[[@B10]\]. hTERT-expressing normal cell clones have been shown to have an extended life span without any change in karyotype \[[@B11]\]. The 76N TERT cells in culture could continuously grow about 60 population doublings \[[@B8]\], and the level of p53 protein has been shown to remain consistent at early or late passages \[[@B9]\]. Unlike the tumor cell lines widely used in breast cancer researches such as MCF-7 and MDA-MD-231 cells, which have undergone several steps in tumorigenesis, the 76N TERT cell line represents a system that is immortal but does not yet have the capacity to form a tumor. Hence, it is potentially a better model to study the genetic changes, and to test the effects of carcinogenic or chemopreventive agents on the development of mammary tumors. In this study, we investigated whether E/P induce growth inhibition in 76N TERT cells; and the molecular mechanisms by which E/P inhibited 76N TERT cell growth. For comparison purpose, the anti-growth effect of both natural and synthetic retinoids was examined in parallel in this immortalized mammary epithelial cell line. We also investigated whether we could increase the responsiveness of retinoids by using retinoids in combination with E/P. Our studies demonstrate that 1) inhibition of cell growth by E/P and retinoids could be partially mediated through a p53-dependent mechanism; 2) induction of p21 expression, inhibition of telomerase activity, or up-regulation of estrogen receptor beta (ERβ) by E/P and retinoids may contribute to their anti-growth effects; 3) combination of retinoids with E/P lead to increased inhibitory effects on cell growth. Results ======= Expression of RARs, RXRs, estrogen receptors and progesterone receptors in 76N TERT cells ----------------------------------------------------------------------------------------- As the ability of estrogen, progesterone and retinoids to influence cell proliferation is mediated by their respective receptors in most cases, we first examined the expression of these receptors in 76N TERT cells. Using Western blot analysis, the proteins of RAR (RARα, RARβ and RARγ), RXR (RXRα, RXRβ and RXRγ), and ER (ERα and ERβ) were observed as suggested by the manufactures (Figure [1](#F1){ref-type="fig"}). The antibody for the progesterone receptor (PR) detected a protein between 85 and 125 KDa (Figure [1](#F1){ref-type="fig"}). The existences of RARβ and ERβ in 76N TERT cells were further confirmed by quantitative RT-PCR at the mRNA level (RARβ data not shown, ERβ data see Figure [6B](#F6){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Expression of RAR, RXR, ER and PR in 76N TERT cells.Whole cell lysates were analyzed by using anti-RAR, RXR, ER, and PR antibodies as described under \"Materials and Methods\". Blots shown are representative of 2--3 experiments with similar results. ::: ![](1475-2867-5-6-1) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Induction of ERβ expression by E/P and retinoids in 76N TERT cells. Cells were treated with indicated retinoids with or without E/P for 72 hours. A.Western blot analysis of ERβ protein. Relative ERβ expression was normalized to actin protein and expressed as fold changes compared to vehicle treatment. Blot shown is representative of 3 experiments with similar results. B.Quantitative RT-PCR analysis of ERβ genes. Data are the means ± SE from 3 experiments. \, significant differences from their own controls. ::: ![](1475-2867-5-6-6) ::: Inhibition of 76N TERT cell growth by E/P and retinoids ------------------------------------------------------- We then tested the influence of E/P or retinoids on 76N TERT cell growth, using \[^3^H\]thymidine incorporation assay. The concentrations of E/P and retinoids used in the experiments were chosen based on previous studies and are clinically or physiologically relevant \[[@B3],[@B4],[@B12]\]. Treatment of cells with E/P or all three retinoids resulted in decreases in cell proliferation. As shown in Figure [2](#F2){ref-type="fig"}, treatment of 76N TERT cells with 1 μM 9-cis RA or 2 μM ATRA significantly decreased the cell growth by 28.8% and 24.5% respectively. In comparison, cells were more responsive to HPR. Treatment with 2 μM HPR exhibit a significant 71.4% decrease in cell growth relative to controls. Combination of 1 ng/ml of β-estradiol and 1 μg/ml of progesterone, a regime that mimics the protective effects of pregnancy, also resulted in a significant 32.2% decrease in cell proliferation. In the presence of E/P, the inhibitory effects on cell growth of 9-cis RA, ATRA and HPR were further increased to a respective 56.6%, 53.3% and 86.8%, indicating that the anti-proliferative effect may be additive between E/P and retinoids. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Inhibition of 76N TERT cell growth by E/P and retinoids. Cells were treated with indicated retinoids with or without E/P for 24 hours and then labelled with \[^3^H\]thymidine as described under \"Materials and Methods\". Data are the means ± SE from 3--4 experiments. \, significant differences from control. ::: ![](1475-2867-5-6-2) ::: Activation of RAREand p53gene by E/P and retinoids ------------------------------------------------------ Many biological responses to retinoids are thought to be mediated through receptors by binding to retinoic acid response elements (RAREs) and regulation of transcriptional activity \[[@B5],[@B6]\]. In addition, cross-talk between ER and RAR pathways has been previously reported \[[@B13],[@B14]\]. Given this, a comparison of RAREgene activation in response to E/P and retinoids was carried out using cells transfected with a RARE-luciferase reporter gene construct. As shown in Figure [3A](#F3){ref-type="fig"}, 76N TERT cells exhibited a respective 3.3-, 5.4-, and 2.5-fold activation of RAREgene in response to 9-cis RA, ATRA or E/P alone. In contrast, HPR caused no significant change in luciferase activity relative to the control. In the presence of E/P, the effects of 9-cis RA and ATRA on RAREgene activation were essentially the same as without E/P. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### *Activation of RAREand p53gene by E/P and retinoids in 76N TERT cells. Cells were transiently transfected with the RARE(A) or p53(B) reporter plasmid, and then treated with retinoids for 24 hours with or without E/P. Luciferase activity was measured and normalized as described under \"Materials and Methods\". Results are the means ± SE from 3 experiments. \, significant differences from control. ::: ![](1475-2867-5-6-3) ::: Functional p53 provides a protective effect against tumor growth \[[@B3],[@B15]\]. We next examined whether E/P and retinoids could enhance the transcriptional activity of p53using 76N TERT cells transfected with a p53-responsive luciferase reporter gene construct. As shown in Figure [3B](#F3){ref-type="fig"}, a 2.3-, 1.8-, 1.8- and 2.2-fold induction of luciferase activity was observed by treatment of cells with 9-cis RA, ATRA, HPR and E/P respectively. Co-treatment with E/P and retinoids showed no additional activation of p53gene as compared to their treatments alone. Induction of p53 and p21 protein expression by E/P and retinoids ---------------------------------------------------------------- We then performed Western blot analysis to test whether increased p53gene activity is paralleled by increased p53 protein expression. Treatment with 9-cis RA, ATRA, HPR or E/P alone slightly increased (about 1.5-fold) the expression of p53 protein. Consistent with the data on p53gene activation, no additive effects between E/P and retinoids on induction of p53 protein were observed (Figure [4](#F4){ref-type="fig"}). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Induction of p53 and p21 protein by E/P and retinoids in 76N TERT cells. Cells were treated with indicated retinoids with or without E/P for 72 hours. Whole cell lysates were subjected to Western blot analysis using anti-p53, p21 and Bax antibodies. Relative expression of each protein was determined using the same membrane, and normalized to actin protein. Data are fold changes compared to vehicle treatment. Blot shown is representative of 4 experiments with similar results. ::: ![](1475-2867-5-6-4) ::: Activated p53 could induce the transcription of either p21 to cause growth arrest, or Bax to induce apoptosis \[[@B15]\]. We therefore investigated whether increased p53 protein expression can modulate the expression level of p21 or Bax proteins in 76N TERT cells. In Figure [4](#F4){ref-type="fig"}, exposure to retinoids or E/P alone did moderately increase the p21 protein level, with a 1.7-, 2.7- and 2.1-fold increase in p21 expression for ATRA, HPR and E/P respectively. The increases of p21 protein expression with combined E/P and retinoids were similar to that observed when E/P and retinoids were used alone. In addition, no significant effects by E/P or retinoids on Bax protein expression were observed in 76N TERT cells. Inhibition of telomerase activity by E/P and retinoids ------------------------------------------------------ Activation of telomerase is an early event in the development of breast cancers that may lead to cellular immortality, a critical and rate-limiting step in oncogenesis \[[@B16],[@B17]\]. Activated p53has been associated with regulation of the telomerase activity \[[@B18]-[@B20]\]. To evaluate the effects of E/P and retinoids on telomerase activity in 76N TERT cells, cells were treated for different time periods with E/P and retinoids, and the levels of telomerase activity were determined by a quantitative real-time PCR method. As shown in Figure [5](#F5){ref-type="fig"}, treatment with E/P decreased telomerase activity in a time-dependent manner with a 63.3% inhibition at 72 hours, whereas vehicle treatment had no effect at any time during the experiment. The maximum inhibition on telomerase activity was observed at 24 hours for both 9-cis RA and ATRA treatments, with a respective of 68.9% and 69.4% decrease. In comparison, the effects of HPR occurred more rapidly, with a complete inhibition at 16 hours, and persisted throughout the treatment. The inhibitory effects of 9-cis RA, ATRA or HPR and E/P seemed to be additive, as in the presence of E/P, 9-cis RA, ATRA or HPR showed increased inhibitions at various time points. These effects correlate well with their observed growth inhibitory effects in 76N TERT cells, suggesting that inhibition of telomerase activity by E/P and retinoids may contribute to their additive effects on inhibition of cell growth. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Inhibition of the telomerase activity by E/P and retinoids in 76N TERT cells. Cells were treated with indicated retinoids with or without E/P for the indicated time periods. Telomerase activity was determined as described under \"Materials and Methods\". Data are the means ± SE from 2--3 measurements. \, significant differences from their own controls. ::: ![](1475-2867-5-6-5) ::: Induction of ERβ expression by E/P and retinoids ------------------------------------------------ There is growing evidence that ERβ could be an inhibitor of tumorigenesis of breast cancer \[[@B21]-[@B23]\]. We examined whether there were any changes in the expression of ERβ in response to E/P and retinoid treatment in 76N TERT cells. After treatment of cells with retinoids or E/P for 72 hours, the amount of ERβ protein was determined by Western blot analysis. As shown in Figure [6A](#F6){ref-type="fig"}, the normalized ERβ protein showed a respective 2.1-, 2.3-, and 1.5-fold increase in response to ATRA, HPR and E/P, as compared to the vehicle treatment. We also carried out a quantitative analysis of ERβ mRNA expression in response to E/P and retinoid treatment in 76N TERT cells using a real-time PCR assay. As shown in Figure [6B](#F6){ref-type="fig"}, there was a 2.0-, 2.3- or 2.8-fold induction of ERβ mRNA levels in HPR, E/P or combination of HPR and E/P treated cells, respectively. Discussion ========== Similar to retinoids, a full-term pregnancy has been associated with beneficial effects on breast cancer risk over the long term \[[@B1],[@B2]\]. The mechanisms by which pregnancy affects maternal breast cancer incidences are not fully understood. Studies have showed that higher concentrations of progesterone elicit a growth-inhibiting response from normal and cancerous breast cells \[[@B24],[@B25]\], and are inversely related to breast cancer incidence \[[@B26]\]. In this study, we examined the ability of pregnancy levels of E/P and retinoids to affect the growth of the immortalized normal mammary epithelial cells. Our results demonstrated that three isoforms for RAR and RXR (α, β, γ), two isoforms for ER (α and β), and PR receptor proteins are expressed by 76N TERT cells. Treatment with 9-cis RA, ATRA, HPR or E/P inhibited 76N TERT cell proliferation and resulted in the activation of p53gene, followed by increased expression of p53 protein and p21 protein, and inhibition of telomerase activity. Additionally, we first report here that the expression of ERβ is induced in response to E/P and retinoid treatment at both the transcriptional and translational levels. Importantly, we demonstrate that the inhibitory effects of retinoids on cell growth are more effective in the presence of E/P, and correlate well with their inhibitory effects on telomerase activity in 76N TERT cells. Our data suggest that the growth inhibitory effects of E/P and retinoids may involve the activation of p53 pathway in 76N TERT cells. First, our results showed that both E/P and retinoid treatments lead to the increased p53gene activity. Secondly, we demonstrated that the protein expression of p53 and p21 were increased following the treatment. It has been shown that p21 can inhibit cyclin A/cdk2 kinase activity and subsequently result in cell cycle arrest \[[@B27],[@B28]\]. Our data is in line with the previous findings that in normal mammary epithelial cells, retinoids induce cell cycle arrest which is associated with an increase in p21 expression \[[@B29]\]; and that in both rats and mice, p53is activated in response to E/P and this activation is sustained to induce p21 \[[@B3]\]. Thirdly, our data showed that treatment with E/P or retinoids decreased the telomerase activity in 76N TERT cells. Although a few reports suggest that telomerase activity appears to be independent of p53expression or mutation \[[@B30],[@B31]\], the majority of the evidence to date support the involvement of p53in regulation of telomerase activity in mammary epithelial cells and breast cancer \[[@B18]-[@B20]\]. The molecular mechanisms of regulation of telomerase activity by p53may involve down-regulation of hTERT transcription or the interaction of p53with other transcription factors \[[@B19]\]. However, our data also suggest the possibility that inhibition of cell growth by E/P and retinoids may be independent of p53 pathway in 76N TERT cells. Our data show that the enhancing effects of retinoids on p53gene activation or on the p53 and p21 protein expression were not further augmented by the addition of E/P, unlike their inhibitory effects on cell growth, indicating that other mechanisms besides the p53 pathway are likely to be involved. A p53-independent cell cycle arrest by retinoids has been previously suggested in a number of breast carcinoma cells \[[@B27],[@B32]\]. The p53-independent inhibitory effects of retinoids on cell growth can be exerted through various mechanisms including regulation of other genes that play critical roles in cell cycle progression such as c-myc \[[@B18]\], inhibition of activator protein mediated transcription \[[@B33]\], or induction of caspase-independent cell death via calcium and calpain \[[@B34]\]. However, the mechanisms of E/P-mediated p53-independent cell growth inhibition are still unknown, and are currently under investigation using cell lines with different functional p53 systems. Clearly, our data suggest that there are some overlaps between E/P- and retinoid-mediated growth inhibition in 76N TERT cells, considering that in response to E/P and retinoid treatments, same effectors such as RAREand p53gene, p53 and p21 protein, and the telomerase activity were affected. Additionally, there also seems to be cross-talk between the E/P- and retinoid-mediated growth inhibition. Previous studies have suggested that there is cross-talk between ERβ and RAR pathways \[[@B13],[@B14],[@B35]\]. Here, we demonstrated that the RAREgene activity was increased in response to E/P treatment. Furthermore, for the first time, we showed that treatment of immortal cells with E/P or retinoids could induce the expression of ERβ, both at the mRNA and protein level. The expression of ERβ often is found to be decreased markedly in the early stages of mammary carcinogenesis \[[@B22]\]. Loss of ERβ expression has been suggested as one of the events leading to the development of breast cancer \[[@B36]\]. Our data may reveal another important mechanism by which E/P and retinoids produce their anticancer function, indicating ERβ may represent a possible therapeutic target in breast cancer prevention. More importantly, our data show that the growth-inhibitory effects of retinoids were potentiated by co-treatment with E/P in 76N TERT cells. These observations indicate that different mechanisms may be involved in E/P- and retinoid-mediated inhibition of cell growth. Our results of their differential inhibitory effects on telomerase activity may provide some explanation for this. Although E/P and all three retinoids inhibited telomerase activity, the time courses of their actions were different. While retinoids produced their maximal inhibitory effects around 16 to 24 hours after treatment, E/P required 72 hours to reach its maximal inhibition, suggesting different mediators may be utilized to decrease telomerase activity by E/P and retinoids. ATRA and 9-cis RA have been previously reported to inhibit cell growth and decrease telomerase activity through down-regulation of the expression of hTERT telomerase gene \[[@B37]\]. On the other hand, progesterone treatment has been shown to down-regulate telomerase activity by modulation of cell cycle phases \[[@B38]\]. Previous studies have also provided evidence that the function of p53in suppression of telomerase activity is separable from its cell cycle checkpoint function \[[@B20]\]. Therefore, it is likely that even though E/P and retinoids treatments both activate p53 pathway, they may use different mechanisms to inhibit telomerase activity. The different mediators involved in the inhibitory effects of E/P and retinoids on telomerase activity may contribute to their additive effects on inhibition of 76N TERT cell growth. The detailed mediator mechanisms down-stream of p53 and up-stream of telomerase activity for both E/P and retinoid pathways remain to be defined. Several lines of evidence suggest that the mechanisms through which HPR regulates cell growth seem different than those by 9-cis RA and ATRA in 76N TERT cells. In the \[^3^H\]thymidine incorporation experiments, our results showed that whereas only 25--30% inhibition was observed for 9-cis RA and ATRA, 70% inhibition was reached by HPR. In addition, in the RARE-luciferase activity assay, 9-cis RA and ATRA induce about 3- to 5-fold activation on RAREgene activity. In contrast, HPR treatment resulted in no significant change. Finally, our data showed that 9-cis RA or ATRA treatment caused a moderate inhibitory effect on telomerase activity. In comparison, the telomerase activity is almost completely abolished by HPR treatment. The time courses of their inhibition of telomerase activity were different as well. While 9-cis RA and ATRA maximally inhibited the telomerase activity around 24 hours after treatment, HPR produced its maximal effect at 16 hours post-treatment. An obvious explanation for these different responses observed between 9-cis RA, ATRA and HPR is that these retinoids most likely possess different mechanisms for their actions. As suggested by numerous investigators, 9-cis RA and ATRA may function through the classical retinoid pathways involving the RARs and RXRs. On the other hand, in addition to activation the retinoid receptors \[7a\], HPR may also function through alternative pathways such as down-regulation of the IGF system \[[@B39]\], activation of TGF-beta \[[@B40]\], induction of genes which have antiproliferative activity \[[@B41]\], inhibition of aromatase activity and expression \[[@B42]\], and involvement of cellular signals such as reactive oxygen species \[[@B43]\] and the sphingolipid ceramide \[[@B44]\]. Conclusion ========== In summary, our data demonstrate that 76N TERT cells express RAR, RXR, ER and PR, and represent a potential useful model to investigate the genetic changes, and the carcinogenic or chemopreventive effects of new agents on the development of mammary tumors. In addition, our data clearly suggest that part of the anti-growth effects of E/P is mediated through a p53-dependent pathway, as well as the involvement of the inhibition of telomerase activity and induction of ERβ. Comparing the E/P- and retinoid-mediated inhibitory effects on cell growth, there are overlaps, cross-talks and distinct effectors between these pathways. Furthermore, our studies suggest that retinoids may be more effective when combined with E/P to prevent breast cancer development. This increased potency and sensitized response to retinoids with combined E/P treatment might have several important clinical implications for anti-cancer agents that mimic E/P effects. Firstly, it might allow the currently used RA regimens to show improved response in cancer prevention. Secondly, it may be sufficient to overcome some acquired or intrinsic RA resistance in cancer cells. Finally, it may lower the required does of either classes of anticancer agents used, leading to less side effects or toxicity. Overall, our studies better the understandings of the common and the unique mechanisms by which E/P and retinoids regulate cell growth, and may help us to design or to improve the clinical applications of anti-cancer agents. Methods ======= Chemicals --------- ATRA, 9-cis RA, HPR, β-estradiol and progesterone were all purchased from Sigma (St. Louis, MO, USA) and dissolved in ethanol. The final concentration was 2 μM for ATRA and HPR, 1 μM for 9-cis RA, 1 ng/ml for β-estradiol and 1 μg/ml for progesterone. These concentrations were chosen based on previous studies and are clinically or physiologically relevant \[[@B3],[@B4],[@B12]\]. Culture of 76N TERT cells ------------------------- Cell line was originally supplied and cultured as described by Band et al. \[[@B8]\]. The culture medium D-MEM/F-12, fetal bovine serum, penicillin, streptomycin, and gentamicin were from Gibco (Carlsbad, CA, USA). All the other cell culture reagents were from Sigma (St. Louis, MO, USA). Cells were grown in D-MEM/F-12 mixture (1:1, vol/vol) containing 15 mM HEPES buffer and 2.5 mM L-glutamine, supplemented with 1% fetal bovine serum, 12.5 ng/ml epidermal growth factor, 10 nM triiodothyronine, 50 μM freshly made ascorbic acid, 2 nM estradiol, 1 μg/ml insulin, 2.8 μM hydrocortisone, 0.1 mM ethanolamine, 0.1 mM phosphorylethanolamine, 10 μg/ml transferrin, 15 nM selenite, 1 ng/ml cholera toxin, 35 μg/ml bovine pituitary extract, 100 units/ml penicillin, 100 mg/ml streptomycin, and 20 μg/ml gentamicin. Cells were maintained in 95% humidified air plus 5% CO~2~and sub-cultured weekly. All experiments were performed on cells with passage numbers from 6 to 15. \[^3^H\]Thymidine Incorporation Assay ------------------------------------- Cells were seeded into 24-well plates at 5 × 10^4^cells per well and incubated at 37°C overnight. Cells were then treated in triplicates with indicated retinoids in the presence or absence of E/P for 24 hours. After labelling cells with 1 μCi/ml of \[^3^H\]thymidine (Amersham, Arlington Heights, IL, USA) for 24 hours, cells were harvested by washing with PBS and 10% TCA, solubilizing with the mixture of 0.1% SDS and 0.1N NaOH. Aliquots were taken for the quantification of radioactivity by the Tri-Carb 2900 TR Liquid Scintillation Analyzer (Perkin Elmer, Wellesley, MA, USA). Incorporation of \[^3^H\]thymidine was expressed as a fold change from vehicle control under the same conditions. Luciferase Reporter Assays -------------------------- Cells were seeded in 24-well plates at 5 × 10^4^cells per well. Cells were transiently transfected with 0.5 μg of either a RARE-luciferase or a p53-luciferase plasmid along with 0.05 μg of pCMV-Renilla luciferase using the SuperFect transfection reagent (QIAGEN, Valencia, CA, USA), following the manufacturer\'s recommended procedure. Twenty-four hours after transfection, triplicate cultures were treated with retinoids for 24 h in the presence or absence of E/P. The cells were then washed and lysed. The luciferase activities were measured using the DUAL-luciferase Assay System (Promega, Madison, WI, USA), and normalized by pCMV-Renilla luciferase activity for each sample. Real-Time PCR Telomerase Activity Assay --------------------------------------- Cells were lysed in CHAPS lysis buffer (Chemicon International, Temecula, CA, USA) and incubated at 4°C for 30 min. The lysate was then centrifuged at 12000 × g*for 20 min at 4°C, and the supernatant was collected. The protein concentration was measured in each extract using the BCA protein Assay Reagent Kit (Pierce, Rockford, IL, USA). Telomerase activity was determined in duplicates by a quantitative real-time PCR telomerase detection Kit (Allied Biotech, Ijamsville, MD, USA) according to the manufacturer\'s protocol, using Mx4000 Multiplex Quantitative PCR System (Stratagene, La Jolla, CA, USA). Quantitative RT-PCR for ERβ gene analysis ----------------------------------------- Cells at subconfluence were treated with retinoids in the presence or absence of E/P for 72 hours. Total RNA was extracted with TRIZOL Reagent from Gibco (Carlsbad, CA, USA) according to the instructions of the manufacture. Single-stranded cDNA was made from 1 μg of total RNA with the Cells-to-cDNA kit (Ambion, Inc., Austin, TX, USA) at 42°C for 15 min. The primers for ERβ were 5\'CGA TGC TTT GGT TTG GGT GAT 3\' (forward) and 5\'GCC CTC TTT GCT TTT ACT GTC 3\' (reverse). The primers for GAPDH were 5\'CCA TGG AGA AGG CTG GGG 3\' (forward) and 5\'CAA AGT TGT CAT GGA TGA CC 3\' (reverse). All primers were from Integrated DNA Technologies, Inc. (Coralville, IA, USA). cDNA (1 μl) was amplified in duplicates in Mx4000 Multiplex Quantitative PCR System (Stratagene, La Jolla, CA, USA) by using Brilliant SYBR Green QPCR Master Mix from Stratagene (La Jolla, CA, USA). The reaction was carried out at 95°C for 10 min to denature, 40 cycles of 95°C for 30 sec, 55°C for 60 sec, 72°C for 60 sec. ERβ gene was quantified and normalized with external standard GAPDH. Western blot analysis --------------------- Cells were treated with retinoids in the presence or absence of E/P for 72 hours. Cell lysates were obtained by incubating cells for 30 minutes at 4°C in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP40, 100 mM NaF, 10 mM MgCl~2~, and protease inhibitor cocktail (Sigma, St. Louis, MO, USA), followed by centrifuged at 12,000 rpm for 20 min. Protein content was determined using the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA). Cell lysates (\~30 μg protein) were separated on 10% polyacrylamide gels in the presence of 0.1% SDS with prestained low-range molecular-weight standards (Biorad, Richmond, CA, USA). After transfer, the membranes were blocked and then probed with antibodies against interested proteins as suggested by manufactures, followed by incubation with a peroxidase-conjugated secondary antibody. Immunoreactive bands were developed with an ECL reagent from Amersham (Arlington Heights, Il, USA). All blots were probed with anti-actin to normalize for loading differences. Quantification of gels was carried out using ImageJ software (NIH, Bethesda, Maryland, USA). The p53 (DO-1), p21 (F-5) and RARγ (G-1) mouse monoclonal antibodies, and RXRα (D-20) polyclonal antibody were from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Anti-ERβ (Ab-2), ER (Ab-1), PR (Ab-1), RARα and RARβ mouse monoclonal antibodies, and Bax (Ab-1) polyclonal antibody were from Calbiochem (San Diego, CA, USA). Monoclonal anti-actin, RXRβ and RXRγ were from Sigma (St Louis, MO, USA). Statistical Analysis -------------------- Statistical differences were analyzed by T-test. Levels of statistical significance were set at p \< 0.05. List of abbreviations ===================== 9-cis RA: 9-cis-retinoic acid; ATRA: all-trans-retinoic acid; E/P: ?β-estradiol and progesterone; ERβ: estrogen receptor beta; HPR: N-(4-hydroxyphenyl) retinamide; PR: progesterone receptors; RARs: retinoic acid receptors; RXRs: retinoid X receptors. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= JZ designed and carried out most of the assays and drafted the manuscript. YT carried out the real-time PCR experiments. SS conceived of the study and participated in its coordination. All authors read and approved the final manuscript. Acknowledgements ================ We are grateful that Dr. Vimla Band (Evanston Northwestern Healthcare Research Institute, Evanston, IL) generously provides us the 76N TERT cells. We thank the helpful suggestions from Dr. Rong Shao (BMC, Springfield, MA) and Dr. Joseph Jerry (Umass, Amherst, MA). The tremendous help and support from Dr. Paul Friedmann (Division of Academic Affairs, BMC, Springfield, MA) is greatly appreciated. This project was funded by the Rays of Hope and the Comprehensive Breast Center, Baystate Medical Center, Springfield, MA, USA.	PubMed Central	2024-06-05T03:55:54.153909	2005-3-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555559/", "journal": "Cancer Cell Int. 2005 Mar 8; 5:6", "authors": [ { "first": "Jiahui", "last": "Zhang" }, { "first": "Yifan", "last": "Tu" }, { "first": "Sallie", "last": "Smith-Schneider" } ] }
PMC555560	Background ========== The incidence of intrapartum asphyxia was recently determined to be 2.5% in a large population of singleton pregnancies \[[@B1]\]. Even so, the occurrence of adverse neurological outcome following delivery is actually much lower. There are undoubtedly a number of reasons why greater numbers of infants do not suffer lasting neurological impairment, but one of the reasons may be the formation of new cells in the damaged tissue. Recent studies have demonstrated the presence of multipotent neural stem cells in the subventricular zone and the subgranular zone of the hippocampus \[[@B2]\]. Hippocampal stem cells from the adult brain have been demonstrated to proliferate and differentiate into neurons, astrocytes, and oligodendrocytes \[[@B3]\], as a response to multiple factors, including hypoxic-ischemic (H-I) injury, or trauma \[[@B4],[@B5]\]. New neuronal growth has been demonstrated in several studies of adult H-I models, including in humans \[[@B6]-[@B10]\], but this finding has received less attention in neonatal animals. The pluripotent stem cells found in the neonatal brain have not been characterized as completely as those in the adult brain. As this process may be relevant to the recovery of the neonatal brain from H-I injury, we sought to determine whether new cells arose in the neonatal brain following this type of injury. Bromodeoxyuridine (BrdU) is a thymidine analog incorporated into the DNA of dividing cells, rendering them detectable by immunohistochemical means \[[@B11]\]. We employed a neonatal mouse H-I brain injury model with BrdU injection to determine what types of BrdU-positive cells are present in the hippocampus after an H-I event and to determine if this response if different than that due to hypoxia alone. Results ======= Figure [2](#F2){ref-type="fig"} shows the area of damage produced in the affected hippocampus. The majority of the BrdU positive/NeuN positive cells were found in the granule cell layer on the experimental side. The side of the hippocampus contralateral to ligation, although also exposed to hypoxia, did not demonstrate the same degree of injury, remaining histologically normal in appearance. The finding that common carotid artery occlusion or hypoxia alone does not cause apparent brain damage has been previously reported \[[@B12],[@B13]\]. Thus, we compared the hypoxia alone side of the brain to the hypoxic-ischemic side of the brain in the statistical analyses. Regional differences -------------------- The DG on the hypoxic-ischemic side of the brain exhibited significantly higher numbers of BrdU-positive cells at P10, P17, and P42 than either CA1 or CA3. BrdU/RCA-positive cells were significantly higher at P10 in the DG. The morphology and location of the BrdU/RCA-positive cells suggested that they were endothelial or microglial cells. BrdU/NeuN-positive cells were higher at P17 and P42 in the DG (Table [1](#T1){ref-type="table"}). Hypoxia alone versus hypoxic-ischemic side differences ------------------------------------------------------ Table [2](#T2){ref-type="table"} shows the trends in the DG at the three sacrifice times for hypoxia alone and hypoxic-ischemic sides of the brain. BrdU-positive cells without co-labeling and BrdU/RCA-positive cells occur first after the H-I injury (significant increase at P10), followed by BrdU/NeuN-positive cells (significant increase at P42). Colabeling of BrdU and NeuN was confirmed by confocal microscopy (Figure [2](#F2){ref-type="fig"}). For BrdU/CNPase-positive cells the only significant difference was seen in the DG at 10 days, where the hypoxia alone side was significantly higher (7.0 ± 24.2 v. 0.1 ± 0.3, p \< 0.0002). No significant differences were seen in the BrdU/GFAP-positive cells when the hypoxic-ischemic side was compared with the hypoxia alone side. Both GFAP positive and CNPase positive cells without BrdU staining were seen in appropriate areas. Discussion ========== The hippocampus has been the focus of much research involving H-I injury, both for its susceptibility to H-I injury and its regenerative capacity due to the adult neural stem cells found there. The bulk of this work has been compiled using adult models of H-I injury. In the neonatal mouse, the degree of damage to the hippocampus from this type of injury has been found to be variable, based on multiple factors, including the strain of mouse used \[[@B16]\]. In addition, the timing of the insult has been demonstrated to affect the severity of damage. The mouse hippocampus is remarkably resistant to H-I injury at 2--3 post-natal days but becomes progressively vulnerable, and by age 13 post-natal days hippocampal damage exceeds that of cortex \[[@B16]\]. Scheepens et al \[[@B17]\] found an increase in proliferation of hippocampal cells following global asphyxia in the newborn rat, much as we found in the model we used, but these investigators did not assess maturation of the proliferating cells into neurons and other cell types. In one study neonatal hypoxia alone triggered neurogenesis in a neonatal rat model \[[@B18]\]. The changes they noted were mainly in the CA1 region, as opposed to the DG, where we found the primary changes. Because the hippocampus is vulnerable to H-I damage and has the potential for neurogenesis, yet has not been extensively examined in a neonatal model, the hippocampus was the focus for this study. We can summarize the regenerative process as follows: the initial phase is characterized by comparatively large numbers of incompletely differentiated cells, primarily in the DG (10 days of age or 3 days after injury), followed rapidly by the appearance of BrdU/RCA-positive cells, again mainly in the DG, and then by BrdU/NeuN-positive cells (putative neurons) developing only in the DG (at 42 days of age). There were minimal numbers of BrdU/GFAP-positive cells observed at all points. Ischemia has been shown to increase neurogenesis in several adult animal models of brain ischemia. Following global cerebral ischemia in adult gerbils, neurogenesis has been shown to increase in the hippocampal SGZ, while newly born neurons were demonstrated in the CA1 pyramidal cell layer \[[@B8]\]. In another study of adult gerbils, after 10 minutes of bilateral carotid occlusion, newborn cells with a neuronal phenotype were first seen 26 days after injury \[[@B5]\]. In the adult rat, ischemia has been shown to increase the incorporation of BrdU-positive cells co-expressing neuronal markers in both the DG and the SVZ \[[@B7]\]. Furthermore, this effect was more prominent on the side of ischemic insult \[[@B7]\]. Similar studies in the adult rat demonstrate that up to 60 percent of the new cells found after this type of ischemic brain damage demonstrate neuronal characteristics. In adult mice, after transient forebrain ischemia, BrdU-positive cells have been demonstrated to significantly increase in the DG \[[@B4]\]. In view of the fact that we observed the BrdU/NeuN-positive cells to increase only in the DG, it seems probable that the regenerative process in neonates is similar to that in adult animals. The data strongly suggested that the development of oligodendrocytes (BrdU/CNPase-positive cells) was impaired in the DG on the hypoxic-ischemic side of the brain, with significantly fewer of these cells observed in the hypoxic-ischemic side as compared to the hypoxia alone side at 10 days. This finding is in contradiction to findings reported by Zaidi et al \[[@B19]\], who demonstrated that neonatal H-I injury in rats led to increased numbers of oligodendrocytes ipsilateral to H-I injury. There are several possible explanations for this discrepancy. In our study, oligodendrocytes were found to be decreased on the side of the brain ipsilateral to carotid ligation at P10. In the Zaidi study, they were found to be increased at P35. It is possible that oligodendrocytes proliferate at the site of damage after P10. It is also possible that the oligodendrocytes are at the site of damage at P10, but are not mature enough to stain with CNPase. Lastly, the Zaidi group evaluated the SVZ, while we reported dual staining in the hippocampus. There could be regional differences between the two. White matter damage in neonates is thought to be in part due to vulnerability of the immature oligodendrocyte to H-I damage \[[@B20]\]. In several neonatal animal models, oligodendrocytes have been demonstrated to be susceptible to H-I injury \[[@B21]-[@B24]\]. This effect seems to be dependent on both the degree of H-I damage \[[@B24]\] and the timing of the injury, with late oligodendrocyte progenitors being more susceptible than early oligodendrocyte precursors or more mature oligodendrocytes \[[@B25]\]. Our results were consistent with these reports. This window of vulnerability of the oligodendrocyte coincides with the human population at high risk for the development of PVL. This vulnerability of the immature oligodendrocytes might explain the paucity of new, mature oligodendrocytes in our model on the hypoxic-ischemic side \[[@B26]\]. We found a significant, early increase in BrdU-positive cells expressing RCA, an endothelial cell marker and microglial marker, on the side of the brain affected by hypoxic-ischemic injury. The mechanisms of angiogenesis in the brain after H-I injury are not well understood, and there is also little known about angiogenesis in neonatal animal models. Vascular endothelial growth factor (VEGF) is endothelial cell specific and has been implicated in hypoxia-mediated angiogenesis \[[@B27]\]. VEGF has also been demonstrated in microglial cells after cerebral infarct in adult rats \[[@B27]\]. In the rat brain, angiogenesis is not completed until about post-natal day 20 \[[@B28]\]. Therefore, it remains a possibility that the increase in cells co-labeling for BrdU and RCA after H-I injury in our study partly represents the normal brain maturation process. However, the increase of these cells on the hypoxic-ischemic side of the brain would imply that the injury led to this effect. Further studies will be needed to more accurately identify if there are changes in angiogenesis following H-I injury. The majority of these BrdU positive/RCA positive cells appeared morphologically to be activated microglia, which are thought to play a role in the damage to immature oligodendrocytes \[[@B20]\]. The present study did not demonstrate an increase in BrdU-positive cells co-labeling for an astrocyte marker, GFAP, after H-I injury. Astrocytes have been demonstrated to express apoptotic enzymes within hours after H-I injury in neonatal rats \[[@B29]\]. In neonatal piglets, GFAP-positive cell bodies were reduced by roughly 50% at 48 hours after H-I injury, but subsets of astrocytes were subsequently shown to proliferate later after the insult \[[@B30]\]. In adult rats, the number of astrocytes was shown to remain unchanged after traumatic brain injury in one study \[[@B31]\], while in another similar study, there was loss of GFAP reactivity in the ipsilateral CA3 region of the hippocampus 30 minutes after injury with progressive astrocyte loss over the next 24 hours \[[@B32]\]. As evidenced by the variability reported in the astrocytic response to H-I brain injury, the response of this cell in this environment needs to be better characterized. Because astrocytes help to maintain the extracellular milieu needed for neuronal formation and function, it is possible that their early loss after H-I injury may contribute to subsequent neuronal degeneration. Future studies in our neonatal mouse model could be geared towards better understanding the early response of astrocytes. Postponing evaluation until three days post-injury in this model likely resulted in underestimation of the astrocytes\' role in this injury repair process. Chronic hypoxia has been demonstrated to induce astrocytes into more immature phenotypes, which do not express normal levels of GFAP \[[@B33]\]. The ability of acute hypoxia to cause this effect is not well understood. Therefore, it remains a possibility that evaluating for immature astrocytes in this model would have resulted in the visualization of more astrocytic cells. Although the BrdU-positive cells in our study were co-labeled with stains identifying four separate types of cells, there were many BrdU-positive cells that were not identified by these four stains. The co-labeling studies we performed accounted for only about 20% of all the BrdU-positive cells. The BrdU labeling protocol we used consisted of BrdU injections over the course of the experiment, rather than a one time injection at the time of H-I injury, which resulted in the staining of more cells than would be found following a single injection. In this manner, we were able to evaluate how each cell type varied its number and location at different intervals after the injury. This provided information about the cellular response to hypoxic-ischemic brain injury in both the acute and long-term settings. The role and identity of the non-co-labeled cells remains unclear. It is probable that these were immature cell types, not yet expressing the markers for which we evaluated. The validity of cells staining for BrdU representing new or dividing cells versus apoptotic or necrotic cells is also an issue to be considered. As a result, TUNEL staining was performed on several hippocampal sections. At P10, three days after H-I injury, TUNEL staining does reveal apoptotic and necrotic nuclei. However, by P42, this finding is virtually non-existent, suggesting that any BrdU-positive cells are present due to cell proliferation or growth. (See Figures [4](#F4){ref-type="fig"} and [5](#F5){ref-type="fig"}.) Previous studies have indicated that neurons subjected to H-I injury do, indeed, incorporate BrdU, but that these injured neurons which undergo apoptosis have disappeared by 28 days \[[@B34]\]. A possible criticism of the present study is that we did not count the cells with stereological methods. As a result, our findings might be due to increased cell density due to surrounding tissue loss as opposed to an increase in the absolute number of new cells present. However, the cell counts were conducted in non-overlapping sections within a very short segment of the anterior hippocampus, in which the sections\' thickness remained constant. No gross differences were observable in brain size in either the coronal or sagittal planes. Furthermore, hippocampal volume on both the hypoxia alone and hypoxic-ischemic sides of the brain did not vary noticeably histologically. Lastly, confocal imaging revealed cell density on each side of the brain to be roughly equivalent, although exact volumetric measurements can not be made without stereologic techniques. Conclusion ========== Neuronal and other cell regeneration was significantly increased in the DG on the side of the brain exposed to H-I injury, as opposed to hypoxic injury alone. This study addresses the neonatal mouse brain\'s ability to repair H-I damage and can serve as a platform to determine if interventions can be made to augment these inherent repair mechanisms. The rate of neuronal regeneration found in our study, while showing a significant increase in the DG, was not robust. Perhaps the stimulus was insufficient to result in a great degree of neurogenesis. The degree of brain injury was not stratified in this study. Neurogenesis might prove to be proportional to the degree of injury. It is also possible that this regeneration could increase further with time, but this is unlikely because new cell production was decreasing by the final time point after the injury in our study. Stem cell transplantation or manipulations of factors affecting intrinsic stem cell activity could be applied to this system in the future. Methods ======= Animal procedures ----------------- This study was performed in accordance with the guidelines provided by the Laboratory Animal Studies Committee of the Veterans Administration Hospital in Augusta, GA. Under 2% isoflurane anesthesia C57 BL/6 mouse pups underwent permanent ligation of the left common carotid artery at post-natal day of life seven (P7). The pups were then placed with the dams for two hours prior to placement in an 8% oxygen chamber partially immersed in a water bath at 37° for 75 minutes. This procedure was described previously in the rat by Levine \[[@B12]\] and Rice et al\[[@B13]\], and then adapted to the mouse by Sheldon et al\[[@B14]\]. Within the literature, there is variation of the neonatal mouse H-I brain injury protocol. Our seventy-five minute hypoxia time was determined by exposing animals to varying intervals of hypoxia. In our laboratory, seventy-five minutes of hypoxia time resulted in reliable, reproducible brain injury, with minimal animal death. Any animal that died during surgery, while in the hypoxia chamber, or prior to sacrifice time was excluded. The time at which the animal would be sacrificed was determined prior to surgery. Forty-eight hours after the H-I injury, the pups were injected intraperitoneally with BrdU, at a dose of 100 mg/kg. This dose was administered twice weekly until the pups were sacrificed at 3, 10, or 35 days after the injury (see Figure [1](#F1){ref-type="fig"}) with 70 mg/kg of ketamine and 15 mg/kg of xylazine, and tissue fixation done by transcardiac perfusion with 0.9% saline, followed by 4% paraformaldehyde in PBS. BrdU injection was continued twice weekly after injury until the time of sacrifice in order to elucidate if the types of new cells found in the hippocampus varied at different time intervals after the injury. This protocol allowed evaluation of an accumulation of cells, reflecting both the acute and long-term cellular response to hypoxic and hypoxic-ischemic injury. Brains were removed and postfixed in 4% paraformaldehyde for 4 hours, cut into 3--5 mm sections, and postfixed in 4% paraformaldehyde for an additional 20 hours. Tissue was then dehydrated in an ethanol series, cleared in xylene, and infiltrated and embedded in PolyFin Embedding and Infiltration Wax (TBS Biomedical Sciences, Durham, NC). Paraffin embedded tissue was sectioned to 5 μ thickness and mounted on Fisher Superfrost Plus slides (Fisher Scientific). Immunohistochemistry and cell counting -------------------------------------- Paraffin was removed from slides using xylene, followed by rehydration in an alcohol dilution series. Slides were soaked in 0.1% Triton-X 100 in PBS for ten minutes to increase permeability of fixed tissue, followed by rinsing in 1X PBS. Antigen retrieval was performed using a microwave method. Slides were incubated for twenty minutes after slow boiling for ten minutes and then rinsed in PBS and blocked using 2% normal calf serum for 20 minutes. Sheep anti-BrdU antibody (Biodesign \#M20105S), diluted 1:200 in PBS, was selected for the visualization of new cells. Co-labeling runs used mouse anti-NeuN (Chemicon \#MAB377) 1:2000 for the visualization of neurons, rabbit anti-GFAP (DAKO \# Z0334), diluted 1:300 in PBS for the visualization of astrocytes, biotinylated RCA (Vector \#B-1085) diluted 1:300, for the visualization of endothelial cells and microglia cells, and CNPase (Sigma \#C-5922), diluted 1:200, for the visualization of oligodendrocytes. All primary antibodies except NeuN were allowed to incubate at room temperature for one hour. Primary NeuN antibody was allowed to incubate for thirty minutes at room temperature, and washed three times in a 1X PBS solution. Biotinylated anti-mouse antibody (Vector EliteABC Kit \#PK-6102), diluted 1:200 in 1.5% normal horse serum, was applied to each section and incubated for thirty minutes at room temperature. Slides were washed in a PBS series before applying Vector EliteABC reagent and incubation for thirty minutes at room temperature. Sections were washed again in PBS and TSA amplification reagent (NEN \#SAT700) with biotinylated tyramide 1:200 in amplification buffer was applied. Sections were incubated for eight minutes at room temperature. Preliminary staining demonstrated that only NeuN antibody required amplification. All other antibodies were used in an indirect staining method, with a secondary, fluorescent antibody. Cy3 anti-Sheep (Jackson \# 713-165-147), diluted 1:400, was used to visualize BrdU in all cases. FITC anti-rabbit (Jackson \# 711-095-152), diluted 1:100, was used to visualize GFAP. Strepavidin FITC (Jackson \#016-010-084), diluted 1:100, was used to visualize biotinylated RCA and NeuN. FITC anti-mouse (Jackson \#715-095-151), diluted 1:100, was used to visualize CNPase. To reduce background, slides were rinsed in 4 changes of PBS for 5 minutes each. Secondary antibodies were applied and incubated for 1 hour. Slides were rinsed in PBS, and immersed in Hoechst Stain for 8 minutes. Another rinse series in PBS was applied before coverslipping, using Vectashield Mounting medium. (Vector \#H-1000) Cells were counted in 4--6 animals at each time interval. For each animal, the cells were counted from the injury area in the anterior hippocampus (equivalent adult mouse interaural coordinates 2.10 to 1.98 mm) \[[@B15]\] in CA1, CA3, and DG. (In Figure [2](#F2){ref-type="fig"}, the circles show the specific fields, CA1, CA3, and the DG, counted under one 40× magnification field in each section of brain. Corresponding areas of hippocampus were estimated in each section of brain when choosing the 40X field.) At least three brain sections from each animal for each stain type were counted. Therefore, each brain had three CA1, three CA3, and three DG 40X fields stained and counted with BrdU, one of the four co-labeling cell markers, and Hoescht stain. In each case, the same observer performed the cell counting in all brain sections. The counter could not be blinded due to the loss of normal architecture on the side of the brain consistent with H-I injury. A separate observer did count numerous sections in order to ensure consistency and accuracy. Each 40X field counted by separate individuals was found to have consistent data. Counting was completed by counting the total number of cells labeled for BrdU only and for BrdU co-labeled with one of the four the cell markers found in one field under 40× magnification in each of the three regions in the hippocampus, on both the hypoxia alone and hypoxic-ischemic side of the brain. Due to the large size of the hippocampus and the H-I injury, analysis was confined to one level of the brain cut through the middle of the injury. Any BrdU-only cells with abnormal morphology and any co-labeled cells were confirmed by corresponding Hoescht staining. Confocal imaging ---------------- To confirm dual labeling, confocal imaging was performed in selected cells with a Zeiss 510 laser scanning confocal microscope with the use of Zeiss software. The objective used was Zeiss63x C-Apochromat 1.2 NA. To excite the FITC fluorochrome (green), a 488-nm laser line generated by an argon laser was used, and for the Cy3 fluorochrome (red), a 543-nm laser line from a HeNe laser was used. Filter sets used were a bandpass 500- to 600- filter (\"green\" channel) and a long-pass 585- to 650-nm filter (\"red\" channel). We identified cells that were labeled for BrdU and the neuronal marker NeuN. These cells were then examined by confocal microscopy, and 1-μm step Z series were obtained. (See Figure [3](#F3){ref-type="fig"}.) TUNEL staining -------------- Staining for the presence of apoptotic cells was completed using the ApopTag Peroxidase Kit (Chemicon \#S7100), according to manufacturer recommendations. Therefore, after rehydration, the tissue was treated with Proteinase K (20 μg/mL) and incubated for 15 minutes. Endogenous peroxidases were quenched with hydrogen peroxide. Sections were then treated with equilibration buffer and TdT enzyme treatment. Next, the sections were stained with anti-dioxigenin peroxidase conjugate for 30 minutes, and the slides were developed with stable DAB. Statistical analyses -------------------- Nested, repeated measures analysis of variance was used to examine differences in number of cells between times of sacrifice (10, 17, or 42 days), side of brain (hypoxia alone or hypoxic-ischemic injury), region of the hippocampus (CA1, CA3, DG) and antibody type (CNPase, GFAP, NeuN, and RCA). A second analysis was performed using only BrdU-positive cells. Post hoc analyses were performed using the adjusted least square means and applying a Bonferroni correction to the overall alpha level. Statistical significance was assessed using an alpha level of 0.05. All analyses were performed using SAS 8.2. Abbreviations ============= hypoxic-ischemic -- H-I, bromodeoxyuridine -- BrdU, subventricular zone -- SVZ, subgranular zone -- SGZ, dentate gyrus -- DG, glial fibrillary acidic protein -- GFAP, 2\'3\'-cyclic nucleotide 3\'-phosphodiesterase -CNPase, Ricinus communisagglutinin I --s RCA, neuronal nuclear marker -- NeuN Acknowledgements ================ James Carroll, MD, NS43439-02; David Hess, MD, NS43487-02, VA Merit Review, William Hill, PhD, VA Merit Review Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Schematic diagram showing BrdU injection schedule ::: ![](1471-2202-6-15-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Hippocampal injury following hypoxic-ischemic injury in a P17 mouse. A -- HI injury (small arrows), triple filter 100X; B -- colocalization with NeuN and BrdU, triple filter, 400X; C -- BrdU label alone, Cy3 filter, 400X; D -- NeuN label alone, FITC filter, 400X; and E -- Hoechst counterstain, DAPI filter, 400X. B through E shows the same field. The large arrows show a neuron with BrdU and NeuN staining, demonstrating that the cell has recently divided. The circles show the three areas where the counting was performed. ::: ![](1471-2202-6-15-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Three dimensional reconstructed confocal microscopy demonstrating colabeling BrdU positive and NeuN positive cell in granule cell layer of HI injured hippocampus (P42). Orthogonal view. The neurons are labeled with NeuN (green), and the BrdU positive cells are shown in red. The square is a the Z slice, the superior rectangle is the X slice, and the right rectangle of the Y slice. A and B show two different cells, both co-labeled with BrdU and NeuN. ::: ![](1471-2202-6-15-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### 200× magnification of TUNEL staining developed with stable DAB in the injured hippocampus of a ten day old animal (P10), showing a small amount of label. Brown coloration indicates 3\'OH DNA termini (apoptotic or necrotic cells). ::: ![](1471-2202-6-15-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### 200× magnification of TUNEL staining developed with stable DAB in the injured hippocampus of a 42 day old animal (P42), showing no labeling of apoptotic or necrotic cells. Brown coloration indicates 3\'OH DNA termini (apoptotic or necrotic cells). ::: ![](1471-2202-6-15-5) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Regional differences in injured side: Numbers of cells ::: Age at sacrifice: stain CA1 CA3 DG ------------------------- -------------- -------------- ----------------- P10: BrdU 120.6 ± 39.7 109.7 ± 37.4 154.5 ± 59.6\* P10: BrdU/RCA 19.8 ± 12.2 15.1 ± 6.1 30.5 ± 17.8\\ P10: BrdU/NeuN 0 0 0.2 ± 0.6 P17: BrdU 61.4 ± 21.7 49.6 ± 17.1 98.7 ± 26.2\* P17: BrdU/RCA 12.5 ± 4.1 6.2 ± 4.5 8.6 ± 3.8 P17: BrdU/NeuN 0.4 ± 0.7 0.3 ± 0.6 11.6 ± 4.4\\ P42: BrdU 31.7 ± 19.4 35.0 ± 21.6 68.9 ± 23.4\* P42: BrdU/RCA 5.7 ± 5.8 2.7 ± 2.0 6.3 ± 4.5 P42: BrdU/NeuN 0 0.3 ± 0.8 15.0 ± 4.6\\ \Significantly greater than CA1 or CA3, p \< 0.0011 \\Significantly greater than CA1 or CA3, p \< 0.0002 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Injured versus non-injured dentate gyrus side differences: Numbers of cells ::: Stain: Side of brain P10 P17 P42 ---------------------- ----------------- --------------- ---------------- BrdU Injured 154.5 ± 59.6\ 98.7 ± 26.2 68.9 ± 23.4\* Non-injured 92.9 ± 32.7 86.7 ± 21.5 52.4 ± 17.1 BrdU/RCA Injured 30.5 ± 17.8\\ 8.6 ± 3.8\\ 6.3 ± 4.5 Non-injured 2.7 ± 2.6 2.4 ± 2.4 1.1 ± 1.1 BrdU/NeuN Injured 0.2 ± 0.6 11.6 ± 4.4 15.0 ± 4.6\\ Non-injured 0.1 ± 0.3 6.6 ± 3.2 5.2 ± 1.6 \Significantly greater than control, p \< 0.0011 \\*Significantly greater than control, p \< 0.0002 :::	PubMed Central	2024-06-05T03:55:54.156389	2005-3-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555560/", "journal": "BMC Neurosci. 2005 Mar 2; 6:15", "authors": [ { "first": "John", "last": "Bartley" }, { "first": "Thomas", "last": "Soltau" }, { "first": "Hereward", "last": "Wimborne" }, { "first": "Sunjun", "last": "Kim" }, { "first": "Angeline", "last": "Martin-Studdard" }, { "first": "David", "last": "Hess" }, { "first": "William", "last": "Hill" }, { "first": "Jennifer", "last": "Waller" }, { "first": "James", "last": "Carroll" } ] }
PMC555561	Background ========== Long-term measurement of human movement in the field is an important need today \[[@B1]\]. For many types of rehabilitation treatment, it is desirable to monitor a patient\'s activities of daily life continuously in the home environment, outside the artificial environment of a laboratory or doctor\'s office \[[@B2]\]. This type of monitoring is quite beneficial to the therapist, allowing a better assessment of human motor control, and tremor or functional use of a body segment, over long periods of time \[[@B1]\]. Evaluating a patient\'s daily life activities allows a more reliable assessment of a patient\'s disabilities, and aids in developing rehabilitation treatments and programs, as well as assessing a treatment\'s effectiveness \[[@B2],[@B3]\]. In addition, the recognition of deviations in joint movement patterns is essential for rehabilitation specialists to select and implement an appropriate rehabilitation protocol for an individual \[[@B4],[@B5]\]. Many specific medical applications benefit from the information provided by continuous human movement monitoring. To better develop and optimize total joint replacements, for instance, a detailed record of a patient\'s daily activities after such a replacement is required \[[@B6]\]. The measurement of tremor and motor activity in neurological patients has long been studied \[[@B7]\]. In pulmonary patients, it is often desirable to precisely quantify the amount of walking and exercise performed during daily living, since this is a fundamental goal in improving physical functioning and life quality \[[@B3]\]. Furthermore, physiological responses, such as changes in heart rate or blood pressure, often result from changes in body position or activity, making the assessment of posture and motion an essential issue in any type of continuous, ambulatory monitoring \[[@B8]\]. Presently, there is no satisfactory solution for long-term, human movement monitoring in the field. The use of video and optical motion analysis systems offer the most precise evaluation of human motion, but obviously restrict measurements to a finite volume \[[@B9]\]. Body mounted sensors such as accelerometers and pedometers are used for monitoring daily physical activity, but those devices are unable to detect the body posture and are often limited in reliability and applicability \[[@B3],[@B7]\]. Even methods of self-report designed to gather information on general daily activity, such as diaries or questionnaires, are time consuming and often unreliable, especially for the elderly relying on their memory \[[@B3]\]. Electrogoniometers are frequently used to measure dynamic, multi-axis joint angle changes in individuals, providing continuous joint movement information. These devices, however, are not desirable for long-term monitoring of daily living, since they are exoskeletal devices that cross the joint, potentially interfering with movement. Furthermore, any shift from their original placement leads to errors in angle estimations \[[@B2]\]. Such commercially available goniometers can produce erratic readings once the device is detached from the patient body and put back on the same joint in a slightly different orientation. It is therefore difficult to use these goniometers at home for long periods of time. Other types of goniometric devices have been developed for measuring particular parts of the body. Electronic gloves \[[@B10]-[@B13]\], for example, can measure the hand posture accurately, but are often cumbersome to wear for long periods of time. Various types of textile fabrics with integrated sensing devices have also been devised \[[@B14],[@B15]\]. In each of these cases, the sensing devices are traditional strain gauges, carefully attached to an article of clothing. One patented device uses conductive fabrics acting as strain gauges on a garment to emit \"effects\" such as light or sound based upon a wearer\'s movements \[[@B16]\]. While this is a novel wearable device, it is not designed, nor is suitable, for long-term accurate joint angle measurement. For all types of body-mounted sensors, the issues of comfort and wearability are of major importance, if a patient has to wear the monitoring device for extended periods of time. Furthermore, such home-use wearable sensors need to be put on and off every day without close supervision of a medical professional. Proper registration of the sensor is therefore a crucial requirement for deploying wearable sensors to the home environment. The goal of this paper is therefore to develop a new method for continuous monitoring of human movement by measuring single or multi-axis joint angles with a wearable sensing garment that is non-intrusive and non-cumbersome and that can be properly registered for reliable monitoring. A new method is presented here for joint monitoring using conductive fibers incorporated into comfortable, flexible fabrics. All that is needed is a one-time calibration with a standard goniometer, and a conductive fiber sensor garment is then able to continuously detect joint movement and measure specific single or multi-axis joint angles. With an array of sensors incorporated into a sensing garment, registration of the sensor occurs automatically each time the garment is worn through only a few simple motions by the wearer. This type of wearable sensor would allow extended home monitoring of a patient, and is no harder to put on than a typical article of clothing. In the following, the principle and design details of this wearable device will be presented, along with effective algorithms for allowing a patient to perform long-term, unsupervised monitoring in the home environment. Experimental feasibility tests will also be presented on a prototype wearable sensor for both single-axis and multi-axis joints. Methods ======= Working Principle ----------------- The basic principle behind the wearable sensors presented in this paper is as follows: when a particular joint on the human body moves, skin around the joint stretches, along with any clothing surrounding the joint as well. A former study by the textile industry has shown that body movements about joints require specific amounts of skin extension. Lengthwise across the knee for example, the skin stretches anywhere from 35--45% during normal joint movement \[[@B17]\]. When a particular joint moves, fabric around the joint will either expand or contract accordingly, assuming the fabric is form fitting to the skin, and has the necessary elastic properties. To assure comfort and freedom of body movement, stretchability of 25 to 30 percent is recommended for fabrics fitting closely to the body \[[@B17]\]. By incorporating conductive fibers into such a fabric surrounding a joint, the conductive materials will necessarily change length with joint movement. The electrical resistance of the conductive material will change as well, and can be directly measured and correlated to changes in the orientation of the joint. Figure [1](#F1){ref-type="fig"} shows how a single conductive fiber is implemented as a sensor. One end of the conductive fiber is permanently attached to the nonconductive, form-fitting fabric substrate at point Ain the figure. Along the conductive fiber, there is a wire contact point at B that is permanently stitched into the fabric. The other end of the conductive fiber, point C, is kept in tension by a coupled elastic cord, which is permanently attached to the remote side of the joint, point D. Therefore, any stretching in this coupled material will take place in the highly elastic cord, CD, and not in the conductive fiber AC. As the joint moves, the elastic cord will change length, causing the coupled conductive fiber to freely slide past the wire contact point at Bthat is stationary. The conductive fiber always keeps an electrical contact with this wire, but the length of conductive thread between points Aand Bwill change as the joint rotates. The resistance, which is linearly related to length, is then measured continuously across these two points Aand B. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Sensor Design Schematic.This particular sensor arrangement shows one sensor thread running lengthwise across a single-axis knee joint. ::: ![](1743-0003-2-7-1) ::: Predictor Design ---------------- Consider Figure [2](#F2){ref-type="fig"}. Shown here are a sensor spanning across a single axis knee joint, and a pair of sensors about a double axis hip joint. The angles of interest are labeled θ~1~, θ~2~, and θ~3~. Our goal is to estimate these joint angles based upon the output of sensors 1, 2, and 3. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Lower Body Sensors.Schematic of three sensors positioned to measure three lower body joint angles. ::: ![](1743-0003-2-7-2) ::: Preliminary experiments have shown a clear relationship between joint angle and sensor output for individual sensors about various joints of the body. Figure [3](#F3){ref-type="fig"}, for instance, shows a typical set of output data from a single sensor thread across a single-axis knee joint with the output \"zeroed\" for a joint angle of 0°. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Sensor Output Curve.Preliminary data showing sensor output vs. knee flexion angle. ::: ![](1743-0003-2-7-3) ::: It is desired to design a filter that receives sensor signals as inputs, and predicts the joint angle(s) of interest. In the proposed method, each joint angle being monitored has a corresponding single sensor that is situated about that particular joint for maximum sensitivity, as in Figure [2](#F2){ref-type="fig"}. Consider Naxis sensors for measuring Njoints, each consisting of a single thread sensor, as shown in Figure [2](#F2){ref-type="fig"}. The simplest predictor model that can be used is a linear regression: ![](1743-0003-2-7-i1.gif) where ![](1743-0003-2-7-i2.gif) is the N× 1 vector of Njoint angle predictions, ![](1743-0003-2-7-i3.gif) is its bias term, y= (y~1~... y~N~)^T^is the N× 1 vector of corresponding sensor readings, and Gand ![](1743-0003-2-7-i3.gif) are, respectively, the N× Nmatrix and the N × 1 vector experimentally determined to relate the inputs and the outputs. Since there is a slight amount of curvature in the preliminary data of Figure [3](#F3){ref-type="fig"}, a nonlinear predictor may be more effective. We will use a second order polynomial model ![](1743-0003-2-7-i4.gif) where ![](1743-0003-2-7-i5.gif) and G\' is an N× N(N-1)/2 experimentally determined matrix. The three terms on the right hand side of the above equation can be incorporated into a homogeneous expression using augmented matrix and vector: ![](1743-0003-2-7-i6.gif) where Wand Yare W= (θ~0~GG\') (5) ![](1743-0003-2-7-i7.gif) To determine the parameter matrix W, a least squares regression is performed using msets of experimental data from a collection of sensors on an individual patient. Let Pbe a N× mmatrix consisting of msets of experimentally measured joint angles, ![](1743-0003-2-7-i8.gif) and Bbe a {1 + N(N+ 1)/2} × mmatrix containing the corresponding sensor outputs and their quadratic terms: B= (Y^(1)^... Y^(m)^) (8) The optimal regression coefficient matrix W\* that minimizes the squared prediction errors is given by W\* = PB^T^(BB^T^)^-1^ (9) if the data are rich enough to make the matrix product BB^T^non-singular. The above expressions are the most general forms for Naxis sensors. In practice, however, they can be reduced to a compact expression with lower orders. First the offset ![](1743-0003-2-7-i3.gif) can be eliminated from the coefficient matrix W, if the sensor outputs are zeroed at a particular posture, e.g. the one where the extremities are fully extended. Second, although the matrix Gcontains off-diagonal elements representing cross couplings among multiple joints, some joints have no cross coupling with other joints. For example, the measurement of the knee joint can be performed separately from that of the hip joints. If the j-th joint is decoupled from all others, it can be treated separately as: ![](1743-0003-2-7-i9.gif) where the offset is eliminated. Third, although multiple joints are coupled to each other having non-zero, first-order off-diagonal coefficients in matrix G, their second-order cross coupling terms, e.g. y~j~y~k~, can be negligibly small with proper design of individual sensors. In such a case, two coupled joints, say jand k, can be written as: ![](1743-0003-2-7-i10.gif) where the offset terms have been eliminated. Thus the number of parameters to identify through calibration experiments is reduced. In consequence, the dimension of the optimal coefficient matrix must be reduced accordingly. The same calibration procedure is performed for both single axis and multiple axis cases, and need be performed only once for a specific set of sensors on an individual. Although one sensor is sufficient to capture single-axis joint motion, any misalignment of such a sensor from use to use will lead to erroneous measurements. From a practical standpoint, it is obvious that a method is needed to adjust for any shifting of a sensor about the joint that will take place from one use to the next. It is both undesirable and impractical to recalibrate the whole sensor every time the patient takes off the sensing garment and places it back again. To take care of such registration problems, an array of multiple sensor threads is used. By incorporating multiple threads in a known pattern, a template-matching algorithm can be performed to determine a sensor\'s offset from calibration. In this way, measurement errors due to sensor misalignment are significantly reduced. The details of this method are described in the next section. Sensor Registration for Single Axis Joints ------------------------------------------ The goal in designing these wearable sensors is to create a device that is ultimately self-registering for subsequent uses after the initial one-time calibration experiments. This means that no additional equipment is needed to register the sensors for each use. Also, it is important that any procedures that are needed for self-registration are simple, and able to be preformed by the patient without supervision. To achieve these goals, a multi-thread sensor array design is presented. First, consider an array of Msensors covering a single-axis joint as shown in Figure [4(a)](#F4){ref-type="fig"}. Each sensor thread is separated from the adjacent sensor thread by a known, constant distance, d. This multi-thread sensor array is used to estimate a single-axis joint angle, θ~j~. To develop a registration procedure let us first calibrate each sensor thread individually. Let ![](1743-0003-2-7-i11.gif) be the estimate of the j-th joint based on the i-th thread sensor given by ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Sensor Arrays.(a) Array of equidistantly spaced sensors over knee joint. (b) Array shifted by an unknown distance, α. ::: ![](1743-0003-2-7-4) ::: ![](1743-0003-2-7-i12.gif) where ![](1743-0003-2-7-i13.gif) and ![](1743-0003-2-7-i14.gif) is the 1 × 2 regression vector that is optimized for the i-th single-thread sensor of the j-th joint placed at a home position. Now consider the situation where the sensor array has been removed, and placed back on the joint for more measurements. The sensor array is now offset an unknown distance, α, from the original position where calibration was performed. See Figure [4(b)](#F4){ref-type="fig"}. Since the individual single-thread sensors in the array are equally spaced, each sensor thread is shifted from its home calibration position by the same distance α. Assuming that the individual sensor threads are identical other than being separated by a distance d, we can conclude that the pattern of the sensitivity array is a shifted version of the calibrated one, as shown in the simplified plots of Figure [5](#F5){ref-type="fig"}. This reduces the self-registration problem to a type of pattern matching problem. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Sensitivity Shifts.(a) Array of equidistantly spaced sensors over knee joint, with each sensor having unique sensitivity in this calibration position. (b) Shifting of array by an unknown distance, α, will lead to a shift in sensitivities. ::: ![](1743-0003-2-7-5) ::: ![](1743-0003-2-7-i14.gif) will no longer be the appropriate regression matrix to estimate θ~j~from Y~j~(i). A new, unknown vector ![](1743-0003-2-7-i15.gif) will instead relate the sensor output to θ~j~: ![](1743-0003-2-7-i16.gif) Although ![](1743-0003-2-7-i15.gif) is unknown, each individual sensor in the array should ideally give the same estimate for the actual joint angle at any time, so that ![](1743-0003-2-7-i17.gif) If the shifting of the sensor array were to happen in a discrete fashion, α= nd (16) where nis an integer value, it is seen that ![](1743-0003-2-7-i18.gif) Since nis an unknown, it is desired to find an nthat satisfies (15) and (17), rewritten as ![](1743-0003-2-7-i19.gif) ![](1743-0003-2-7-i20.gif) where nis assumed to be \\|n\\| \<M- 1. Namely, the sensor array, although shifted, can still cover the joint, having an overlap with the original sensor at the home position. In the ideal, theoretical case, there will exist an integer nthat can be found to exactly solve (18). Unfortunately, for practical usage, nwill not be a discrete integer. Furthermore, ncannot be explicitly found since process and measurement noise will cause the sensor outputs to deviate from their \"ideal\" values. With the knowledge of ![](1743-0003-2-7-i14.gif) for i= 1 \~ M, however, it is possible to find the optimal integer nthat bestsolves (18). Let us first define the average joint angle estimate for M threads of sensor outputs for a given integer nas follows (with Yand H\* reducing to scalars for the linear case): ![](1743-0003-2-7-i21.gif) ![](1743-0003-2-7-i22.gif) The best estimate for nis found by minimizing the average squared error between each sensor\'s estimate and the average estimate with respect to n(i.e. reducing the variance in the estimated angle as a function of n): ![](1743-0003-2-7-i23.gif) ![](1743-0003-2-7-i24.gif) ![](1743-0003-2-7-i25.gif) Equations (20a) and (20b) are solved for n= -M+2, -M+3, \..., M-3, M-2. The value for ![](1743-0003-2-7-i26.gif) found from (20c) is then used in (17) to approximate each sensor\'s predictor regression matrix for this new offset position of the array. In the ideal discrete case, where α= n~o~d, n~o~is the discrete offset of the sensor array, ![](1743-0003-2-7-i26.gif) = n~o~, and R~j~(![](1743-0003-2-7-i26.gif)) = 0. For the non-ideal case, where ais not a discrete multiple of d, the minimum variance is not zero, R~j~(![](1743-0003-2-7-i26.gif)) ≠ 0, but it will decrease as Mincreases, and ddecreases. Creating a denser sensor array in this way leads to more accurate estimates of sensor sensitivities, which in turn leads to more accurate estimates of θ~j~. Furthermore, since ![](1743-0003-2-7-i15.gif) can always be approximated using this algorithm, a one-time calibration is all that is needed for these wearable sensors to be used by a patient. The registration algorithm takes place in real time as the sensor is in use. All that is needed for a patient to begin using these sensors is to first \"zero\" the sensor output with the joint fully extended in the 0° position, and then freely move the joint to obtain non-zero data. This non-zero data will then allow the self-registration to take place. While registration is not needed at all times, it should be performed during initial operation until an appropriate ![](1743-0003-2-7-i26.gif) is converged upon. Again, the denser the array of sensors used, the better the estimate obtained. Following this, the algorithm need not be performed as often, as long as the sensor array remains stationary for an individual use. To begin monitoring, it is assumed that ![](1743-0003-2-7-i26.gif) = 0. Sensor Registration for Double Axis Joints ------------------------------------------ In the double axis case, two sensor arrays are placed around a predominantly two-axis joint such as the hip. As in the single-axis case, each array contains M sensor threads equally spaced by a distance d. The j-th joint array is placed so that it is most sensitive to changes in θ~j~, while the k-th joint array is situated so that it is most sensitive to changes in θ~k~. For registration, let the patient move only one axis at a time. As illustrated in Figure [6-(a)](#F6){ref-type="fig"}, the patient is instructed to move axis θ~1~alone. This hip flexion/extension causes significant changes to sensor array 1, y~1~(i), i= 1 \~ M. Next the patient is instructed to make hip abduction/adduction (θ~2~) alone, which causes significant changes to sensor array 2, as shown in Figure [6-(b)](#F6){ref-type="fig"}. Until registration has been completed, the estimate of the joint angles is not accurate. However, it is possible to distinguish which joint, θ~1~or θ~2~, has been moved, since sensor array 1 is most sensitive to θ~1~, and sensor array 2 for θ~2~. Once the individual axis movements are detected, the same registration procedure as that of a single axis can be applied to determine the misalignment of each sensor array. Once the misalignment is determined, the corrected, optimal predictor can now be used for verifying whether the registration has been performed correctly based on individual axis movements. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Registration Procedure for Hip Sensor Array.For registration of individual sensor arrays, the patient moves only one axis at a time (a) flexion/extension, and (b) abduction/adduction. ::: ![](1743-0003-2-7-6) ::: This registration method reduces the multi-axis problem to individual single axis procedures. However, the single axis procedures do not have to be repeated for all axes, if they are tightly related. For the two hip axes in Figure [6](#F6){ref-type="fig"}, a shifting of one sensor array around the body will be accompanied by a nearly identical shift in the second array. Therefore, registering one array will also register the other. In this case, it is required that a patient performs only one simple movement when first putting on the sensors -- extending the joint about a single axis over a sufficient range. Results ======= All experiments have been conducted under a protocol approved by the Massachusetts Institute of Technology Committee on the Use of Humans as Experimental Subjects (Approval No. 0411000960). Wearable Prototype Garment -------------------------- Figure [7](#F7){ref-type="fig"} shows a prototype pair of spandex pants with conductive fibers incorporated into the fabric to measure lower body movement. Spandex was chosen due to its favorable qualities: very stretchable, elastic, fits closely to the skin, and is able to withstand normal body movements and return to its original shape with no permanent distortions \[[@B17]\]. Furthermore, it is a comfortable material, able to be worn on a daily basis since it does not restrict movement in any way. Thus it is quite suitable for this sensor design. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Prototype Sensing Garment.Spandex pants with conductive fiber sensors for lower body monitoring. ::: ![](1743-0003-2-7-7) ::: In these particular pants, an array of eleven sensors spans across the knee joint, each separated by a distance of 5 mm, and each with an unstretched length of 55 cm. The sensors threads were silver plated nylon 66 yarn, which had an impedance of approximately 3.6 Ω/cm. Single sensors span both the posterior and side of the hip as well to capture two axes of hip motion. These single sensors are not seen in the view of Figure [7](#F7){ref-type="fig"}, but the locations are the same as those shown for sensors 1 and 2 in the schematic of Figure [2](#F2){ref-type="fig"}. This is the sensing garment used for all experimental tests. Preliminary Experiments ----------------------- To get an idea of the capabilities of existing technology available for joint monitoring, tests were initially performed using a standard electrogoniometer. Figure [8](#F8){ref-type="fig"} shows the set-up of the preliminary experiments. The goniometer used was a BIOPAC TSD130B Twin Axis Goniometer that consisted of two telescoping end-blocks that were taped to the side of the leg on either side of the knee joint. A strain gauge between these blocks was the device that measured the joint angle. The goniometer was used to measure knee flexion angle for two discrete positions. An untrained professional attached the goniometer to the leg, but followed the recommended attachment procedures as described by the vendor in the instruction manual. This was to simulate the knowledge of a typical patient who would be using such a device on his or her own, outside a carefully controlled setting. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Preliminary experiments set-up.Measurements were taken from a standard electrogoniometer at Position 1 (0°) and Position 2 (50°) ::: ![](1743-0003-2-7-8) ::: The goniometer was taken off and placed back on the knee joint eight separate times. Each time the goniometer was put on, the leg was extended (Position 1) and the goniometer output was set to 0°. The leg was then slowly bent to Position 2 (50°) and the goniometer output was recorded. The average rms error between the goniometer output, and the known joint angle (50°) for these tests was 3.5° with a standard deviation of 2.6°. Even with the goniometer placed on the same joint by the same person, these results illustrate the fact that slight changes in how the goniometer is attached can lead to varying measurements. It will be important to keep errors such as these in mind when the results from the conductive fiber sensor are analyzed. Having just discussed the possible errors introduced by a standard electrogoniometer, it is important to also highlight the possible errors introduced by a conductive fiber thread sensor. Consider again Figure [3](#F3){ref-type="fig"}, which shows sensor output vs. knee flexion angle for one thread sensor on the pants garment when the knee was randomly swung over a large range of motion. As can be seen, there is a significant amount of variation possible in sensor output for a given joint angle. In particular, for threads over the knee joint, the average rms error between curves such as those shown in Figure [3](#F3){ref-type="fig"}, and the calibrated predictor curves from (10) was approximately 3°-5° over the many tests performed. Therefore, it is noted upfront that errors will be introduced based solely on the type of measuring device being used due to hysteresis, material uncertainties, and other processes that cannot be accurately modeled. This should be kept in mind when using such a wearable device. Single Axis Results ------------------- The pants sensing garment was first used to estimate single-axis knee angle measurements. For the following single-axis experiments, a rotary potentiometer firmly attached to the leg was used as a goniometer, and this was the standard for which to compare joint angles. In each experiment, the potentiometer was \"zeroed\" with the leg in the full extension position. A calibration was performed to find the optimal regression matrix for both the linear and nonlinear predictors, and a sequence of knee movements was then monitored with the sensors. Figure [9](#F9){ref-type="fig"} shows the results of a typical sequence of these knee measurements, comparing the estimated angle from the conductive fiber sensors using the predictor models to that of the rotary potentiometer firmly attached to the leg. ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Sensor outputs-- Comparison of goniometer measured knee joint angle and estimated angles from wearable conductive fiber sensor. ::: ![](1743-0003-2-7-9) ::: The performance of the pants sensors can be seen to be quite good, accurately capturing the joint movement patterns over time. The average rms error between the pants sensor estimate and the potentiometer using the linear predictor was 5.4°, while that for the quadratic predictor was significantly better, at just 3.2°. It is important that these sensors are able to measure all types of motion, including higher frequency motion. To determine the frequency capabilities of the prototype fiber sensors, tests were performed where the leg was swung back and forth at different frequencies. The resulting sensor estimations, and errors when compared to the potentiometer, are summarized in Figure [11](#F11){ref-type="fig"} and Table [1](#T1){ref-type="table"} respectively. ::: {#F11 .fig} Figure 11 ::: {.caption} ###### Self-Registration Results.Joint angle measurements with sensing garment taken off and put back on before each test. ::: ![](1743-0003-2-7-11) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Sensor Frequency Capability Results ::: Approximate Frequency (Hz) Average RMS Error (degrees) ------------------------------ ------------------------------- 0.1 3.8 0.5 6.6 1 5.5 1.5 4.9 2 7.1 Fiber sensor thread errors for various frequencies of joint motion. ::: From these results, it is seen that the sensors are able to track the joint motion for frequencies as high as 2 Hz, but significantly larger errors result as the frequency is increased. Since most gross human motion takes place below these frequencies in a typical day, these sensors are suitable for everyday measurements, but such limitations should be considered if more accurate measurements are desired. Since these sensors are to be worn multiple times by a user, the reliability of registration is important every time the sensors are worn. Therefore, it is important that using the template-matching algorithm with an array of sensors will give an accurate registration each time the sensors are taken off and put back on. To verify this, an initial repeatability test was performed on the prototype sensor pants. The pants sensors were taken off and put back on four separate times to simulate four future uses of the sensors after an initial calibration test. The knee joint was moved over a wide range of motion in each instance. The joint angles measured by the fiber sensors for each test are shown in 11. The errors between these measurements and the potentiometer measurements are summarized in Table [2](#T2){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Sensor Self-Registration Results ::: Test Number Average RMS Error (degrees) --------------- ------------------------------- 2 5.7 3 8.6 4 8.5 5 11.6 Fiber sensor thread errors for successive tests where pants have been taken off and put back on. ::: Again, the sensors are able to capture the overall motion of the knee in each case, but appear to give less accurate results each time the pants are worn. For this reason, while a completely self-calibrating sensor is always desirable, it may be necessary to re-calibrate the sensors after many uses for more accurate measurements. Double Axis Results ------------------- Figure [12](#F12){ref-type="fig"} shows sensor outputs for a sequence of semi-random leg movements. In this case, output was captured from sensors y~1~and y~2~, spanning the posterior and lateral side of the hip, respectively (see Figure [2](#F2){ref-type="fig"}). In the first segment of motion, the leg was kept fully extended in the sagitall plane, and the subject performed a flexion/extension three times (θ~1~varies, while θ~2~= 0). In the second segment, leg movement was allowed only in the frontal plane, while the subject performed an abduction/adduction movement three times (θ~2~varies, θ~1~= 0). ::: {#F12 .fig} Figure 12 ::: {.caption} ###### Multi-Axis Sensor Outputs.Hip sensor outputs for two distinct leg motions. ::: ![](1743-0003-2-7-12) ::: In each case, the sensor spanning the axis in which the angle changes took place was the most sensitive to change, as expected. Each joint motion also produced small, but not insignificant, cross-coupling outputs in the \"remote\" sensors as well, showing that a single sensor output is dependent on multiple joint angles, and not one single angle. The pants sensor threads about the hip joint were then calibrated with the twin-axis goniometer. Table [3](#T3){ref-type="table"} shows the calibration matrix obtained per (9) using the predictor expression of (11). As can be seen, the first-order diagonal terms are dominant, with the cross-coupling terms significant, but not as dominant. The third and higher-order non-linearities were found to be insignificant compared to the values shown, and thus a second order predictor of the form of (11) seemed sufficient. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Calibration Matrix ::: y~1~ y~2~ -- -------- -------- ------- ------- 2.86 0.27 0.04 -0.24 1.32 3.83 -0.29 0.17 Calibrated parameter matrix for two hip sensor threads on one individual. ::: After initial calibration, random leg movements were then monitored with the sensors. Figure [13](#F13){ref-type="fig"} shows the results of a typical sequence of the resulting hip angle measurements. Again, the estimated angles from the conductive fiber sensors using both a linear and quadratic predictor are compared to that of a twin-axis goniometer. ::: {#F13 .fig} Figure 13 ::: {.caption} ###### Hip Joint Measurement Results.Comparison of goniometer measured hip joint angles and estimated angles from wearable conductive fiber sensors: (a) Hip flexion/extension, (b) Hip abduction/adduction. ::: ![](1743-0003-2-7-13) ::: The pants sensors were again able to capture the joint movement patterns over time, in this case for two axes of motion. The average rms error between the pants sensors\' estimate of hip flexion angle and the goniometer\'s was 2.5° using the linear predictor and 2.4° using the quadratic predictor. For hip abduction, these errors were 2.1° and 1.7° respectively. In this double axis case, the differences between the linear and quadratic predictors were not very significant over the typical ranges of hip joint angles measured. Previously, the assumption was made for the double axis hip joint that both sensor arrays would be offset from their calibration position by the same amount for each use. This allowed the double axis registration to be reduced to a single axis registration. To verify this assumption, a simple experiment was performed on the pant\'s hip sensors. The pants were taken off and put back on ten times. Each time, the distance around the waist between the sensor thread on the side of the hip, and the sensor thread on the rear of the hip was measured (distance between Point A and B in Figure [2](#F2){ref-type="fig"}). The average distance measured on a single individual in this way was 12.5 cm, with a standard deviation of 0.1 cm. The greatest discrepancy between any of these ten measurements was 0.6 cm (Maximum was 12.8 cm, minimum was 12.2 cm), which is approximately the same distance that separated the single threads in the array over the knee joint. Therefore, slight errors may result from making this assumption, but overall these errors should not contribute much due to the small variation in this experimental data. Discussion ========== For continuous joint monitoring, it should be noted that there are at least three fundamental sources of uncertainty in sensor output. The resistance measures across a section of conductive fiber, while ideally linearly related to length, may differ from an expected value due to the following factors: 1) movement of the fiber across the wire contact point may affect sensor output due to uncertainty in the area being contacted, and dynamic effects of the constant rubbing action; 2) although the elastic cord takes up a majority of the sensor tension, slight changes will also take place in the fiber tension as the joint is moved, and this will affect fiber resistance; and 3) different sections of even the same fibers will exhibit slightly different resistance characteristics due to the slightly inhomogeneous nature of such fibers. In spite of all these sources of uncertainty, it is still possible to accurately calibrate a set of sensors, and achieve acceptable joint measurements with minimal errors. These effects are minimized through careful selection of the particular fibers used as sensors, and in manufacturing the garment. While two specific predictor models have been presented for the calibration of a set of sensors, there are of course many more candidates that could be used as well. The linear and quadratic models used in this paper were the simplest choices, and the experimental results showed no advantage to adding more terms. Doing so only increased the computational requirements unnecessarily. This is why the models were presented as they were. A few more words should also be said about the registration algorithm. As presented, this algorithm only accounts for shifting of a set of sensors in one direction (particularly, in the \"horizontal\" direction). It is felt that this is appropriate due to the construction of the sensing garment. With the sensors instrumented in a \"vertical\" fashion, the user is responsible for visually checking that they put the garment on with no twist. This is relatively easy to do with the fibers oriented vertically. Furthermore, as long as the sensors span well beyond the local effects of skin movement around a joint, small shifts in the vertical direction will theoretically have little to no effect on the sensor output. Requiring a patient to \"zero\" the sensor output with all joints in the 0° position each time the garment is worn further eliminates any errors due to sensor drift. Finally, the wearability of the pants sensing garment must be addressed. What makes this sensing garment \"more wearable\" than existing joint measurement devices is that it is simply a pair of pants that people already wear on a regular basis. The extra sensors and wires added to these pants are compact and lightweight, almost negligible to the wearer. These sensors are easy to use, requiring much less skill and carefulness by the user, in general, than a typical goniometer. Conclusion ========== A wearable joint movement sensor design has been presented that uses conductive fibers incorporated into a fabric that is form fitting to a joint. Resistance changes in the fibers caused by fiber movement as the joint is moved can be related to angular joint position. Using multiple fiber sensors, multi-axis joint angles can be determined, in addition to single-axis angles, after a one-time calibration procedure performed by a therapist/physician. Implementing a nonlinear predictor model, continuous joint angle measurements can be made during daily activities, with the sensor able to be taken off and put back on at any time with no need for manual recalibration. Sensor offsets due to misregistration can be accounted for through the use of a sensor array spanning the joints of interest. This allows the sensors to self-calibrate, with only a few simple motions of the patient. After preliminary experiments involving a pants sensing garment for lower body monitoring, it has been seen that this methodology is feasible for monitoring joint motion of the hip and knee. Multiple sensor arrays are used at multi-d.o.f. joints, where each sensor output is coupled to multiple joint angle changes. This design therefore produces a robust, comfortable, truly wearable joint monitoring device. This paper outlines the development of this sensor from initial idea to working prototype. Future effort is needed in developing a completely wearable, highly accurate sensor, though. This would include making the sensors wireless, and therefore \"tether-free.\" More precise textile manufacturing techniques would also be needed to further reduce measurement errors. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= PTG developed the ideas discussed in this paper under the guidance of HHA. PTG carried out all experiments. Both authors read and approved the final manuscript. ::: {#F10 .fig} Figure 10 ::: {.caption} ###### Frequency Variation Results.Joint angle estimations for various frequencies of joint motion. ::: ![](1743-0003-2-7-10) ::: Acknowledgements ================ This material is based upon work supported by the National Science Foundation under Grant: NSF 0097700.	PubMed Central	2024-06-05T03:55:54.159183	2005-3-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555561/", "journal": "J Neuroengineering Rehabil. 2005 Mar 2; 2:7", "authors": [ { "first": "Peter T", "last": "Gibbs" }, { "first": "H Harry", "last": "Asada" } ] }
PMC555562	Background ========== The immobilization of biomolecules onto insoluble supports is an important tool for the fabrication of a diverse range of functional materials or devices \[[@B1]\]. Enzyme immobilization for example, is a desired biological procedure because of the possible application of immobilized enzymes in continuous operations, product purification, and catalyst recycling \[[@B2]\]. Furthermore, immobilization provides many advantages such as enhanced stability, easy separation from reaction mixture, possible modulation of the catalytic properties, and easier prevention of microbial growth \[[@B3]\]. In the last decade, nanosize materials have been widely used as support for this purpose. Among these materials, magnetic nanoparticles are very popular when used in conjunction with biological materials including proteins, peptides, enzymes \[[@B4]-[@B9]\] antibodies and nucleic acids \[[@B8]\], because of their unique properties \[[@B4]-[@B9]\]. The ability to tract magnetically labeled entities or target organelles using magnetic force offer the opportunity to conduct biological operations with increased specificity. Magnetite (Fe~3~O~4~) are biocompatible superparamagnetic materials that have low toxicity and strong magnetic properties \[[@B5]\]. They have been widely used for invivo examination including magnetic resonance imaging, contrast enhancement, tissue specific release of therapeutic agents, hyperthermia \[[@B10],[@B11]\], magnetic field assisted radionucleide therapy \[[@B11]\], as well as invitro binding of proteins and enzymes \[[@B4]-[@B8]\]. Magnetite nanoparticles have been used as support material for binding of enzymes including yeast alcohol dehydrogenase \[[@B4]\] and lipase \[[@B5]\] directly via carbodiimide activation. This method brought about considerable promise because of its simplicity and efficiency. Recently, γ-Fe~2~O~3~magnetic nanoparticles were used for binding Candida rugosalipase after acetylation of thiophene functionalized nanoparticles, or through nitroso-derivative formed on the surface of the particles by reacting nitroso tetrafluoroborate in methylene chloride. Both methods and more effectively lipase immobilized on acetylated nanoparticles exhibited long term stability. Glucose oxidase (GOX, β-D-glucose oxygen 1-oxidoreductase, EC 1.1.3.4) is a homodimer flavoprotein containing two active sites per molecule \[[@B12],[@B13]\]. It catalyses the oxidation of β-D-glucose to gluconic acid, concomitant with the reduction of oxygen to hydrogen peroxide. Glucose oxidase has been used to test various types of enzyme immobilization, and is the most commonly studied in the construction of biosensors for glucose assay development \[[@B12],[@B14],[@B15]\]. A more recent study \[[@B16]\] examined the activity of cholesterol oxidase activity using carbodiimide activation. Here, we report the stability and enzymatic activity of glucose oxidase immobilized onto Fe~3~O~4~magnetic nanoparticles using two binding methods, the direct binding via carbodiimide activation of amino functionalized particles and binding to thiophene-functionalized acetylated nanoparticles. A comparison of the stability and activity of glucose oxidase immobilized to magnetite using different protocols will lay the foundation for magnetic immunoassays. The size and structure of the nanoparticles were characterized using Transmission electron microscopy (TEM) and Fourier Transform Infrared (FTIR) spectroscopy, respectively. The stability, activity, and kinetic behavior of bound glucose oxidase were also examined. Materials and methods ===================== Glucose oxidase (specific activity 200 units/mg protein) from Aspergillusniger was purchased from VWR international (Pittsburgh, USA). Carbodiimide-HCl (1-ethyl-3-(3-dimethyl-aminopropyl), ammonium hydroxide, sodium hydroxide, acetic anhydride, glucose, bovine serum albumin (BSA), iron (II) chloride tetrahydrate 97 % and iron (III) chloride hexahydrate 99%, were obtained from Sigma-Aldrich St Louis (USA). 11-bromoundecanoic acid was obtained from TCI America Portland, USA. The Biorad Protein Assay Reagent Concentrate was purchased from Biorad Laboratories (Hercules, CA). Thiophene-2-thiolate was obtained from Alfa Aesar MA, USA. Iodine was obtained from Mallinckrodt Kentucky, USA, and acetonitrile was obtained from EMD Chemicals (New Jersey, USA). Sodium phosphate monohydrate and potassium phosphate dihydrate were acquired from EM Science (New Jersey, USA). Sodium carbonate was obtained from Orion Research Inc. (Beverly, USA). Magnetic nanoparticles (Fe~3~O~4~) were prepared by chemical co-precipitation of Fe^2+^and Fe^3+^ions in a solution of ammonium hydroxide (magnetic nanoparticles I or Fe~3~O~4~I), or sodium hydroxide (magnetic nanoparticles II or Fe~3~O~4~II) followed by a treatment under hydrothermal conditions \[[@B4],[@B5]\]. Iron (II) chloride and iron (III) chloride (1:2) were dissolved in nanopure water at the concentration of 0.25 M iron ions and chemically precipitated at room temperature (25°C) by adding NH~4~OH solution (30%) or NaOH 3 M at a pH 10. The precipitates were heated at 80°C for 35 min under continuous mixing and washed 4 times in water and several times in ethanol. During washing, the magnetic nanoparticles were separated from the supernatant using a magnetic separator of strength greater than 20 megaoersted (MOe). The particles were finally dried in a vacuum oven at 70°C. The dried particles exhibited a strong magnetic attraction. Magnetic nanoparticles I (50 mg) produced, using a solution of ammonium hydroxide were added to 1 mL of phosphate buffer (0.05 M. pH 7.4). After adding 1 mL of carbodiimide solution (0.02 g/mL) in phosphate buffer (0.05 M. pH 7.4), the mixture was sonicated for 15 min. Following the carbodiimide activation, 2 mL of glucose oxidase (1000 units /mL) was added and the reaction mixture was sonicated for 30 min at 4°C in a sonication bath. The magnetic nanoparticles were separated from the mixture using a magnetic separator. The precipitates containing Fe~3~O~4~nanoparticles I and Fe~3~O~4~bound glucose oxidase (GOX-Fe~3~O~4~I) were washed with phosphate buffer pH 7.4 and 0.1 M Tris, pH 8.0, and then used for activity and stability measurements. NaCl was added to enhance the separation of the magnetic nanoparticles \[[@B4]\]. A second functionalization protocol using a modification of the strategy adopted to immobilize Candida rugosalipase on the γ-Fe~2~O~3~\[[@B12]\] was implemented (Magnetic nanoparticles II). Briefly, 1.5 g Fe~3~O~4~was added to 5 g of 11-bromoundecanoic acid dissolved in 15 mL of ethanol. 11-bromoundecanoic acid was covalently linked to the nanoparticles surfaces by heating the mixture with microwave irradiation for 10 min. Functionalization of the particle was achieved through nucleophilic substitution with the 2-thiophene thiolate. In practice, 2-thiophene thiolate (7 mL) was added to the mixture containing the particles and heated in microwave for 5 minutes. The mixture was washed with ethanol and transferred in a round bottom flask. Acetic anhydride (4 mL) and 34.6 mL of iodine (0.01N) were successively added to the particles and agitated. The mixture was heated for 1 h under reflux condition \[[@B17]\]. The particles were washed several times with water, once with 10% sodium carbonate solution and finally with ethanol. The acetylated particles were reacted directly with the enzyme covalently linked to the particles via C = N bond \[[@B11]\]. For the attachment of glucose to nanoparticles, 2 mL of the GOX solution (1000 units/mL) was added to 50 mg of functionalized magnetic nanoparticles in a test tube and sonicated at 15°C for 3 h. The supernatant containing unbound enzymes was separated from the magnetic nanoparticles using the magnetic separator, and the enzymes bound to magnetic nanoparticles were then used for activity determination. A schematic of the procedures used for both attachments are presented in Figure [1](#F1){ref-type="fig"}. The amount of protein in the supernatant was determined by a colorimetric method at 595 nm with the Biorad Protein Assay Reagent Concentrate using bovine serum albumin (BSA) as the protein standard. The amount of bound enzymes was calculated from: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Description of GOX attachment procedures. (I) procedure employed for GOX-Fe~3~O~4~I attachment, (II) thiophene functionalization and the acetylation of particles for GOX-Fe~3~O~4~II attachment. ::: ![](1477-044X-3-1-1) ::: A= (C~i~- C~s~)\* V (1) Where Ais the amount of bound enzyme, C~i~and C~s~are the concentration of the enzyme initially added for attachment and in the supernatant, respectively (mg/mL) and Vis the volume of the reaction medium (mL). The size of Fe~3~O~4~magnetic nanoparticles, GOX-Fe~3~O~4~I and GOX-Fe~3~O~4~II were characterized by transmission electron microscopy (TEM, JEM 1200 EXII, JEOL) and structure by FTIR spectroscopy (Biorad FTS 6000, Cambridge, MA). The samples for TEM analysis were prepared as follows: a drop of magnetic nanoparticles was dispersed in nanopure water. The resulting solution was sonicated for 4 min to obtain better particle dispersion. A drop of the dispersed solution was then deposited onto a copper grid and dried overnight at room temperature. The binding of GOX onto the magnetic nanoparticles was investigated using FTIR spectroscopy. Samples for FTIR analysis were prepared in phosphate buffer pH 7.4. The activity of bound GOX was determined by measuring the initial rate of formation of hydrogen peroxide at a given temperature following the formation of a red quinoneimine dye. The principle of enzymatic determination of the activity of glucose oxidase is described as follows: Glucose is oxidized by glucose oxidase to gluconate and hydrogen peroxide. Phenol + 4-AAP, in the presence of peroxidase (POX), produces a quinoneimine dye that is measured at 500 nm using a Beckman Du Spectrometer to provide an absorbance that is proportional to the concentration of glucose in the sample. The reaction is described as follows: ![](1477-044X-3-1-i1.gif) ![](1477-044X-3-1-i2.gif) The activity of glucose oxidase was measured as follows. An assay mixture was prepared by mixing 500 U of horseradish peroxidase, 0.015 mmol of 4-aminoantipyrine (4-AAP), 0.025 mmol of phenol and 5 mmol of glucose in 50 mL of phosphate buffer solution (0.05 M. pH 7.4) to result in a glucose concentration of 0.1 M. To start the enzymatic reaction, 2 mL of the assay solution was added to 15 mL centrifuge test tubes containing GOX-Fe~3~O~4~and mixed by vortex. A solution of free GOX of the same molar concentration was used to evaluate the activity of the free enzyme for comparison. The solution was incubated at various temperatures (37--80°C) at specific intervals of time (30 min) and the supernatant was separated from GOX-Fe~3~O~4~using a magnetic separator. 10 μL aliquots of the supernatant were then taken for determining the concentration of hydrogen peroxide following the procedure by Trinder \[[@B18]\]. The activity of the enzyme can be calculated using the following equation: ![](1477-044X-3-1-i3.gif) Where ABSsample denotes the absorbance of the sample, ABS Stdis the absorbance of the standard solution, and C the concentration of glucose in the sample. The effect of temperature on the free GOX, GOX-Fe~3~O~4~I and GOX-Fe~3~O~4~II was estimated by determining the concentration of glucose in the sample at various temperatures. A solution of the assay mixture was added to the various centrifuge test tubes containing bound or free enzymes. The samples were stored in a water bath at specific temperatures (37, 50, 60, 70 and 80°C) and the absorbance was monitored at fixed time intervals to determine the glucose content. The effect of pH on GOX was monitored by measuring the initial rate of glucose oxidation by glucose oxidase in different phosphate and carbonate buffer solutions of pH (5--10) at 25°C. The thermal stability of free GOX, GOX-Fe~3~O~4~I, and GOX-Fe~3~O~4~II were determined by measuring the residual activity of the enzyme at 25°C, after being exposed to different temperatures (37--80°C) in phosphate buffer (0.05 M, pH 7.4) for 30 min. Aliquots of the reacting solutions were taken at periodic intervals (every 30 min for 6 h) and assayed for enzymatic activity as described above. The first-order inactivation rate constant, kwas calculated from the equation: ln A= ln A~0~- kt (5) where A~0~is the initial activity, Ais the activity after time t (min) and kis the reaction constant. The storage stability was examined by measuring the change in the concentration of glucose at room temperature at different time intervals (4 days). Test tubes with samples of GOX-Fe~3~O~4~I, GOX-Fe~3~O~4~II, or free GOX solutions were stored at 25°C in phosphate buffer (0.05 M. pH 7.4) for 33 days. Thereafter, 3 mL of the assay solution was added, and the residual activity of GOX was assayed. The kinetic parameters of free GOX, GOX-Fe~3~O~4~I and GOX-Fe~3~O~4~II, K~m~and V~max~were determined by measuring the initial rates of glucose oxidation (0.2--1 mM) by glucose oxidase (0.25 mg/mL) in phosphate buffer (pH 7.4) at 25°C. Results and discussion ====================== Synthesis of magnetic nanoparticles with equivalent amounts of ferric and ferrous chlorides resulted in 66% of magnetic nanoparticles I and 73% of magnetic nanoparticles II. These yields suggested that the synthesis using NaOH is more advantageous for large scale production of magnetic nanoparticles. \"Bare\" Fe~3~O~4~I and Fe~3~O~4~II, and their GOX bound counterparts shown in the TEM micrographs in Figures ([2A, B, C](#F2){ref-type="fig"} and [2D](#F2){ref-type="fig"}), respectively reveal that the particles are fine and spherical. The sizes of the particles of each sample were evaluated from 2 different TEM images. The diameter was in the range between 9 and 13 nm. The coefficient of variation between different measurements was less than 7%. There was no significant change in the size of the \'bare\" particles and GOX bound particles. However, signs of agglomeration of the particles were visible in the samples. The agglomerates were not considered in the examination of the size distribution of the magnetic particles because of the assumption that agglomerated particles do not describe the original size of the particles. Figure [3](#F3){ref-type="fig"} shows the distribution of the particles sizes. Fe~3~O~4~nanoparticles 14.5 mg/mL corresponding to GOX/Fe~3~O~4~weight ratios of 0.2 was used for the binding process. The amount of unbound enzyme was determined by assaying the protein content in the supernatant. The amount of enzymes bound to the magnetic nanoparticles in each binding procedure is given in table [1](#T1){ref-type="table"}. Average binding efficiencies of 38.4 units/mg particles for GOX-Fe~3~O~4~I and 27.6 units/mg particles for GOX-Fe~3~O~4~II were noted. The overall percentage of binding efficiency was between 94 and 100% for GOX-Fe~3~O~4~I and 66 to 72% for GOX-Fe~3~O~4~II. These results show that the binding was successful in both cases, particularly with Fe~3~O~4~I which uses the carbodiimide activation. The percentage of binding of GOX to Fe~3~O~4~II was significant but still below the level obtained through carbodiimide activation with the Fe~3~O~4~I. However, despite the difference in the level of GOX bound, the two procedures adopted were successful. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Transmission electron micrographs of Fe~3~O~4~magnetic nanoparticles I (A), Fe~3~O~4~magnetic nanoparticles II (B), GOX-Fe~3~O~4~I (C), and GOX-Fe~3~O~4~II (D). ::: ![](1477-044X-3-1-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Distribution of particles on the electron micrographs of Fe~3~O~4~magnetic nanoparticles I and GOX-Fe~3~O~4~I (A), and Fe~3~O~4~magnetic nanoparticles II and GOX-Fe~3~O~4~II (B). ::: ![](1477-044X-3-1-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Summary of binding efficiencies of enzyme-functionalized systems (n = 9). ::: Bound enzymes (units/mg nanoparticles) ----------------- ---------------------------------------- GOX-Fe~3~O~4~I 38.4 ± 0.8 GOX-Fe~3~O~4~II 27.6 ± 0.6 ::: The FTIR spectra of magnetic Fe~3~O~4~I, Fe~3~O~4~II, GOX-Fe~3~O~4~I and GOX-Fe~3~O~4~II are shown in Figure [4(A, B, C, D)](#F4){ref-type="fig"}, respectively. The characteristic bands of proteins at 1541 and 1645 cm^-1^assigned to amide I and amide II, respectively were visible in the spectra of GOX-Fe~3~O~4~I and GOX-Fe~3~O~4~II. These peaks are associated with two sharp peaks in the region 1420-1300 cm^-1^typical of carboxylate groups, from the enzyme. A weak peak at 1900 cm^-1^appeared in the spectra of GOX-Fe~3~O~4~I and GOX-Fe~3~O~4~II and could be assigned to the C-O bonds in the enzyme molecule. These peaks were absent in the spectra of Fe~3~O~4~I and Fe~3~O~4~II. In the 1100-1000 cm^-1^region in all spectra appeared a characteristic adsorption spectra typical to phosphate ion that can be assigned to the phosphate buffer used in the samples preparation. The occurrence of negative peaks in the spectra of Fe~3~O~4~I, and GOX-Fe~3~O~4~II is possibly due to the reduced amount of phosphate in comparison to the amount of phosphate used to background subtraction. The characteristic peaks of proteins found in the spectra of GOX-Fe~3~O~4~I and GOX-Fe~3~O~4~II were not visible in the spectra of Fe~3~O~4~I and Fe~3~O~4~II, indicating enzyme attachment onto the particles. The amino groups on the surface of the particles resulted from the use of concentrated ammonia solution during the co-precipitation of Fe^2+^and Fe^3+^as demonstrated by \[[@B5]\] and \[[@B19]\]. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### FTIR spectra of Fe~3~O~4~magnetic nanoparticles I (A) and Fe~3~O~4~magnetic nanoparticles II (B), GOX-Fe~3~O~4~I (C), and GOX-Fe~3~O~4~II (D). ::: ![](1477-044X-3-1-4) ::: The most important aspect of this study is related to the retention of biocatalytical activity of GOX after binding to magnetic nanoparticles. The amount of bound GOX was estimated using the UV-vis spectrophotometer and the catalytic activity of free and bound GOX was compared. Kinetic parameters (V~m~and K~m~) were estimated from the double reciprocal plots of the initial rates of glucose oxidation by GOX. The double reciprocal plots are presented in Figure [5](#F5){ref-type="fig"}. The Michaelis-Menten constants V~max~and K~m~for GOX are shown in Table [2](#T2){ref-type="table"}. V~max~of free GOX, GOX- Fe~3~O~4~I and GOX-Fe~3~O~4~II were 0.731, 0.803, and 0766 μmol/min mL and, the corresponding K~m~values were, 0.383, 0.208, and 0.237 mM, respectively. The V~max~value of GOX immobilized on magnetic nanoparticles (I and II) was higher than that of the free enzyme. The highest V~max~was obtained with GOX-nanoparticles I. Since a low K~m~indicates a high degree of affinity of the enzyme to substrate \[[@B5]\], calculations showed that the affinity of the enzyme to the substrate increased in the order of free GOX, GOX-Fe~3~O~4~II and GOD-Fe~3~O~4~I, respectively. The high affinity of the enzyme to the substrate may be explained by a favorable change in the structural organization of the enzyme due to the immobilization procedure \[[@B20]\]. Consequently, the active sites of the enzymes could be more readily available for enzymatic interactions. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Double reciprocal plots of the initial rates of free GOX (■), GOX-Fe~3~O~4~I (▲) and GOX-Fe~3~O~4~II (●) at pH 7.4, from experimental data. ::: ![](1477-044X-3-1-5) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Kinetic parameters of free GOX, GOX-Fe~3~O~4~I, and GOX-Fe~3~O~4~II determined from the double reciprocal plots. ::: ----------------- --------------------- ------------ V~max~(μmol/min mL) K~m~(mM) Free GOD 0.731 0.383 GOD-Fe~3~O~4~I 0.803 0.208 GOD-Fe~3~O~4~II 0.766 0.237 ----------------- --------------------- ------------ ::: The effect of pH on the activities of the free and bound GOX was investigated in the pH range of 5--10 at 25°C (Figure [6](#F6){ref-type="fig"}). Each data point was the average of two measurements. The coefficient of variation between measurements was between 2 and 5%. In the pH range between 6 and 7.4, the enzyme activity increased in all the systems. However, the enzyme activity was higher in GOX-Fe~3~O~4~I than in GOX-Fe~3~O~4~II and free GOX. The activity reached 100% at pH 7.4, and decreased above this pH value to 43% for GOX-Fe~3~O~4~I, and to 26% for GOX-Fe~3~O~4~II and 9% for the free GOX at pH 10. The free GOX experienced a more severe loss in activity, while GOX-Fe~3~O~4~I retained greater activity as the pH increased. In this system, the binding process occurred directly upon activation of the particle surface using carbodiimide, while the binding of GOX-Fe~3~O~4~II involved a C = N bond formed on the acetylated thiophene. It can be argued that the direct binding between the protein and the amino bond in the former case exhibited a greater resistance to a medium with higher alkalinity. This medium appeared even more constraining to the free enzyme and placed the enzyme in an electrostatic state that might affect the activity. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Effect of pH on the activities of free GOX (■), GOX-Fe~3~O~4~I (▲) and GOX-Fe~3~O~4~II (●). ::: ![](1477-044X-3-1-6) ::: The effect of temperature on the activity of free GOX was examined by measuring its relative activity when stored at various temperatures. Figure [7a](#F7){ref-type="fig"} and [7b](#F7){ref-type="fig"} shows the effect of temperature on GOX-Fe~3~O~4~I and GOX-Fe~3~O~4~II at various temperatures. Each data point represents the average of duplicate measurements (coefficient of variation was less than 6 %). It can be observed that at 37°C, the enzyme retained its activity for about 80 minutes before showing a slight decrease. At 50, 60, 70 and 80°C the activity decreased as the temperature increased in both systems. In GOX- Fe~3~O~4~, the remaining activity was 23% at 50°C and 15% at 60°C after 270 min. For this time period and duration, the remaining activities were 9% and 0%, respectively for GOX-Fe~3~O~4~II. A similar trend was observed at 70 and 80°C with a more drastic loss of activity from the GOX-Fe~3~O~4~II protocol. Loss of activity occurred more rapidly in GOX-Fe~3~O~4~II indicating that direct binding through carbodiimide activation provides a greater thermal stability to GOX. The effect of temperature on the activities of free, GOX-Fe~3~O~4~I, and GOX-Fe~3~O~4~II at pH 7.4 are presented in the Arrhenius plots (Figure [8](#F8){ref-type="fig"}). The activation energies were calculated as 1.4, 0.9, and 1.1 kJ/mol for free-GOX, GOX-Fe~3~O~4~I, and GOX-Fe~3~O~4~II, respectively. These results show that the unbound enzyme has the highest activation energy, while GOX-Fe~3~O~4~I had the lowest. The low activation energy of GOX associated with binding to magnetic nanoparticles suggests that the energy requirement on the surfaces of the nanoparticles for enzymatic activity is reduced. Table [3](#T3){ref-type="table"} shows the inactivation rates constants (k) at 50, 60 70, and 80°C. The rate constants increased with increasing temperature in the order GOX-Fe~3~O~4~I, GOX-Fe~3~O~4~II and free GOX. Here again, binding GOX to magnetic nanoparticles minimized structural denaturation due to heat treatment. Covalent binding was expected to provide the enzyme with the protection against structural denaturation due to the unfavorable solvent-protein interactions, and thus result in activation effect \[[@B21]\], a possible reason for a better activity of the bound enzyme compared with the free enzyme after heat treatment. GOX-Fe~3~O~4~II had a lower stability at higher temperatures compared to GOX-Fe~3~O~4~I. The reason for this difference could reside in the stability of the binding, since the binding methods are so far the major difference between these systems. Indeed with GOX-Fe~3~O~4~I, the binding of the enzyme occurred through the amino groups on the surfaces of the particles and the carboxylic groups of proteins in the enzymes \[[@B5]\] which is a natural way for protein binding, while in the case of GOX-Fe~3~O~4~II the N atom to which the enzyme is attached shared a double bond with the carbon atom which is less stable than the amide bond. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Effect of temperature on the activity of GOX-Fe~3~O~4~I (A) and GOX-Fe~3~O~4~II (B) at pH 7.4. The samples were stored at 37, 50, 60, 70 and 80°C for 30 min and the activities were measured at 25°C. ::: ![](1477-044X-3-1-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Arrhenius plots of the initial oxidation rates of glucose by free GOX (■), GOX-Fe~3~O~4~I (▲) and GOX-Fe~3~O~4~II (●) for samples at 37, 50, 60, 70, and 80°C. ::: ![](1477-044X-3-1-8) ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Inactivation rate constants (k) of the free-GOX, GOX-Fe~3~O~4~I, and GOX-Fe~3~O~4~II at various temperatures. ::: Temperature (°C) Free-GOX k(min^-1^) GOX-Fe~3~O~4~I k(min^-1^) GOX-Fe~3~O~4~II k(min^-1^) ------------------ ----------------------- ----------------------------- ------------------------------ 50 3.42. 10^-2^ 7.10.10^-4^ 2.87.10^-3^ 60 9.36.10^-2^ 1.49.10^-3^ 3.50.10^-2^ 70 2.81.10^-1^ 6.24.10^-2^ 1.92.10^-2^ 80 9.3210^-1^ 4.52.10^-2^ 5.46.10^-1^ ::: Loss of storage stability is a major concern in enzyme preservation. The storage stability of the enzyme was examined for 33 days. Figure [9](#F9){ref-type="fig"} shows the storage stabilities of free GOX, GOX-Fe~3~O~4~I, and GOX-Fe~3~O~4~II at 25°C at pH 7.4. Each data point was the average of duplicate measurements (coefficient of variation of the measurements was between 1 and 5%). The activity decreased with time in all the systems. Total loss of activity was observed after 20 days for the free GOX and 28 days for GOX-Fe~3~O~4~II while GOX-Fe~3~O~4~I retained 26% activity after 33 days of storage under identical conditions. The stability of the enzyme was found to improve upon binding to the magnetic nanoparticles but the most significant improvement in stability was observed for the GOX-Fe~3~O~4~I nanoparticle complex. The fixation on the surface of the magnetic nanoparticles has been a tangible argument supporting the prevention of auto-digestion of the enzyme and lysis, and the subsequent conservation of its activity \[[@B4]\]. This argument supports our results and justifies the long term stability of GOX-nanoparticles I and GOX-nanoparticles II over the free GOX. The efficiency of binding of GOX via carbodiimide activation over the binding by thiophene acetylation may be attributed to the potential of carbodiimide to activate the carboxylic acid side chains partially buried at the surface or in active sites of the enzyme, as well as the amino groups on the nanoparticles, favoring the formation and the stability of the amide bond \[[@B22]\]. This may explain why the amount of bound enzymes is higher in the binding via carbodiimide than with the thiophene acetylation. Furthermore, carbodiimide might cause cross-linking of the enzyme providing a better stability to its quaternary structure \[[@B23]\] and a subsequent improvement in stability. ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Storage stability of free GOX (■), GOX-Fe~3~O~4~I (▲), and GOX-Fe~3~O~4~II (●) measured at a pH of 7.4 at 25°C. ::: ![](1477-044X-3-1-9) ::: Conclusion ========== Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chlorides using two different base solutions. GOX was bound to the particles by direct attachment via carbodiimide activation and chemically via covalent attachment onto thiophene acetylated magnetic nanoparticles. Confirmation of the binding was demonstrated by FTIR spectroscopy and the sizes of the particles were characterized by TEM. The direct binding of GOX via carbodiimide activation was more effective and resulted in binding efficiency in the range between 94--100% while the binding efficiency was only between 66--72% for the GOX-Fe~3~O~4~II complex. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when the complex was subjected to thermal and pH variations. This study shows that binding onto magnetic nanoparticles can allow the enzyme to acquire the conformational and structural arrangement for a better activity and stability, and suggests that binding of enzyme onto magnetic nanoparticles via carbodiimide activation was efficient for creating bioconjugates for a variety of applications in health and food safety. Authors\' contributions ======================= Drs. Gilles Kouassi and Joseph Irudayaraj were the primary authors. They were responsible for the concept and experimental plan of the article. Dr Gregory MacCarty was the secondary author and contributed to the overall effort. Acknowledgements ================ The authors acknowledge the USDA challenge grant program for partial funding of this research. Dr Chen Yu is acknowledged for his help in TEM image acquisition.	PubMed Central	2024-06-05T03:55:54.162975	2005-3-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555562/", "journal": "Biomagn Res Technol. 2005 Mar 11; 3:1", "authors": [ { "first": "Gilles K", "last": "Kouassi" }, { "first": "Joseph", "last": "Irudayaraj" }, { "first": "Gregory", "last": "McCarty" } ] }
PMC555563	Introduction ============ Dengue is the most important arthropod-borne viral disease of public health significance. Compared to nine reporting countries in the 1950s, today the geographic distribution includes more than 100 countries worldwide. Many of these had not reported dengue for 20 or more years and several have no known history of the disease. The World Health Organization (WHO) estimates that more than 2.5 billion people are at risk of dengue infection. Most will have asymptomatic infections. The disease manifestations range from an influenza-like disease known as dengue fever (DF) to a severe, sometimes fatal disease characterised by haemorrhage and shock, known as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which is on the increase. Dengue fever and dengue haemorrhagic fever/dengue shock syndrome are caused by the four viral serotypes transmitted from viraemic to susceptible humans mainly by bites of Aedes aegyptiand Aedes albopictusmosquito species. Recovery from infection by one serotype provides lifelong immunity against that serotype but confers only partial and transient protection against subsequent infection by the other three. First recognised in the 1950s, it has become a leading cause of child mortality in several Asian and South American countries. The average number of DF/DHF cases reported to WHO per year has risen from 908 between 1950 and 1959 to 514,139 between 1990 and 1999. The real figure is estimated to be closer to 50 million cases a year causing 24,000 deaths. Of an estimated 500,000 cases of DHF/DSS requiring hospitalisation each year, roughly 5% die according to WHO statistics. Regional distribution of dengue and its serotypes are described elsewhere \[[@B1],[@B2]\]. In summary, DF/DHF/DSS is an immediate problem in south and southeast Asia and Central and South America. Although DF is present in the African region, there are no cases or outbreaks reported to WHO \[[@B3]\]. Half the world\'s population lives in countries endemic for dengue, underscoring the urgency to find solutions for dengue control. The consequence of simple DF is loss of workdays for communities dependent on wage labour. The consequence of severe illness is high mortality rates, since tertiary level care required for DHF/DSS management is beyond the reach of most of the persons at risk. This paper reviews the changing epidemiology of the disease, focusing on host and societal factors and drawing on national and regional journals as well as international publications. It does not include vaccine and vector issues. Although each one of the issues taken up below merits an independent, in-depth treatment, we have selected only those issues where the literature raises challenges to prevailing views and therefore require further research, particularly given that most of these issues are key for improved service delivery in poor countries. Analysis ======== Clinical presentation --------------------- Dengue infection can cause a spectrum of illness ranging from mild, undifferentiated fever to illness up to 7 days\' duration with high fever, severe headache, retro-orbital pain, arthralgia and rash, but rarely causing death. Dengue Haemorrhagic Fever (DHF), a deadly complication, includes haemorrhagic tendencies, thrombocytopenia and plasma leakage. Dengue Shock Syndrome (DSS) includes all the above criteria plus circulatory failure, hypotension for age and low pulse pressure. DHF and DSS are potentially deadly but patients with early diagnosis and appropriate therapy can recover with no sequelae. Case management for DF is symptomatic and supportive. DHF requires continuous monitoring of vital signs and urine output. DSS is a medical emergency that requires intensive care unit hospitalisation \[[@B4]\]. The increase in dengue mortality is considered to be a reflection of the increase in the proportion of DF patients who develop DHF/DSS. The pathogenesis of DHF/DSS is widely considered to be antibody-dependent enhancement in secondary infection with a virus of different serotype \[[@B5]\]. Evidence in support of this comes from many studies including from the Cuban epidemics of 1981 and 1997 \[[@B5],[@B6]\] and a five-year study of Yangon (Myanmar) \[[@B7]\]. However, absence of a significant association between secondary infection or co-circulation of different serotypes and DHF/DSS has also been noted \[[@B8],[@B9]\]. The disease is widely considered to be associated with secondary infection and co-circulation of several serotypes. Alternative or additional factors associated with severe illness, such as high viraemia titres, have also been suggested \[[@B10]\]. So far, this has been associated with secondary infection as demonstrated by Vaughn et al., and Libraty et al. \[[@B11],[@B12]\]. On the other hand, one expression of higher viral virulence could be higher viraemia leading to greater severity, but this has not not yet been demonstrated (Guzmán, 2003 personal communication). Viral virulence \[[@B13]\], immunological responses and increased pathogenicity of specific serotypes \[[@B14]\] have been implicated as critical for the appearance of DHF. This has been found for the three serotypes DEN1, \[[@B13]\] DEN 2 \[[@B15]\] and DEN 3 \[[@B8],[@B10],[@B16],[@B17]\], but so far not for DEN 4 \[[@B18]\]. The evidence from different studies also shows that the pathogenesis of DHF/DSS may be multi-factorial and understanding remains incomplete. Epidemiological changes ----------------------- Demographic, economic, behavioural and social factors are often keys for effective communicable disease control and underpin successful public health programmes. Despite promising indications in the literature, these factors have remained poorly understood in the case of dengue. Furthermore, recent field evidence raises some questions regarding widely accepted characteristics of dengue that need review and confirmation. ### Shift in modal age DF is typically acknowledged to be a childhood disease and is an important cause of paediatric hospitalisation in southeast Asia. There is, however, evidence of increasing incidence of DHF among older age groups. Since the early 1980s, several studies in both Latin America and southeast Asia have reported a higher association of DHF with older ages. The earliest studies were by Guzmán (1981) in Cuba and Rigau-Pérez in Puerto Rico \[[@B6],[@B19]\]. Later on similar observations were noted in Nicaragua and Brazil. In some southeast Asian countries where dengue has been epidemic for several years, this age shift is clearly observed, indicating an epidemiological change in dengue infection in those locations \[[@B20]-[@B22]\]. Three studies in Asia using surveillance data report increasing age of infected patients. In Singapore, surveillance data showed a shift in peak dengue mortality from paediatric ages (1973--1977) to adults in 1982, since which year more than 50% of the deaths occurred in patients older than 15 years. From 1990--96, the highest age-specific morbidity rates were in the 15 to 34 year age groups \[[@B23]\]. In Indonesia, surveillance data from 1975 to 1984 showed an increase in incidence rates among young adults in Jakarta as well as in the provincial areas \[[@B24]\]. Adults have accounted for proportions as high 82% of all cases in the hospital-based surveillance study during the 2000 epidemic of dengue in Bangladesh \[[@B25]\]; the highest proportion of cases occurred in the 18 to 33 year age group. All deaths in the Bangladesh outbreak in 2000 were in persons older than 5 years. In Puerto Rico, surveillance data analysis showed the highest incidence rate (11.8/1000) in the 10--19 year age group during an outbreak in 1994 and 1995 \[[@B26]\]. Hospital-based studies have similarly reported increasing infection rates among adults, mentioning that it is contrary to the popular belief that dengue is a paediatric disease \[[@B27],[@B28]\]. The trend for increased incidence among young adults has important implications for control and prevention. Whether these are real increases (based on population distributions), increases in the proportion of DHF/DSS (and, hence, the proportion hospitalised) but not DF, or the result of improved classification and diagnosis needs clarification. Comparative incidence and case fatality ratios (CFRs) of severe illness in adults and children and the economic implications are discussed later. ### Racial predisposition Race-related susceptibility to dengue has been observed in a few studies and merits further investigation. In a retrospective seroepidemiologic study Guzmán reported that blacks and whites were equally infected with DEN-1 and DEN-2 viruses during the Cuban epidemics of 1977 and 1981, while severe dengue disease was observed less frequently in dengue-infected black persons than whites \[[@B5],[@B6]\]. A study in Haiti observed that despite virologic pre-conditions hypothesised to be precursors for DHF (i.e. the evidence of previous infection by DEN virus types 1, 2 and 4), local children did not develop severe illness \[[@B29]\]. The authors concluded that this finding provides further evidence of a dengue-resistant genotype in black populations. In 1998 the Los Angeles County vital registration system reported DF/DHF incidence, but only among Hispanic and white ethnic groups (0.1 and 0.07/100,000) \[[@B30]\]. Genetic polymorphism in cytokine profiles and coagulation proteins has been proposed as a factor protecting persons of African origin \[[@B31]\]. Evidence for this hypothesis has been found in meningococcal disease, in which a genetic polymorphism in the gene encoding an essential protein involved in coagulation is a predictor for developing severe disease with lethal outcome. In Asia, two studies report racial differences in disease incidence. A 15-year study of the epidemiology of dengue reports a significantly higher incidence of DHF among Chinese compared to Malaysian males \[[@B32]\]. This finding was supported by a six-year surveillance data study in Singapore, which found the race-specific morbidity rate among the Chinese to be three times that of the Malays and 1.7 times that of Indians \[[@B23]\]. Although none of the above constitutes convincing evidence for the hypotheses, they highlight a useful area for better understanding of dengue pathogenesis and health service planning. ### Sex differences Understanding male-female differences in infection rates and severity of disease is important for public health control programmes. A few hospital-based studies and surveillance data show a male-female difference in infection rates and in severity of disease. Three independent studies from epidemics in India and Singapore found nearly twice the number of male patients compared to females (Lucknow and Singapore both report male to female ratios of 1.9:1 and Delhi 1:0.57) \[[@B33]-[@B35]\]. In his hospital-based study during the 1996 epidemic in Delhi, Wali reported an even higher ratio of 2.5:1 \[[@B27]\]. Another study during the same epidemic found a male to female ratio of 1:0.25 cases for DSS. However, of the three deaths in this sample, two were female \[[@B35]\]. Surveillance data from Malaysia revealed a male preponderance among Indian and Malay patients (1.5:1), but the ratio was almost equal for those of Chinese origin \[[@B32]\]. The Ministry of Health, Bangladesh reported a hospital patient DF/DHF male to female ratio of 1.5:1 during an outbreak in Chittagong in 1997 \[[@B36]\], although a later study of DHF only during the 2000 outbreak found no differences between sexes \[[@B37]\]. With the exception of the study by Shekhar, all the others were hospital-based and may represent those who sought care rather than the infected population \[[@B27]\]. Studies in South America generally report that both sexes are equally affected \[[@B26],[@B38]\] although a male to female ratio of 0.65:1 was described as \"typical\" for dengue \[[@B9]\]. Kaplan, in a rare study testing for significance, found a higher proportion of women in all of his four Mexican samples (p \< 0.001) \[[@B39]\]. Of significance are two studies in Asia by Kabra and Shekhar where severe illness and CFR were consistently higher among females despite higher incidence in males \[[@B32],[@B40]\]. Halstead \[[@B41]\] had pointed out as early as 1970 that males predominate among those with milder disease but females account for more severe illness. He suggested that either immune responses in females are more competent than in males, resulting in greater production of cytokines, or the capillary bed of females is prone to increased permeability. Kaplan in Mexico suggests that an incidence bias in favour of females is related to the timing of the survey interviews, while Goh puts forward that low incidence among women occurs because they stay at home and are less exposed to infection \[[@B39],[@B42]\]. No studies suggest gender bias in home care and male preferences in health care seeking, still prevalent in many Asian and other traditional societies. It is widely recognised that in many of the Asian communities, lower disease incidence in women may be a statistical artefact related to lower reporting and care-seeking for women from traditional practitioners who do not report to public surveillance systems. By the same token, women are less likely to be taken for care at a hospital when ill or are taken at late stages of disease, when no other options are available. Determining sex differences, both in infection and severity of disease, requires well-designed and targeted studies to capture both biological and social factors that drive disease patterns in a community. ### Rural spread Historically, DF/DHF has been reported as occurring predominantly among urban populations where density of dwellings and short flying distance of the vector create the right conditions for transmission. However, the literature shows that dengue transmission and, in some cases, outbreaks occur in rural settings in both Asia and Latin America. In the WHO Western Pacific region, WHO has confirmed that disease spread into rural areas from where it had not been reported previously \[[@B20]\]. Rural epidemics occurred as early as 1976 in Indonesia, and in 1994 the outbreak in Laos began in a remote, rural district of Nasaithong \[[@B43],[@B44]\]. Today, Thailand has an incidence rate that is higher in rural (102.2 per 100,000) than urban areas (95.4 per 100,000) \[[@B28]\]. Similarly, in India, entomological investigation showed a widespread distribution of Aedes aegypti, both in rural and urban areas during an outbreak in Gujarat in 1988 and 1989 \[[@B45]\]. Increase in DF/DHF among rural populations is also observed in Central and South America and identical rates in both populations are reported \[[@B9]\]. Among jungle dwellers in Peru, antibody prevalence up to 67% compared to 66% among the urban population have been found \[[@B46]\]. In industrialised settings, the Centers for Disease Control and Prevention (CDC) reported an outbreak of DF among residents of the rural towns of Hana and Nahiku in Hawaii in 2001. The outbreak was historically unusual because infection occurred among residents who have no history of recent travel and the Aedes aegyptimosquito has not been seen in Hawaii since it was supposedly eradicated by pesticide spraying in 1943 \[[@B47]\]. Increased transport contact, mobility and spread of peri-urbanisation have been the most frequently cited reasons for spread of dengue to rural areas \[[@B48]\]. While some rural incidence linked to travel contact with urban areas is conceivable, outbreaks and infection rates equal to those in urban areas warrant further investigation. Improved reporting could also be a factor, but it would be less likely in areas such as Hawaii, USA. Standard epidemiological techniques such as spatial studies of cases and careful patient histories could shed further light into transmission patterns in rural populations. Health service structures and utilisation patterns differ substantially between urban and rural areas in many tropical countries and contextually appropriate strategies will be required for effective impact. ### Seasonality and climate variability The incidence and, in particular, epidemics of dengue have been commonly associated with the rainy season, and the El Niño phenomenon has been incriminated in the increases of certain vector-borne diseases, including dengue \[[@B49],[@B50]\]. Despite the number of studies, convincing data or models supporting these hypotheses are scarce. The relationship between temperature, rainfall and vector-borne disease are increasingly seen as oversimplifications. A study modelling DF transmission and seasonal temperature on data from Puerto Rico from 1988 to 1992 revealed weak relationships between monthly mean temperature and incidence of DF \[[@B51]\]. The study concluded that factors related to history of herd immunity, introduction of new serotype or demographic transitions influence transmission. More recently, long-term meteorological trends were studied in four high-altitude sites in East Africa, where increases in malaria have been reported in the past two decades \[[@B52]\]. They did not observe any significant change in temperature, rainfall, vapour pressure and the number of months suitable for P. falciparumtransmission in the past century or during the period of reported malaria resurgence. Others have questioned models linking global temperatures and disease incidence, stating that, historically, climate has rarely been the principal determinant of vector-borne disease prevalence. Neither does the literature provide an adequate evidence base establishing the impact of climate change on vector-borne disease \[[@B53],[@B54]\]. The \"bandwagon\" of El Niño \[[@B55]\] and dengue incidence is now placed under scrutiny and further research will have to be done before climate variations can be nailed down as a culprit. Health systems issues --------------------- ### Socio-economic context Social and economic factors play an essential role in the incidence and prevalence of DF and DHF. Air conditioning, screens and safe water supplies in wealthier countries help prevention and better health services reduce or eliminate mortality from DHF. Unplanned urbanisation and inadequate resources for vector control are factors that promote transmission and are characteristic of poor rather than richer countries. Reiter et al. (2003) studied dengue transmission on the Mexico-USA border and found higher rates in the Mexican city compared to the American one \[[@B56]\]. However, some anomalies persist despite the rich/poor divide in disease incidence. Despite energetic control programs in the wealthier endemic countries of southeast Asia such as Singapore, Malaysia and parts of China (eg. Hong Kong), dengue continues to be a problem. Malaysia reports some of the highest numbers of cases during epidemics compared to other countries in the region. In some of these cases, particular traditional practices, such as rainwater storage on roofs, expose them to higher risk. The major epidemic in Puerto Rico in 1977 serves as a reminder that advanced public health capacities and economic development may not guarantee protection against massive epidemics \[[@B9]\]. Despite high quality of health services and richer circumstances, complacency in endemic countries may lead to increased rates without continued vigilance. On an individual level, evidence points to greater susceptibility among well-nourished or middle-class communities rather than malnourished and poorer patients commonly associated with other tropical diseases. A case-control study of serologically-confirmed DHF patients, other infectious diseases patients and healthy children in the Children\'s Hospital in Bangkok showed that malnutrition amongst DHF patients was significantly lower \[[@B57]\]. In India, a hospital-based study found no association between nutritional status and severity of illness \[[@B40]\]. Middle classes have been specifically noted as the proportionally predominant group during the epidemic in Dhaka Bangladesh \[[@B25]\] and upper social classes had statistically higher sero-infection rates in Fortaleza and San Luis epidemics in Brazil \[[@B38]\]. Confounding factors for the preponderance of DF/DHF among the upper classes or well-nourished dengue patients were not discussed in any of these studies. Few studies specifically measure and test socio-economic determinants of exposure at community levels. Heukelbach in Fortaleza, Brazil did examine socio-economic variables but their study did not show an association with DF \[[@B58]\]. Since all 34 cases selected were from a shanty town (favela), a lack of heterogeneity may have been a factor for this result rather than a real absence of difference. In Taiwan, Ko, also in a case-control study, observed that patients who lived near markets and/or open sewers or ditches had a risk of contracting disease 1.8 times higher than those who did not \[[@B59]\]. Since housing near sewers and ditches is likely to comprise poorer families, the analysis should have tested for house site while controlling for use of screens, which were significantly associated with incidence. ### Costs On a macro level, the impact of dengue on the economy is likely to be increasingly similar to that of malaria. Prevalent in communities characterised by subsistence or daily wage labour, a week\'s illness can be catastrophic for poor families. As a primarily paediatric disease in the past, the active labour force or the family wage earners were less affected. Now, as the modal age of illness and incidence increases, losses in productivity and earning capacity may be expected. The economic lesson from malaria was learnt late and when the resurgence was already in full swing. Dengue fever risks the same fate. With regard to costs of care, few economic studies exist and most estimate economic loss to range in millions. Von Allmen et al. undertook a cost analysis of the epidemic of DF/DHF in Puerto Rico in 1977 using upper and lower limits of incidence \[[@B60]\]. They estimated the direct costs (medical care and epidemic control measures) to range between US\$2.4 and \$4.7 million and indirect costs (lost production of patients and parents of children) between US\$6 and \$15 million. Another economic study, still in Puerto Rico, assessed the loss in terms of DALYs due to dengue \[[@B61]\]. At 658 DALYs per year per million population, the study concluded that, in terms of its magnitude, DF ranks with TB, STDs (including HIV), childhood diseases or malaria. On a micro level, a detailed study on costs of care in 3 hospitals in Bangkok estimated direct adult patient costs at US\$67. Including opportunity costs, this figure increased to US\$161.49. The net hospital cost for each DHF patient was US\$54.60 and the public sector cost of prevention and control of the outbreak was US\$4.87 million. The total expenditure for DHF in 1994 was estimated to be at least US\$12.56 million, of which 45% was borne by the patients \[[@B62]\]. These figures are reminders that most of the countries subject to DF/DHF cannot realistically afford a US\$5 million prevention and control budget for a single disease and that the monthly income of many families in these countries is less than the direct cost of US\$70 a month. ### Knowledge, attitude and practice (KAP) Much needs to be done in finding effective strategies for behaviour change. Since mothers are the first-line care-givers, this aspect is key, particularly for childhood diseases. KAP studies are rare and therefore little is known regarding knowledge and attitude of the exposed population towards dengue. However, the little that is known is encouraging. Straightforward community education to reduce breeding sites for mosquitoes performed better than chemical spraying in a controlled experiment in Mexico \[[@B63]\]. However, housewives, the unemployed and the elderly had significantly lower levels of knowledge of the disease compared to students and persons of younger ages (odds ratio (OR) = 0.44, 95% confidence interval (CI): 0.31--0.64). Other KAP studies have found that radio and television are very effective channels for knowledge dissemination. Nevertheless, these same studies found that while communities can score well in knowledge of the disease, they perform less well in attitude and practice, indicating that behaviour change is one area to target in social mobilisation programmes \[[@B64]-[@B67]\]. Treatment-seeking behaviour and lay symptom assessment is the first step in the chain to early diagnosis and was found to have an impact on duration of illness in Thailand \[[@B68]\]. In that context, it is discouraging to note that 45% of individuals in a population-based survey (23,970 households) in the urban municipality of Vientiane did not know what action to take when their children are diagnosed with dengue or what they should do for prevention \[[@B44]\]. Finally, reducing mortality from DHF and strengthening its control and prevention clearly cannot be done by the population alone. In most circumstances, these are poor populations with other pressing agendas. The programme requires public sector leadership with strong intersectoral collaboration. The WHO has made important progress to determine ways and mechanisms through which to achieve collaboration between sectors and state policy directions for control. ### Trends in case fatality rates Two aspects present themselves for useful discussion in this area. One relates to wide variations in CFRs between countries, sub-national units and hospitals under similar virological conditions. The other relates to differential risks of severe illness and mortality between children and adults. The global case-fatality rate (CFR) for DHF/DSS has been declining in most of the endemic countries according to government statistics. The overall CFR in the southeast Asia region is now less than 1% \[[@B20]\]. However, disaggregated data reveal a different picture. Rates vary significantly between countries, provinces and hospitals, pointing to a more complex situation. From 1995--2000, the CFR in the countries of WHO Western Pacific Region ranged from 0.06% in Singapore and 0.17% in Malaysia to 3.4% in Cambodia. Hong Kong reported no deaths \[[@B69]\]. In Vietnam, province-based 1998 data for DHF show CFR ranging from nearly 13% in Ha Tinh to 0.5% in Quang Tri \[[@B70]\]. Although the four provinces with the highest CFR were at some distance from Ho Chi Minh City or Hanoi, the four of the lowest were not particularly closer to these centres of tertiary care. In Laos, on the other hand, CFR for DHF during 1998 reached a high of 9.7% in Champassak province compared to 1.4% in Municipality of the capital city, Vientiane \[[@B44]\]. Wide variation in CFRs ranging from 0.1% to 5%, was also noted between the first administrative divisions in the Philippines \[[@B71]\]. During the 1998 epidemic in Cambodia the CFR in Kantha Bopha, a private, charitable hospital, was substantially lower (1.96%) than the national average (2.91%) \[[@B70]\]. Inter-district and inter-hospital variation is generally indicative of quality of care. Availability of medical supplies, equipment and economic status of patients can explain some differences but analyses to distinguish between the performances of provinces and countries in comparable settings would be useful for designing more effective disease control. Secondly, studies have postulated higher risk of DF/DHF morbidity and mortality among children compared to adults \[[@B15]\]. Recently, increasing reports of severe illness among adults and in some cases higher CFRs (e.g. age-specific CFRs from San Lazaro hospital over one year were 3.8% for 35--39 year olds, 8% for over 45s compared with 2% and 2.6% for 0--4 and 5--9 year age groups) merit closer looks at determinants of adult mortality \[[@B37],[@B71],[@B72]\]. ### Case management and early detection In addition to vector control, widely recognised as a preventive strategy of choice, key health sector response for reduction of mortality and morbidity lies primarily in two areas: early detection (including care-seeking behaviour change and better surveillance) and improved case management of patients. Mortality in excess of 1% may be considered the consequence of inadequate care, late diagnosis and delayed hospitalisation. A hospital-based study during the dengue outbreak in Delhi revealed that mortality could be very low in patients who came early to the hospital \[[@B73]\]. Late presentation was also strongly associated with increased mortality in children with DHF in the Philippines \[[@B74]\]. The short interval between onset of haemorrhage and death, especially in young children, makes rapid medical intervention for DHF/DSS a critical factor for survival. For most communities at highest risk of disease, intensive care facilities are only available at distant capitals requiring motorised transport, usually beyond the reach of many. Early diagnosis and leading indicators for DHF/DSS can ensure the availability of travel time to transfer the patient for effective treatment. Case-control studies have shown that low-normal hematocrit count at time of shock is a significant risk factor for haemorrhage \[[@B75]\] and potential predictors for clinical outcome, such as decrease in total plasma cholesterol, and high- and low-density lipoprotein, were associated with the severest cases \[[@B76]\]. However, research into predictive factors for severe illness is neither abundant nor conclusive. Moreover, as Van Gorp concludes, low capacity and lack of resources at secondary levels of health services limit the operational use of many of these findings \[[@B76]\]. At this time, the WHO classification of dengue diseases is often not feasible in many countries because of lack of trained health professionals, inadequate laboratories, and radiological support. Neither are facilities to detect DHF by using hematocrit and plasma leakage signs readily available in many tropical countries. As successful treatment of dengue depends on symptom recognition and careful fluid management, a simpler dengue disease classification scheme, realistic in poor, provincial conditions and better training of district-level personnel is needed. A few creative approaches to primary health care to improve quality of care and case management at primary health care levels have been reported in the literature. For example, encouraging results have been found in Vietnam where they reduced dengue mortality rates by 64% through innovative primary healthcare concepts, including paediatric priority training units for medical staff, health education for patient carers and promotion of outpatient treatment to avoid unnecessary admissions \[[@B77]\]. Reduction of CFRs from 10--15% (40% in some areas) in the early 1950s to less than 0.5% today in east Asian referral hospitals have been attributed to better training of the hospital staff \[[@B78]\]. The effect of strengthened health systems is recognised by public health authorities including WHO but is missing operational research and policies to put them into effect. ### Surveillance and reporting Unreliable statistics are an extremely serious weakness from many perspectives. Estimates of DHF/DSS CFR from surveillance data are consistently lower than those from single sample study data suggesting under-reporting or misclassification of deaths. Inadequate knowledge of case definitions among district health personnel compromise complete reporting even within the public health service system. Inappropriate denominators further add to the confusion in estimating prevalence and incidences. Reporting deviations can lead to seriously misleading CFRs in countries where reliable estimates are urgently needed for effective resource programming. In Laos, for instance, 8197 DHF cases and 24 deaths were registered by the WHO in 1996, compared to 2563 cases and 23 deaths registered by the Institute of Malariology, Parasitology and Entomology (IMPE) for a CFR of that is 3 times higher than WHO statistics \[[@B22]\], Most national surveillance data rely only on public sector institution reporting. An evaluation of the dengue reporting system in Bandung, Indonesia (covering private and public hospitals) found that only 31% of hospitalised DHF/DSS cases were reported to the Municipal Health Authorities \[[@B79]\]. In Puerto Rico, a hospital record review revealed a ratio of 3:1 total DHF cases compared to those detected by surveillance \[80\]. More alarmingly, in Texas, USA, an assessment of underdiagnosis of dengue was undertaken motivated by an outbreak in a town across the border in Mexico. A review of medical records between 23 July and 20 August 1999 found that 50% of suspected cases had undiagnosed dengue infection. \[81\]. Based on the above studies, a conservative estimate would be that a third of the total cases are captured by surveillance systems, indicating that the global incidence rate could be around 1.5 million cases of DHF on an average year rather than the 0.5 million estimated by WHO. While complete surveillance data may be an unrealistic option in many the affected countries, sentinel surveillance and sample surveys using reliable methodologies could be undertaken to provide more accurate estimates of the disease burden and fill in the gaps. Occasional sample surveys of the private sector could help better estimate the bias in disease burden. Conclusion ========== On 18 May 2002, the WHO General Assembly confirmed dengue fever as a matter of international public health priority through a resolution to strengthen dengue control and research. Today, changing characteristics of the disease deserve serious research attention. Shifts in modal age, rural spread, social and biological determinants of race- and sex-related susceptibility have major implications for health service planning and control strategies. Behavioural risk factors, individual determinants of outcome and leading indicators of severe illness are poorly understood, compromising the effectiveness of control programmes. Early detection and case management practices have been noted as a critical factor for survival. Yet well-targeted operational research in these areas is rare. Population-based epidemiological studies with clear operational objectives should be launched as concerted efforts at regional levels. A major weakness is the inadequacy of sound statistical methods in some of the reviewed studies. Samples are exceedingly small in many cases, selection methods are often inadequately described or are self selecting, tests of significance are frequently not undertaken or not reported and denominators are not clearly described. Conclusions therefore do not have the full benefit of objective statistical analyses, reducing the scientific strength of the results. Furthermore, conclusions regarding case fatality or disease-specific mortality rates are hard to draw since they are frequently based on hospitalised patients who had actively sought care in tertiary centres. However, a systematic approach and a clear international research agenda can quickly bring forward the frontiers of knowledge. Better understanding of the above will not only feed into operational policy for dengue control, but also provide fertile terrain for vaccine application strategies in the future. Today, dengue control and prevention requires thinking outside the tropical disease box. Many of the affected countries are some of the poorest. Approaches that are realistic for their infrastructure need to be urgently developed. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= Debarati Guha-Sapir set out the plan of the paper, its focus areas and main messages. Barbara Schimmer carried out the literature search, summarised the studies and their results. She helped D. Guha-Sapir with the writing of the text. Acknowledgements ================ Grateful acknowledgements are due to M. Nathan, S. Macfarlane and A. Karaoglou for keeping us informed, involved and encouraged and Wilbert Van Panhuis for general research assistance. The European Union Fifth Framework programme Contract no ICA4-CT-1999-50008 permitted us to undertake some of the basic epidemiological data reviews on the ground.	PubMed Central	2024-06-05T03:55:54.165377	2005-3-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555563/", "journal": "Emerg Themes Epidemiol. 2005 Mar 2; 2:1", "authors": [ { "first": "Debarati", "last": "Guha-Sapir" }, { "first": "Barbara", "last": "Schimmer" } ] }
PMC555564	Background ========== Light is the principal zeitgeber that entrains circadian rhythms of physiology and behaviour \[[@B1],[@B2]\]. The major light input pathway to the suprachiasmatic nucleus (SCN) is the retinohypothalamic tract \[[@B3]\], which arises from a population of retinal ganglion cells \[[@B4]\]. Recent studies have demonstrated that melanopsin-containing retinal ganglion cells, rods, and cones all convey photic information to the SCN, and mice lacking these photoreceptive systems cannot be entrained by light \[[@B5]-[@B11]\]. Excellent progress has been made in the understanding of circadian photic entrainment \[[@B12]-[@B15]\]. This includes light-induced transcriptional activation of core clock genes in the SCN, such as Per1and Per2, as well as immediate-early gene c-fos. Exposure to light pulses at night induces expression of these genes in the SCN, and this light induction mechanism has been suggested as a critical pathway for the resetting of circadian clock in response to changes in light/dark conditions \[[@B16]-[@B19]\]. Intercellular signalling mechanisms between SCN neurons are also important in circadian photic entrainment, as mice with mutation in a neuropeptide receptor for VIP (Vasoactive Intestinal Peptide) and PACAP (Pituitary Adenylate Cyclase Activating Peptide) are unable to sustain normal circadian behaviour and exhibit loss of sensitivity to light \[[@B20]\]. In addition to the effect of light on circadian entrainment, light also has a direct effect on physiology and behaviour, generally termed as \"masking\" \[[@B21],[@B22]\]. For instance, light pulses given at night acutely suppress the locomotor behaviour of nocturnal rodents \[[@B21],[@B22]\], and this can occur without functional clockwork \[[@B23]-[@B27]\]. Masking may account for the maintenance under normal light/dark conditions of wheel-running rhythms in cryptochrome-deficient (Cry1-/-Cry2-/-) mice, which are behaviourally arrhythmic under constant darkness. The contribution of masking to normal locomotor activity rhythms is unclear, as is the participation of the SCN in masking effects of light. Vitaterna et al (1999) first observed a light-driven Per2rhythm in the SCN in Cry1-/-Cry2-/-mice, and have suggested that the light-driven molecular rhythm in the SCN may be related to the preservation of their locomotor rhythm \[[@B25]\]. We previously found that prokineticin 2 (PK2) is a first order clock-controlled gene, whose expression in the SCN is regulated by CLOCK and BMAL1 acting on the E-boxes in the gene\'s promoter \[[@B28]\]. We have also demonstrated that PK2 may function as a SCN output molecule that transmits circadian locomotor rhythm via activation of a G protein-coupled receptor \[[@B28],[@B29]\]. Interestingly, we also observed that PK2expression is rapidly induced by light pulses administered at night \[[@B28]\], a characteristic that is usually seen with core clockwork genes but not clock-controlled genes. Here we further investigated the light regulation of the rhythm of PK2expression in the SCN. In particular, we investigated the photoreceptive mechanisms responsible for the light-induced PK2expression in the SCN. Utilizing Cry1-/-Cry2-/-mice, we also determined whether light can drive PK2expression in the SCN independent of a functional circadian clock. Results ======= PK2responds differentially to the delay and advance of light/dark cycles -------------------------------------------------------------------------- We first examined the effects of abrupt shifts of light/dark cycles on PK2mRNA rhythm in the SCN. Animals were first entrained for two weeks under 12 hour light: 12 hour dark (LD), then subjected to either a 6 hour delay (6hrD) shift or 6 hour advance (6hrA) shift of light/dark cycles. We measured PK2mRNA in the SCN of these animals to examine how quickly the PK2mRNA rhythm re-entrains to the shifted light/dark cycles. Under LD, PK2mRNA peaks during the day and remains low or undetectable during the night. During the first cycle of the delayed shift (6hrD), the PK2mRNA rhythm responds quickly: the rising phase of PK2expression adjusts rapidly to the delayed light/dark cycles, while the falling phase still resembles that of the unshifted light/dark cycles (Figure [1A](#F1){ref-type="fig"}). In contrast, the PK2mRNA rhythm responds very little to a 6 hour advance shift (6hrA). During the first cycle of the advance shift, the PK2oscillation pattern remains similar to that of the unshifted LD (Figure [1B](#F1){ref-type="fig"}). These changes in PK2expression during 6hrD or 6hrA shift indicate that the endogenous circadian clock exerts dominant control over the PK2rhythm, as PK2expression cannot respond immediately and completely to the shifts of light/dark cycles. As it normally takes about 1--2 days for locomotor rhythms to stably entrain to phase delays and about 5--6 days to entrain to phase advances \[[@B30],[@B31]\], we next examined the timecourse of shifts of the PK2rhythm to 6 hour phase advances and delays. Consistent with the animal\'s locomotor behaviour, the PK2mRNA rhythm reaches stable phase within 2 days of 6hrD shift (Figure [1C](#F1){ref-type="fig"}). In contrast, only the rise of PK2reaches stable phase within 2 days of 6hrA shift, while the fall of PK2takes longer (Figure [1D](#F1){ref-type="fig"}). Thus, we further examined whether the PK2rhythm is stably entrained after 6 days of 6hrA shift. As expected, the PK2rhythm is completely entrained to 6hrA shift after 6 days (Figure [1D](#F1){ref-type="fig"}). Together, the differential responses of PK2rhythm to a 6hrD or 6hrA shift indicate that the endogenous circadian clock predominantly controls PK2rhythm, as circadian oscillators typically show rapid phase delays but advance with transients \[[@B31],[@B32]\]. The entrainment patterns of PK2during phase shifts are consistent with behavioural studies in animals and human subjects \[[@B30],[@B31]\]. PK2rhythm is entrained by different photoperiods -------------------------------------------------- We next examined the effect of photoperiod on the PK2molecular rhythm in the SCN. PK2mRNA was measured in the SCN of mice entrained under different photoperiods: 8 hour light: 16 hour dark (8L:16D), 16 hour light: 8 hour dark (16L:8D), or 20 hour light: 4 hour dark (20L:4D). During 12L:12D, PK2mRNA is highly expressed during the 12 hour light phase with peak level at ZT4 (Figure [1A](#F1){ref-type="fig"}, Figure [3A](#F3){ref-type="fig"}). Under 16L:8D, PK2mRNA expands to the entire 16 hour light phase and is essentially undetectable during the 8 hour dark period (Figure [2B](#F2){ref-type="fig"}). However, the expression of PK2mRNA is not confined to the light phase of the shorter photoperiod (8L:16D), as PK2mRNA rises before lights on and persists after lights off (Figure [2A](#F2){ref-type="fig"}). The temporal profile of PK2mRNA under this short photoperiod (8L:16D) is very similar to that observed under 12L:12D (Figure [1A](#F1){ref-type="fig"}, Figure [3A](#F3){ref-type="fig"}) or constant darkness (2DD) \[[@B28]\]. Thus, although light can induce PK2mRNA and expand the duration of PK2expression, the phase angle of PK2expression is determined by the circadian clock, and its duration cannot be further compressed under shorter photoperiods. Interestingly, the peak of PK2mRNA expression was significantly higher in long days (16L:8D) than in shorter days (8L:16D) (Figure [2A--B](#F2){ref-type="fig"}), further indicate the enhancing effect of light on PK2expression. However, a significant reduction in the PK2peak level is observed under a very long photoperiod (20L: 4D) (Figure [2C](#F2){ref-type="fig"}). We also noticed that under 20L:4D, PK2mRNA is further expanded and becomes detectable even in dark phase (Figure [2C](#F2){ref-type="fig"}). Under this long photoperiod (20L:4D), the difference between the peak and basal level of PK2is only about 4 fold (Figure [2C](#F2){ref-type="fig"}). As it has been reported that the rhythms of mPer1and mPer2mRNAs in the SCN are also entrained with different phase angles under a variety of photoperiods \[[@B33]-[@B35]\], we have also examined Per1and Per2rhythm in our photoperiod studies (see [Additional file 1](#S1){ref-type="supplementary-material"}). The Per1and Per2rhythm we observed under these photoperiods are consistent with previous findings \[[@B35]\]. Taken together, these results indicate that changes in photoperiod alter PK2rhythm in the SCN, and the amplitudes of PK2mRNA oscillation are greatly reduced in very long photoperiods. Light inducibility of PK2is eliminated in mice that lack melanopsin, rod and cone phototransduction system (Opn4-/-, Gnat1-/- Cnga3-/-mice) ----------------------------------------------------------------------------------------------------------------------------------------------- As melanopsin has been implicated in circadian photoreception \[[@B5]-[@B11]\], we examined whether the PK2molecular rhythm is normally entrained in melanopsin-deficient (Opn4-/-) mice. Figure [3](#F3){ref-type="fig"} shows that the oscillation profile of PK2in the SCN of Opn4-/-mice is essentially identical to that observed in the wild type mice under LD. This normal temporal profile of PK2mRNA corresponds with the normal locomotor rhythm of Opn4-/-mice under light/dark conditions \[[@B7],[@B8]\]. As Opn4-/-mice display attenuated phase resetting in response to light pulses and exhibit impaired light masking responses to bright light \[[@B36]\], we also examined whether light inducibility of PK2is blunted in Opn4-/-mice. Figure [3B](#F3){ref-type="fig"} shows that light pulse-induced PK2in the SCN of Opn4-/-mice was significantly reduced by about 50% and 60%, one and two hours after the light pulse, respectively. The Opn4-/-light pulse studies show that a residual PK2expression is still present after a light pulse, suggesting that without melanopsin, other phototransduction system can still transmit light information to induce PK2expression. Thus, we decided to examine the light inducibility of PK2in triple knockout mice lacking melanopsin, rod and cone phototransduction systems (Opn4-/- Gnat1-/- Cnga3-/-mice), as these animals free run under light dark conditions (LD) and lack masking responses to light \[[@B10]\]. Figure [3C](#F3){ref-type="fig"} shows that the light pulse-induced PK2in the SCN is completely eliminated in these triple knockout mice, consistent with their malfunctioning photoentrainment systems and their lack of masking responses to light \[[@B10]\]. In addition, we also observed that PK2mRNA followed the free-running locomotor rhythms in these triple knockout mice (Figure [3D](#F3){ref-type="fig"}), with high levels of PK2during the inactive phase (CT3) and low levels during active phase (CT15). Together, these results suggest that melanopsin contributes to the light inducibility of PK2, and intact melanopsin with functional rod/cone phototransduction systems are required for the light inducibility of PK2. A low amplitude PK2rhythm is preserved in cryptochrome-deficient (Cry1-/-Cry2-/-) mice under light/dark conditions ---------------------------------------------------------------------------------------------------------------------- Previous studies have shown that the light-regulated Per2rhythm is maintained in the SCN of cryptochrome-deficient (Cry1-/-Cry2-/-) mice that lack functional circadian clock \[[@B25],[@B37]\]. In order to determine whether the regulation of PK2, Per1, Per2and Bmal1expression by light requires an intact circadian pacemaker, we systematically assessed the temporal mRNA profiles of clockwork genes in Cry1-/-Cry2-/-mice under both light/dark (LD) and constant dark (DD) conditions. Figure [4](#F4){ref-type="fig"} shows that the molecular rhythm of Per2remained largely intact in Cry1-/-Cry2-/-mice entrained under12L:12D, with levels about 4-fold higher during the light phase than the dark phase. This amplitude of the Per2oscillation profile was similar to that observed in wild type mice \[[@B18],[@B38]\]. A low amplitude Per1rhythm in Cry1-/-Cry2-/mice was also apparent under LD, but not DD (Figure [4B](#F4){ref-type="fig"}). We further detected a light-driven Bmal1rhythm in the SCN of Cry1-/-Cry2-/-mice under LD, but not DD (Figure [4C](#F4){ref-type="fig"}). Interestingly, this Bmal1rhythm in Cry1-/-Cry2-/-mice peaked during light phase, opposite from the Bmal1rhythm in wild type mice and in phase with Per1\[[@B39],[@B40]\]. As it has been suggested that PER2 can positively regulate Bmal1expression via inhibition of the orphan nuclear receptor REV-ERBα \[[@B41],[@B42]\], it is possible that this Bmal1rhythm is secondary to the light-driven Per2rhythm. Further studies are required to clarify this observation. We also examined the molecular rhythm of PK2in Cry1-/-Cry2-/-mice. Figure [4D](#F4){ref-type="fig"} shows that PK2mRNA rhythm in the SCN of Cry1-/-Cry2-/-mice was apparent under LD, with the presence of a low level PK2during light phase and absence of PK2during dark phase (see [Additional file 2](#S2){ref-type="supplementary-material"}). Similar to wild type mice, the peak level of this low amplitude PK2rhythm was around ZT4, although its peak was only about 8% of that observed in wild type mice (Figure. [4D](#F4){ref-type="fig"}, Figure [1A](#F1){ref-type="fig"}, Figure [3A](#F3){ref-type="fig"}). No PK2rhythm was evident when Cry1-/-Cry2-/-mice were placed under DD (Figure [4D](#F4){ref-type="fig"}). Furthermore, the inducibility of PK2to nocturnal light pulses is also maintained in Cry1-/-Cry2-/-mice. PK2mRNA increased one and two hours after a brief light pulse at ZT14 (Figure [4E](#F4){ref-type="fig"}). Nevertheless, light-induced PK2was still detected in Per1,2,3-/-mice and Clk-/-mice that lack functional circadian clock (Cheng, Weaver & Zhou, unpublished observations). As PK2remains responsive to light in these clock mutant mice that lack functional circadian clock, it is likely that the low amplitude PK2rhythm in Cry1-/-Cry2-/-mice under LD is directly driven by light. In order to test whether this light-driven PK2rhythm may be related to the maintenance of behavioural rhythms observed in Cry1-/-Cry2-/-mice under LD, we studied the responses of Cry1-/-Cry2-/-mice to a 6 hour advance of lighting schedule. In contrast to the transients of entrainment of locomotor rhythms in wild type mice (which takes about 4-5 days to re-entrain to phase advance), the locomotor activity of Cry1-/-Cry2-/-mice adjusted rapidly to 6 hr advance (Figure [4F](#F4){ref-type="fig"}). Such a rapid response is characteristic of masking. A correlative rapid adjustment of PK2was also observed in the SCN of Cry1-/-Cry2-/-mice (Figure [4G](#F4){ref-type="fig"}). As Cry1-/-Cry2-/-mice lack functional circadian clock and their locomotor behaviour and PK2expression patterns are completely light driven, our results suggest that this low amplitude, light-driven rhythm of PK2may contribute to or underlie the masking of locomotor behaviour in these animals. Discussion ========== Our studies indicate that the molecular rhythm of PK2in the SCN is predominantly controlled by the circadian clock, with light playing a modulatory role. Abrupt shifts of light/dark cycles significantly altered the phase of the PK2rhythm. While PK2expression re-entrained rapidly to phase delays, it takes several cycles of transients for PK2to be stably entrained to phase advances (Figure [1](#F1){ref-type="fig"}). The rate of re-entrainment of PK2molecular rhythms to these shifts is consistent with that of behavioural adaptation of animals and human subjects \[[@B30],[@B31]\]. Our photoperiod studies indicate that PK2expression in the SCN responds differentially to changes in photoperiod length (Figure [2](#F2){ref-type="fig"}). Although increasing light period can induce PK2expression and expand the duration of PK2rhythm (Figure [2B](#F2){ref-type="fig"}), shortening of the light period does not lead to corresponding reduction of the duration of PK2expression (Figure [2A](#F2){ref-type="fig"}). It appears that a minimal duration of PK2expression is maintained under short photoperiod (Figure [2A](#F2){ref-type="fig"}) and constant darkness \[[@B28]\], which further indicate the dominant control of PK2expression by the circadian clock. Interestingly, the amplitude of the PK2oscillation was greatly reduced under very long photoperiod (20L:4D) (Figure [2C](#F2){ref-type="fig"}). As the amplitude of both Per1and Per2rhythms were also reduced during 20L:4D (see [Additional file 1](#S1){ref-type="supplementary-material"}), it is likely that these depressed rhythms of clockwork genes may contribute to the depressed PK2rhythm observed. Whether reduction in the amplitude of expression in any of these genes is related to arrhythmicity in LL deserves further examination. Our studies with Cry1-/-Cry2-/-mice revealed the presence of a light-driven PK2molecular rhythm in the SCN under LD, indicating that light can drive PK2rhythm independent of functional circadian clock. Interestingly, the molecular rhythms of some clockwork genes such as Per2, Per1, and Bmal1were also partially maintained in the SCN of Cry1-/-Cry2-/-mice under LD (Figure [4](#F4){ref-type="fig"}). Thus, light-driven molecular oscillations of clockwork or clock-controlled output genes exist in the absence of functional circadian clock. Vitaterna et al (1999) first noticed such light-regulated Per2molecular rhythm in the SCN of Cry1-/-Cry2-/-mice, and suggested the term of \"light-driving\" effect \[[@B25]\]. As Cry1-/-Cry2-/-mice lack functional circadian clocks and their locomotor behaviour remains rhythmic under LD, but not under DD conditions \[[@B24],[@B25]\], it is likely that these light-driven molecular rhythms may drive the locomotor rhythms in these animals. As we have previously shown that PK2may be a critical output molecule responsible for circadian locomotor rhythms, the presence of this light-driven PK2rhythm in Cry1-/-Cry2-/-mice may thus contribute to or underlie masking as well as the free running behavioural rhythms in these animals. It is well established that an intact SCN is necessary for the preservation of free running locomotor rhythms \[[@B43]\]. The role of the SCN in masking of locomotor activity by light is controversial, with similar studies having produced contradictory results \[[@B23],[@B44]\]. Thus, it is possible that there might be common signal molecule(s) that mediate(s) the light-masking and the circadian clock-controlled locomotor behaviour. Construction of PK2-deficient mice will be necessary to resolve the exact role of PK2 in the light-driven locomotor rhythms. The light inducibility of PK2in the SCN is an unusual characteristic for a clock-controlled gene. Our results demonstrate that melanopsin-positive retinal ganglion cells, in conjunction with rods and cones, are responsible for the light-inducibility of PK2(Figure [3](#F3){ref-type="fig"}). The same photoreceptive system has been shown responsible for the entrainment of locomotor rhythm \[[@B5]-[@B11]\]. The light inducibility of PK2may be related to the presence of a putative cyclic AMP response element (CRE) in the promoter of the PK2gene \[[@B28]\]. CRE-dependent activation is critical for light-induced gene expression in the SCN \[[@B45]-[@B48]\]. The reduced light inducibility of PK2in mutant mice that lack functional clock may indicate that CRE-dependent pathway and CLK/BMAL1 transcriptional factors may interact in the light-induced PK2expression in the SCN. Accumulative data have implicated the photic regulation of the transcription of clock genes such as Per1and Per2in the entrainment of behavioural rhythms \[[@B30],[@B34]\]. The phase of the core SCN clock gene expression determines the timing of clock-controlled SCN output signals that ultimately regulate physiology and behaviour. Unlike the Per1promoter, whose activation in the SCN shifts rapidly when the LD cycle is advanced \[[@B31]\], PK2exhibits transients during phase advance, more similar to those of Cry1and Cry2\[[@B30],[@B31]\]. This is consistent with the role for PK2 as a clock-controlled gene and thus is downstream from the light-regulated expression of Per1or Per2. The presence of E box motifs in the PK2promoter suggests that light-regulated Per1(and perhaps Per2) expression can influence PK2expression. However, the light inducibility of PK2indicates that PK2 may have a more direct and central role in entrainment in addition to its putative role as an SCN output signal. In other words, whether PK2 functions completely outside the central circadian loops or partly within them has yet to be determined. It is well established that the activation of glutamate receptor and its downstream actions are critical for the retinohypothalamic inputs of light to the SCN \[[@B49]\]. As receptor for PK2 is highly expressed in the SCN \[[@B28]\] and activation of the PK2 receptor triggers similar signalling pathways as that of glutamate receptors \[[@B29]\], it is possible that the circadian clock and/or light-driven PK2 may feed back to the core circadian loops in the SCN. In addition, PK2 has recently been shown to excite neurons that express PK2 receptor \[[@B50]\], further suggesting that PK2 may activate the firing of SCN neurons, and thus possibly participate in the synchronization of the circadian clock. Thus, the light inducibility of PK2may be relevant to both the phase resetting of the core circadian loops and critical SCN output signals. Conclusion ========== Our studies demonstrate that PK2is predominantly driven by the circadian clock, as PK2expression exhibits circadian transients in response to phase advances. Furthermore, shortening of the light period does not result in corresponding reduction of the phase of PK2rhythm, also consistent with the dominant control from the circadian clock on PK2expression. However, light also modulates PK2rhythm. Nocturnal light pulses can directly induce PK2expression in the SCN. Studies with Cry1-/-Cry2-/-mice revealed that light can drive a low amplitude PK2molecular rhythm in the SCN in the absence of functional circadian oscillators. These studies demonstrate that PK2molecular rhythm in the SCN is controlled by dual mechanisms: dominantly by the circadian transcriptional loops but also directly by light. The light inducibility of PK2in the SCN suggest that in addition to PK2\'s role as a SCN output signal, PK2 may also participate in the photic entrainment of circadian clock and perhaps in masking. Methods ======= Experiments of light/dark cycle shifts -------------------------------------- Male adult C57BL/6 mice (Taconic Farms, New York) were entrained under 12 hour light: 12 hour dark (12L:12D, lights on at 0700 h) cycle for two weeks with food and water available ad libitum. Light phase was either delayed by 6 hours (lights on at 1300 h) or advanced by 6 hours (lights on at 0100 h) and samples were taken every three hours for the 24 hour period (Zeitgeber time, ZT, ZT1-22). To examine PK2expression two days after the shift, animals were placed in two additional light/dark cycles and brain samples were collected. All animal procedures were approved by the Institutional Animal Care and Use Committee and consistent with Federal guidelines. In situ hybridization was used in all studies to examine PK2mRNA expression in the SCN \[[@B28]\]. Antisense and sense riboprobes containing the coding region of mouse PK2(accession number AF487280 1-528 nt), mouse Per1(accession number AF022992 340-761nt), mouse Per2(accession number AF035830 9-489 nt) and mouse Bmal1(accession number AB015203 864-1362 nt) were generated. Photoperiod studies ------------------- Animals were initially entrained under 12L:12D for one week, followed by placement in different photoperiods (light intensity \~400 lux) for three to four weeks: 8 hour light:16 hour dark (8L:16D, lights on at 0900 h, lights off at 1700 h), 16 hour light: 8 hour dark (16L:8D, lights on at 0500 h, lights off at 2100 h). For the 20 hour light: 4 hour dark (20L:4D, lights on at 0300 h, lights off at 2300 h), animals were first placed in 14L:10D for one week, transferred to 16L:8D for another week, followed by two weeks in 20L:4D. All brain samples were taken every two hours throughout the 24 hour cycle, except the first and the last two time points which were sampled every three hours. Studies of melanopsin-deficient mice and mice that lack melanopsin, rods and cones ---------------------------------------------------------------------------------- Wild type and melanopsin-deficient (Opn4-/-) mice (on C57BL/6:129 hybrid background) \[[@B5]\] were entrained to 12L:12D and sampled every three hours for the 24 hour period (ZT1-22). For light pulse studies, wild type, Opn4-/-mice and triple knockouts (Opn4-/- Gnat1-/- Cnga3-/-mice) that lack melanopsin, rod and cone phototransduction systems were used \[[@B10]\]. Animals received a 15 min light pulse (\~200 lux) at ZT14 and brains were sampled one or two hours after light pulse. Dark control animals did not receive a light pulse. Studies of cryptochrome-deficient (Cry1-/-Cry2-/-) mice --------------------------------------------------------- Cryptochrome-deficient (Cry1-/-Cry2-/-) mice on a C57BL/6:129 hybrid background were kindly provided by Dr. Aziz Sancar (University of North Carolina at Chapel Hill). Wild type and Cry1-/-Cry2-/-mice were entrained to 12L:12D and sampled every three hours for the 24 hour period (ZT1-22). A second group of Cry1-/-Cry2-/-mice were placed into two days of constant darkness (2DD) (Circadian time, CT, CT1-22). The mRNA levels of PK2, Per2, Per1and Bmal1were measured in the SCN. For light pulse experiments, Cry1-/-Cry2-/-mice received a 15 min light pulse (\~400 lux) at ZT14, and sampled one or two hours after light pulse. Dark control Cry1-/-Cry2-/-mice did not receive a light pulse. For the shifting experiments, wildtype and Cry1-/-Cry2-/-mice were initially entrained under 12L:12D, then subjected to an acute 6 hour advance of lighting schedule. Running-wheel activities of these mice were monitored 10 days before and 10 days after the 6 hour advance shift. The 6 hour phase advance was then repeated and brains were collected at ZT4 and ZT16 on the day of the shift. Authors\' contributions ======================= ELB sampled the Cry1-/-Cry2-/-mice and performed behavior experiments on Cry1-/-Cry2-/-mice. SH sampled the melanopsin-deficient mice and triple knockout mice. MYC performed the tissue sectioning, in situ hybridizations and all quantitative analyses. MYC, ELB and QYZ drafted the manuscript. ELB, SH, MYC and QYZ designed the studies. All authors read and approved the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Effect of different photoperiods on molecular rhythms in the SCN. Temporal profiles of Per1 (a) and Per2 (b) mRNA under 8L:16D, 16L:8D, 20L:4D. Open and filled bars indicate light and dark periods, respectively. The zeitgeber time (ZT) on the x-axis reflects the timescale for each photoperiod. Each value represents the mean ± SEM of 3--4 animals. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 PK2 mRNA expression in Cry1-/-Cry2-/- and wildtype mice. Representative autoradiograms of PK2 mRNA in the SCN of Cry1-/-Cry2-/- mice (Cry) and wild type mice (WT) under LD (ZT1-22) are shown (top and bottom row, respectively). Scale bar = 1 mm. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We thank David R. Weaver and King-Wai Yau for providing helpful discussions, in situ probes and critical comments on the manuscript. We would also like to thank Aziz Sancar for providing access to Cry1-/-Cry2-/-mice, Frances Leslie for helpful discussions and equipment use, James Belluzzi for help with statistical analysis, Alex Lee for mice running-wheel setup and data analysis, Clayton Bullock and Jia-da Li for technical assistance and discussions. The research is partially supported by NIH grants (to QYZ and ELB). MYC was supported in part by a NIDA training grant and by a predoctoral fellowship from PhRMA Foundation. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### *Temporal profiles of PK2mRNA in the SCN in response to abrupt shifts of light/dark cycles.Animals were entrained to 12L:12D (LD) and subjected to either 6-hour delay of light/dark cycles (6hrD), 6-hour advance (6hrA), 6-hour delay followed by adaptation of 2 additional LD (6hrD+2LD), or 6-hour advance followed by adaptation of 2 additional LD (6hrA+2LD) or 6 additional LD (6hrA+6LD). Open and filled horizontal bars indicate light and dark periods, respectively. The LD data is doubled plotted as dashed line (open square) in all graphs. The zeitgeber time (ZT) on the x-axis reflects the timescale for LD, 6hrD or 6hrA. Please note that the additional LD adaptation groups use the same timescale as the 6hrD or 6hrA. (A) Temporal profiles of PK2mRNA under 6hrD and LD. Note that PK2mRNA responds quickly to the 6hrD shift. (B) Temporal profiles of PK2mRNA rhythm under 6hrA and LD. Note that PK2mRNA did not adjust to the 6hrA shift. (C) Temporal profiles of PK2mRNA rhythm under 6hrD, 6hrD+2LD and LD to indicate adaptation of PK2rhythm under 6hrD. Note that PK2rhythm is stably entrained to 6hrD after two days. (D) Temporal profiles of PK2mRNA rhythm under 6hrA, 6hrA+2LD, 6hrA+6LD and LD to illustrate adaptation of PK2rhythm under 6hrA. Note that PK2rhythm did not stably entrained to 6hrA until after 6 days. Each value is the mean ± SEM of 3 animals. ::: ![](1471-2202-6-17-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Effects of photoperiods on PK2mRNA rhythm in the SCN.Temporal profiles of PK2rhythm under 8L:16D (A), 16L:8D (B) and 20L:4D (C). Open and filled bars indicate light and dark periods, respectively. The zeitgeber time (ZT) on the x-axis reflects the timescale for each photoperiod. Each value represents the mean ± SEM of 3--4 animals. One-way ANOVA indicated that peak levels of all groups are significantly different from each other, p \< 0.05--0.001 using Bonferroni\'s post-hoc test. Autoradiographic images show representative mRNA expression of PK2during these photoperiods. ::: ![](1471-2202-6-17-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### *PK2mRNA rhythm in the SCN of melanopsin-deficient (Opn4-/-) mice and triple knockout mice (Opn4-/- Gnat1-/- Cnga3-/-mice).*(A) Temporal profiles of PK2mRNA rhythm in wildtype (filled squares) and Opn4-/-mice (open triangles) under LD. Open and filled bars indicate light and dark periods, respectively. Each value represents the mean ± SEM of 3--4 animals. Two-way ANOVA indicated that there is no significant difference between genotypes. (B) Light pulse-induced PK2mRNA expression in wildtype (shaded bars) and Opn4-/-mice (filled bars). PK2mRNA was measured one and two hours after brief light pulse at ZT14. Each value represents the mean ± SEM of 6--8 animals. \p \< 0.05, \\p \< 0.01, Student\'s t-test. (C) Light-pulse induced PK2mRNA expression in triple knockout mice that lack melanopsin, rod and cone photoreceptive system (Opn4-/- Gnat1-/- Cnga3-/-mice). Dark controls received no light pulse. Each value represents the mean ± SEM of 3 animals. (D) PK2mRNA expression in triple knockout mice at circadian time (CT) 3 and 15. ::: ![](1471-2202-6-17-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### *Light-driven molecular rhythms in the SCN of Cry1-/-Cry2-/-mice.Temporal mRNA profiles of Per2(A), Per1(B), Bmal1(C) and PK2(D) in Cry1-/-Cry2-/-mice under 12L:12D (LD) or two days constant darkness (2DD). Each value represents the mean ± SEM of 3--4 animals. Two-way ANOVA with Bonferroni\'s posthoc analysis was used to test for significant interactions between expression across time of sampling and under different lighting conditions (LD vs 2DD). p \< 0.0001 (Per2), p \< 0.002 (Per1), p \< 0.0001 (Bmal1) and p \< 0.0001 (PK2). (E) Light pulse-induced PK2mRNA in Cry1-/-Cry2-/-mice. PK2mRNA was measured one and two hours after brief light pulse at ZT14 (shaded bar). Black bar represents dark controls that did not receive light pulse. Each value represents the mean ± SEM of 5--6 animals. Two-way ANOVA indicates a significant difference in PK2expression between light and dark treatment (p \< 0.05), however, the PK2induction is not significantly different between the two timepoints (1 hr vs 2 hr). (F) Locomotor behavioural rhythms of wild type (left) and Cry1-/-Cry2-/-mice (right) in response to 6 hour advance of light/dark cycle. Open and filled bars indicate light and dark periods, respectively. Black arrow indicates the day of 6 hour advance shift (6hrA). Numbers above and below the actograms represent timescale in zeitgeber time (ZT) for LD and 6hrA. (G) Rapid adjustment of PK2rhythm in Cry1-/-Cry2-/-mice to 6 hour advance (6hrA). PK2mRNA was quantitated in the SCN of wildtype and Cry1-/-Cry2-/-mice under LD (shaded) or 6hrA (black). Each value represents the mean ± SEM of 3--4 animals. Three-way ANOVA with Bonferroni\'s post hoc analysis indicates a significant interaction between light/dark cycle (LD vs 6hrA), timepoint (ZT4 vs ZT16) and genotype (wildtype vs Cry1-/-Cry2-/-mice), p \< 0.001. Two-way ANOVA with Bonferroni\'s post hoc analysis show that there is significant difference in wildtype PK2expression level between LD and 6hrA, (ZT4, \\\p \< 0.001; ZT16, \\p \< 0.01), but not in Cry1-/-Cry2-/-mice (ZT4, p = 1.000; ZT16, p = 1.000). ::: ![](1471-2202-6-17-4) :::	PubMed Central	2024-06-05T03:55:54.168988	2005-3-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555564/", "journal": "BMC Neurosci. 2005 Mar 11; 6:17", "authors": [ { "first": "Michelle Y", "last": "Cheng" }, { "first": "Eric L", "last": "Bittman" }, { "first": "Samer", "last": "Hattar" }, { "first": "Qun-Yong", "last": "Zhou" } ] }
PMC555565	Background ========== Worldwide, over one million people die of lung cancer each year \[[@B1]\]. In the US, lung cancer is the most common cause of cancer deaths in both sexes \[[@B2]\], and mortality rates in women have risen 500% since 1950 \[[@B3]\]. In the European Union countries, although age-standardized mortality rates have decreased for most cancer sites, lung cancer mortality rates have significantly risen in women \[[@B4]\]. A rising death rate from lung cancer has also been observed in Taiwan. Between 1971 and 2001, age-standardized lung cancer mortality rates per 100 000 per year in Taiwan have increased sharply, from 12.66 to 32.93 among men and from 7.83 to 14.94 among women \[[@B5]\]. Today, in Taiwan, lung cancer is the leading cause of cancer death in women and the second leading cause in men \[[@B5]\]. Epidemiological studies have shown that cigarette smoking is the major cause of lung cancer in both sexes \[[@B6]-[@B8]\]. However, smoking habits do not seem to be the main explanation of the epidemiological characteristics of female lung cancer mortality in Asian countries \[[@B9]-[@B13]\], where the prevalence of smoking is relatively low but lung cancer mortality rates are relatively high. Factors other than smoking habits might contribute to the variability in lung cancer mortality. Long-term geographical trends in cancer mortality can provide useful information to assist etiological research. However, Asian countries are often excluded from studies of geographical differences in trends in lung cancer mortality. In order to clarify the changing patterns of lung cancer mortality worldwide, we examined lung cancer trends from 1971 to 1995 among men and women for 23 countries including four from Asia -- Taiwan, Japan, Singapore, and Hong Kong. In addition, we plotted the pattern of mortality rate in these countries by using the age-period-cohort (APC) model. Methods ======= We used data from the World Health Organization (WHO) and Taiwan to analyze secular trends from 1971--1995 in lung cancer mortality in both men and women. Mortality data provided by WHO were relatively incomplete in some countries, so we analyzed data from 22 countries and Taiwan. The twenty-two countries -- Hong Kong, Singapore, Japan, Portugal, Poland, Italy, Cuba, Spain, Hungary, France, Greece, Finland, United States, England and Wales, Netherlands, Belgium, Canada, Australia, New Zealand, Denmark, Norway and Sweden -- are members of WHO. The data for Taiwan came directly from the Office of Statistics, Department of Health in Taiwan. Since rates for the under 30-year age group are often based on few deaths, and rates for the over 80-year group might be affected by competitive death effects, only rates for the age range 30 to 79 were considered, so as to ensure adequate reliability of the estimates. Lung cancer mortality rates between 1971 and 1995 were analyzed in five consecutive five year periods (1971--1975, 1976--1980, 1981--1985, 1986--1990, and 1991--1995) and in five year age groups. Statistical methods =================== Age-standardized mortality rates (ASMR) were calculated using the world population for 1976 as the reference \[[@B14]\]. Percent changes in the ASMR were calculated as \[(ASMR~1991--1995~- ASMR~1971--1975~) / (ASMR~1971--1975~)\] × 100. In order to apply the APC model, the matrix of age-specific death rates was calculated for each 5-year calendar period (from 1971--1975 to 1991--1995) and age group (from 30--34 to 75--79). The effect of period of death in the APC model was evaluated by a log-linear Poisson model with a modified method as described by Osmond and Gardner \[[@B15]\]. Briefly, the estimate of period effect results from minimizing the weighted sum of the Euclidean distances from the three possible two-factor models (age/period; age/cohort; period/cohort). The weights used in the minimization process were based on the goodness-of-fit measures of each two-factor model. In this study, these were taken as the inverse of the deviance statistics. The sum of period effects were constrained to be zero. These \"effects\" can be interpreted as logarithms of \"relative\" risks. These relative risks were estimated separately for men and women. A computer program written in the SAS/IML language \[[@B16]\] was developed to perform the above calculations. Results ======= Age-standardized mortality rates from lung cancer per 100 000 population per year in 23 countries for 1971 to 1995 are listed for men in Table [1](#T1){ref-type="table"} and for women in Table [2](#T2){ref-type="table"}. Trends in the ASMR varied by sex. From 1971 to 1995, in men, the rates progressively increased in nine countries (Portugal, Hungary, Taiwan, Spain, Poland, Japan, Norway, France and Greece), progressively decreased in two countries (England and Wales and Finland) and increased then declined in the others. In women, rates increased between 1971--1975 and 1991--1995 in 23 countries except for Hong Kong, Cuba, and Spain, with the highest increasing rate observed in the Netherlands (223.46%). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Age-standardized mortality rate (per100 000 person years) from lung cancer in males in 23 countries, 1971--1995 ::: ASMR ------------------- ---------- -------- -------- -------- -------- -------- ---- Portugal 31.88 40.51 45.96 54.40 59.48 86.57 1 Hungary 97.10 116.05 138.49 161.34 180.91 86.31 2 Taiwan 32.08 39.80 49.95 54.24 58.44 82.17 3 Spain 56.92 68.84 80.68 93.80 101.49 78.30 4 Poland 93.91 113.65 135.11 152.23 158.34 68.60 5 Japan 38.88 46.55 53.40 56.96 59.44 52.87 6 Norway 45.20 53.24 62.61 65.43 67.36 49.03 7 France 74.81 87.45 93.16 99.32 100.08 33.77 8 Greece 79.55 93.63 99.23 103.77 105.44 32.54 9 Italy 94.59 110.47 123.02 125.63 117.10 23.80 10 Hong Kong 94.96 118.63 116.24 116.89 110.13 15.97 11 Canada 96.68 108.38 116.23 118.13 107.71 11.42 12 USA 109.60 117.87 121.16 120.33 115.23 5.13 13 Denmark 101.45 109.36 116.83 113.21 103.74 2.26 14 Sweden 48.34 52.24 50.10 48.56 48.21 -0.27 15 Singapore 92.34 114.24 115.29 102.90 91.47 -0.95 16 Belgium 146.33 163.41 166.59 155.70 144.87 -1.00 17 Cuba 75.86 76.35 76.59 76.16 72.90 -3.90 18 Netherlands 151.10 162.14 160.49 148.73 129.40 -14.36 19 Australia 99.14 100.74 100.11 90.81 80.86 -18.44 20 New Zealand 99.81 104.55 100.52 93.07 79.31 -20.54 21 England and Wales 159.59 152.45 136.82 120.40 101.21 -36.58 22 Finland 142.74 142.51 126.97 105.58 90.20 -36.81 23 \percent increase (%) = 100 × (ASMR~1991--1995~- ASMR~1971--1975~) / (ASMR~1971--1975~) ^\$^Rank by percent increase ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Age-standardized mortality rate (per100 000 person years) from lung cancer in females in 23 countries, 1971--1995 ::: ASMR* ------------------- ---------- ------- ------- ------- ------- -------- ---- Netherlands 8.42 11.06 15.46 20.80 27.24 223.46 1 Norway 8.86 11.02 15.61 21.21 26.42 198.24 2 Denmark 21.25 29.28 40.86 49.78 58.41 174.91 3 Canada 17.94 25.44 34.93 43.53 49.13 173.81 4 USA 24.96 33.85 43.41 51.50 56.51 126.46 5 Hungary 16.75 19.34 22.99 29.11 36.83 119.89 6 Sweden 11.36 13.47 16.96 20.68 24.50 115.72 7 Poland 11.40 13.66 16.41 19.68 22.90 100.83 8 Australia 14.91 19.70 23.61 26.47 28.47 90.99 9 Belgium 10.74 12.31 13.79 16.45 19.86 84.90 10 New Zealand 22.58 26.88 30.83 36.53 38.00 68.29 11 France 7.07 7.44 8.42 9.97 11.89 68.19 12 Finland 8.71 11.35 12.39 13.48 14.54 67.02 13 Taiwan 16.03 19.55 23.13 25.69 26.43 64.94 14 Italy 10.69 12.12 13.51 15.09 16.23 51.86 15 England and Wales 30.45 35.75 40.06 43.52 42.84 40.68 16 Portugal 6.74 7.14 7.97 8.56 9.43 39.97 17 Japan 11.90 13.60 14.97 15.35 15.73 32.25 18 Greece 12.64 13.57 13.21 13.86 14.28 12.97 19 Singapore 29.66 34.34 37.07 36.01 31.25 5.36 20 Hong Kong 44.18 48.99 48.01 48.32 43.14 -2.34 21 Cuba 27.87 26.63 26.90 27.77 27.03 -3.02 22 Spain 8.54 7.99 7.32 7.01 7.58 -11.27 23 \* percent increase(%) = 100 × (ASMR~1991--1995~- ASMR~1971--1975~) / (ASMR~1971--1975~) ^\$^Rank by percent increase ::: Table [3](#T3){ref-type="table"} shows the sex ratio (male:female) of the ASMR for lung cancer for five consecutive five- year periods in 23 countries. The sex ratio was greater than one in each five-year period, indicating that the ASMR from lung cancer was higher in men than in women. Among the 23 countries, the trend in the sex ratio gradually decreased for the whole period in most countries except for Spain, France, Italy, Poland, Greece, Portugal, Hungary, Cuba and the Asian countries. For example, in 1971--1975, the highest sex mortality ratio was seen in the Netherlands with a sex ratio of 17.95, the ratio then gradually decreasing to a value of 4.75 by 1991--1995. The change in the sex ratio of mortality in the Netherlands might be due to the increase and then decrease in male lung cancer mortality and simultaneously to the increase in female lung cancer mortality. On the other hand, the ratio gradually increased in Spain. This might be due to Spain having the the fourth highest increase in male lung cancer mortality accompanied by a progressive decrease in female lung cancer mortality from 1971--1975 through 1986--1990, followed by a slight increase. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Sex ratio of the age-standardized mortality rate from lung cancer in 23 countries, 1971--1995 ::: Ratio ------------------- ----------- ------- ------- ------- ------- ------- ------- ---- Belgium 13.62 13.27 12.08 9.46 7.29 11.15 6.33 1 Netherlands 17.95 14.67 10.38 7.15 4.75 10.98 13.20 2 Finland 16.40 12.56 10.25 7.83 6.20 10.65 10.20 3 Spain 6.66 8.62 11.02 13.39 13.39 10.62 6.73 4 France 10.58 11.76 11.06 9.97 8.42 10.36 3.34 5 Italy 8.85 9.12 9.11 8.33 7.21 8.52 1.91 6 Poland 8.24 8.32 8.23 7.73 6.91 7.89 1.41 7 Greece 6.30 6.90 7.51 7.49 7.39 7.12 1.21 8 Portugal 4.73 5.67 5.77 6.36 6.31 5.77 1.63 9 Hungary 5.80 6.00 6.03 5.54 4.91 5.66 1.12 10 Australia 6.65 5.11 4.24 3.43 2.84 4.45 3.81 11 Norway 5.10 4.83 4.01 3.09 2.55 3.92 2.55 12 England and Wales 5.24 4.26 3.42 2.77 2.36 3.61 2.88 13 Canada 5.39 4.26 3.33 2.71 2.19 3.58 3.20 14 Japan 3.27 3.42 3.57 3.71 3.78 3.55 0.51 15 New Zealand 4.42 3.89 3.26 2.55 2.09 3.24 2.33 16 Denmark 4.77 3.74 2.86 2.27 1.78 3.08 2.99 17 Sweden 4.26 3.88 2.95 2.35 1.97 3.08 2.29 18 Singapore 3.11 3.33 3.11 2.86 2.93 3.07 0.47 19 USA 4.39 3.48 2.79 2.34 2.04 3.01 2.35 20 Cuba 2.72 2.87 2.85 2.74 2.70 2.78 0.17 21 Hong Kong 2.15 2.42 2.42 2.42 2.55 2.39 0.40 22 Taiwan 2.00 2.04 2.16 2.11 2.21 2.10 0.21 23 \Rank by mean ::: The mean values of the sex ratio of the ASMR from lung cancer in Taiwan, Hong Kong, Cuba, Singapore, and Japan were 2.10, 2.39, 2.78, 3.07, and 3.55, respectively, with a range of 0.17 to 0.51 over the five-year periods. These values were not only relatively constant over time, but were also lower than seen in the western countries. For example, the lowest sex ratio of 2.10 was seen in Taiwan, with the sex ratio remaining a relatively constant value over the entire period. Only age is adjusted when the ASMR is calculated. However, both age and cohort effect are adjusted in the APC model. The period effects for males and females from the APC model applied to the data from the 23 countries could be classified into seven patterns: 1) an increasing trend in both sexes, seen in Taiwan (Figure [1](#F1){ref-type="fig"}), Norway, Japan, Hungary, and Portugal; 2) a sharply increasing trend in women, with little change seen in men seen in USA (Figure [1](#F1){ref-type="fig"}), Sweden, Poland, Italy, Canada, Belgium, Denmark and France; 3) a sharply increasing trend in women, and a sharply decreasing trend in men, seen in New Zealand (Figure [1](#F1){ref-type="fig"}), Finland, Australia and Netherlands; 4) a more gradual increasing trend in women, but a sharply declining trend in men, seen only in England and Wales (Figure [1](#F1){ref-type="fig"}); 5) a decreasing trend in both sexes, seen in Singapore (Figure [1](#F1){ref-type="fig"}) and Hong Kong; 6) a decreasing and then a gradually increasing trend in women, but a sharply increasing trend in men, seen in Spain (Figure [1](#F1){ref-type="fig"}) and Greece; and 7) a relatively steady trend in both sexes, seen only in Cuba (Figure [1](#F1){ref-type="fig"}). In most countries, the long-term trend in the period effect as derived from the APC model was similar to the trend of ASMR. It is worth noting that the trend in the ASMR for female lung cancer increased and then declined in Singapore and Hong Kong. After adjusting for the cohort effect, however, a decreasing trend in the period effect was observed in Singapore and Hong Kong. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Secular trend in the relative risk (RR) of dying from male and female lung cancer, 1971--1995, based on analyses using the age-period-cohort model in Taiwan, England and Wales, New Zealand, USA, Singapore, Spain and Cuba ::: ![](1471-2458-5-22-1) ::: Discussion ========== We found that the sex ratio in lung cancer mortality varied over time and geographically. After adjusting for age and cohort effects, seven patterns could be identified using the APC model, indicating that some countries had a similar trend in lung cancer mortality. Koo and Ho \[[@B17]\] indicated that smoking was a strong risk factor in the west and worldwide where there were high rates of smoking in men. We appreciate that lung cancer mortality rates for a given year depend on smoking habits over a period before that year; however, it is not possible to get data on smoking prevalence before 1975 from WHO. Therefore, based on the smoking prevalence obtained from World Health Organization (Table [4](#T4){ref-type="table"}), the first, second, third, and fifth highest sex ratios (male:female) of smoking prevalence among the 23 countries were in Taiwan, Hong Kong, Singapore, and Japan, respectively. However, the sex ratios of ASMR from lung cancer in the four Asian countries were significantly lower than in the western countries. That is, the four Asian countries have a relatively low sex ratio in lung cancer mortality and a relatively high sex ratio in smoking prevalence. This fact is of particular interest. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Smoking prevalence in males and females, and their sex ratio, in 23 countries ::: Country* Prevalence ------------------- ---------------- ------ ------ ---- -------------------------------------- Taiwan 55.1 3.3 16.7 1 Adult (18 years & older), 1996 Hong Kong 27.1 2.9 9.3 2 Adult (15 years & older), 1998 Singapore 26.9 3.1 8.7 3 Adult (18--64 year olds), 1998 Portugal 30.2 7.1 4.3 4 Adult (15 years & older), 1995--1996 Japan 52.8 13.4 3.9 5 Adult (15 years & older), 1998 Poland 39.0 19.0 2.1 6 Adult, 1998 Italy 32.2 17.3 1.9 7 Adult (14 years & older), 1998 Cuba 48.0 26.3 1.8 8 Adult (15 years & older), 1995 Spain 42.1 24.7 1.7 9 Adult (16 years & older), 1997 Greece 46.0 28.0 1.6 10 Adult, 1994--1998 Hungary 44.0 27.0 1.6 11 Adult (18 years & older), 1998--1999 France 39.0 27.0 1.4 12 Adult (18 years & older), 1997 Finland 27.0 20.0 1.4 13 Adult (15--64 year olds), 1999 USA 27.6 22.1 1.3 14 Adult (18 years & older), 1997 Netherlands 37.0 30.0 1.2 15 Adult (15 years & older), 1998 Belgium 31.0 26.0 1.2 16 Adult (15 years & older), 1999 Canada 27.0 23.0 1.2 17 Adult (15 years & older), 1999 Australia 27.1 23.2 1.2 18 Adult (16 years & older), 1995 New Zealand 26.0 24.0 1.1 19 Adult (15 years & older), 1998 Denmark 32.0 30.0 1.1 20 Adult (14 years & older), 1998 England and Wales 29.0 28.0 1.0 21 Adult (16 years & older), 1996 Norway 33.7 32.3 1.0 22 Adult (16--74 year olds), 1998 Sweden 17.1 22.3 0.8 23 Adult (16--84 year olds), 1998 \Rank by Male: female ratio ^\$^Data were obtained from the World Health Organization ::: Dietary fat consumption has been found to be positively related to lung cancer mortality \[[@B18]-[@B20]\]. Our data from Japan, Taiwan and Cuba women (on Tables [2](#T2){ref-type="table"} and [5](#T5){ref-type="table"}) also indicated that the percent increase of fat consumption was positively related to the percent increase of ASMR by using the Spearman\'s rank correlation coefficient. Further study of factors other than smoking, like fat intake, on lung cancer mortality seems warranted, especially for women in Asian countries (Japan and Taiwan). ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Changes in annual per caput fat consumption in 23 countries ::: Fat Consumption\** ----------------- ----------------------- ----------- -------- ---- Taiwan 38.0 136.8 260.0% 1 Hong Kong 71.4^\$^ 135.6^\#^ 89.9% 2 Spain 88.9 137.0 54.1% 3 Portugal 78.6 120.6 53.4% 4 Japan 54.6 79.3 45.2% 5 Greece 101.9 138.4 35.8% 6 Hungary 115.3 153.5 33.1% 7 Italy 114.4 151.0 32.0% 8 France 126.4 161.3 27.7% 9 Cuba 67.6 85.1 25.9% 10 New Zealand 115.4 134.7 16.7% 11 USA 119.6 138.8 16.1% 12 Canada 113.7 127.1 11.8% 13 Australia 117.8 130.6 10.9% 14 Netherlands 132.0 140.9 6.7% 15 Poland 103.9 110.3 6.2% 16 Sweden 116.8 122.6 5.0% 17 Finland 123.6 124.2 0.5% 18 Norway 131.6 127.7 -3.0% 19 England & Wales 141.7 135.8 -4.2% 20 Denmark 140.7 132.6 -5.8% 21 Belgium \- \- \- \- Singapore \- \- \- \- \*Data were obtained from FAO ^&^Rank by percent increase ^\$^:1961,^\#^: 1995 ::: Conclusion ========== Period effects for both men and women in 23 countries, as derived using the APC model, could be classified into seven patterns. The four Asian countries have a relatively low sex ratio in lung cancer mortality and a relatively high sex ratio in smoking prevalence. Factors other than smoking might be important, especially for women in Asian countries. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= YPL was responsible for the development of intellectual content and the study design, collected and analyzed the data, interpretation of the results, manuscript drafting and the critical revisions of manuscript. YCH was responsible for the development of intellectual content, interpretation of the results and manuscript drafting. GWL was responsible for data coding and entry and statistical analyses. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/5/22/prepub> Acknowledgements ================ This study is funded by the National Science Council grant NSC 92-2320-B-040-050. The authors thank Dr. Wen-Chung Lee for providing the program of the APC model.	PubMed Central	2024-06-05T03:55:54.171764	2005-3-5	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555565/", "journal": "BMC Public Health. 2005 Mar 5; 5:22", "authors": [ { "first": "Yung-Po", "last": "Liaw" }, { "first": "Yi-Chia", "last": "Huang" }, { "first": "Guang-Wen", "last": "Lien" } ] }
PMC555566	Background ========== Fish oil, rich in n-3 polyunsaturated fatty acids (PUFA), is considered beneficial in lowering the risk of coronary heart disease \[[@B1],[@B2]\]. The beneficial effects of n-3 PUFA are mainly due to reduction in plasma triacylglycerol and very-low-density lipoprotein (VLDL) levels \[[@B3],[@B4]\]. However, the effect of fish oil on low-density lipoprotein (LDL)-cholesterol concentration is inconsistent \[[@B5]\]. High levels of LDL are strong predictors of coronary heart disease. Normolipidaemic subjects show reduction in plasma LDL-cholesterol concentrations following intake of fish oil diet \[[@B6],[@B7]\], however, fish oil supplementation to hyperlipidaemic subjects causes an increase in LDL-cholesterol concentrations \[[@B8],[@B9]\]. The fish-oil-induced increase in LDL-cholesterol concentration is also observed in animal studies \[[@B10]\]. One of the factors that determine LDL concentrations is reverse cholesterol transport pathway, which removes cholesterol from peripheral tissues and returns to the liver. Cholesterol ester transfer protein (CETP) plays a major role in this pathway to transfer cholesterol esters from high-density lipoproteins (HDL) to VLDL and LDL, in exchange for triacylglycerols \[[@B11],[@B12]\]. Thus, increased CETP activity can cause elevation of LDL-cholesterol concentration while decreasing the HDL-cholesterol concentrations. A positive correlation between plasma CETP concentration and LDL-cholesterol concentration has been observed in primates \[[@B11]-[@B13]\]. Thus, fish-oil-induced elevation of LDL-cholesterol concentration might be explained by changes in plasma CETP concentration. CETP mediated exchange of cholesterol esters for triacylglycerols occurs mainly through an equimolar heteroexchange mechanism, which leads to alterations in cholesterol ester: triacylglycerol ratio. In addition to plasma CETP concentration, fatty acid composition of lipoproteins also modifies reverse cholesterol transfer. Cholesterol esters with n-3 polyunsaturated fatty acyl groups are more likely to transfer compared to cholesterol esters with saturated and monounsaturated fatty acyl groups \[[@B14]\]. This effect might be attributed to change in transition temperature of the lipid core of lipoproteins with change in lipid composition \[[@B15]\]. We previously observed fish-oil-induced elevation of VLDL-and LDL-cholesterol concentrations and decrease in HDL-cholesterol concentration in F1B hamsters \[[@B16]\]. In this study, we examined whether the effects of dietary fish oil on plasma lipoprotein concentrations are due to fish-oil-induced alterations in plasma cholesterol ester transfer protein activity. Results ======= LDL-lipid composition --------------------- The LDL-cholesterol, free cholesterol, cholesterol ester, phospholipid and triacylglycerol concentrations were measured in Bio F1B hamsters treated with fish-oil or MIX diet (Table [1](#T1){ref-type="table"}). Fish-oil diet led to significantly higher concentrations of total cholesterol, cholesterol ester, free cholesterol, triacylglycerol and phospholipids compared to MIX diet. Increasing the dietary fat levels of fish oil from 5% w/w (low fat) to 20 % w/w (high fat) caused a significant increase in free cholesterol and LDL-triacylglycerol concentrations. On the contrary, LDL-cholesterol ester concentrations decreased significantly (p \< 0.0001) when the amount of fat was increased in fish oil diet. There was no effect of increase in dietary fat levels on LDL-cholesterol, cholesterol ester, triacylglycerol or phospholipids concentrations in the hamsters fed with MIX diet. Cholesterol supplementation of the diet led to an increase in LDL-cholesterol, cholesterol ester, triacylglycerol and phospholipids concentrations in hamsters fed with fish-oil diet. However, cholesterol supplementation to the MIX diet had no effect on these parameters. Furthermore, the effect of dietary cholesterol was greater in low fat fish-oil diet fed hamsters compared to the high fat fish oil fed hamsters (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### LDL-lipid composition of hamsters fed with various diets. ::: ------------------------------------------------------------------------------------------------------------------------------- LDL-Lipids Diet Chol Low Fat 5% High Fat 20% Statistical significance\ (3 way ANOVA) ---------------------------- ---------- ---------- ---------------- ------------------ ------------------------------- -------- Total Cholesterol (mmol/l) FO \- 4.84 ± 0.61 4.57 ± 0.36 DT 0.0001 \+ 8.84 ± 2.06 5.10 ± 1.00 DT × DL 0.0020 MIX \- 1.54 ± 0.32 1.41 ± 0.25 DT × CHOL 0.0007 \+ 1.27 ± 0.25 1.63 ± 0.48 DL × CHOL 0.0200 Free Cholesterol (mmol/l) FO \- 0.45 ± 0.05 1.53 ± 0.46 DL 0.0001 \+ 0.69 ± 0.17 1.99 ± 0.45 MIX \- 0.28 ± 0.10 0.70 ± 0.13 \+ 0.38 ± 0.14 0.58 ± 0.15 Cholesterol Ester (mmol/l) FO \- 4.08 ± 0.43 2.62 ± 0.84 DT 0.0001 \+ 8.05 ± 1.96 3.07 ± 1.00 CHOL 0.0001 MIX \- 0.87 ± 0.30 0.88 ± 0.29 DT × DL 0.0300 \+ 0.80 ± 0.17 0.79 ± 0.17 DL × CHOL 0.0004 Triacylglycerol (mmol/l) FO \- 0.65 ± 0.08 1.62 ± 0.27 DT 0.0001 \+ 1.01 ± 0.34 1.53 ± 0.22 DT × DL 0.0001 MIX \- 0.24 ± 0.03 0.22 ± 0.04 DL × CHOL 0.0200 \+ 0.29 ± 0.07 0.17 ± 0.01 Phospholipids (mmol/l) FO \- 0.69 ± 0.10 0.66 ± 0.11 DT 0.0001 \+ 0.98 ± 0.18 0.73 ± 0.10 CHOL 0.03 MIX \- 0.37 ± 0.07 0.36 ± 0.06 DT × DL 0.03 \+ 0.33 ± 0.05 0.36 ± 0.04 DT × CHOL 0.009 ------------------------------------------------------------------------------------------------------------------------------- FO, fish oil; MIX, MIX diet; Chol, cholesterol; DT, diet type; DL, diet level; CHOL, Cholesterol For details of diets and procedures, see \"Materials and methods\" section. Animals were fed the indicated diets for 2 weeks. All values represent means ± SD. ::: Change in dietary fat level altered the LDL-cholesterol ester: triacylglycerol ratio (Figure [1](#F1){ref-type="fig"}). There was a significant decrease in LDL-cholesterol ester: triacylglycerol ratio in fish-oil diet fed hamsters when the dietary fat level was increased from 5 % to 20 % (Figure [1A](#F1){ref-type="fig"}). However, there was no significant effect of dietary fat level on LDL-cholesterol ester: triacylglycerol ratio in the MIX diet fed hamsters (Figure [1B](#F1){ref-type="fig"}). Addition of cholesterol to the high fat fish-oil diet caused a slight but significant increase in LDL-cholesterol ester: triacylglycerol ratio compared to the high fat fish oil diet alone (Figure [1A](#F1){ref-type="fig"}). However, addition of cholesterol to the MIX diet had no significant effect on cholesterol ester: triacylglycerol ratio compared to hamsters fed the MIX diet alone (Figure [1B](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Cholesterol ester: triacylglycerol ratio in fish-oil (A) and MIX-diet (B) fed hamsters. Hamsters were fed fish oil (FO) or MIX diet at a low fat (5% w/w) or a high fat (20% w/w) level in the absence (pink) or presence (blue) of 0.25% w/w cholesterol. Lipids were analyzed as described in the methods. Values are means for 12 animals with standard deviations shown by vertical bars. Differences between groups were evaluated using Student\'s ttest. ::: ![](1743-7075-2-8-1) ::: HDL-lipid composition --------------------- Changes in HDL-lipid composition in Bio F1 B hamsters on different diets are given in Table [2](#T2){ref-type="table"}. Fish-oil diet fed hamsters had significantly lower concentrations of HDL-cholesterol, free cholesterol, cholesterol ester and phospholipids compared to MIX-diet fed hamsters. However, there was no significant effect of fish-oil on HDL-triacylglycerol concentrations. Increasing the dietary fat levels from low fat to high fat caused a decrease in cholesterol ester concentrations in fish-oil diet fed hamsters. Cholesterol supplementation caused an increase in HDL-cholesterol and cholesterol ester concentrations irrespective of the diet type in low fat fed hamsters. However, there was no effect of dietary cholesterol on HDL-cholesterol concentration in high fat fed hamsters. Cholesterol supplementation led to a significant increase in phospholipids concentration for all type of diets, both at low and high fat levels. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### HDL-lipid composition of hamsters fed with various diets. ::: HDL-Lipids Diet Chol Low Fat 5% High Fat 20% Statistical significance (3 way ANOVA) ---------------------------- ---------- ---------- ---------------- ------------------ -------------------------------------------- -------- Total Cholesterol (mmol/l) FO \- 2.90 ± 0.29 2.19 ± 0.48 DT 0.0001 \+ 3.96 ± 0.87 2.24 ± 0.56 CHOL 0.0020 MIX \- 4.74 ± 0.07 5.86 ± 0.37 DT × DL 0.0001 \+ 6.00 ± 1.23 6.78 ± 0.45 Free Cholesterol (mmol/l) FO \- 0.18 ± 0.08 0.31 ± 0.07 DT 0.0001 \+ 0.31 ± 0.08 0.25 ± 0.16 MIX \- 0.60 ± 0.17 0.57 ± 0.03 \+ 0.60 ± 0.17 0.59 ± 0.12 Cholesterol Ester (mmol/l) FO \- 2.67 ± 0.29 1.88 ± 0.41 DT 0.0001 \+ 3.88 ± 0.71 1.98 ± 0.49 CHOL 0.0009 MIX \- 3.55 ± 0.61 4.34 ± 0.42 DT × DL 0.0001 \+ 4.33 ± 0.80 5.34 ± 0.96 Triacylglycerol (mmol/l) FO \- 0.32 ± 0.03 0.44 ± 0.12 DT × DL 0.0400 \+ 0.39 ± 0.14 0.33 ± 0.10 DL × CHOL 0.0400 MIX \- 0.37 ± 0.03 0.32 ± 0.03 \+ 0.40 ± 0.09 0.30 ± 0.04 Phospholipids (mmol/l) FO \- 0.57 ± 0.12 0.59 ± 0.16 DT 0.0001 \+ 1.62 ± 0.43 1.27 ± 0.23 DL 0.01 MIX \- 3.43 ± 0.26 2.91 ± 0.33 CHOL 0.0003 \+ 4.20 ± 0.45 4.67 ± 0.46 DT × DL 0.0005 DT × CHOL 0.05 DL × CHOL 0.0007 FO, fish oil; Mix, MIX diet; Chol, cholesterol; DT, diet type; DL, diet level; CHOL, cholesterol. For details of diets and procedures, see \"Materials and methods\" section. Animals were fed the indicated diets for 2 weeks. All values represent means ± SD. ::: Surface lipid to core lipid ratio for LDL and HDL ------------------------------------------------- The LDL surface lipids (free cholesterol and phospholipids) to core lipids (cholesterol esters and triacylglycerols) ratio was higher in MIX diet fed hamsters compared to hamsters fed the fish oil diet, at both low and high fat levels (Table [3](#T3){ref-type="table"}). Increasing the quantity of fat in the diet increased the surface to core lipid ratio, whereas addition of dietary cholesterol had no significant effect, in both fish oil and MIX diet fed hamsters (Table [3](#T3){ref-type="table"}). The HDL surface lipid to core lipid ratio was also higher in MIX diet fed hamsters compared to the fish oil fed hamsters, at both low fat and high fat levels (Table [3](#T3){ref-type="table"}). Addition of cholesterol to the fish oil diet caused a significant increase in surface lipid to core lipid ratio, at both low and high fat levels, whereas no significant effect of dietary cholesterol was observed for the MIX diet (Table [3](#T3){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Surface lipid to Core lipid ratio of LDL and HDL particles from hamsters fed with various diets. ::: Surface lipid: Core lipid --------------------------- ----------- ---------------- ---------------- ---------------- ---------------- Particle Type Fat level Fish Oil Mix \- Cholesterol \+ Cholesterol \- Cholesterol \+ Cholesterol LDL 5% 0.24 0.18 0.59 0.65 20% 0.52 0.59 0.96 0.98 HDL 5% 0.25 0.45 1.02 1.01 20% 0.39 0.69 0.75 0.93 Surface lipids are composed of free cholesterol and phospholipids. Core lipids are composed of cholesterol esters and triacylglycerols. The surface lipid to core lipid ratio was calculated by dividing the mean surface lipids with mean core lipids. ::: CETP activity and CETP mass --------------------------- It has been shown previously that there is a correlation between the level of CETP and that of LDL-cholesterol. To further investigate our observation of fish-oil-induced elevation of LDL-cholesterol concentrations and a decrease in HDL-cholesterol concentration in F1B hamsters, we measured CETP mass as well as CETP activity. The changes in CETP mass and CETP activity are shown in Figure [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"} respectively. Treatment of hamsters with fish-oil diet led to a decrease in CETP mass (p \< 0.0001) and CETP activity (p \< 0.002) compared to MIX diet, at low fat level. Increasing the dietary fat levels from low fat to high fat caused a further decrease in CETP mass in fish-oil diet fed hamsters, whereas same change brought an increase in CETP mass in MIX diet fed hamsters (DT × DL interaction, p \< 0.002). On the other hand, plasma CETP activity increased in both fish-oil and MIX-diet fed hamsters by increasing the dietary fat levels. Dietary cholesterol caused an increase in CETP mass (p \< 0.0002) and CETP activity (p \< 0.0001) in both fish-oil and MIX-diet fed hamsters at low fat level. However, cholesterol mediated increase in CETP activity was not observed at high fat levels for both fish oil and MIX diets. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Plasma CETP mass in fish-oil and MIX-diet fed hamsters. Plasma CETP mass in hamsters fed with a fish-oil (FO) or a MIX-diet at a low fat (5% w/w) or a high fat (20% w/w) level in the absence (pink) or presence (blue) of 0.25% w/w cholesterol. Plasma was collected and assayed for CETP mass using ELISA as described in the methods. Values are means for 12 animals with standard deviations shown by vertical bars. Differences between groups were evaluated using 3-way ANOVA. Values without a common superscript are significantly different from each other. ::: ![](1743-7075-2-8-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Plasma CETP activity in fish-oil and MIX-diet fed hamsters. Plasma CETP activity in hamsters fed with a fish-oil (FO) or a MIX-diet at a low fat (5% w/w) or a high fat (20% w/w) level in the absence (pink) or presence (blue) of 0.25% w/w cholesterol. Plasma was collected and assayed for CETP activity using a radioisotope method as described in the methods. Values are means for 12 animals with standard deviations shown by vertical bars. Differences between groups were evaluated using 3-way ANOVA. Values without a common superscript are significantly different from each other. ::: ![](1743-7075-2-8-3) ::: Correlation between CETP mass and CETP activity ----------------------------------------------- CETP mass was significantly and positively correlated with CETP activity in low fat fish oil (r = 0.81, p \< 0.03) and MIX diet (r = 0.96 p \< 0.0003) fed hamsters (Figure [4A](#F4){ref-type="fig"}) and high fat MIX diet fed hamsters (r = 0.84, p \< 0.02) (Figure [4B](#F4){ref-type="fig"}). The correlation between CETP mass and CETP activity in high fat fish-oil fed hamsters was not significant. This indicates that CETP activity is an accurate reflection of CETP mass in both fish-oil and MIX-diet fed hamsters at low fat levels. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Correlation between CETP activity and CETP mass in fish-oil and MIX-diet fed hamsters. Correlation between CETP activity and CETP mass in hamsters fed with a fish-oil (blue) or a MIX (red) diet, supplemented with low fat (5% w/w) (panel A) or high fat (20% w/w) (panel B). Plasma was collected and assayed for CETP activity and CETP mass as described in the methods. ::: ![](1743-7075-2-8-4) ::: Correlation between CETP and LDL-lipid levels --------------------------------------------- The correlations between CETP mass, CETP activity and LDL-cholesterol are shown in Figure [5](#F5){ref-type="fig"} and [6](#F6){ref-type="fig"} respectively. There was no significant correlation between CETP mass and LDL-cholesterol concentrations in fish-oil diet (r = 0.56) or MIX diet (r = 0.48) fed hamsters. Similarly, the CETP activity was not significantly correlated with LDL-cholesterol concentrations in fish-oil (r = 0.48) or MIX diet (r = 0.45) fed hamsters. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Correlation between CETP mass and LDL-cholesterol in fish-oil and MIX-diet fed hamsters. Correlation between CETP mass and LDL-cholesterol in hamsters fed with a fish-oil (blue) or a MIX (red) diet. Plasma CETP mass and LDL-cholesterol concentration were assayed as described in the methods. ::: ![](1743-7075-2-8-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Correlation between CETP activity and LDL-cholesterol in fish-oil and MIX-diet fed hamsters. Correlation between CETP activity and LDL-cholesterol in hamsters fed with a fish-oil (blue) or a MIX (red) diet. Plasma CETP activity and LDL-cholesterol concentration were assayed as described in the methods. ::: ![](1743-7075-2-8-6) ::: Discussion ========== CETP plays an important role in maintaining the levels of plasma LDL and HDL \[[@B12]\]. Increased levels of plasma CETP cause an increase in the transfer of cholesterol esters from HDL to LDL, thereby raising the levels of LDL. In recent years, antibodies have been raised against CETP as a therapy to lower plasma LDL-levels and to prevent cardiovascular disease \[[@B12]\]. Fish oil has also gained much interest in the recent years as a therapy against cardiovascular disease. Earlier, we observed that fish oil caused an increase in plasma lipid and lipoprotein levels in a unique animal model, the F1B hamster \[[@B16]\]. In this study, we investigated whether the increase in plasma LDL-levels in fish-oil fed F1B hamsters is due to an increase in plasma CETP activity. Fish oil caused an increase in LDL-cholesterol concentration and a decrease in HDL-cholesterol concentration compared to hamsters fed a MIX diet (Table [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}). However, CETP activity and CETP mass were lower in fish-oil fed hamsters compared to the MIX-diet fed hamsters. Earlier studies in human subjects also show fish-oil-induced reduction in CETP activity compared to safflower oil fed counter parts \[[@B17],[@B18]\]. Moreover, we did not find significant correlation between CETP mass as well as CETP activity and LDL-cholesterol in this study. Data presented here suggests that cholesterol ester transfer between HDL and LDL is not likely to play a major role in determining fish-oil-induced changes in LDL- and HDL-cholesterol concentrations. Cholesterol esters are more liable to transfer in fish-oil fed subjects since the core of the lipoprotein particles from these subjects have lower transition temperature \[[@B15]\] due to abundance of long chain polyunsaturated fatty acids. This theory is in agreement with the finding that there is increased cholesterol ester mass transfer in eicosapentaenoic acid (omega-3 20:5, EPA) fed rabbits while there was no change in plasma CETP activity \[[@B14]\]. We did not measure cholesterol ester mass transfer to determine the contribution of HDL-and LDL-fatty acid composition in order to explain fish-oil- induced elevation of LDL-cholesterol concentration. However, our data shows that the LDL and HDL surface lipid to core lipid ratio is significantly lower in fish oil fed hamsters than the MIX diet fed hamsters, at both low and high fat levels. Thus, the composition of the LDL and HDL particles appears to be significantly different between the fish oil and MIX diet fed hamsters. It has previously been shown that the LDL particle composition is an important determinant of LDL clearance \[[@B19]\]. In our previous study \[[@B16]\] we have shown that hepatic LDL-receptor mRNA levels were significantly low in fish-oil fed hamsters. In addition there are reports that in cases of CETP deficient subjects, LDL-particles have reduced affinity for LDL-receptor \[[@B20]\]. One can hypothesize that the increase in both LDL-cholesterol and triacylglycerol concentrations (Table [1](#T1){ref-type="table"}) in fish-oil fed hamsters may points towards suppression of LDL clearance rather than increased cholesterol ester/triacylglycerol exchange as the cause. Increasing dietary fat level of fish oil decreased cholesterol ester transfer as reflected by decrease in CETP mass (Figure [2](#F2){ref-type="fig"}). Thus, dietary fat level dependent reduction in LDL-cholesterol ester: triacylglycerol ratio in fish-oil diet fed hamsters (Figure [1A](#F1){ref-type="fig"}) might be due to the decrease in cholesterol ester transfer. However, in contrast to CETP mass, CETP activity slightly increased with the increase of dietary fat level. The plasma used for CETP activity assay in high fat fish-oil fed hamsters contained very high levels of chylomicrons, VLDL and LDL, which are potential acceptors of cholesterol esters from radiolabeled exogenous HDL \[[@B21]\]. Thus, CETP activity of high fat fish-oil fed hamsters might be an exaggerated reflection of CETP mass. HDL-total cholesterol and cholesterol ester concentrations were significantly lower in fish-oil diet fed hamsters compared to MIX-diet fed hamsters, while there was no difference in HDL-triacylglycerol concentrations (Table [2](#T2){ref-type="table"}). Previous studies using hamsters \[[@B22],[@B23]\] and non-human primates \[[@B24]\] have shown decreased HDL-cholesterol concentrations following fish oil feeding, which support our observation. Plasma HDL-cholesterol concentration is mainly regulated by reverse cholesterol transport pathway and cellular cholesterol efflux \[[@B25],[@B26]\]. The reduction of HDL-cholesterol concentration, while decrease in cholesterol ester transfer in fish-oil fed hamsters compared to MIX-diet fed hamsters implicates that fish-oil-induced HDL-cholesterol lowering effect is not due to the changes in cholesterol ester transfer, but might be attributed to decreased efflux of cholesterol from peripheral cells. Increasing the dietary fat level from low fat to high fat caused a decrease in HDL-total cholesterol and cholesterol ester levels (Table [2](#T2){ref-type="table"}), which might be due to poor esterification of HDL-free cholesterol-to-cholesterol esters by lecithin-cholesterol-acyl-transferase (LCAT) as n-3 PUFA are known to be less utilized by LCAT for the formation of cholesterol esters \[[@B27],[@B28]\]. Dietary cholesterol supplementation led to an increase in HDL-cholesterol concentration and also caused an increase in CETP mass/activity in both fish-oil and MIX-diet fed hamsters. This finding suggest that cholesterol mediated increase in HDL-cholesterol was not associated with changes in cholesterol ester transfer, but might be due to other factors, possibly due to an increased cholesterol efflux from peripheral cells. The interactive effect of dietary cholesterol and n-3 PUFA on plasma cholesterol ester transfer is not yet known. Cholesterol supplementation caused an increase in plasma CETP activity in both fish-oil and MIX-diet fed hamsters at low fat levels (Figure [3](#F3){ref-type="fig"}), which is consistent with other reports \[[@B29]\]. These observations suggest that the regulation of CETP is dependent on the presence of dietary cholesterol. Dietary cholesterol is known to increase plasma CETP concentrations \[[@B29]\]. The increase in plasma CETP concentrations in response to dietary cholesterol is due to an increase in CETP mRNA levels in adipose tissue and liver \[[@B30],[@B31]\]. In hamsters, CETP is mainly expressed in the adipose tissue, and the cholesterol mediated increase in plasma CETP activity is directly related to an increase in adipose tissue CETP mRNA levels \[[@B31]\]. Our findings are consistent with the previous observations that the regulation of CETP is dependent on dietary cholesterol. However, our observations show that the supplementation of cholesterol to the high fat fish oil and MIX diet had no significant effect on CETP activity as compared to high fat fish oil and MIX diet alone (Figure [3](#F3){ref-type="fig"}). These findings suggest that high fat diets interfere with cholesterol to regulate CETP, which is similar to the observations made for the regulation of the human CETP gene (under publication). Conclusion ========== In summary, fish oil induced increase in LDL-cholesterol concentration in F1B hamsters, as well as effects of diet type, diet fat level and dietary cholesterol level on HDL-lipids were not associated with changes in plasma cholesterol ester transfer activity. It is likely that the dietary fat composition altered the LDL-core lipid composition, which in turn inhibited the uptake of LDL particles. This in combination with decrease of LDL-receptor mRNA levels may be the likely cause of increased plasma LDL-levels in fish-oil fed F1B hamsters. Methods ======= Animals and diets ----------------- The F1B hamsters (7 weeks old) were obtained from Bio Breeders Inc. (Water Town, MA) and kept on chow diet for one week prior to feeding specific diets. After this equilibration period, hamsters were divided into 8 groups (n = 12) and each group was fed with one of the specified diets. The specified diets consisted of fat free semi-purified diet (ICN Biomedical Inc., OH) that was supplemented with either fish oil (menhaden oil, Sigma Chemical Co., St. Louis, MO) or a mixture of lard and safflower oil in 1.5:1 ratio (MIX diet) \[[@B16]\]. The fat content of the diets was either 5% w/w (low fat) or 20% w/w (high fat). Due to the presence of cholesterol in fish oil, the low fat fish oil diet contained 0.025% w/w of cholesterol, and the high fat fish-oil diet contained 0.1% w/w of cholesterol. Thus, the same amount of cholesterol was added to the low fat and the high fat MIX diets to keep cholesterol content similar. For the high cholesterol diets, the fish oil and MIX diets were supplemented with additional cholesterol to bring the final concentration of cholesterol to 0.25% w/w. All diets were stored at -20°C and animals were given fresh diets each day. The animals were maintained on specific diets for 2 weeks ad libitum. The food intake was measured daily during the study period, and the body weight was checked at the beginning of the study period, one week later and at the conclusion of the study. There was no difference in food intake and body weight gain between different diet groups. All animals were housed in individual cages in a single room with enriched environment. The Institutional Animal Care use Committee (IACC) approved all experimental procedures, which are in accordance with the principles and guidelines of the Canadian Council on Animal Care. After two weeks on specified diets the animals were sacrificed after 14 hrs of fasting. Blood was collected by cardiac puncture into tubes containing EDTA and centrifuged immediately to separate plasma. Plasma samples were stored at 4°C on ice until further use. Lipoprotein separation and analysis ----------------------------------- Plasma was centrifuged at 15,500 gfor 20 min at 12°C \[[@B32]\] to separate chylomicrons. The infranatant was separated and used for isolation of other lipoproteins fractions i.e. VLDL, LDL and HDL by sequential density ultra centrifugation \[[@B33]\]. Isolated individual lipoprotein fractions i.e. VLDL, LDL and HDL were stored at 4°C for further analysis. Total cholesterol concentration was assayed in all lipoprotein fractions using cholesterol assay kit \# 402 (Sigma Diagnostics Inc, St. Louis, MO). Total triacylglycerol concentration of lipoprotein fractions was assayed using triglyceride assay kit \# 344 (Sigma Diagnostics Inc, St. Louis, MO). Free cholesterol in plasma and individual lipoprotein fractions was assayed using free cholesterol assay kit (Wako Chemicals, VA). Cholesterol ester concentration was determined by subtracting the free cholesterol concentration from total cholesterol concentration. Phospholipids were analyzed using the method of Bartlett \[[@B34]\]. Cholesterol ester transport protein (CETP) activity assay --------------------------------------------------------- CETP in the plasma samples was assayed by a radioactive method \[[@B21]\] that was modified from a previously published method \[[@B35]\]. LDL- and HDL-lipoprotein fractions were isolated from human plasma \[[@B36]\] and the HDL-fraction was radiolabeled, using ^14^C-cholesterol oleate \[[@B35]\]. Plasma (5 μl) was combined with radiolabeled HDL (5 μg), LDL (50 μg) and 115 μl of incubation buffer (10 mM Tris, 150 mM NaCl, 2 mM EDTA) and incubated at 37°C for 1 hr. The LDL fraction was separated by heparin-manganese precipitation. The radioactivity in the supernatant and the precipitate was counted and the results were expressed as percent cholesterol ester(CE) transferred/hour. CETP mass --------- CETP mass in plasma samples was quantitatively assayed using CETP ELISA- DAIICHI kit (Daiichi Pure Chemicals Co., LTD, Tokyo) as previously published \[[@B21]\]. Concentrations of the samples were calculated using a standard curve developed using a CETP stock solution with known concentration. Statistical analysis -------------------- The effect of diet type, dietary fat level and dietary cholesterol was determined using 3-way analysis of variance, and a Tukey\'s post hoc test was used to test significant differences revealed by the ANOVA. Values are group means ± SD, n = 12; Differences were considered to be statistically significant if the associated Pvalue was \<0.05 \[[@B37]\]. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= PPD conducted all the experiments in this study. PPD and AAM analyzed and interpreted the data, as well as drafted the manuscript. PJD is a collaborator on this project. SKC is the Principal investigator, has conceived the study, participated in its design and final approval of the version to be published. All Authors read and approved the final manuscript. Acknowledgements ================ This research was supported by a grant from NSERC, Canadian Institutes of Health Research and Canadian Innovation Fund. SKC holds a New Investigator Award from the Canadian Institutes of Health Research. We thank Amanda Bennett and Stephanie Tucker for expert technical support.	PubMed Central	2024-06-05T03:55:54.174939	2005-3-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555566/", "journal": "Nutr Metab (Lond). 2005 Mar 11; 2:8", "authors": [ { "first": "Pujitha P", "last": "de Silva" }, { "first": "Alka", "last": "Agarwal-Mawal" }, { "first": "Phillip J", "last": "Davis" }, { "first": "Sukhinder Kaur", "last": "Cheema" } ] }
PMC555567	Background ========== Osteoid osteoma is a rare benign osteoblastic lesion usually involving the long bones of the lower limbs. Cranial involvement has been mainly localised to the skull vault. Osteoid osteoma of the skull base is a rare entity \[[@B1]\]. Surgical management of skull base osteoid osteoma may be challenging due to its proximity to vital structures, access and hard consistency. This report deals with a case of an ethmoidal osteoid osteoma invading the adjacent orbit and anterior cranial fossa and the team approach employed to achieve radical excision. The radiological findings and the surgical procedure employed are presented. Case presentation ================= Clinical presentation --------------------- A 33 year old male presented with decreased vision in the left eye and left sided headache of 3 months duration. Examination revealed left eye blindness with primary optic atrophy and no other neurological deficit. Imaging revealed a bony tumor in the left ethmoid sinus invading the left orbit and compressing the left optic nerve. Intracranial extension into the anterior cranial fossa on the left side was noted (Figure [1](#F1){ref-type="fig"}). Core biopsy of the mass showed an osteoid osteoma. Surgical management ------------------- A multispeciality team approach was devised to achieve radical excision of the tumor. Bicoronal scalp flap and pericranial flaps were raised separately. A single burr hole left fronto orbital bone flap was raised including the orbital roof and left zygomaticofrontal process (Figure [2](#F2){ref-type="fig"}). Dura was retracted and the bony hard whitish tumor visualised. This was excised using the high speed drill (Figure [3](#F3){ref-type="fig"}). Weber Ferguson incision was used to access the orbital portion of the tumor. Medially the tumor could be felt in the orbit but retraction of the globe was difficult. Hence inferior orbitotomy was done by removing the lower and lateral orbital margins. The intraorbital contents could now be retracted laterally and the tumor visualized (Figure [4](#F4){ref-type="fig"}). The tumor was then detached from the ethmoid sinus and the intraorbital extension excised. The ethmoidal portion was drilled and radical excision achieved (Figure [5](#F5){ref-type="fig"}). Dural tears were covered with temporalis fascia and glue. The ethmoidal sinus was packed with free temporalis muscle graft. Vascularised pericranial graft was used to cover the anterior skull base. The frontal sinus was exenterated and packed with gelfoam. The bone flap and orbital margins were replaced. Postoperative period and follow up ---------------------------------- Post-operatively patient had a frozen left eye (possibly due to retraction of the orbital contents) with no improvement in the left eye vision in the immediate postoperative period. His postoperative period was otherwise uneventful. Histopathological examination revealed an osteoid osteoma (Figure [6](#F6){ref-type="fig"}). On follow up after 12 months patient was disease free (Figure [7](#F7){ref-type="fig"}). Discussion ========== Osteoid osteoma is a benign osteoblastic lesion and constitutes 1% of all bone tumors and 11% of benign bone lesions \[[@B1]\]. It is usually seen in the second and third decades and a male preponderance has been noted. It can occur throughout the skeleton but the long bones of the lower extremities and the vertebrae are most commonly affected. They are usually metaphyseal but may be epiphyseal occasionally. It is frequently localized to the cortex (85%) but may also occur in spongiosa (13%) and subperiosteal region (2%) \[[@B2]\]. Cranial cases are seen to generally arise from the skull vault. Skull base osteoid osteomas are extremely rare and occur in the frontal or ethmoidal sinuses \[[@B1],[@B3]\]. It usually presents with sharply localized pain and tenderness especially at night. In our case the osteoid osteoma was seen to originate from the ethmoid sinus and pain was not a presenting feature. The radiological diagnosis rests on Computerised Tomography and isotope bone scan \[[@B1]\]. Radiographically osteoid osteoma appears as a radio opaque lesion with a nidus which has a radiolucent centre surrounded by dense sclerosis \[[@B2]\]. This may at times be mistaken for Garre\'s osteomyelitis. Occasionally the nidus may have a radio opaque centre with a surrounding radiolucent area. In our patient no definite nidus could be visualized probably due to the large size and unusual location. The treatment generally consists of en bloc resection or curettage of the tumor. Recurrence rate after incomplete resection may be upto 10% \[[@B1]\]. If asymptomatic and small, the lesion may be left alone and observed. However a rare complication of a large pneumocephalus has been reported by Ferlito et al from a frontoethmoidal osteoid osteoma \[[@B4]\]. Our patient presented with loss of vision due to a large osteoid osteoma of the ethmoid invading the left orbit. Anterior skull base lesions have been approached through a frontoorbitotomy which is usually removed as two separate parts. We have found that a single burr hole frontoorbitotomy flap gives excellent exposure to the anterior skull base without excessive retraction on the brain and is also cosmetically superior. Combining the craniotomy with a Weber Ferguson incision and orbitotomy made a single stage radical excision possible. Histologically the nidus is sharply delineated from the surrounding variably thick layer of dense bone. The nidus is composed of more or less calcified osteoid lined by plump osteoblasts within a highly vascularised connective tissue stroma \[[@B2]\] (Figure [6](#F6){ref-type="fig"}). It does not invade the adjacent tissue. No malignant transformation has been reported \[[@B1]\]. A differential diagnosis of benign osteoblastoma may be entertained. However, in the case of the latter, active osteoblasts are more numerous and the stroma is richly vascularized and extravasated blood with large number of multinucleated giant cell macrophages are noted \[[@B5]\]. Several authors have stressed the fact that the two are identical histologically and the differentiation between them if any can only be on the basis of size \[[@B5]\]. In our patient although the tumor was large, none of the above mentioned histological features of a benign osteoblastoma could be noted. Conclusion ========== Osteoid osteoma of the skull base is rare and anecdotally reported. Radical excision is difficult especially if the tumor involves major blood vessels and cranial nerves. The surgical team constituted the neurosurgeon, surgical oncologist and plastic surgeon. A craniofacial approach made radical single stage excision of this multicompartmental osteoid osteoma possible with an uneventful postoperative period. Competing interests =================== All the authors of the article \"ETHMOIDAL OSTEOID OSTEOMA WITH ORBITAL AND INTRACRANIAL EXTENSION -CASE REPORT\" hereby declare that there are no competing interests -- financial and non financial. Authors\' contributions ======================= SBPperformed the craniotomy, excised the intracranial extension of the tumor, assisted in excising the orbital portion, drilled the ethmoidal portion and drafted the manuscript. KHexcised the orbital portion and helped draft the manuscript. MSV and USperformed the orbitotomy, helped in tumor excision and closure. DJdid the histopathological examination and helped in drafting the manuscript. All authors have read and approved of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6815/5/2/prepub> Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Preoperative CT scan.Preoperative coronal CT scan which shows a large bony tumor arising from the left ethmoid sinus with orbital and intracranial extension. ::: ![](1472-6815-5-2-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Elevation of pericranial flap and craniotomy.Pericranial flap being raised after elevation of the scalp flap (B) followed by a single burr hole frontoorbitotomy (A). ::: ![](1472-6815-5-2-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Intracranial portion of the tumor.Dura being retracted after the craniotomy and visualization of the intracranial portion of the tumor. The tumor core has been drilled. The shell was subsequently nibbled away. ::: ![](1472-6815-5-2-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Facial approachWeber Ferguson incision (B) followed by inferior orbitotomy (A) which enabled the retraction of the eyeball. ::: ![](1472-6815-5-2-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Specimen.Specimen after it was excised in 2 pieces with part of the ethmoid sinus mucous membrane attached to one of the pieces. The ethmoidal portion was subsequently drilled away. ::: ![](1472-6815-5-2-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Microphotograph of the tumor.Microphotograph (250X) of the specimen in H & E stain showing new osteoid lined by plump osteoblasts (solid arrow) in a highly vascularised stroma (broken arrow). ::: ![](1472-6815-5-2-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Postoperative CT.Postoperative CT scan of the patient showing total excision of the tumor and the presence of free temporalis graft in the ethmoid and sphenoid sinus (arrow). ::: ![](1472-6815-5-2-7) :::	PubMed Central	2024-06-05T03:55:54.178179	2005-3-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555567/", "journal": "BMC Ear Nose Throat Disord. 2005 Mar 11; 5:2", "authors": [ { "first": "S Balaji", "last": "Pai" }, { "first": "K", "last": "Harish" }, { "first": "MS", "last": "Venkatesh" }, { "first": "Deepthi", "last": "Jermely" } ] }
PMC555568	Background ========== A diaphragmatic hernia is defined as a defect in part of the diaphragm through which abdominal contents can protrude into the thorax. It may be congenital or acquired, usually through trauma \[[@B1]\]. Congenital diaphragmatic hernias usually occur in the posterolateral portion of the diaphragm (Bochdalek\'s hernia) and are on the left side in 90% of cases. Loops of bowel, even most of the abdominal contents, may protrude into the hemithorax on the involved side \[[@B1],[@B5]\]. Most congenital diaphragmatic hernias are detected in the newborn whereby, after delivery, as the infant cries and swallows air, the loops of bowel quickly fill with air and rapidly enlarge, causing acute respiratory embarrassment as the heart and mediastinal structures are pushed to the right, compressing the right lung. Respiratory distress is immediate in severe cases; a scaphoid abdomen (due to displacement of abdominal viscera into the chest) is likely. Bowel sounds (and an absence of breath sounds) may be heard over the involved hemithorax. In less severe cases, mild respiratory difficulty develops a few hours or days later as abdominal contents progressively herniate through a smaller diaphragmatic defect \[[@B1]\]. Urgent surgery is usually required to repair the defect \[[@B1]\]. Acquired diaphragmatic hernias are relatively rare and result from either blunt or penetrating trauma. Blunt trauma typically produces large radial tears measuring 5--15 cm, most often at the posterolateral aspect of the diaphragm. Penetrating injuries to the diaphragm can follow accidental trauma, knife or gunshot wounds. Typically, the defect is small, less than two centimeters in size and may present late after years of gradual herniation and enlargement. Occasionally a shotgun blast causes a large defect \[[@B3],[@B4]\]. Diaphragmatic hernias are usually congenital in nature often requiring early corrective surgery for future survival. There have been three recent publications \[[@B5]-[@B7]\] in the literature relating Marfan\'s syndrome to right sided diaphragmatic hernias. Jacobs et al\[[@B5]\] described an association between unique FBN1 gene mutations in neonates and the presence of large unilateral diaphragmatic hernias, while Subirats et al\[[@B7]\] demonstrated a correlation between patients exhibiting positive Marfan\'s syndrome features and having unilateral diaphragmatic hernias. Yetman et al\[[@B5]\] recently described a case of acute dyspnoea in a child with Marfan\'s syndrome secondary to bowel herniation into the thoracic cavity. In these published cases, the diaphragmatic hernias were described in neonates or children and always required corrective surgery for survival, unsuccessfully in two cases. Case presentation ================= A 43 year old female district nurse presented to the accident and emergency department with a 6 hour history of initially generalized abdominal pain which was localizing to the right. This was associated with nausea and loss of appetite, made worse by movement and not relieved with intramuscular morphine injection. There was no history of recent or past trauma to the chest or abdomen. On examination, she was tender in the right upper quadrant and right iliac fossa with rebound and guarding, with a Rovsing positive sign and normal bowel sounds. She was incidentally found to have some marfanoid features including the long span of upper limbs, a high arched palate and the very soft early diastolic murmur of aortic regurgitation. The patient had a positive family history of Marfan\'s syndrome but had never undergone genetic testing to confirm the diagnosis. A likely diagnosis of appendicitis was made on the clinical picture associated with a pyrexia and raised white cell count and C -- reactive protein. Her abdominal X-ray at that time showed absence of gas in the right side of her bowel. Her chest X-ray did not show any obvious abnormality (Figure [1](#F1){ref-type="fig"}) ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Chest radiograph demonstrating no obvious abnormality ::: ![](1471-2482-5-4-1) ::: She was taken to theatre the same day for appendicectomy through a standard right iliac fossa incision. There was a small amount of free fluid but the right iliac fossa was filled with a normal looking gall bladder and small bowel only with no sign of the caecum, appendix, ascending or proximal transverse colon. The terminal ileum was found to run up under the right lobe of the liver towards the hilum. The right iliac fossa wound was closed and we proceeded to an exploratory laparoscopy. At laparoscopy, the liver was retracted to see under the right liver lobe. The proximal transverse colon was found to be running up towards the liver hilum were it felt to be tethered. This suggested a possible herniation of the right colon into the chest. The procedure was abandoned and a CT thorax and abdomen were performed to define the anatomy involved. This (Figure [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}) demonstrated bilateral pleural effusions of moderate size with underlying unexpanded lungs. The liver had what appeared to be a large Reidl\'s lobe. Behind the right liver lobe, the right kidney was markedly elevated and the ileo-caecal junction appeared to lie between the liver and the kidney. The right side of the colon appeared to lie above the liver. Three dimensional reconstruction of the scans demonstrated absence of the right hemidiaphragm. (Figure [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}) ::: {#F2 .fig} Figure 2 ::: {.caption} ###### CT scan of thorax and abdomen demonstrating the abnormal anatomy ::: ![](1471-2482-5-4-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### CT scan of thorax and abdomen demonstrating the abnormal anatomy ::: ![](1471-2482-5-4-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Three-dimensional CT reconstruction demonstrating the diaphragmatic defect ::: ![](1471-2482-5-4-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Three-dimensional CT reconstruction demonstrating the diaphragmatic defect ::: ![](1471-2482-5-4-5) ::: The symptoms and signs persisted and a laparotomy was performed. At laparotomy, there was some free fluid. There was definitely a large defect in the right hemidiaphragm. The right liver lobe had grown right up into the right intrathoracic space. There was a large hernial sac beneath the right lobe of the liver between the common bile duct, duodenum and liver, displacing the right kidney medially and containing transverse colon, terminal ileum, caecum, appendix and free fluid. The contents of this sac were successfully reduced and a perforated gangrenous appendix with pus was found within the intrathoracic hernial sac. A standard appendicectomy was performed. The right colon was formally mobilized, fully reduced and the caecum was fixed within the right iliac fossa. The patient was admitted to the intensive care unit postoperatively where she made a good recovery. She had instant relief of her abdominal symptoms and post-operative contrast study demonstrated the presence of the right colon within the abdomen (Figure [6](#F6){ref-type="fig"}). She was discharged a week post-operatively. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Abdominal radiograph demonstrating the large colon present in the abdominal cavity ::: ![](1471-2482-5-4-6) ::: Conclusion ========== We have demonstrated a new case of an adult patient, with Marfan\'s syndrome clinical features and a positive family history of the syndrome, presenting with a large congenital diaphragmatic hernia, compatible with life and undiagnosed into adulthood, requiring an emergency admission into hospital with a perforated gangrenous intrathoracic appendix. Abbreviations ============= CT = computerized tomography Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= MJB designed the case report, researched the article and drafted the manuscript. Both MJB and JHV carried out the surgery and were involved in all investigations. 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/1471-2482/5/4/prepub> Acknowledgements ================ Many thanks to all the general surgical team contribution. Thanks to the radiological and pathological departments at Weston General Hospital for providing all the results required.	PubMed Central	2024-06-05T03:55:54.179158	2005-3-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555568/", "journal": "BMC Surg. 2005 Mar 11; 5:4", "authors": [ { "first": "Mohannad J", "last": "Barakat" }, { "first": "Jon H", "last": "Vickers" } ] }
PMC555569	Background ========== Rearrangements of the cortical actin cytoskeleton are essential for numerous cell processes such as cell migration, phagocytosis \[[@B1],[@B2]\], and adhesion \[[@B3]\], in which formation of dendritic networks of polymerised actin plays a key part. The Arp2/3 complex is required in the formation of dendritic networks to provide sites for de novonucleation of actin filaments and to form branch points from existing filaments \[[@B4]-[@B6]\]. The mammalian Arp2/3 complex can be activated downstream of Rho family small GTPases by several known interacting proteins, in particular members of the Wiskott Aldrich Syndrome Protein Family (haematopoietic WASP, ubiquitous N-WASP, and Scar/WAVE 1, 2, and 3 \[Suppressor of Cyclic AMP Receptor mutation/Wiskott Aldrich VErprolin homologous protein\]) \[[@B7],[@B8]\]. Homology between these proteins consists of a core proline rich region, and C-terminal WH2 (Wiskott Homology 2) and Acidic (A) domains, of which the WH2 and A domains together are sufficient to activate the Arp2/3 complex \[[@B9]-[@B11]\]. N-WASP and the Scar/WAVE proteins differ the most at the amino-terminus, designated the Wiskott-Homology 1 (WH1) domain and the Scar/WAVE homology domain (SHD) \[[@B9]\]. Regulation of WASP-family proteins involves many interactions and is still the subject of intensive research. WASP and N-WASP are found in an inhibitory complex with WIP/CR16 proteins and can be activated by the small GTPase Cdc42 and a co-activating protein TOCA-1, recently described by Ho et al. \[[@B12]\]. Phosphorylation on tyrosine residues in the N-terminal half of WASP/N-WASP can also enhance the activation and perhaps serve to prolong its duration (\[[@B13]-[@B15]\]). Original reports had suggested that WASP/N-WASP were autoinhibited in their pure form, but whether this form is ever present in cells is unclear, so the physiological relevance of autoinhibition is unclear as well (\[[@B16]\]). Recombinant Scar/WAVE proteins were constitutively active toward Arp2/3 complex in vitro, but have been postulated to be regulated by the small GTPase Rac in vivo\[[@B17],[@B18]\]. In 2002, Eden et al purified Scar/WAVE1 from bovine brain extracts in association with a complex of four other proteins; p140-SRA1, p125 Nck-Associated Protein (NckAP1), HSPC300, and Abi2 \[[@B19]\]. They proposed a model whereby active Rac could bind to p140-SRA1 and cause it plus NckAP1 and Abi2 to dissociate, releasing active Scar/WAVE in a complex with HSPC300. Some of the members of this so-called \"Scar/WAVE complex\" had been previously studied by other groups and implicated in signalling. SRA1 (also called Cytoplasmic FMRP Interacting protein \[[@B20]\]) is a Rac associated protein which also binds to the SH3 domain containing adapter protein Nck, and the Fragile-X Mental Retardation Protein (FMRP) \[[@B20]-[@B23]\]. NckAP1 also interacts with active Rac and Nck, although the interaction appears to be indirect in both cases \[[@B22],[@B23]\]. HSPC300 is a small, ubiquitously expressed, uncharacterised Haematopoeitc Stem/Progenitor Cell protein, bearing considerable homology to the Maize actin cytoskeletal associated protein Brk1 \[[@B24]-[@B26]\]. Abi2 (Abl interactor 2) is a neuronally expressed SH3 domain containing protein associated with Abl tyrosine kinase \[[@B27]\]. It has a close relative, Abi1, also referred to as e3B1 (eps8 binding protein 1) in early literature \[[@B28],[@B29]\]. Since Eden et al., a similar complex has also been isolated in association with Scar/WAVE2 \[[@B30],[@B31]\]. This complex contains the more ubiquitously expressed Abi1 (as opposed to Abi2) consistent with the more ubiquitous expression of Scar/WAVE 2 compared to the other Scar/WAVE isoforms \[[@B31]-[@B33]\]. In these studies, the complex does not dissociate upon Rac activation and is not inhibitory during in vitroactin polymerisation assays \[[@B31]\]. Recombinant Abi1 is rather found to increase the Arp2/3 complex activating ability of recombinant Scar/WAVE 2 \[[@B31]\]. Following these studies, the mechanism for regulation of the different Scar/WAVE proteins has been called into question. It has been proposed that differences between Eden et al. and the other two studies may be due to experimental conditions or to genuine differences between Scar/WAVE isoforms \[[@B19],[@B30],[@B31],[@B34],[@B35]\]. Knockdown of Abi1, SRA1 and NckAP1 in mammalian tissue culture cells causes severe defects in formation of lamellipodia \[[@B31],[@B36]\], similar to loss of Scar/WAVE 2 activity \[[@B37],[@B38]\]. Mutations or RNAi knockdown of the only DrosophilaScar/WAVE protein severely affects the ability of cultured cells to produce lamellipodia, ruffles and filopodia \[[@B39]-[@B41]\]. Mutational and RNAi studies used to produce Drosophilacells deficient in the NckAP1 homologue, Kette, reveal a lack of actin based protrusions in the absence of functional protein \[[@B42],[@B43]\]. RNAi knockdown of DrosophilaAbi1 and SRA1 also prevents the formation lamellipodia in tissue culture cells \[[@B39],[@B41],[@B43]\]. Dictyosteliumknock out of Scar protein exhibits severe defects in chemotaxis and motility, but cells can still extend pseudopods and migrate directionally \[[@B44],[@B45]\], whereas knockout of the PirAgene encoding an homologue of SRA1 causes an excessive lamellipodial protrusion phenotype, thought to be due to unregulated Scar protein activity \[[@B46]\]. While the Scar/WAVE complex is currently thought to be the most likely regulator of Scar/WAVE activity in cells, several other Scar/WAVE binding proteins have been identified and proposed to regulate its activity. IRSp53, for example, binds to Scar/WAVE2 and also to Rac and was proposed to be a Scar/WAVE2 regulator prior to Eden et al. (\[[@B47]-[@B50]\]). It was unclear whether IRSp53 binding was specific to Scar/WAVE 2, or ubiquitous among the Scar/WAVEs and where/when Scar/WAVE was associated with IRSp53 as opposed to the other binding partners. IRSp53 is also a scaffold protein, with several partners, raising the possibility of at least two different types of large protein complexes in association with Scar/WAVE proteins \[[@B49],[@B51]-[@B55]\]. Scar/WAVE3 is the most tissue-specific of the three mammalian Scar/WAVE isoforms, being found in haematopoietic cells and brain tissue, but not yet being characterised in cells \[[@B9],[@B56]\]. It has not previously been shown whether Scar/WAVE3 interacts with either Abi1/2 or HSPC300. This report presents data indicating that the interaction of Scar/WAVE proteins with Abi1, HSPC300 and IRSp53 is conserved among all three Scar/WAVE proteins, suggesting that multiple different protein complexes likely exist in cells that contain different Scar/WAVE isoforms. This observation suggests that the different Scar/WAVE isoforms are not regulated by exclusive participation in specific complexes, but rather that the regulation is likely to be more subtle, at the level of affinity or modifications/binding partners not yet discovered. Alternatively, Scar/WAVEs may have largely overlapping functions when present in the same cell type, although there is already some evidence against this idea \[[@B57]\]. Results ======= Association of the Scar/WAVE complex with Scar/WAVE 3 ----------------------------------------------------- Protein sequence conservation between the three Scar/WAVE proteins in the N-terminal Scar Homology Domain (SHD) indicates a potential for association of binding partners with all three members of the family. Scar/WAVE1 and 2 have been shown to associate with the key adapter protein Abi1 through the SHD, so it is possible that Abi1 would also associate with Scar/WAVE3 \[[@B19],[@B30]\]. To test for an interaction between Scar/WAVE3 and Abi1, immunoprecipitations were performed with antibodies recognising Abi1 (Fig. [1A](#F1){ref-type="fig"}) and antibodies recognising Scar/WAVE3 (Fig. [1B](#F1){ref-type="fig"}). Anti-Abi1 immunoprecipitates co-precipitate Scar/WAVE1 and 3 with endogenous Abi1 from mouse brain extracts, while Abi1, Scar/WAVE1 and Scar/WAVE3 are not present in immunoprecipitates with an unrelated control antibody or without antibody (Fig. [1A](#F1){ref-type="fig"}). Abi1 also co-immunoprecipitates with Scar3, but is not detected in the absence of antibody or in unrelated control immunoprecipitates (Fig. [1B](#F1){ref-type="fig"}). Since Abi1 is thought to bind directly to Scar/WAVE proteins and to connect them with the rest of the Scar/WAVE complex, interaction with Abi1 is a strong indicator Scar/WAVE3 associates in an analogous complex to Scar/WAVE1 and 2 isoforms \[[@B35]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Co-immunoprecipitation of Scar3 and Abi1 from mouse brain extract. Protein G beads were used to precipitate Abi1 from mouse brain extracts in the presence or absence of (A) anti-Abi1 or an unrelated control antibody or (B) anti-Scar3 or an unrelated control antibody. (A) The beads fractions were probed with anti-Abi1, anti-Scar/WAVE1 and anti-Scar/WAVE3. Anti-Abi1 immunoblotting confirmed precipitation of Abi1 only with specific antibody and immunoblotting with anti-Scar/WAVE1 shows association with a known binding partner. Immunoblotting with anti-Scar/WAVE3 revealed Scar/WAVE3 to also be present in Abi1 immunoprecipitates. Anti-Abi1 pulls down both Scar1 and Scar3 together with Abi1. (B) Beads fractions from anti-Scar/WAVE3 immunoprecipitates were probed with both anti-Scar/WAVE3 and anti-Abi1. Both Scar3 and Abi1 are detected in anti-Scar/WAVE3 immunoprecipitates, but not with controls. ::: ![](1471-2121-6-11-1) ::: Conservation of Scar complex association between all three human Scar/WAVE isoforms ----------------------------------------------------------------------------------- Our data and previously published studies indicate that Abi1 exhibits the potential to bind to all three mammalian Scar/WAVE isoforms. To test whether the association of Scar/WAVE1, 2, and 3 with Abi1 is mediated by the Scar Homology Domain, Myc-Scar/WAVE SHD of each of the three family members were transiently expressed in Cos cells with HA-Abi1 or HA-HSPC300 and immunoprecipitated using a 9E10 anti-myc monoclonal antibody. HA-Abi1 precipitated with each of the three SHDs, but not in the absence of antibody or Myc-SHD (Fig. [2A](#F2){ref-type="fig"}). HA-HSPC300 also co-immunoprecipitated with all three SHDs, but not the negative controls (Fig [2B](#F2){ref-type="fig"}). Pull-down experiments performed from HA-Abi1 or HA-HSPC300 transfected Cos cell lysates using GST-fusion proteins of Scar/WAVE1 and 2 (Fig. [3A](#F3){ref-type="fig"}) and Scar/WAVE3 (Fig. [3B](#F3){ref-type="fig"}) also show conservation of interaction of all human Scar/WAVE isoforms with Abi1 and HSPC300. Supporting data showing the specific interaction of Abi1 with Scar/WAVE1 SHD is shown in Table [1](#T1){ref-type="table"} and [Additional file 1B](#S1){ref-type="supplementary-material"}. Data supporting interaction of Scar/WAVE1 SHD with HSPC300 is shown in Table [1](#T1){ref-type="table"} and in [Additional file 1C](#S1){ref-type="supplementary-material"}. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Co-immunoprecipitation of Abi1 and HSPC300 with Scar Homology Domain. Cos 7 fibroblasts transiently transfected as indicated. Protein G beads were used to immunoprecipitate Myc-Scar1 SHD, Myc-Scar2 SHD or Myc-Scar3 SHD from lysates in the presence (+) or absence (-) of anti-Myc (9E10) monoclonal antibody. Empty pRK5-Myc vector was used as an additional negative control. (A) HA-Abi1 was detected in the beads plus antibody fractions for Myc-Scar1 SHD, Myc-Scar2 SHD, and Myc-Scar3 SHD, but not in the negative controls. (B) HA-HSPC300 was detected in the beads plus antibody fraction of immunoprecipitations from cells co-transfected only with Myc-Scar1 SHD, Myc-Scar2 SHD, and Myc-Scar3 SHD, but not empty vector controls or in the absence of 9E10 antibody. ::: ![](1471-2121-6-11-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Recombinant SHD pulls down Abi1 and HSPC300. Lysate of Cos7 fibroblasts transfected with HA-Abi1 or HSPC300 was incubated as indicated with GST alone, GST-Scar1 SHD (A), GST-Scar2 SHD (A), or GST-Scar3 SHD (B) on glutathione-s-agarose beads. Beads bound fractions were analysed by SDS-PAGE and immunoblotting with a monoclonal anti-Myc (9E10) antibody. HA-Abi1 and HA-HSPC300 were found in pull-downs with all three Scar Homology Domains, but not with GST alone. ::: ![](1471-2121-6-11-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Yeast two-hybrid analysis of interactions between Abi1, HSPC300, and various domains of Scar1. (+) Indicates a positive interaction backed up by a beta-Gal reporter gene assay. (-) Indicates negative for an interaction between the constructs. ::: pAct-Scar1 PYTH9 ------------------ ------- ----- Full Length (FL) \+ \+ SB \+ \+ SP \+ \+ BPWA \- +/- PWA \- \- ::: Conserved interaction of Scar/WAVE isoforms with IRSp53 ------------------------------------------------------- Conservation of Scar Homology Domain binding partners raises the question of whether other binding partners of Scar/WAVE are also capable of interacting with the entire Scar/WAVE family. IRSp53 has been implicated in regulation of actin dynamics through binding to Scar/WAVE proteins \[[@B49],[@B50],[@B54]\]. GST-IRSp53 was used for pull down experiments to test for binding to Scar/WAVE1, 2, and 3 (Fig. [4](#F4){ref-type="fig"}). None of the Scar/WAVE isoforms were detected in the beads fraction of a negative control (constitutively active GST-L61 Cdc42), but all were detected in the beads fraction of GST-IRSp53. An ability to bind to IRSp53 is conserved between all three human members of the Scar/WAVE family, although the binding appears to be strongest for Scar/WAVE2. Conservation of this interaction may indicate that all three Scar/WAVE proteins are regulated by common mechanisms, but the increased binding of Scar/WAVE2 to IRSp53 highlights the possibility that differing affinities for binding partners between the three Scar/WAVE isoforms may affect their roles in cells. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### IRSp53 interacts with Scar/WAVE1, 2 and 3. Equivalent amounts of GST-IRSp53 or constitutively active GST-L61 Rac were used for pull-down assays from lysates of Cos cells transfected with Myc-Scar1, 2 or 3. Bead fractions and whole cell lysates, indicating loading with each Scar/WAVE isoform, were analysed by SDS-PAGE and immunoblotting with monoclonal (9E10) anti-Myc antibody. None of the Scar/WAVE isoforms bound to active Cdc42, but all were detected in GST-IRSp53 bead fractions. ::: ![](1471-2121-6-11-4) ::: Cellular localisation of Scar/WAVE3 ----------------------------------- As an activator of the Arp2/3 complex, Scar/WAVE3 would be expected to localize to the same areas of the cell as polymerized actin, subunits of the Arp2/3 complex, and members of the Scar/WAVE complex. C2C12 cells were used for immunocytochemistry with anti-Scar3 polyclonal antibody (Fig. [5Ai, Bi, Ci](#F5){ref-type="fig"}), phalloidin to detect filamentous actin (Fig. [5Aii](#F5){ref-type="fig"}), anti-Arp2/3 monoclonal antibody (Fig. [2Bii](#F2){ref-type="fig"}), and anti-Abi1 antibody (Fig. [5Cii](#F5){ref-type="fig"}). C2C12 cells were used because they were the only standard tissue culture cell line that we found to contain a detectable amount of Scar/WAVE3 protein (see Methods). Scar/WAVE3 mainly exists in a peri-nuclear pool with enrichment to areas of polymerised actin (Fig. [5Aiii](#F5){ref-type="fig"}). Scar/WAVE3 co-localises with the Arp2/3 complex subunit, Arp3 (Fig. [5Biii](#F5){ref-type="fig"}), and with Abi1 (Fig. [5iii](#F5){ref-type="fig"}) in areas of lamellipodial protrusion. Our results show Scar/WAVE3 to localise to areas of polymerised actin near the cell periphery and to co-localise with a known component of the Scar/WAVE complex, indicating that Scar/WAVE3 may have a similar role and method of regulation to Scar/WAVE1 and 2. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Cellular localisation of Scar3. C2C12 cells were stained with anti-Scar/WAVE3, anti-Arp3, anti-Abi1 and fluorescently labelled phalloidin to investigate the cellular localisation of endogenous proteins. (A) C2C12 cells stained with (i) anti-Scar/WAVE3 (green), (ii) phalloidin (red) (iii) reveal co-localisation of Scar/WAVE3 with cortical polymerised actin in membrane ruffles. Scar/WAVE3 also colocalises with other areas enriched in polymerised actin, but not stress fibres. (B) Co-staining with (i) anti-Scar/WAVE3 and (ii) anti-Arp3 reveals co-localisation of Scar/WAVE3 (green) with the Arp2/3 complex (red) at protruding areas of the cell membrane. (C) Cells stained with (i) anti-Scar/WAVE3 and (ii) anti-Abi1. (iii) Scar/WAVE3 (green) co-localises with Abi1 (red) in some areas of membrane protrusion. A (i), B (i), and C (i) all show a cytoplasmic and perinuclear and cytoplasmic pool of Scar/WAVE3, with enrichment at areas of lamellipodial protrusion. B (ii) and C (ii) show similar patterns of staining for Arp3 and Abi1. Scale bars are equal to 20 μm in all pictures. ::: ![](1471-2121-6-11-5) ::: Cellular localisation of Scar/WAVE SHD, Abi1, and HSPC300 --------------------------------------------------------- If the SHD of Scar/WAVE protein mediates the interaction with a regulatory complex it can be expected that the SHD will co-localise with binding partners in cells. Ectopic expression of full length Scar/WAVE isoforms in cells reveals a peri-nuclear and cytoplasmic pool, causing an abundance of polymerised actin in the cytoplasm, but with enrichment of Scar/WAVE protein in lamellipodia \[[@B50],[@B58]\]. A sizeable amount of Myc-Scar 1 SHD (Fig [6Aiii](#F6){ref-type="fig"}), Myc-Scar 2 SHD (Fig [6Aiv](#F6){ref-type="fig"}), and Myc-Scar 3 SHD (Fig. [6Av](#F6){ref-type="fig"}) localises to the cell periphery in lamellipodia, as well as in a cytoplasmic pool similar to full length Scar/WAVE proteins (see Figure [5Ai](#F5){ref-type="fig"} for comparison). HA-Abi1 is present throughout the cytoplasm in spots (Fig [6Aii](#F6){ref-type="fig"}), previously described as characteristic of the reticulovesicular system \[[@B59]\]. HA-HSPC300 exhibits a similar cellular localisation to ectopically expressed Scar/WAVE1, 2 and 3 being abundant in and around the nucleus, and diffusely around the cytoplasm with enrichment at lamellipodia (Fig [6Ai](#F6){ref-type="fig"}). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Cellular localization of Abi1, HSPC300, and the Scar homology domain. Transfected Cos cells were stained with a monoclonal anti-Myc antibody and polyclonal anti-HA antibodies to detect over-expressed proteins. (A) Cos7 cells were co-transfected with (i) HA-HSPC300, (ii) HA-Abi1, (iii) Myc-Scar1 SHD, (iv) Myc-Scar2 SHD, or (v) Myc-Scar3 SHD to examine localization of these proteins. HSPC300, and all three SHDs localise in a diffuse cytoplasmic pool with some enrichment at protrusive edges of cells. Abi1 is not detected at the edges of cells, but appears in vesicle-like spots throughout the cytoplasm. (B) Cos7 cells were co-transfected with Myc-Scar1 SHD (i) and HA-HSPC300 (ii) shown as red and green respectively in a merged image (iii). Scar1-SHD and HSPC300 exhibit the same diffuse staining throughout the cytoplasm but also co-localize at protruding edges of cells. (C) Cos7 cells were co-transfected with Myc-Scar1 SHD (i) and HA-Abi1 (ii). (iii) Shows a merge image with myc-Scar1 SHD in red and HA-Abi1 in green. Scar1 SHD and Abi1 both colocalize to punctate spots similar to those seen for Abi1 alone. (D) Cos7 cells co-transfected with Myc-Scar2 SHD (i) and HA-HSPC300 (ii) shown as red and green respectively in a merge image (iii). Both Scar2 SHD and HSPC300 exhibit peri-nuclear staining and localization to protrusive edges of cells. (E) Cos7 cells co-transfected with Myc-Scar2 SHD (i) and HA-Abi1 (ii). (iii) Scar2 SHD (red) and Abi1 (green) show co-localization to cytoplasmic spots and the edges of cells. (F) Cos7 cells co-transfected with (i) Myc-Scar3 SHD (red) and (ii) HA-HSPC300 (green), in a merged image (iii). HSPC300 and Scar3 SHD show diffuse cytoplasmic staining with enrichment at protrusive edges of cells. (G) Cos7 cells co-transfected with (i) Myc-Scar3 SHD and (ii) HA-Abi1. (iii) In a merge image Abi1 (green) and Scar3 SHD (red) co-localize to punctate cytoplasmic spots and to the edges of lamellipodia. Scale bars in all panels are 20 μm. ::: ![](1471-2121-6-11-6) ::: When expressed with Myc-Scar 1 SHD (Fig. [6Bi](#F6){ref-type="fig"}), HA-HSPC300 (Fig. [6Bii](#F6){ref-type="fig"}) co-localises (Shown as yellow in merge pictures \[6Biii\]) with enrichment in ruffles. Myc-Scar/WAVE 2 (Fig. [6Di](#F6){ref-type="fig"}), and 3 (Fig [6Fi](#F6){ref-type="fig"}) SHD also co-localises with HA-HSPC300 (Fig. [6Dii, 6Fii](#F6){ref-type="fig"}) in the nucleus, cytoplasm, and particularly strongly at areas of ruffles, shown as yellow in merge images (Fig. [6Diii, 6Fiii](#F6){ref-type="fig"}). All three human isoforms behave the same in the presence of HSPC300 consistent with a conservation of the interaction between Scar/WAVEs and HSPC300. HA-Abi1 (Fig. [6Cii](#F6){ref-type="fig"}) strongly co localises with Myc-Scar 1 SHD (yellow, Fig [6Ciii](#F6){ref-type="fig"}), in unidentified punctate spots (Fig [6Ci](#F6){ref-type="fig"}). Myc-Scar 2 SHD (Fig. [6Ei](#F6){ref-type="fig"}) shows a similar co localisation with Abi1 (Fig. [6Eii](#F6){ref-type="fig"} and [6iii](#F6){ref-type="fig"}), whilst Myc-Scar 3 SHD (Fig. [6Gi](#F6){ref-type="fig"}) shows partial co-localisation to Abi1 (Fig. [6Gii](#F6){ref-type="fig"} and [6iii](#F6){ref-type="fig"}). Although Myc-Scar3 SHD (Fig. [6Gi](#F6){ref-type="fig"}) co-localises to the HA-Abi1 spots (Fig. [6Giii](#F6){ref-type="fig"}) to a lesser extent than Scar/WAVE1 and 2 SHD, leaving some diffuse staining in the cytoplasm and nucleus. Abi1 expression affects the localisation of all three human Scar/WAVE isoforms in Cos7 cells, providing further evidence for a role of the Scar/WAVE complex in regulation of the activity and localisation of Scar/WAVE proteins. The staining pattern seen for overexpressed HA-Abi1 (Fig. [6Aii, Cii, Eii](#F6){ref-type="fig"} and [6Gii](#F6){ref-type="fig"}) does not strongly correlate with the cellular localisation of endogenous Abi1 (Fig. [5Cii](#F5){ref-type="fig"}). Although the punctuate staining pattern seen in cells over-expressing Abi1 has been described as being characteristic of the reticulovesicular system (Ziemnicka-Kotula, 1998), it is also possible that these spots are protein aggregates induced by overexpression of Abi1. The relocalisation of Scar/WAVE1, 2 and 3 SHDs to these vesicles or aggregates may or may not be biologically important, but it does demonstrate the possibility that Abi1 can affect Scar/WAVE protein localisation in cells. Discussion ========== We show that Abi1 and HSPC300 interact with three human Scar/WAVE isoforms, via the Scar Homology Domain. Conservation of Abi1 binding between all three Scar/WAVEs is indicative of the association of the \"Scar/WAVE complex\" with all three isoforms, as Abi1 seems to be the link between Scar/WAVE and NckAP1 and SRA-1/PIR121 in previous reports \[[@B30],[@B31]\]. Association of the complex with all three Scar/WAVE proteins is a possibility, as all the complex members are widely expressed throughout the body and seem to be found in all tissues where a Scar/WAVE protein is expressed \[[@B26],[@B60]\]. Little is known about the role of Scar/WAVE3 in cells. Expression of the mammalian Scar/WAVE3 gene is largely limited to the brain and lung with relatively little expression in other adult tissues \[[@B9],[@B32],[@B56]\]. The major suggestion has been a role in lamellipodial and filopodial protrusion indicated by localisation in neuronal growth cones and interaction with the Arp2/3 complex \[[@B9],[@B57]\]. Association with a Abi1-NckAP1-SRA1 does indicate a further potential role for Scar/WAVE3 in lamellipodial protrusion given the requirement for these proteins in the ruffling process \[[@B31],[@B36]\]. Scar/WAVE3 is also implicated as a potential tumour suppressor protein in some ganglioneuroblastomas when the gene is down-regulated \[[@B56]\]. The role of interaction of HSPC300 with the Scar/WAVE3 complex is unknown. Loss of function of the brk1gene product, the Maize homologue of HSPC300, causes aberrations in cellular morphology and filamentous actin distribution of leaf epidermal cells \[[@B24],[@B25]\]. This could indicate a role in regulating the localisation of actin polymerisation machinery, but this idea awaits further testing. HSPC300 is also implicated in the frequency of occurrence of renal cell carcinoma \[[@B26]\]. Association of all three Scar/WAVE isoforms with an Abi-NckAP1-SRA1 complex may show that there is some functional redundancy between Scar/WAVE family members but data from mice deficient in a Scar/WAVE isoform indicates that Scar/WAVE1 and 2 do not have completely overlapping functions \[[@B37],[@B38],[@B57],[@B61]\]. The identification of specific binding partners for Scar/WAVE1, such as WRP, BAD or the RII subunit of PKA suggests a potential for differing roles between Scar/WAVE proteins \[[@B62]-[@B64]\]. The conservation of the Abi1-NckAP1-SRA1 complex between three Scar/WAVE isoforms suggests that regulation of individual Scar/WAVE proteins is likely to involve components that we have not yet studied \[[@B37],[@B57],[@B58],[@B61]\]. Other proteins that associate with the complex may perform this regulation, or tissue specificity may occur between the complexes formed where differentially expressed related proteins, such as Abi1 or 2, bind to alternative Scar/WAVE isoforms with different affinities. Further study of the Scar/WAVE family and associated proteins will be required to fully understand the true roles and mechanisms of function of these complexes in cells. Conclusion ========== We conclude that Scar/WAVE3 is likely to participate in similar signalling complexes to Scar/WAVE1 and 2 and that the differences between these Scar/WAVE proteins is likely to be at the level of tissue expression, differences in affinity for certain binding partners and possibly interaction with yet undiscovered binding partners. Methods ======= Reagents and chemicals ---------------------- All chemicals were purchased from Sigma-Aldrich, UK unless otherwise specified. Antibodies were from the following sources: 9E10 anti-Myc monoclonal (Cancer Research UK), 12CA5 anti-HA monoclonal (Cancer Reaearch UK), anti-Myc polyclonal 1:500 anti-HA polyclonal (Santa Cruz), 1:500 anti-Scar/WAVE3 (Upstate), 1:100 anti-Abi1, or 1:100 anti-Scar/WAVE1 \[[@B65]\], anti-Abi1 (gift from Giorgio Scita, Instituto Europeo di Oncologia, Milan, Italy), HRP-conjugated secondary antibody anti-mouse or anti-rabbit (Jackson labs), anti-Arp3 (Sigma Israel), Goat anti-mouse or rabbit, FITC, TRITC, Alexa-488 or Alexa-546 Conjugates (Molecular Probes). The specificity of the anti-Scar/WAVE3 antibody for Scar/WAVE3 has been demonstrated by Oda and colleagues \[[@B47]\]. Vectors and cloning ------------------- HSPC300 and Abi1 expression vectors were created using the Gateway Cloning system (Invitrogen). Open reading frames for HSPC300, and Abi1, derived from I.M.A.G.E. clones 4519512 (HSPC300) and 4158413 (Abi1) (UK-Human Genome Resource Centre, Babraham), were amplified by PCR and ligated into the pENTR-Topo entry vector (Invitrogen). N-terminally HA- and Myc- tagged HSPC300, and Abi1, were created in pRK5 DEST-Myc or pRK5 DEST-HA Gateway destination vectors by recombination from entry vectors. pRK5 DEST-Myc or pRK5 DEST-HA were created by ligation of the Gateway Vector Conversion System (Invitrogen) reading frame A cassette into pRK5-myc or pRK5-HA cut with SmaI. Scar/WAVE1, Scar/WAVE2, and Scar/WAVE3 full length and deletion constructs were created by PCR and cloned into pRK5-myc or pGEX4T-2. Scar/WAVE-1 constructs were derived from KIAA00429\[[@B7]\]; Scar/WAVE2 constructs were derived from pDSRed Scar2, a kind gift from Giorgio Scita (Innocenti et al., 2004); Scar/WAVE3 constructs were derived from pcDNA Scar3, a kind gift from John Scott, Portland, U.S.A. L61 Cdc42-pGex2T and IRSp53-pRK5-Myc were a gift from Alan Hall (LMCB, London) \[[@B54]\]. IRSp53 was subcloned into pGex4T-2 using BamH1 and EcoR1 restriction sites. For Yeast Two-Hybrid analyses, Abi1 and HSPC300 were fused in-frame to the C-terminus of the GAL4 DNA-binding domain in pYTH9. Myc-tagged Scar/WAVE1 deletion constructs have been previously described\[[@B7]\]. Scar1 deletion mutants (described in [Additional file 1](#S1){ref-type="supplementary-material"}) were fused to the C-terminus of the GAL4 activation domain in pACT-II \[[@B66]\]. Yeast 2-hybrid analyses were performed as previously described \[[@B66]\]. Cell culture, transfection, and lysis ------------------------------------- Cos 7 and C2C12 cells were grown in DMEM plus 10% Foetal Calf Serum and antibiotics. Transfections were performed using Genejuice transfection reagent (Novagen) following the manufacturers instructions. Cell lysis was performed from confluent cells in a 1% Triton X-100 lysis buffer (1% Triton X-100, 50 mM Tris-HCl pH 7.5, 150 mM NaCl,, 1 mM EDTA, 10% Glycerol, 1 mM PMSF, and 1 μg/ml each of chymostatin, aprotinin, leupeptin, and pepstatin). Lysates were cleared by centrifugation and equalised to 1 mg/ml total protein concentration. Preparation of mouse brain extract ---------------------------------- Mouse brains were homogenised on ice in 2 μl of 1% Triton X-100 lysis buffer per mg of tissue using a fine gauge needle. Homogenates were centrifuged once for 10 minutes at 13,000 rpm in a microcentrifuge, then for 30 minutes at 100,000 g in a Beckmann TLA-100.2 rotor. Supernatants were equalised to 1 mg/ml total protein concentration with 1% Triton X-100 lysis buffer. Immunoprecipitations -------------------- Lysates or brain extracts (500 μg of total protein) were incubated with 30 μl bed volume of pre-washed protein G beads (Cancer Research UK) for 1 hour, designated beads minus antibody controls. Beads were precipitated and supernatants removed. 5 μg of antibody monoclonal 9E10 anti-myc, 12CA5 anti-HA, Rabbit anti-Scar/WAVE3, or Mouse anti-Abi1 or an irrelevant control antibody (rabbit or mouse anti-myc) was added to the supernatant and incubated for 1 hour, before the addition of 30 μl bed volume of pre-washed protein G beads, designated beads plus antibody, for 1 hour. Beads were precipitated, and the supernatant removed. Beads were washed 3 times in 30-fold bed volume of lysis buffer. Beads were resuspended in an equal volume of 2× SDS-PAGE loading buffer \[[@B67]\]. 10 μl of beads sample and supernatant was analysed by SDS-PAGE and western blotting. GST-production and pull downs ----------------------------- GST-fusion proteins were expressed in BL21 E.coliinduced for 3 hours with 0.2 mM IPTG, or 15 hours at 25°C for GST-IRSp53. Cells were spun down and sonicated in 1 ml of 1% Triton PBS (1% Triton X-100, PBS pH 7.5, 1 mM PMSF, and 1 μg/ml each of chymostatin, leupeptin, aprotinin, and pepstatin) per 100 ml bacterial culture. GST fusion proteins were batch purified on 100 μl Glutathione-s-agarose (Sigma) per 100 ml culture and washed 5 times with 10-fold bed volume of sonication buffer. Beads were resuspended in an equal volume of 1% Triton lysis buffer. SDS-PAGE analysis and coomasie staining were used to qualify GST fusion proteins. 500 μg total protein of transfected Cos7 cell lysate was incubated with GST-fusion protein or GST alone for 1 hour. Beads were precipitated, lysates removed and the beads were washed 3 times in 1% Triton lysis buffer. Beads were resuspended in and equal volume of 2× SDS-PAGE loading buffer. Samples were analysed by SDS-PAGE and western blotting. SDS-PAGE and Western blotting ----------------------------- Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane by western blotting. Blots were saturated with 5% dried milk in PBS 0.2% Tween-20, probed with 1:500 9E10 anti-Myc monoclonal, 1:500 12CA5 anti-HA monoclonal, 1:500 anti-Myc polyclonal 1:500 anti-HA polyclonal, 1:500 anti-Scar/WAVE3, 1:100 anti-Abi1, or 1:100 anti-Scar/WAVE1 primary antibody in 2.5% BSA 0.2% Tween-20 PBS followed by 1:10\'000 dilution of HRP-conjugated secondary antibody diluted in 0.2% Tween-20 PBS. Bound secondary antibody was visualised using SuperSignal (Pierce) according to the manufacturers instructions. Immunofluorescence microscopy ----------------------------- Cells cultured on glass coverslips were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton PBS. Cells were stained with 1:200 Rb anti-HA polyclonal, 1:200 12CA5 anti-HA monoclonal, 1:200 9E10 anti-myc monoclonal, 1:100 anti-Scar\\WAVE3, 1:200 monoclonal anti-Arp3, or 1:25 anti-Abi1 primary antibodies diluted in 1% BSA PBS. Secondary antibodies used were Goat anti-mouse or rabbit, FITC, TRITC, Alexa-488 or Alexa-546 Conjugates at 1:200 dilution in 1% BSA PBS. Filamentous actin was visualised using Alexa-546 conjugated phalloidin (Molecular Probes) diluted 1:500 in 1% BSA PBS. Slides were viewed using a BioRad MRC100 confocal laser scanning microscope. For endogenous Scar/WAVE3 labelling, it was important to test which cell lines expressed Scar/WAVE3. Among the following cell lines: NIH 3T3, N1E 115 neuroblastoma, N19 neuroblastoma, PC6 neuronal precursor, J774.A1 bone marrow macrophage, RAW 264.7 alveolar macrophage and C2C12 myoblast, we found that only C2C12 expressed endogenous Scar/WAVE3 by western blotting of whole cell lysates (M. Vartiainen, unpublished results). We thus used C2C12 for localisation of endogenous Scar/WAVE3 protein. Abbreviations ============= GST glutathione-S-transferase, WH2 WASP-homology 2, A acidic sequence motif, SHD Scar homology domain, NckAP1 Nck-associated protein 1, HSPC300 haematopoeitc stem/progenitor cell protein, Abi 2 abelson tyrosine kinase interactor 2 Authors\' contributions ======================= C.F.S. performed most of the experiments and contributed intellectually to the design and interpretations. C.F.S. also drafted the manuscript. T.H.M. performed the experiments shown in Figure [4](#F4){ref-type="fig"} and contributed intellectually to the project as a whole. L.M.M. Contributed to the design and analysis of the experiments and data and edited the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Characterization of HSPC300 and Abi1 binding domain of Scar/WAVE1. (A) Schematic representation of the domain architecture of Scar/WAVE family proteins and the deletion constructs used in these experiments. Numbers indicate start or end point of Scar/WAVE1 deletion mutants in amino acid residue position. Start/End points for Scar/WAVE2 deletion mutants are FL = 498, delta A = 456, SPW = 455, SP = 435, SB = 245, SHD = 170, BPWA = 171, PWA = 246. Start/End points for Scar/WAVE3 deletion mutants are FL = 499, delta A = 488, SPW = 457, SP = 439, SB = 238, SHD = 171, BPWA = 172, PWA = 239. FL; Full length. SHD; Scar Homology Domain. B; Basic Rich Region. PRR; Proline rich region. W; Wiskott Homology 2 domain. C; Central or connecting region. A; Acidic Rich Region. (B) Co-immunoprecipitation of HA-Abi1 with Myc-Scar/WAVE deletion constructs. (C) Co-immunoprecipitation of HA-Abi1 or HA-HSPC300 respectively with Myc-Scar/WAVE deletion constructs. Protein G beads were used to immunoprecipitate protein from transiently transfected Cos 7 fibroblasts before (-Ab) and after addition (+Ab) of an anti-Myc (9E10) monoclonal antibody. Bead bound fractions and supernatants (Sup) were analysed by SDS-PAGE followed by immunoblotting. Blots were probed with an anti-HA polyclonal antibody for the presence of HA-Abi1 or HA-HSPC300 in immunoprecipitated complexes. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ C.F.S. is supported by a CASE studentship in collaboration with Wyeth Research UK, L.M.M. and T.H.M. are supported by an MRC Senior Fellowship to L.M.M. G117/379. We thank Giorgio Scita and John Scott for reagents.	PubMed Central	2024-06-05T03:55:54.180406	2005-3-7	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555569/", "journal": "BMC Cell Biol. 2005 Mar 7; 6:11", "authors": [ { "first": "Craig F", "last": "Stovold" }, { "first": "Thomas H", "last": "Millard" }, { "first": "Laura M", "last": "Machesky" } ] }
PMC555570	Background ========== Understanding how patients would like to be notified of laboratory test results is important for improving provider-patient communication, patient satisfaction, and patient safety. Data from Applied Strategies for Improving Patient Safety (ASIPS), a primary care practice-based study of medical errors, indicate a high frequency of errors in laboratory testing and patient notification\[[@B1]\]. Other studies in primary care show similar safety concerns with laboratory testing\[[@B2],[@B3]\]. Preliminary ASIPS data show that laboratory errors were commonly discovered by patient-initiated requests for results. A study of patient notification of emergency department test results found that passive notification -- the \"no news is good news\" approach -- was ineffective and potentially dangerous\[[@B4]\]. Thus, ensuring that patients receive all lab results -- even normal results -- may be an important and last safety net for identifying missing or mishandled laboratory results. Patients want to be notified of all test results, regardless of whether the results are abnormal \[[@B4]-[@B8]\] A few studies have explored patient\'s preferences for being notified of specific test results, reporting that patients prefer to be notified by telephone for breast biopsy results\[[@B9]\] and normal mammograms\[[@B6]\], and wish to receive timely, detailed, written information for normal pap smear results\[[@B10]\]. We found no studies from primary care concerning notification procedures and patient preferences. To inform future interventions in this area, we elicited patients\' thoughts, needs, and preferences regarding test result notification. Methods ======= Sample ------ This study was conducted within the Colorado Research Network (CaReNet) and High Plains Research Network (HPRN), two primary care practice-based research networks. CaReNet practices are located mostly in urban / suburban cities in Colorado; HPRN practices are in rural northeast Colorado. We intentionally used these settings as the most optimal example of the setting (primary care clinics) in which we would see the need for normal lab result notification. We purposively sampled study participants based on emerging themes from our analysis. Thus, we intentionally recruited participants who were 18 years of age or older, able to speak and understand English or Spanish, and who had at least one laboratory test at a participating practice within the last year. Laboratory tests were defined as blood or urine tests, pap smears, and biopsies. The aim of our strategy was not to maximize generalizability, but rather to understand the context and conditions under which normal lab result notification does or does not occur. We recruited to the point of saturation or replication of data\[[@B11]\]. All final 20 participants spoke English and gave verbal telephone consent. Procedures ---------- Patients were recruited via posters and business cards placed in 12 primary care practices participating in the ASIPS study. ASIPS was a multi-institutional, primary care practice-based project to collect, codify, and analyze data on medical errors\[[@B1],[@B12]\]. Interested patients called an automated call center and left their names and telephone numbers. A research assistant called patients to determine eligibility, obtain consent, and schedule a 30--45 minute interview. An interview was completed by telephone (19 cases) or face-to-face (1 case) and was audio taped. This study was approved by the University of Colorado Health Sciences Center\'s institutional review board. Measures -------- ### Demographic information We collected the patient\'s age, gender, race, ethnicity, highest level of education, if they had a permanent home address, and access to the Internet and e-mail. Patients were asked if they had personally received results of a test by message left on answering machine, through phone conversation, mail, e-mail, automated telephone call-up system, or via web-based system. Patients ranked these notification methods by their preference. ### Semi-structured interview The interview began with questions about the patient\'s most recent experiences with test result notification (who, what, when, etc.). The interview then shifted to preferences of notification and patient-doctor discussion about notification preferences. Inquiry also elicited patient factors that may affect test result notification. An in-depth interview provides a narrative understanding of how particular individuals arrive at their experience. Our purpose was to construct a meta-narrative of the interviewees\' many stories. This on-going interpretive process informed each subsequent interview in an iterative fashion. For example, during early interviews it appeared that different preferences might be expressed by people with different educational levels. We then stratified our subsequent sample by educational level (more than a high school education vs. high school education or less). Data analysis ------------- We used mixed methodology to analyze the data. Quantitative descriptive statistics (frequencies, proportions, etc.) were generated for our demographic and preference variables. All coding and analysis was done by a three-member team that included a physician, a doctorate researcher experienced in qualitative methodology, and a professional research assistant. The use of a multidisciplinary team approach helped limit any personal biases, subjectivity, and preconceptions as well as enhanced our reflexivity process\[[@B11]\]. A commitment to reflexivity resulted in ongoing assessment of subjectivity by the team in all steps of the analyses\[[@B13]\]. Our qualitative analysis was initially guided by multiple styles of qualitative analyses including an initial template approach as outlined by Crabtree and Miller\[[@B11]\] using already published literature on lab result notification as guides. We created an initial a priori template of codes (code manual) and then applied it to the text data. This approach was then followed by editing approach, a technique derived from grounded theory, to identify emerging themes. Members of the research team independently coded a number of pages of the same text to test for both the utility and appropriateness of the codes and the intercoder reliability, which measures correspondence between two or more coders\' assessments (84%)\[[@B14]\]. Achieving an acceptable level of intercoder reliability is important for providing basic validation of coding scheme. We modified the code manual to correct for discrepancies and deficiencies. Research team members then identified and sorted segments of text, which allowed further abstractions and emerging codes. ATLAS.ti software facilitated the iterative coding and sorting process\[[@B15]\]. We further sorted related text, producing connections and interpretations. Results ======= In the end, we found no differences qualitatively or quantitatively across participants\' educational levels. Thus, all results are reported as a single group. Most (75%) participants were female (see Table [1](#T1){ref-type="table"}); 90% indicated they expected to be notified of all test results, regardless if normal or abnormal. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Demographics of participants ::: Demographic Characteristics TOTAL % -------------------------------------------------- ----------- ------- Female 15 75% Race White 15 75% Black 4 20% Asian 1 5% Age ≤ 30 4 20% 31--40 0 0% 41--50 8 40% 51--60 5 25% 61--70 2 10% ≥ 71 1 5% Education Some or graduate of high school 10 50% Some college 5 25% College graduate 1 5% Post graduate education 4 20% Have access to e-mail & Internet 19 95% Location of frequent access to e-mail & Internet Home 9 45% Library 4 20% Multiple sites 5 25% ::: When exploring the possible modes of lab result notification (phone, message left on an answering machine, mail, e-mail, automated telephone call up system, and web-based systems) we found no differences in preferences by age groups, educational levels, or access to e-mail and the Internet (table [1](#T1){ref-type="table"}). Many participants who had access to the Internet or e-mail were open to the idea of retrieving results via this medium only if the security was assured; however, most patients felt that web-based systems and e-mail are not secure. Only a minority of patients interviewed were willing to try an automated telephone call, e-mail, or web-based system. Patients\' preferences seemed to reflect their most recently experienced method of notification. Table [2](#T2){ref-type="table"} shows total number of patients who experienced a particular method of notification within the past year and their preferred method of notification. Most patients experienced and preferred phone or mail notification. All interviewees stated that a message left on an answering machine was not appropriate. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Participants\' experience with and preference for notification methods ::: Selected Methods Experienced Method % (n = 20) Preferred Method % (n = 20) ----------------------------------- ------------------------ ---------------- ---------------------- ---------------- Message left on answering machine 9 45% 0 0% Telephone 16 80% 12 60% Mail 11 55% 3 15% Automated telephone call-up 0 0% 2 10% E-mail 0 0% 2 10% Web-based system 0 0% 1 5% ::: We identified three emerging themes: (1) Important Characteristics of Notification:most notable factors influencing patient preferences in notification; (2) Patient/Provider Discussion:the lack of communication between patients and providers around notification preferences; and (3) Communication Frustration:challenges encountered during attempted communication between the patient and the practice regarding notification of their results. Below we briefly describe these themes with illustrative quotations from the participants. Important characteristics of notification ----------------------------------------- Important factors that defined patients\' concerns around notification were always being notified of results, timeliness, details of the results, responsive and interactive feedback, who should provide the notification, convenience, and assured security/confidentiality. ### Always notify Not surprising, almost all patients responded that they wanted to be notified of all results. The \"no news is good news\" approach is unacceptable to patients. \"Obviously there was a reason to have that diagnostic test, so I\'d at least like to know whether it was normal or what.\" \"To me, no news is worrisome.\" ### Timeliness Patients wanted to receive their results in a timely manner -- shortly after the physician or provider receives the results. \"Let me know right away. Don\'t keep me hanging. Do the test on Friday and if I don\'t know till Tuesday, I want to know Monday morning. As soon as you find out the results, you let me know the results because this is my body.\" ### Details of test results The amount and detail of information is important in providing the context in which the patient can interpret the results. \"I would like to know what normal means in relation to the general population, so I would certainly like some reference ranges\...If there weren\'t any reference ranges, then I would certainly like to know that a test\...came back negative and what negative meant.\" ### Responsiveness & interactive feedback The test result was only half of the information participants want. They also wished to discuss what the results mean for them. In some cases, patients are left wondering about the \"next steps.\" A patient may feel confused if someone is not available during the notification process who can discuss what the test means. \"Well, the information on what I can and what I can\'t do, you know. I mean I don\'t know what I can do at this point.\" \"The thing I do like is that we can actually talk about the numbers and I can see where it\'s at because he just gives me, we just look at the sheet together\...I like that.\" ### Who should notify Most patients recognize that providers are too busy to attend to all normal test results. They were comfortable having someone else notify them, but preferred that this person be knowledgeable enough to answer questions. Many patients indicated that notification by receptionists, who they felt were not knowledgeable enough to answer questions, is unacceptable. When asked specifically the preferred role of a notification person the responses were: 65% provider and 15% nurse; 20% were not concerned with who notifies. \"Ok, this test result is normal but I still have this pain, what alternative do I have now. Where do I go from here and a physician would be the best person to be able to explain it to me, rather than a physician assistant or a nurse or an administrator or something like that.\" \"Well, it doesn\'t have to be the actual doctor. It could be the RN\...Somebody who knows about what is going on and if I have a question I could ask that person.\" ### Convenience Patients identified convenience as important to their satisfaction. Calling the office for results can mean long waits to reach a person who notifies them of their result and then longer waits when the results prompt a patient question that cannot be immediately answered. \"That way (using web-based system) I could do it at anytime and it seems more personal and confidential to me because\...you can draw up that information at any time when you\'re ready.\" \"That\'s why the mail is so good because I always get the mail everyday.\" ### Security/confidentiality The most persistent issue we uncovered was a patient\'s privacy and assured confidentiality of test results and diagnoses. Participants seemed hesitant to experiment with alternative notification methods (i.e., web-based methods) if they perceived a possibility of a breach in this trust. \"Who wants the public to read what their values are?\...Even if it\'s normal, I\'d rather have it personalized in a sealed envelope.\" Provider discussion ------------------- Most patients assumed that their office used a specific system for notification; however, they were not aware of the details of this system. Often patients were told to call for results if not received within specified time, leaving the patients to close the feedback loop. Other patients were told to rely on \"no news is good news,\" although they were uncomfortable with this policy. The majority of patients indicated that they had not discussed their notification preferences with their provider. \"No, I haven\'t. I just assumed that was normal and customary procedure\...That they only notify you if something is wrong.\" \"I didn\'t know they were just going to send me a letter. In the past, I\'ve had the doctors call me. Well, they automatically call \[ed\].\" Communication frustration ------------------------- Two areas of frustration related to communication were identified: lack of follow-through and confusion within the office. ### Follow-through Communicating how the provider will notify the patient is important, but equally important is follow-through. Unmet expectations result in patient frustration. \"I told him that he could call me, or the nurse. Either one would be ok, but\...it doesn\'t happen. Even though I asked them face to face, it still hasn\'t happened.\" ### Office confusion Patients are also frustrated and worried when they try to complete the feedback loop, but find that the practice is unable to provide them the information they need. \"I have to sometimes call in to find out about my results\...I wasn\'t actually notified of the results\...so obviously it was my responsibility. I felt it was my responsibility anyway. But\...they \[the results\] seemed to have \[been\] misplaced. The parties concerned didn\'t seem to be aware that I had had a particular test and so they couldn\'t provide me with information\...It wasn\'t until I went back recently and asked about that result, that I was told it was normal.\" Discussion ========== Our results reinforce other literature suggesting patients want timely\[[@B9]\] and detailed\[[@B10],[@B16]\] information, and want to be notified of all test results, even if normal\[[@B5],[@B7]\]. These results also support the idea that patients prefer clinicians telephone them with lab results\[[@B6]\]. We, other researchers\[[@B17],[@B18]\], and the patients in our study recognize that this plan is often too costly to be practical. A number of our findings, however, suggest that more than preferring a particular channel of communication, patients prefer a specific manner of communication, features of which could be incorporated into newer communication channels. For example, our participants overwhelmingly preferred a personal telephone call -- but only from someone who was knowledgeable enough to answer their questions. They wanted responsive and interactive feedback, personalizedto their situations. Many of the patient identified characteristics of notification could be more easily incorporated into a system that allowed for asynchronous communication (communication that doesn\'t rely on immediate person to person transfer of information.) Nonetheless, few of our participants were willing to try the computerized asynchronous communication methods we asked about: an automated telephone call, e-mail, or web-based system. This finding is similar to others\[[@B7]\], although Ridgeway and colleagues report that patients used and were generally satisfied with an automated phone call up system\[[@B19]\]. Our patients said they would be willing to try a web-based system if they were convinced of security and confidentiality, suggesting that concerns with web systems lie not with the technology, itself, but with the privacy of the information. Notably, privacy was one reason patients wanted a personal phone call. Thus, we suspect that their stated preferences for telephone calls are related to the perceived high level of privacy and interaction available through a synchronous telephone call, while their distrust of websites and e-mail indicate underlying discomfort with the perceived privacy of the technology. Recognizing these tradeoffs may be useful for those who are designing systems to provide test result notification to patients. Our patients emphasized the importance of receiving results in a timely fashion. Timeliness is a critical feature of notification systems that can significantly affect patient safety; timely recognition of mishandled or misplaced results will increase the practice\'s ability to correct or mitigate an error. However, timeliness is not an inherent feature of patients\' preferred channel of communication: a telephone call. In our experience, providers\' and patients\' hectic schedules often mean it can take days for successful telephone contact between providers and patients. Again, our patients\' focus on timeliness suggests that they are not wedded to the concept of telephone communication; rather, they prefer the perceived timeliness of communication by telephone. Similarly, our participants indicated that convenience was important to them. Again, waiting for a telephone call is not intuitively convenient, though the dramatic increase in cell phones may help alleviate this problem. Perhaps our study participants could not envision a convenient communication method other than phone calls that provide secure, personalized, interactive communication. A traditional mail-based system (a low-tech asynchronous communication system) was ranked second highest among preferences for notification. This finding detracts from our assumptions that patients focus on notification methods that are timely and interactive; however, it supports our idea that patients want convenient, private, personalized information. Perhaps the three patients who preferred mail notification were more concerned with privacy and convenience than with timeliness and interactivity. Further study is needed to elucidate these findings. Finally, we found that patients do not discuss with their provider their preferences for notification. Our patients indicated that it never occurred to them that their health care provider lacked a standard procedure for communicating test results to patients. Our findings may be a manifestation of poor communication between patients and providers, which has been shown to be a related to poor patient outcomes and safety issues\[[@B20]\]. More study is needed to explore this example of poor interpersonal communication. A potential limitation of this study relates to the recruitment, which was limited to patients who had access to call our research line after seeing an advertisement. However, qualitative inquiry rarely uses random sampling. Rather, samples are selected more purposefully and not by the need to generalize or predict but by a need to create deeper understanding or meaning\[[@B11]\]. Thus, studying the narratives of people who called our research line to talk about their experiences is appropriate and adequate. Additionally, we sampled to the point of redundancy; no new information was coming forth by the end of 20 interviews. A second limitation may be a gender bias. Most of our respondents were female. Male experiences may differ and may not be represented by our results. However, considering that the majority of health care utilizers are women, and women are critical in maintaining the health and health care consumption within families \[[@B21]-[@B23]\] their experience becomes crucial for primary care service delivery. Finally, the most significant limitation of this study relates to the participants\' lack of experience using a web-based or automated telephone system to receive test results. While we could discuss how patients\' preferences may possibly be met by such a system, we could not comment on how previous experiences might affect their preferences. Conclusion ========== The results of this study provide us with a better understanding of how patients experience notification of laboratory tests within the primary care setting. Notifying patients of test results is important for laboratory information management, and ultimately, patient safety. We believe patients can play an important role in ensuring that laboratory tests results are obtained and reviewed by providers. Asking for all laboratory tests results is a recommended strategy\[[@B24]\] for improving patient safety that draws patients into the feedback loop and provides a last safety net for identifying misplaced or mishandled results. Learning patients\' preferences for result notification is merely one step in this important patient safety area. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= All authors contributed to the study design and conceptualization, discussed the drafts of the paper, and read and approved the final manuscript. Additionally: DB developed the study idea and conducted the data analysis. JQ conducted the interviews. CD provided technical assistance in protocol development and data analyses, and contributed to the development of the manuscript. ES contributed to the development of the manuscript. WP obtained funding for the study, helped develop the study methods, and contributed to the development of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2296/6/11/prepub> Acknowledgements ================ This study was supported in part by the AHRQ grant 5U18HS011878-02, Wilson D. Pace, MD, Principal Investigator. The authors thank the clinicians and staff of practices participating in ASIPS, as well as the members of the ASIPS Collaborative. Preliminary findings from this study were presented at the North American Primary Care Research Group Annual Meeting, Banff, Canada, October, 2003.	PubMed Central	2024-06-05T03:55:54.183640	2005-3-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555570/", "journal": "BMC Fam Pract. 2005 Mar 8; 6:11", "authors": [ { "first": "Donna M", "last": "Baldwin" }, { "first": "Javán", "last": "Quintela" }, { "first": "Christine", "last": "Duclos" }, { "first": "Elizabeth W", "last": "Staton" }, { "first": "Wilson D", "last": "Pace" } ] }
PMC555571	Background ========== Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycaemia. It requires life long treatment with drugs coupled with diet control and exercise. It may be due to decrease in the synthesis of insulin (Type-I diabetes) or due to decrease in the secretion of insulin from β-cells of islets of Langerhans of pancreas (Type-II diabetes). Insulin is the drug of choice in type -- I diabetes and sulfonylureas are the drugs of choice in type II. Among sulfonylureas, tolbutamide is the drug of choice for geriatrics because of its short duration of action and lower incidence of hypoglycaemia in early hours of night. Diabetes is one of the stress related disorder. Diabetic subjects are shown to have increased oxidative stress and decreased antioxidant levels \[[@B1]-[@B3]\]. It was also shown that tight control of blood glucose is possible with decrease in oxidative stress \[[@B4]\]. Antioxidants are claimed to work as antistress agents by decreasing oxidative stress. L-ascorbic acid used in therapy for disorders like scurvy produces antioxidant activity. Earlier reports show that the relationship between scurvy and diabetes mellitus indicates the low levels of plasma ascorbic acid in diabetic rats compared to control rats \[[@B5],[@B6]\]. Hence the present study was conducted to find the influence of L-ascorbic acid, a water soluble antioxidant and a free radical scavenger on the hypoglycaemic and antihyperglycaemic activities of tolbutamide in normal and diabetic rats. Methods ======= All animal experiments were performed in accordance with our institutional animal ethics committee. Albino rats of either sex (Mahaveer Enterprises, Hyderabad) weighing between 125 -- 175 g were used in the study. They were housed five per cage at temperature 22 ± 2°C and 12/12 h light/dark under controlled environment. Rats were fed with standard pellet diet. (Mahaveer Enterprises, Hyderabad) and water ad libitum. They were divided into 3 groups of five each. They were fasted for 18 h prior to the experiment allowing access to water only, and the water was withdrawn during the experiment. Blood samples were collected from the retro-orbital plexus of each rat at 0, 0.5, 1, 1.5, 2, 4, 6 hr after drug administration. Blood glucose levels were determined by using GOD -- POD method \[[@B7]\]. Group I received L-ascorbic acid 60 mg / kg body weight, Group II received tolbutamide 20 mg/kg body weight and Group III was given L-ascorbic acid (60 mg / kg body weight) prior to the administration of tolbutamide 20 mg/kg body weight in normal rats. In clinical practice tolbutamide and vitamin C are administered orally hence in our study also this was administered orally. Induction of diabetes --------------------- Albino rats of either sex weighing between 125 to 175 g were fasted overnight before injection with alloxan. Alloxan monohydrate was dissolved in saline solution and was administered at a dose of 100 mg/kg body weight intraperitonially. Animals were treated with 10% dextrose orally to combat the early phase of hypoglycaemia. Rats showing fasting blood glucose levels above 150 mg/dl were selected for the study. They were divided into 3 groups of five each. Group I received L-ascorbic acid 40 mg/kg body weight and Group II received tolbutamide 20 mg/kg body weight while Group III was given L-ascorbic acid 40 mg/kg prior to tolbutamide administration (20 mg/kg). L-ascorbic acid dose was fixed based on its response, which produced above 40%. Statistical analysis -------------------- The significance of blood glucose reduction produced by L-ascorbic acid with tolbutamide compared to tolbutamide control was determined by applying students unpaired t-test and the significance is indicated by \* mark. Results ======= The presence of L-ascorbic acid upto 20 μg did not interfere with the blood glucose estimation when tested with different quantities in in vitro studies. In normal rats L-ascorbic acid at the dose of 60 mg/kg body weight administered orally produced 50.91% blood glucose reduction at 0.5 h and 20 mg/kg body weight of tolbutamide produced 33% at 4 h as peak effects. In the presence of L-ascorbic acid (60 mg/kg), the action of tolbutamide was early in onset and maintained for 6 h. In diabetic rats, oral administration of L-ascorbic acid alone at the dose of 40 mg/kg body weight produced 42.53% blood glucose reduction at 1.5 h and tolbutamide 20 mg/kg body weight produced 45.09 at 4 h. Administration of L-ascorbic acid 40 mg/kg body weight prior to tolbutamide produced antidiabetic activity at 0.5 h and was maintained for 6 h. The percent blood glucose reduction with L-ascorbic acid / tolbutamide/ L-ascorbic acid + tolbutamide in normal rats and diabetic rats were given in table [1](#T1){ref-type="table"} &[2](#T2){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Percent blood glucose reduction (Mean ± SEM) with L-ascorbic acid/ tolbutamide / L-ascorbic acid + tolbutamide in normal rats (n= 5) ::: Time in hours L-ascorbic acid 60 mg/kg bd.wt. Tolbutamide 20 mg/kg bd.wt. L-ascorbic acid 60 mg/kg bd.wt. + Tolbutamide 20 mg/kg bd.wt. ------------------- ------------------------------------- --------------------------------- ------------------------------------------------------------------- 0 \- \- \- 0.5 50.91 ± 0.49 10.08 ± 1.08 53.57 ± 2.01\\\* 1 20.26 ± 3.14 15.56 ± 1.48 28.70 ± 1.75\\\* 1.5 6.06 ± 1.3 18.75 ± 2.1 23.25 ± 1.72 2 1.65 ± 0.93 22.22 ± 2.13 29.78 ± 2.57 4 -2.83 ± 0.4 33.0 ± 0.69 45.21 ± 2.79\\ 6 \- 10.28 ± 1.02 12.54 ± 2.5 Significance \\\* P \< 0.001, \\P \< 0.01 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Percent blood glucose reduction (Mean ± SEM) with L-ascorbic acid/ tolbutamide / L-ascorbic acid + tolbutamide in diabetic rats (n = 5) ::: Time in hours L-ascorbic acid 40 mg/kg bd.wt. Tolbutamide 20 mg/kg bd.wt. L-ascorbic acid 40 mg/kg bd.wt. + Tolbutamide 20 mg/kg bd.wt. ------------------- ------------------------------------- --------------------------------- ------------------------------------------------------------------- 0 \- \- \- 0.5 18.23 ± 1.88 3.03 ± 0.8 23.55 ± 2.37\\\* 1 35.45 ± 3.26 7.83 ± 1.84 35.59 ± 4.48\\\* 1.5 42.53 ± 1.78 18.58 ± 2.49 57.49 ± 1.63\\ 2 34.04 ± 2.22 37.97 ± 6.40 59.74 ± 1.22\* 4 20.02 ± 3.32 45.09 ± 4.95 62.55 ± 0.64\\ 6 \- 21.6 ± 1.94 39.43 ± 2.15\\\* Significance \\\* P \< 0.001, \\P \< 0.01, \*P \< 0.05 ::: Discussion ========== The drug interaction studies are usually conducted in animal models to find out the mechanism before they are conducted in humans. We have selected rat as animal model since it is one of the animal, which synthesize ascorbic acid and can be maintained easily in the laboratory conditions. Reactive oxygen species (ROS) are thought to be implicated in the pathogenesis of diabetes as well as other diseases\[[@B8]\]. Reactive oxygen species usually comprise radicals that have the ability to oxidize and damage DNA, proteins and carbohydrates. Hyperglycaemia appears to induce oxidative stress on cells and this can cause an increase in the production of free radicals\[[@B9]\]. Human antioxidant enzymes are mobilized during hyperglycaemia, but they cannot meet the continued demand due to increased oxidative stress\[[@B10]\]. This problem is either due to decreased intake of needed precursors or an inability to synthesise the antioxidant enzymes\[[@B11]\]. Antioxidant supplementation may provide the only means to reverse this process\[[@B12]\]. Use of typical antioxidants alone or in combination may retard or even prevent the normal progression of diabetic complications. It was reported that L-ascorbic acid levels were decreased in diabetic patients and rats\[[@B13]\]. So it is felt that L-ascorbic acid supplementation may help in the treatment of diabetes mellitus. In the present study L-ascorbic acid and tolbutamide reduced blood glucose levels in normal & diabetic rats in a dose dependent manner. L-ascorbic acid when administered alone produced an early onset of action 0.5 & 1.5 h in normal and diabetic rats respectively. This early onset may be due to increase in the insulin secretion which support earlier reports that L-ascorbic acid supplementation increase the plasma insulin concentration\[[@B14]\]. Tolbutamide when administered in therapeutic dose produced the maximum effect at 4 h and was maintained up to 6 h in both normal and diabetic rats. In the presence of L-ascorbic acid the onset of action of tolbutamide was early and maintained for longer duration compared to tolbutamide control. Tolbutamide acts by stimulating insulin secretion (pancreatic)\[[@B15]\] and also by increasing tissue uptake of glucose (extra pancreatic)\[[@B16]\]. The early onset of action was noticed to be due to L-ascorbic acid, which was maintained later due to tolbutamide activity since both are reported to have influence on insulin secretion \[[@B14],[@B15]\]. Conclusion ========== The study indicates that additive action of L-ascorbic acid on pharmacodynamic response of tolbutamide may be useful to improve the tolbutamide activity in insulin resistant cases and to postpone the occurrence of diabetic complications. However further work on human patients is required to confirm the observation in diabetic condition and usefulness of L-ascorbic acid as supplemental agent for improved control of blood glucose levels when administered along with sulfonylureas. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= SS Conceived of the study, participated in the design of the study, performed the statistical analysis and drafted the manuscript. EK Participated in the design and coordination and in the standardization of methods. RJ Carried out the study in normal rats. VA Carried out the study in diabetic rats. All authors read and approved the final manuscript. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Percent Blood Glucose Reduction with L-ascorbic acid / tolbutamide / L-ascorbic acid + tolbutamide in normal rats (n = 5) ::: ![](1472-6823-5-2-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Percent Blood Glucose Reduction with L-ascorbic acid / tolbutamide / L-ascorbic acid + tolbutamide in diabetic rats (n = 5) ::: ![](1472-6823-5-2-2) ::: Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6823/5/2/prepub> Acknowledgements ================ The authors thank the UGC for sanctioning research fellowship (JRF) to J. Rajasekhar for pursuing Ph.D programme. The authors also thank M/s Hoechst Marion Roussel Ltd., Mumbai for supplying tolbutamide as gift sample.	PubMed Central	2024-06-05T03:55:54.186172	2005-3-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555571/", "journal": "BMC Endocr Disord. 2005 Mar 3; 5:2", "authors": [ { "first": "Satyanarayana", "last": "Sreemantula" }, { "first": "Eswar K", "last": "Kilari" }, { "first": "Vishnu A", "last": "Vardhan" }, { "first": "Rajasekhar", "last": "Jaladi" } ] }
PMC555572	Background ========== Clinical practice guidelines (CPGs) are one option for promotion of quality in health services \[[@B1]-[@B6]\] Many countries are faced with common challenges in delivering high-quality health care with available resources and have pursued the development of CPGs as a means to optimize effective and efficient care. As a result there is a need to evaluate CPGs guidelines to assess their quality and their impact on practice. The Appraisal of Guidelines for Research and Evaluation (AGREE) instrument was developed by a group of researchers from 13 countries to provide a systematic framework for assessing guideline quality\[[@B7],[@B8]\] This instrument was thoroughly evaluated and refined and is now a commonly used assessment instrument for CPGs\[[@B2],[@B3],[@B9]-[@B13]\] A large-scale validation study focussing primarily on medical (i.e. physician) guidelines, was conducted supporting the reliability and validity of this instrument\[[@B14]\]. The AGREE Collaboration published the development process and associated reliability and validity data in 2003\[[@B14]\] This report outlined the rigorous process undertaken to develop the AGREE instrument which included item generation, selection and scaling followed by field-testing and refinement. This process resulted in the final instrument with 23 items distributed across six subscales termed \"domains\", for which reliability and validity data were presented. Reliability was determined by calculating the internal consistency of each domain within the final instrument and assessing the agreement between different appraisers. A total of 33 guidelines were evaluated by the four appraisers. Internal consistency ranged from 0.64--0.88. \'Scope of Purpose\' and \'Rigour of Development\' were the most homogeneous domains. The inter-rater reliability exhibited a wide range from 0.25--0.91. Reliability was higher with four appraisers and the most reliable domain was \'Rigor of Development\'. Higher reliability within the domain of \'Rigor of Development\' is a positive finding, as this domain should contain items that are more objective than items contained on other subscales of the AGREE instrument. That is, because the \'Rigor of Development\' questions relate to the methodology of developing a CPG and thus there are optimal criteria that would be expected regardless of the content of the CPG. In measurement terms, it is more likely that a \"true\" score exists for elements within this domain. Therefore, variability observed on repeated assessments of the same CPG should reflect measurement error between appraisers. Other scales such as stakeholder involvement and applicability might reasonably have different criteria depending on clinical expertise or application. In measurement terms, no single true score may exist for these items. Therefore, variability observed between appraisers on these domains might reflect a combination of measurement error, as well as true variations in perspective. This concept is important when assessing and interpreting the reliability of evaluation instruments like the AGREE. The AGREE Collaboration also assessed face, construct and criterion validity of the AGREE Instrument. Face validity was determined by surveying appraisers attitudes and opinions about the instrument and its associated user guide. Construct validity was determined by comparing scores of guidelines in different subgroups to determine whether they fit three specific constructs. The constructs tested included whether established quality guideline programs produced guidelines with higher domain scores than those developed outside of established systems; whether guidelines supported by well-documented technical information had higher domain scores than those without such documentation; and finally whether guidelines developed as national policies were higher quality than regional or local CPGs. The first hypothesis was supported with respect to editorial independence, but not other domains. The second and third hypotheses were supported with respect to the domain Rigor of Development, but not other domains. Finally, criterion validity was determined by assessing the rank correlation between appraisers domain scores and their overall assessment scores (final item on the AGREE instrument). These correlations were all highly significant (range Kendall\'s Tau-b = 0.67 -- 0.88). Physical therapy and other health care disciplines shares common challenges in providing effective care within limited resources. While many disciplines currently use the AGREE the validation paper emphasized medical practice and practitioners. The nature of physical therapy practice differs substantively from medical practice in a number of ways including access, funding, the nature of interventions, research systems and professional associations; all of these might affect the type of CPGs, developed. Differences in training between disciplines might also cause variations in how the AGREE was interpreted. Additional validation with other settings or users would strengthen the validation of the AGREE across a variety of applications. CPGs have arisen within the field of physical therapy from a variety of sources\[[@B15],[@B16]\] Professional associations have assisted physical therapy practitioners in becoming aware of the existence of such guidelines through websites and newsletters. Ideally, information on the quality of such guidelines should also be available to assist practitioners in selecting between available guidelines. Currently, this is not the case in physical therapy. While professional associations may help co-ordinate the evaluation of CPGs, they must inevitably must make decisions about this process including which members of the profession are able to evaluate CPGs, how many appraisers should be selected and which instrument should be used. For this reason, our purpose was to determine whether the AGREE instrument is a reliable and valid tool when used by physical therapists to assess CPGs that pertain to physical therapy practice. Our hypothesis was that the AGREE instrument would be a valid tool when used by physical therapists to evaluate CPGs. Our secondary question was whether an ideal number or type of physical therapist appraiser would be evident from reliability data. We hypothesized that 4 appraisers might be best, as the AGREE Collaboration recommends \" al least two appraisers and preferably four as this will increase the reliability of the assessment\". \[[@B7]\] Methods ======= This study was a cross-sectional study conducted to evaluate inter-appraiser reliability and validity of quality appraisal of CPGs performed by physical therapists using the AGREE instrument Permission to use the AGREE instrument was obtained from the AGREE Collaboration. Ethics approval for this study was received from the University of Toronto, Office of Research Services. Clinical practice guidelines ---------------------------- Clinical practice guidelines (CPGs) are \"systematically developed statements to assist practitioner and patient decisions about appropriate healthcare for specific clinical circumstances\"\[[@B17]\]. The CPGs evaluated during this study were identified through an inventory that was created by the study authors from a series of systematic searches that included electronic databases, websites, contact of professional associations and guideline developers. The inventory included all located documents that were identified by authors as Clinical Practice Guidelines. This inventory was completed in 2002 and updated yearly and subsequently posted on the website for the Canadian and Ontario Physiotherapy Associations (members only access). Within this database, guidelines were categorized according to the area of physical therapy practice (e.g., musculoskeletal, neurological, cardiorespiratory). Sixty guidelines published in the last five years were selected from this database for inclusion in the present study. Four of these CPGs were excluded because they were not actually CPGs (e.g. systematic reviews included in error) and one CPG was excluded because it was not relevant to physical therapy. Thus in total, a sample of fifty-five guidelines were evaluated in the present study. Participants/training --------------------- All participants were physical therapists who were recruited through advertisements in professional newsletters. A total of 72 therapists responded to advertisements and agreed to participate, 69 actually participated in training and study evaluations -- two others had personal circumstances that prevented them from attending training and one failed to respond further. The participants were classified according to the following criteria: 1. Clinical Specialists were Physical Therapists who were currently practicing in a specific area, had a minimum of three years experience and had participated in at least one post-graduate course per year in their area of clinical expertise. 2. Generalists were Physical Therapists practicing in a variety of areas of physical therapy or an ongoing general practice, which covered a broad-spectrum of neurological, orthopaedic and cardiorespiratory health problems. 3. Researchers were Physical Therapists with or approaching completion of a graduate research degree (M.Sc. and/or PhD) with experience in conducting clinical trials or outcomes research and experience in formal critical appraisal of clinical research. Demographic data was collected on all participants. Eligible participants were provided with training materials which included the AGREE instrument (form and associated interpretation guidelines)\[[@B7],[@B18]\] as well as a multiple-choice test that required participants to answer questions on the content and structure of the AGREE (see [Additional file 1](#S1){ref-type="supplementary-material"}). In addition, a sample guideline was provided to participants with instructions to read the guideline (on management of lymphedema following treatment for breast cancer\[[@B19]\]) and appraise it using the AGREE instrument and associated documentation. Subsequently, all participants met by teleconference for one hour with a subgroup (4--8) of study participants. The sessions (a total of 6 teleconferences conducted) were led by a single facilitator (first author). During this session participants reviewed their responses to each question and discussed their rationale or concerns regarding scoring. The facilitator did not indicate a correct score for any individual item. Participants were instructed that the group facilitator would not indicate whether a given score was correct, as this was not possible for many of the items. Participants were directed to appreciate the difference between items that had clear answers because they inquired about specific factual information versus those that had a subjective element where responses might vary according to context. Participants were corrected if they incorrectly interpreted the meaning of a given item on the AGREE instrument. Although consensus was not the objective of the training sessions, participants tended to come to consensus after discussing items with colleagues and a facilitator. In total, 69 appraisers attended the training sessions. Appraisal instrument -------------------- The appraisal instrument used to evaluate the CPGs was the AGREE \[[@B18]\]. This instrument consists of 23 items organized in six domains ; each domain is intended to capture a separate dimension of guideline quality. The following domains are included: 1\. Scope And Purpose:3 items that address the overall aim of the guideline, the clinical question and the target population 2\. Stakeholder Involvement:4 items that address the composition, expertise and representation of the development group 3\. Rigor Of Development:7 items that evaluate the process used to locate and synthesize the evidence and to formulate and update the recommendations, 4\. Clarity And Presentation:4 items that address language and format 5\. Applicability:3 items that address the potential organizational, behavioural and cost implications of implementation and 6\. Editorial Independence:2 items that address potential conflicts of interest. Items are rated on a 4-point scale with endpoints of 4 \'strongly agree\' and 1 \'strongly disagree\'; the two midpoints are 3 \'agree\' and 2 \'disagree\'. A section for overall assessment is included at the end of the instrument that requires the appraiser to make a judgment about the overall quality of the CPG. Appraisers are asked whether they would \'strongly recommend\', \'recommend (with provisos or alterations)\', \'would not recommend\' or are \'unsure\' if they would recommend the CPG for practice. Evaluation process ------------------ All participants completed the training program and proceeded to evaluate a set of 6 CPGs. These CPGs, six copies of the AGREE instrument, and a pre-paid return were provided by mail. Each guideline was evaluated independently by three pairs of appraisers who were randomly picked from the three pools of Physical Therapists (i.e. two clinical specialists in the area of the CPG, two generalists and two researchers). All appraisers returned their packages, although up to 3 reminders calls were required for late returns. Participants who completed the study were provided with an honorarium (\$100). Data analysis ------------- Data analysis was conducted to verify the quality of data, assess instrument reliability and determine the validity of the AGREE instrument for physical therapy practice. SPSS statistical software for Windows (Version 11.0; SPSS Inc, Chicago, Illinois) was used for all statistical analyses. P-values of 0.05 or less were considered significant. Data Entry/Quality ------------------ Data entry was completed by a single research assistant who inspected data for errors once the data file was complete. The first author conducted random checks of data entry against original data sheets. Descriptive statistics were conducted to identify outliers or unusual values. Domain scores of each CPG were calculated as recommended by the AGREE Collaboration. The scores of the individual items in the domain were summed and standardized as a percentage of the maximum possible score for that domain (AGREE Collaboration, 2001). ![](1472-6963-5-18-i1.gif) Reliability analyses -------------------- The internal consistency of each domain was evaluated using Cronbach\'s Alpha. The reliability between appraisers was determined for each question and each domain of the AGREE. Intraclass correlation coefficients (ICC 1,1) were calculated within each pair of appraisers and across the pool of appraisers. A one-way random effects model was used as pairs of appraisers were randomly selected from our pool of physical therapist appraisers. An unweighted and quadratic weighted kappa were calculated to indicate the agreement within pairs of appraisers on whether a CPG was appropriate for clinical utilization. ICCs or kappa values above 0.75 were considered to represent good, 0.40--0.75 moderate and \<0.40 poor reliability\[[@B20]\] Validity analyses ----------------- ### Face validity The face validity of the instrument for physical therapy practice was determined from feedback provided on the instrument from two sources. Participants (experienced physical therapists) in the study were invited to provide feedback at the training sessions (open-ended questions regarding the training session and the AGREE itself -verbal response). They were also asked to add comments about any items, any issues with clarity or concerns directly on their AGREE form when they were using the AGREE on their assigned CPGs. These were returned, by mail, with their ratings. In addition, over the course of two years, a sample of 102 entry-level-masters trainees at McMaster University were provided the training materials (except for multiple-choice questionnaire) and were required to complete an assignment where they evaluated the same CPG\[[@B19]\] used during the study training session. This assignment consisted of a facilitated group component where students worked in groups of 4--6 to complete the AGREE evaluation for the assigned CPG. The individual component of this assignment required each student to write a 1--2 page essay evaluating the instrument itself in terms of its relevance to clinical practice, validity and their personal preference about whether they would use it again. This information was summarized by the course instructor (first author) and the percentage of students who responded that they would use the instrument again was tabulated. ### Factor (domain) validity The validity of the domain structure was evaluated using a principal components, varimax rotated factor analysis. Item means across all 6 appraisers were entered into the analysis. Coefficients were evaluated to determine whether they supported the domain structure and followed a similar pattern as to that reported for a previous factor analysis published by the AGREE Collaboration. ### Construct Validity Construct validity was assessed by evaluating 2 hypotheses. The first hypothesis was selected to match the hypothesis tested by the AGREE Collaboration guidelines\[[@B14]\] and supported by others\[[@B21],[@B22]\], that CPGs developed by established guideline developers should have higher quality scores than those created outside of established system. All guidelines in the database were classified as having been developed by established guideline developers if it could be identified that an experienced guideline developer or development group was responsible for a specific guideline. A CPG was classified as having \"Experienced Guideline Developers\" if it 1) had more than 3 authors (also fulfilled by an agency) and 2) at least one team member could be identified an a methodologist experienced in CPG development -- either by descriptions contained within the body or the CPG or after a review of listed authors (conducted by JM and DB). If this could not be verified the CPG was classified as \" No or Uncertain Methodology Expertise\". Our second hypothesis was that physical therapists would be more likely to recommend a guideline that was rated as having higher Rigor of Development scores. An independent t-test was used to evaluate the scores obtained for Rigor of Development for guidelines judged as acceptable versus those that were not. This hypothesis test is not ideal, as we are testing whether this subscale contributes to the overall rating within the same instrument. Nevertheless, in the absence of an external criterion, we choose to use this analysis given that it was also conducted in the original validation paper and there was an advantage to having a comparable analysis. ### Criterion validity Finally, criterion validity was assessed in the same manner as reported by the AGREE Collaboration in their validation study. Again we recognize we did not have an external criterion. Kendall Tau B Rank correlation coefficients were calculated between the appraisers domain scores and the overall assessment score. Results ======= Participants ------------ Sixty-nine physical therapists were recruited and were categorized as clinical specialists (n = 29), generalists (n = 21) or researchers (n = 19) (Table [1](#T1){ref-type="table"}). Generalists and specialists reported similar years of clinical experience with generalists ranging from 3--33 (mean ± SD,16 ± 11) years and specialists 3--35 (15 ± 8) years. Researchers reported 3--21 (7 ± 6) years of research experience. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Demographic description of physical therapy evaluators ::: Group Age Mean (SD) Gender % Highest Degree % ------------------------- ------------------- -------------- ---------------------- ------------- --------------- ------------- --------------- Female Male Diploma Bachelors Masters Doctorate Overall(n = 69) 40 (8) 96 4 9 58 32 1 Generalists(n = 21) 39 (9) 86 14 19 71 10 0 Specialists(n = 29) 39 (7) 100 0 7 79 14 0 Researchers(n = 19) 41 (9) 100 0 0 11\* 84 5 \* these two participants had significant research experience and were close to completion of Masters degree ::: The majority of specialists were orthopaedic physical therapists (55%), followed by neurological (24%) and cardiorespiratory (10%). One participant reported to specialize in paediatric physical therapy. Two participants did not specify their area of specialization. Reliability ----------- Analysis of reliability of individual items indicates a trend for higher reliability in items within the domain from Rigor of Development (See Table [2](#T2){ref-type="table"}). Intraclass correlation coefficients (ICCs) for each domain of the AGREE instrument for pairs of appraisers are presented in Table [3](#T3){ref-type="table"}. No systematic differences were observed that would indicate that type of appraisers had any substantial impact on the reliability obtained. Variation in reliability was observed across domains with Rigor of Development demonstrating the highest level of reliability. Few ICCs reached the excellent benchmark of 0.75, if a single appraiser performed the evaluation. ICC models that estimate the reliability when appraisers were averaged using models (1,2) for pairs or (1,6) across all six appraisers indicated substantial improvement in reliability if appraisals were averaged across appraisers. When comparing the reliability across different numbers of appraisers (Table [4](#T4){ref-type="table"}) the improvement in reliability was most notable when going from two to three appraisers, with the exception of editorial independence). Additional benefit for adding additional appraisers was inconsistent. Agreement on the overall assessment of the CPG had low reliability for generalists and specialists and moderate reliability for researchers. Quadratic weighting demonstrated some improvement in reliability coefficients for generalists and researchers, but not for specialists. (Table [5](#T5){ref-type="table"}) ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Inter-rater reliability of AGREE instrument -- individual items ::: Generalists Clinical Specialists Researchers Overall ------------- ----------------- -------------------------- ----------------- ------------- ----------- ----------- ---------- ---------- Item 1 0.34 0.51 0.50 0.66 0.58 0.73 0.46 0.84 Item 2 0.28 0.44 0.03 0.05 -0.09 -0.19 0.25 0.67 Item 3 0.45 0.62 0.23 0.37 0.19 0.31 0.22 0.63 Item 4 0.58 0.74 0.65 0.79 0.75 0.86 0.67 0.92 Item 5 0.57 0.73 0.37 0.54 0.55 0.71 0.45 0.83 Item 6 0.43 0.60 0.62 0.76 0.69 0.82 0.55 0.88 Item 7 0.48 0.65 0.18 0.30 0.36 0.53 0.36 0.77 Item 8 0.88 0.93 0.77 0.87 0.78 0.88 0.83 0.97 Item 9 0.72 0.84 0.84 0.91 0.69 0.82 0.72 0.94 Item 10 0.61 0.76 0.54 0.70 0.66 0.80 0.62 0.91 Item 11 0.45 0.63 0.13 0.23 0.32 0.49 0.39 0.79 Item 12 0.61 0.75 0.46 0.63 0.55 0.71 0.63 0.91 Item 13 0.74 0.85 0.66 0.79 0.56 0.72 0.64 0.92 Item 14 0.57 0.73 0.61 0.76 0.70 0.82 0.65 0.92 Item 15 0.31 0.47 0.30 0.46 0.25 0.40 0.38 0.79 Item 16 0.39 0.56 0.49 0.66 0.46 0.63 0.43 0.82 Item 17 0.31 0.48 0.31 0.48 0.41 0.58 0.35 0.76 Item 18 0.32 0.48 0.60 0.75 0.42 0.59 0.52 0.87 Item 19 0.53 0.69 0.50 0.67 0.45 0.62 0.43 0.82 Item 20 0.48 0.65 0.40 0.57 0.49 0.66 0.43 0.82 Item 21 0.34 0.51 0.37 0.54 0.20 0.34 0.28 0.70 Item 22 0.53 0.69 0.15 0.27 0.42 0.60 0.26 0.68 Item 23 0.58 0.73 0.53 0.69 0.72 0.84 0.40 0.80 ICC -- Intraclass correlation coefficient Results statistically significant at the p \< 0.05 level except where indicated by bold. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Inter-rater reliability of AGREE instrument domain scores ::: Scope & Purpose Stakeholder Involvement Rigor of Development Clarity & Presentation Applicability Editorial Independence -------------------------- --------------------- ----------------------------- -------------------------- ---------------------------- ------------------- ---------------------------- ------------------- ------------------- ------------------- ------------------- ------------------- ------------------- Generalists 0.37 (0.11--0.59) 0.54 (0.19--0.74) 0.71 (0.54--0.83) 0.83 (0.70--0.90) 0.81 (0.68--0.89) 0.89 (0.81--0.94) 0.41 (0.15--0.62) 0.58 (0.26--0.76) 0.65 (0.46--0.79) 0.79 (0.63--0.88) 0.60 (0.39--0.75) 0.75 (0.56--0.86) Clinical Specialists 0.35 (0.10--0.56) 0.52 (0.18--0.72) 0.59 (0.39--0.74) 0.74 (0.56--0.85) 0.65 (0.47--0.78) 0.79 (0.64--0.88) 0.51 (0.29--0.69) 0.68 (0.45--0.81) 0.43 (0.19--0.63) 0.61 (0.32--0.77) 0.32 (0.06--0.54) 0.49 (0.11--0.70) Researchers 0.17 (0.14--0.46) 0.30(-0.32--0.63) 0.73 (0.54--0.85) 0.84 (0.70--0.92) 0.77 (0.61--0.87) 0.87 (0.76--0.93) 0.47 (0.18--0.69) 0.64 (0.31--0.81) 0.47 (0.19--0.68) 0.64 (0.32--0.81) 0.59 (0.34--0.77) 0.75 (0.51--0.87) Overall 0.40 (0.25--0.58) 0.80 (0.66--0.89) 0.67 (0.54--0.80) 0.93 (0.87--0.96) 0.79 (0.68--0.88) 0.96 (0.93--0.98) 0.55 (0.40--0.72) 0.88 (0.80--0.94) 0.50 (0.35--0.68) 0.86 (0.76--0.93) 0.35 (0.19--0.54) 0.76 (0.59--0.88) CI -- confidence interval; ICC -- intraclass correlation coefficient Results statistically significant at the p \< 0.5 level except where indicated by bold. ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Intraclass correlations for each AGREE instrument domain as a function of the number of raters ::: Scope & Purpose Stakeholder Involvement Rigor of Development Clarity & Presentation Applicability Editorial Independence -------------- --------------------- ----------------------------- -------------------------- ---------------------------- ------------------- ---------------------------- ------ ------ ------ ------ ------ ------ 2 Raters 0.37 0.54 0.71 0.83 0.81 0.89 0.41 0.58 0.65 0.79 0.60 0.75 3 Raters 0.38 0.64 0.71 0.88 0.82 0.93 0.54 0.78 0.66 0.85 0.38 0.65 4 Raters 0.41 0.73 0.64 0.88 0.77 0.93 0.51 0.80 0.54 0.82 0.35 0.69 5 Raters 0.42 0.78 0.69 0.92 0.79 0.95 0.51 0.84 0.57 0.87 0.38 0.76 6 Raters 0.40 0.80 0.67 0.93 0.79 0.96 0.55 0.88 0.51 0.86 0.35 0.76 ICC = Intraclass correlation coefficient ::: ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Agreement on whether a CPG would be recommended or not ::: Pair of Raters Kappa (unweighted) Kappa (quadratic weights) ---------------- -------------------- --------------------------- Generalists 0.20 0.34 Specialists 0.25 0.22 Researchers 0.39 0.47 A Kappa was calculated on the final overall rating question whether or not a CPG should be using with the data dichotomized as YES (strongly recommend or recommend with provisos) or NO (Would not recommend or unsure) or by using quadratic weighting to compare the strength of recommendation (Strongly, with provisos, would not, unsure). ::: Validity -------- ### Face Validity/ User Feedback Study participants provided feedback during training sessions primarily with respect to the training session itself. They found the opportunity to discuss the results with others to be useful as a means of understanding the intent of individual items. Only three participants had previously been exposed to the AGREE instrument and that majority expressed positive comments about the value of learning about the AGREE instrument. Some clinicians expressed some anxiety about the role for CPGs and how they might be used. None of the study participants provided any feedback when returning their mail packages. The entry-level physical therapists (students) uniformly agreed that the AGREE instrument provided a useful structure and guidance in the evaluation of the CPG. Students compared the AGREE instrument to evaluation instruments they had used for critical appraisal of different study designs, such as clinical trials and systematic reviews. Students frequently commented that these previous instruments had a more open-ended format and expected the reviewer to understand issues in critical appraisal with little direction as to expectations or scoring criteria. Thus, they found the concrete nature of the AGREE instrument and the clear instruction on interpretation to be a useful framework for the evaluation process. Students stated that this direction increased their confidence that they had addressed all important issues. Although students differed on their ratings for individual questions, as well as the overall usefulness of the CPG evaluated, the majority of students understood and correctly interpreted the intent of the items from all of the AGREE domains. The majority of the students, 96 %, stated that they would use the AGREE instrument in other situations. A concern raised by the remaining 4% and other students who would continue to use the instrument was the length of the form and the amount of time required to complete the evaluation, given the busy nature of clinical practice. ### Factor analysis The factor analysis supported a 4-factor solution. The first factor explained 45% of the variance and contained items primarily from the Scope and Purpose or Rigor of Development domains. The second component explained 12% of the variance and contained items primarily from the Clarity and Presentation or Applicability domains. The third factor explained 7.7% of the variance and contained all of the items from the Stakeholder Involvement domain, all of the Editorial Independence items and question 13 from Rigor of Development, which pertains to whether the guideline has been externally reviewed by experts. The fourth component explained 5.6 percent of the variance and contained item 11 regarding health benefits/side effects and item 15 regarding whether recommendations were specific and unambiguous. Item means and their loadings are presented in Table [6](#T6){ref-type="table"}. ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Results of factor analysis (principal components with varimax rotation) ::: Item Mean Std. Deviation Components -------------------------- ----------- ---------------- ------------ ---------- ---------- ---------- 1 2 3 4 Scope and Purpose Q1 3.4003 .72614 .690 .339 .271 .217 Q2 2.9681 .68948 .803 .213 .129 .296 Q3 3.3961 .51941 .393 .489 .016 .430 Stakeholder Involvement Q4 2.6814 1.07262 .336 .422 .661 .093 Q5 1.7517 .89553 .179 .173 .739 .058 Q6 2.8331 .89717 .195 .294 .726 .259 Q7 1.6664 .75710 .255 .290 .675 .175 Rigour of development Q8 2.7344 1.21287 .902 .096 .247 -.087 Q9 2.6533 1.20139 .913 .112 .229 -.123 Q10 2.6314 1.05247 .800 .132 .384 .066 Q11 2.9492 .76769 .059 .339 .302 .682 Q12 2.9811 1.00409 .626 -.058 .223 .514 Q13 2.3556 1.16306 .422 .273 .617 .213 Q14 1.9425 .99370 .323 .668 .090 .044 Clarity and presentation Q15 3.2250 .73262 .132 .200 .149 .864 Q16 3.2269 .77070 .022 .607 .241 .322 Q17 3.1631 .71880 .093 .606 .453 .400 Q18 2.3342 .98180 .091 .671 .449 .273 Applicability Q19 2.2072 .94797 .158 .830 .231 .005 Q20 1.9500 .83211 .172 .818 .176 -.073 Q21 2.2336 .80322 -.017 .775 .299 .237 Editorial Independence Q22 2.1453 .90158 .451 .108 .669 .045 Q23 1.7575 .92802 .178 .305 .452 -.436 This table presents the results of the final 4 factor solution to factor analysis. Boldedcells shown the factor for which each item loaded most strongly. Results are grouped according to the Domains of the AGREE. ::: ### Construct validity The construct that the CPGs developed by expert guideline development groups would have a higher score on the domain Rigor of Development was supported (See Table [7](#T7){ref-type="table"}). The construct that therapists would be more likely to recommend for usage a CPG with a higher quality on the domain Rigor of Development was also supported (mean of 76 vs. 58 p \<0.001). ::: {#T7 .table-wrap} Table 7 ::: {.caption} ###### Hypothesis test: rigour of development is greater where panel is known to have methodology expertise ::: Rater Expert Panel No or Uncertain Expertise p --------------------------- -------------- --------------------------- --------- Generalist \#1 79 58 0.002 Generalist \#2 79 57 0.001 Specialist \#1 73 58 0.015 Specialist \#2 75 65 0.09 Researcher \#1 72 57 0.02 Research \#2 80 61 0.014 All 6 appraisers combined 78 59 \<0.001 This table contains the scores for the Agree Domain on Rigour of Development. CPGs were classified as having an expert panel if 1) there were more than 3 authors listed (or an agency) and 2) there was an experienced CPG methodologist clearly identified as a panel member or if one of the study investigators was recognized as such. All others were classified as \"No or Uncertain Expertise\". The p value for the independent samples t-test is shown. ::: ### Criterion validity The correlation between overall assessment and the domain scores ranged from low (0.38) to moderate (0.64), with the highest correlation being observed for the Rigor Of Development domain. (Table [8](#T8){ref-type="table"}) ::: {#T8 .table-wrap} Table 8 ::: {.caption} ###### Correlation of domain scores with overall assessment of AGREE ::: Domain Correlation with Overall Rating -------------------------- --------------------------------- Stakeholder Involvement 0.59 Scope and Purpose 0.52 Rigour of Development 0.64 Clarity and Presentation 0.62 Applicability 0.49 Editorial Independence 0.38 Kendall\'s Tau-b correlations were conducted between the mean rating of the over assessment of the CPG across all raters as compared to the mean of each Domain score. As hypothesized the correlation was highest with Rigour of development. ::: Discussion ========== The study findings suggest that the AGREE instrument is reliable and valid when used by physical therapists to evaluate CPGs. While some differences exist between the results reported in the original validation study authored by the AGREE Collaboration, the similarities far outweigh the differences. This would suggest that the process of evaluating CPGs using the AGREE instrument can be transferred to physical therapy practice to support the translation of higher quality CPGs into physical therapy health services. Typically, individual item reliability is of little relevance when evaluating the properties of a measurement scale, as items are not intended to be used in isolation. However, it may be useful from a practical point of view to form hypotheses about were further training might be necessary, if it appears that appraisers have particular difficulty with certain items. As discussed above, one must be careful in interpreting reliability coefficients in isolation, particularly for instruments like the AGREE where some items have \"relatively true\" scores and others have a spectrum of true scores. For the AGREE this spectrum might exist on application items. Thus, we would expect low item reliability might result from items where there is large measurement error, but also from items where there is substantial variability in how the item might apply in different circumstances. Our data support this. Low reliability was evident on items such as whether the guideline was editorially independent (2/8 ICCs were not greater than 0) and whether key review criteria for monitoring or audit had been provided (2/8 ICCs were not greater than 0). Study participants also appeared to have difficulty interpreting these items during the training session and thus these disagreements may reflect a lack of consensus about the meaning of these items. In some CPGs information on Editorial Independence is contained in footnotes and was missed by some evaluators. Improvements in reliability on these items might be anticipated with further training. Other items where low reliability was observed included whether the clinical question was specifically described (6/8 ICCs were not greater than 0) and whether the patients to whom the guideline applied were specifically described (2/8 ICCs were not greater than 0). During training, participants appeared to understand these questions, although expectations about what constituted an appropriate description of the clinical question or patient population tended to vary according to the participant\'s level of expertise in that given clinical area. Thus, disagreements on these items may partially relate to differences in priorities or familiarity with relevant issues between participants. Further training is unlikely to enhance reliability in this case, but raises the question about the importance and specificity of relevant clinical expertise when selecting evaluators. Although our intent was to evaluate the importance of clinical expertise, we classified participants quite broadly. Participants classified as clinical specialists were provided CPGs within their broad area of practice; but, we did not ascertain whether in fact the specific topic of the CPG was an area in which they actually did have knowledge or experience. For example, a clinician with expertise in musculoskeletal practice might practice primarily in a narrower area within the field, such as, rheumatology, upper extremity, lower extremity, joint replacements etc. In such cases, their familiarity with the salient features of the specific conditions might be less detailed and result in a differential evaluation regarding whether the clinical population had been appropriately defined. Future investigations should focus on content knowledge in a more specific sense to determine the importance of this issue when selecting evaluators. It is not uncommon for the policy developers to use reliability data to set standards for the number of appraisers required when establishing guideline evaluation processes. Our data provide little direction in this regard, except to suggest that more than two appraisers are advisable. This is consistent with the minimum recommendation of 2 made by the AGREE Collaboration. \[[@B7]\] Measurement theory suggest that additional appraisers/ratings should produces higher levels of reliability\[[@B20]\] as do the AGREE Collaboration when stating that 4 appraisers is preferable. Our data did not follow this trend, beyond three appraisers. Our study evaluated the reliability of appraisers performing their assessment in isolation. This process replicates that which might be used by a working committee independently assessing quality scores. However, individual therapists might also need to evaluate CPG when no committee has been established to do so. In these cases, we would recommend three or more appraisers should still be recruited. We recognize that when evaluating CPGs, it is not just the score, but the process that is important. Adding additional appraisers with different perspectives may increase the variability/disagreements. In fact, our data support this, as quadratic weighting of disagreements improved reliability coefficients in generalists and researchers, but not specialists. Specialists tended to disagree on whether a CPG should be used are not, but were less uncertain about that recommendation. We suggest the ideal approach to evaluation of CPGs is one where a group of potential end-users with clinical and guideline expertise work together to review a potential CPG by using the AGREE to facilitate a consensus process to determine both the quality and relevance to practice. These quality ratings should be disseminated to the relevant clinical communities. We recognize that the ideal process will not always be possible. Individual clinicians who must make decisions about utilization of CPGs should recruit colleagues to assist with the evaluation process before modifying their clinical practice based on CPGs with unknown validity. Although interpretation of validity analyses is complex and requires some subjective decisions, overall our validity analyses are supportive of the AGREE instrument and are substantively similar to that reported by the AGREE Collaboration for 33 medically based CPGs\[[@B14]\] A factor analysis structure which suggests that the concepts measured by the AGREE are similar to the Domains described by the AGREE underlies the content and structural validity of the scale. The factor analysis was strongly supportive of the domains of Stakeholder Involvement and Editorial Independence. This would support uniqueness of items within these domains. The largest factor contained items from the domain Rigor of Development as well as the first two items in Scope and Purpose. Conceptually these two items, which require specific objectives and a well-defined clinical question, fit well with a process of rigorous development. That the largest factor relates to methodological issues supports the AGREE as an evaluative tool. The second factor contained the items regarding whether the patients were specifically described, whether procedures for updating the guideline were provided, all of the items from the applicability domain and 3/4 items from the clarity and presentation. Clarity of presentation, applicability and a defined patient population may all relate to the ability to implement CPGs and thus all retain a conceptual relationship to the domains Applicability or Clarity of Presentation described by the AGREE Collaboration. The third factor contained all items of the stakeholder involvement as well as the additional question in rigor of development addressing whether experts had externally reviewed the guideline. In general, these results support stakeholder involvement as a subscale and include external involvement within this distinct domain. The fourth factor contained only two items, one the item on health benefits/ side effects and a second whether recommendations were specific and unambiguous. This factor explained a small percentage of the variance and contained only two items, making it difficult to relate these results to the factor analysis presented by the AGREE Collaboration. While the results vary somewhat from those reported by the AGREE Collaboration, there is substantial similarity, particularly when one examines the concepts that are represented by the items that clumped together. We view our factor analysis results with caution given the small sample sizes. The AGREE Collaboration study use 100 guidelines and conducted this analysis during the field-testing prior to inclusion of item 23. Our analysis included only 56 guidelines. We did not conduct formal sample size calculations, as practical limitations on guideline availability determined our sample size. Rules of thumb suggest ten \"subjects\" per item for factor analysis, requiring 230 CPGs for a well-powered analysis. Thus, some inherent instability should be expected in the factor analyses conducted to date. With the proliferation of CPG, we expect that larger samples might be evaluated in future studies and provide more definitive results on the factor validity of the AGREE instrument. Construct validity tests also supported the validity of the AGREE instrument. Constructs were developed to replicate those used in the previous validation of the AGREE\[[@B14]\] and provided similar findings. Despite our inability to be confident about whether some CPGs had a methodologist, we still found large differences between the Rigour of Development Domain score obtained where we could identify a team containing a methodologist as compared to where this was not present or unclear (21 point difference). The AGREE Collaboration found a 9 point advantage in this domain for guidelines developed within a guideline development program and 19 points for those developed as national initiatives. Similarly, our correlations between overall assessment scores and those reported for Rigor of Development (0.64) concurred with those reported by AGREE Collaboration data (0.87) in that both suggest a substantive relationship. Overall, our findings were remarkably consistent with those reported by the AGREE collaboration when validating the AGREE as used by medical practitioners\[[@B14]\] Validation by independent groups in different settings provides a stronger foundation for any instrument. These findings can be used to increase confidence in the current practice of different health care disciplines to use the AGREE to facilitate the evaluation of CPGs. Conclusion ========== 1\. The AGREE instrument is reliable and valid when used by physical therapists to assess the quality of clinical practice guidelines 2\. A minimum of three appraisers should be used to optimise reliability, although issues on effecting knowledge transfer or maximizing validity might increase these requirements. 3\. There is no evidence that specific types of physical therapists provide more reliable scores. As this study did not address whether familiarity with the actual content of the CPG influenced reliability, this should be studied in the future. 4\. There is evidence that guidelines developed with the assistance of experienced guideline developers are more rigorous. 5\. The AGREE instrument was found to be useful in assisting both novice and experienced therapists in evaluation of clinical practice guidelines. The majority of therapists would continue to use the instrument. 6\. Validity analysis supported the majority of results reported by the AGREE Collaboration)\[[@B14]\] The type of CPGs and evaluators were different in the current study supporting the validity of this instrument across a spectrum of circumstances. Competing interests =================== IG is an associate member of the AGREE Collaboration. Authors\' contributions ======================= JM, DB, SS, SSW formed the original study team which conceived of the research question, obtained the ethics approval and obtained grant funding. JM developed the research design, conducted statistical analyses, developed and facilitated the training sessions, developed and evaluated the student evaluations and drafted the manuscript. DB managed grant funds, selection of clinical practice guidelines, appraiser recruitment and data collection. All authors approved the final study protocol, contributed to interpretation of the study results and participated in revisions of the manuscript. IG provided consultation with respect to CPG research and health care policy. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6963/5/18/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional File 1 The training test ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We are grateful for financial support received from the Physiotherapy Foundation of Canada, Canadian Physiotherapy Association and Ontario Physiotherapy Association to support this project. Joy MacDermid, Dina Brooks and Ian Graham hold New Investigator Awards from the Canadian Institute for Health Research. Joy MacDermid was a Career Scientist of the Ontario Ministry of Health and Long-Term Care, Health Research Personnel Development Program when this project was conducted. Thanks to Teresa Small for data entry/quality checking and to Maria Juricic for her assistance with study coordination/data collection. The authors appreciate the contribution of the study participants who analysed CPGs using the AGREE.	PubMed Central	2024-06-05T03:55:54.187701	2005-3-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555572/", "journal": "BMC Health Serv Res. 2005 Mar 2; 5:18", "authors": [ { "first": "Joy C", "last": "MacDermid" }, { "first": "Dina", "last": "Brooks" }, { "first": "Sherra", "last": "Solway" }, { "first": "Sharon", "last": "Switzer-McIntyre" }, { "first": "Lucie", "last": "Brosseau" }, { "first": "Ian D", "last": "Graham" } ] }
PMC555575	Introduction ============ Allergic diseases are characterized by elevated serum IgE, an inflammatory reaction with increased number of eosinophils, mast cells and an adaptative immune responses orchestrated by Th2-like CD4+ memory T cells secreting an array of cytokines such as IL-4, IL-5 and IL-13. Thus, there are major efforts focused on a therapeutic treatment which will decrease the Th2 profile and/or re-direct the immune response from a Th2, IgE-mediated allergic hypersensitivity reaction towards the more favorable Th1 response. IL-12 and IFN-γ are of primary importance in modulating the Th1/Th2 balance. IFN-γ has been shown to attenuate eosinophil recruitment\[[@B1]\], and also inhibit the development of secondary allergic response \[[@B2]-[@B4]\]. There has also been extensive research into therapeutic use of IL-12\[[@B5]\]. However, difficulties with precise dosing and toxicity associated with the direct administration of these cytokines may preclude their therapeutic application. Another approach is to use natural up-regulators to elevate endogenous levels of IL-12 or IFN-γ. Many microbial products, including heat-killed bacteria and CpG motifs can up-regulate Th1 cytokines. Oligodeoxynucleotides (ODN) containing unmethylated cytosine-guanine motifs (CpG) have powerful immunomodulatory activity in human and murine lymphocytes in both Th1 and Th2 associated diseases \[[@B6]-[@B12]\]. It is believed that CpG exert their effect through antigen presenting cells by inducing cytokines such as TNF-alpha, IL-12, IL-18, and IFNs \[[@B9],[@B13],[@B14]\]. Type I IFNs have been proposed as mediators of immunomodulatory effects of CpG oligonucleotides \[[@B15]\]. Importantly, some studies have suggested that endogenous type I IFN might contribute to the downregulation of eosinophil infiltration in murine asthma model \[[@B16]\]. Furthermore, reduced inflammatory infiltration and IgE production have been shown after administration of recombinant IFN-β\[[@B17],[@B18]\]. We have recently demonstrated that lung eosinophilic inflammatory response was exacerbated by the lack of IFN-β gene\[[@B19]\]. Even though it is believed that immunomodulatory effects of CpG-ODN are mediated by type I IFNs, the relative role of IFN-β has not been defined. In this report, we examined the role of IFN-β in the immune response after CpG treatment in a murine model of allergic inflammation. Our results indicate that induction of Th1 response by therapy with CpG-ODN is partially dependent on IFN-β, while IFN-β is not an absolute requirement for suppression of eosinophilia and IgE. Materials and methods ===================== Animals ------- Groups of pathogen-free female\[[@B20],[@B21]\] 8-10-week-old, 17-20 g, B10.RIII mice (n = 5 mice per group) were used in the experiments. IFN-β deficient mice (IFN-β-/-) were kindly provided by Dr Leanderson\[[@B22]\]. Genotyping of the offspring has been described before\[[@B23]\]. All animal care and experimentation were conducted at the animal unit of Medical Inflammation Research in Lund in accordance with the current protocols in Lund University. Induction of disease and treatment protocol ------------------------------------------- Immunization and allergen challenge of the mice were carried out according to a short term allergy model protocol by Sur and colleagues \[[@B24]\] with slight modification. Mice were sensitized by i.p. injection on days 0 and 4 with OVA 50 μg (Sigma Chemical Co., St Louis, Mo), with 5 mg alum (Sigma Chemical Co.). At day 14 and 16 after immunization, mice were challenged with 50 μg of OVA plus 5 μg of CpG-ODN (Scandinavian Gene Synthesis AB, Köping, Sweden) delivered through the airways as intranasal drops after light anesthesia. Control mice were immunized with 5 mg alum with PBS, and challenge with PBS using the same schedule as OVA immunized mice. Our previous studies have confirmed that control mice did not show any remarkable allergy changes\[[@B19]\]. The ODNs were designed using published sequences\[[@B8],[@B25]\] consisting of a single-stranded phosphorothioate-modified ODNs with 22 bases containing two CpG motifs (5\'-TGACTGTGAACGTTCGAGATGA-3\'), highly purified with undetectable levels of LPS (detection limit: 1 ng/mg DNA): and were dissolved in PBS with a final concentration of 1 μg/μl \[[@B11]\]. Mice received either 5 μg of CpG-ODN in PBS or PBS alone intranasally in conjunction with OVA challenges. On day 17 (i.e. 24 h after the last challenge) mice were assessed for lung allergic inflammatory response. In the prevention study (vaccination), mice were pretreated i.p. with 5 μg of CpG-ODN in PBS on day 0. On the same day, mice were sensitized by i.p. injection with OVA complexed with 5 mg alum (Sigma). On day 4 mice were injected i.p. OVA (50 μg) in Alum (5 mg). On days 14 and 16 after immunization mice were challenged with 50 μg of OVA delivered through the airways as intranasal drops after light anesthesia. On day 17 mice were assessed for lung allergic inflammatory response, 24 hours (h) after the last challenge. Bronchoalveolar lavage Fluid (BALF) ----------------------------------- Mice were deeply anesthetized with an ip injection of 0.2 ml avertin (20 mg/ml; 2,2,2 tribromoethanol, Sigma-Aldrich) and sacrificed 24 hours after the last OVA exposure. After thoracotomy, the trachea was cannulated and BAL was collected twice with 0.5 mL of PBS and the collected fluid was pooled. Total cell counts were determined using an automated hemocytometer (Sysmex CDA-500, Toa Medical Electronics CO., Ltd., Kobey, Japan), and the fluid was centrifuged (1.000 rpm, 4°C, 10 min). The supernatant was used to determine the airway cytokine and IgE levels contents. The cells were applied to slides using a cytospin apparatus (Auto-smear CF-12DE, Sakura Finetek Europe BV, Zoeterwoude, The Netherlands) and were stained with May-Grunwald-Giemsa staining. Eosinophils were specifically detected by histochemical staining of cyanide-resistant eosinophil peroxidase activity (CREPA) using as substrate 3,3 diaminobenzidine tetrahydrochlorhid (DAB), as described before\[[@B26]\]. Briefly, samples were dried overnight at room temperature and fixed with 4% paraformaldehide for 5 min and PBS for 2 min. Then, samples were incubated in PBS buffer with DAB 60%, H~2~O~2~30% and NaCN 120% for 7 min. After washing with PBS, samples were counterstained with hemtoxiline 30\" and mounted with Kaiser medium (Merck, Darmstadt, Germany). Eosinophils were easily detected by its dark brown color. The slides were examined by light microscopy (×40 magnification) in a blinded fashion counting at least 400 cells per slide Allergen specific T cell proliferation -------------------------------------- At the time of sacrifice spleens were dissected and a single cell suspensions from each mouse was prepared in DMEM with glutamax I (Gibco BRL, Life Technologies), supplemented with 10% heat-inactivated fetal calf serum, 10 mmol/l HEPES, 50 mmol/l β-mercaptoethanol, 100 U/ml penicillin G, and 100 μg/ml streptomycin. Cells were cultured (5 × 10^6^/ml) in triplicates in 96-well flat-bottomed plates at 37°C, 5% CO2 in a humidified incubator. Cells were cultured in absence or presence of OVA (111 μM), CpG-ODN (1 μg/ml) or concavalin A (4 μg/ml). ^3^H-thymidine (100 μCi/ml) was added 54 h later, and after a further 18 hr later incubation, a beta-scintillation counter measured incorporation. Cytokine Assays --------------- Splenocytes were isolated as described and incubated for 48 h with or without OVA (Sigma-Aldrich) (111 μM) in 48-well plates. Enzyme immunoassays were performed as described before\[[@B23],[@B27]\] using monoclonal Ab (anti-IL-2, anti-IL-4, anti-IL-5, anti-IL-12, anti-IFN-γ (BD Pharmingen, San Diego, CA, USA) and reading by chemiluminescence (Victor^®^; 1420 Multilabel Counter ^©^, Wallac Oy; EG & G Turku, Finland). Determination of total and OVA-specific IgE levels -------------------------------------------------- Mice were bled at the time of sacrifice. A sandwich ELISA (BD Pharmingen) was used to measure levels of IgG and IgE as described previously\[[@B28]\]. To determine OVA-specific IgE plates were incubated with OVA 10 μg/ml in PBS buffer (pH 7.\'5). Procedure was the same as total IgE. Standard curve was performed with sera with known levels of specific IgE as it has been published before \[[@B29]\]. Briefly, real concentration of specific IgE in ng/ml of a pooled serum was determined indirectly by absorption of 50 μl of serum with either conjugated BSA in Sepharose (Pharmacia, Uppsala, Suecia) or conjugated OVA in Sepharose. Total IgE ELISA, as mentioned before, determined the level of not absorbed specific IgE. The percentage of OVA-specific IgE was calculated by reciprocal value of: (IgE not absorbed by OVA-Sepharose/IgE not absorbed by BSA-Sepharose) × 100. The result of a pool of sera from several immunized mice by this method was 402 ng/ml of OVA-specífica IgE. In next experiments this serum was used as standard pattern. For that, plates were coated with OVA (10 μg/ml) overnight 4°C and blocked with 1% BSA in PBS 1 h room temperature. The remainder steps were performed as total IgE ELISA, as described before. Flow cytometry -------------- At time of sacrifice spleens were removed and a single cell suspension was made, cells were then lysed with 0.84% NH~3~Cl~2~and washed in PBS with 1% BSA and 0.01% sodium azide. After blocking Fc receptors, using 24.G2 (from our hybridoma collection), cells were stained with the following antibodies (BD PharMingen); PE conjugated anti-B7.1 (clone 16-10A1), FITC conjugated anti-B7.2 (GL1), cytochrome conjugated anti-B220 (RA3-6B2), APC conjugated anti-Thy1.2 (53-2.1), PE conjugated anti-CD4 (H129.19), cytochrome conjugated anti-CD8 (53-6.7). The cells were then analyzed by flow cytometry FACSort (Becton Dickinson, Franklin Lakes, NJ, USA), using the BD Cell-Quest™ Pro, Version 4.0.1 software (Becton Dickinson). Three individuals per time point and group were analyzed. The program then displays the percentage of events, which express the CD86 molecule and this percentage is the compared between the groups. Clinical and Histological analysis of joints for arthritis ---------------------------------------------------------- Seventeen days post CpG-ODN or control vaccination, paws were visually assessed looking for swelling or deformation with redness in one joint, several joints or severe swelling of the entire paw and/or ankylosis\[[@B30]\]. Then, mice were sacrificed and paws were dissected and were fixed in 4% formaldehyde, decalcified with EDTA (for 2--3 weeks), embedded in paraffin, sectioned at 5μm and stained with hematoxylin and erythrosine. Approximately, 20--30 sections were made from each paw (2 paws per mouse, i.e. front and back paws). The sections were then evaluated blindly for pathological changes in joints (synovitis, erosion or destruction)\[[@B31]\]. Statistic analysis ------------------ The significance of changes was evaluated using Mann-Whitney Utest. Significance was assumed at pvalues ≤ 0.05. Results ======= Treatment with different dose of intranasal CpG-ODN showed similar results -------------------------------------------------------------------------- The percentage of local eosinophils in airways was increased after immunization and challenge with OVA in BALF of WT and IFN-β-/- compared to non immunized mice. Preliminary data with different dose of CpG administered intranasally with OVA (5 μg, 10 μg or 20 μg) to both strain of mice resulted in similar reduction of percentage of infiltrating eosinophils in BALF (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Eosinophils in airways with different dose of intranasal CpG-ODN ::: Treatment Genotype Eosinophils --------------- -------------- ----------------- PBS B10.RIII 0.5 % IFN-β^-^/- 0.7 % OVA B10.RIII 55 % IFN-β^-^/- 62 % OVA+CPG 5 μ B10.RIII 2.1 % IFN-β^-^/- 9.2 % OVA+CPG 10 μ B10.RIII 1\. 9 % IFN-β^-^/- 9.4 % OVA+CPG 20 μ B10.RIII 1.9 % IFN-β^-^/- 9.0 % B10.RIII/WT (□) and IFN-β^-^/- (■) mice were sensitized to OVA by intraperitoneal injection and subsequently challenged with OVA either alone or with different dose of CpG-ODN by intranasal drops on days 14 and 16. Eosinophil percentage in bronchoalveolar lavage with different dose of intranasal CpG-ODN were similar in all IFN-β^-^/- treated mice. ::: Treatment with CpG-ODN inhibits total number of infiltrating cells in airways in WT but not in IFN-β-/- mice ------------------------------------------------------------------------------------------------------------ The treatment with 5 μg of CpG administered intranasally with OVA resulted in significant reduction of total number of infiltrating cells in BALF in WT group while it had no effect in IFN-β-/- group (Figure [1A](#F1){ref-type="fig"}). We examined the number of recruited cells in lung airways after administration of PBS, OVA or CpG-ODN plus OVA and challenge with OVA. We found that OVA nasal challenge increased significantly the number of cells recruited in airways of OVA-primed mice compared to PBS group. CpG-ODN vaccinated mice had reduced the number of cells in OVA-sensitized B10.RIII mice but not in IFN-β-/-. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Effects of treatment with CpG-ODN on total BALF cell recruitment (A), eosinophils (B). B10.RIII/WT (□) and IFN-β^-^/- (■) mice were sensitized to OVA by intraperitoneal injection and subsequently challenged with OVA either alone or with CpG-ODN by intranasal drops on days 14 and 16. Cells were harvested on day 17^th^. n= 5/group, \*P\< 0.05 vs. OVA groups. † P\< 0.05 vs OVA-treated WT mice. ::: ![](1465-9921-6-25-1) ::: Suppression of eosinophilia by CpG-ODN in airways is only partially dependent on IFN-β gene ------------------------------------------------------------------------------------------- Next, we were interested in the effect of CpG-ODN treatment on eosinophilia. As expected, we found that OVA-sensitized/OVA-challenge WT mice had a dramatic increase in numbers of eosinophils compared with non-treated WT. Vaccination with CpG-ODN diminished dramatically the number of eosinophils in WT mice while it was only partially effective in prevention of eosinophilia in IFN-β^-^/- mice, and the difference between the CpG-ODN vaccinated and PBS vaccinated mice was statistically significant for both WT and IFN-β^-^/- (figure [1B](#F1){ref-type="fig"}). IFN-γ induction in the airways by CpG-ODNs vaccination is impaired in IFN-β-/- mice ----------------------------------------------------------------------------------- We were interested in investigating if disease mediated Th2 cytokines or disease counter-acting cytokine, IFN-γ, was effected by the CpG-ODN vaccination. We observed that the level of IL-4 in BALF was reduced from 65 ± 7 pg/ml to 43 ± 6 pg/ml (33% of reduction) in WT mice and from 62 ± 8 pg/ml to 46 ± 87 pg/ml (26%) in IFN-β-/- mice respectively after CpG-ODN vaccination. The levels of IL-5 were significantly reduced in both groups with no difference between groups (figure [2A](#F2){ref-type="fig"}). IFN-γ production in airways of WT mice was enhanced upon CpG-ODN vaccination and it was dependent on IFN-β gene since its induction was impaired in IFN-β-/- mice (figure [2B](#F2){ref-type="fig"}). Hence, the ratio IFN-γ/IL-4 determining the Th1/Th2 ratio, was skewed to a Th1 response in both groups although much stronger in WT mice (figure [2C](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### BALF cytokine (protein) concentrations after intranasal CpG-ODN. BALF were collected 24 h after the last challenge from each group (n= 5/group) and cytokine levels determined by ELISA in non-immunized, OVA-challenged, and OVA-challenged/CpG-treated B10.RIII (□) and IFN-β^-^/- (■) mice at days 14 and 16. IL-5 (A) levels were significantly augmented after OVA challenge and diminished after CpG vaccination in both strains similarly. IFN-γ (B) was not induced in OVA/primed-OVA/challenge, but was induced after CpG vaccination. IFN-γ was stronger induced in B10.RIII than in IFN-β^-^/- mice. Th1/T2 ratio was stronger skewed to Th1-profile in B10.RIII than in IFN-β^-^/- mice. Data are given as mean ± SEM, \*P\< 0.05 vs. OVA groups. † P\< 0.05 vs B10.RIII mice treated with CpG ::: ![](1465-9921-6-25-2) ::: Vaccinated with CpG-ODN induces CD86 expression on B cells in IFN-β-/- mice --------------------------------------------------------------------------- In order to observe any differences between cell surface markers between IFN-β^-^/- and wild type mice treated with CPG-ODN or with PBS, splenocytes were analyzed by flow cytometry. We could not see any difference in T cell population, in regards to both CD4:CD8 ratio and expression of CD86 (B7.2) on T cells. However, there was a significant difference in CD86 (B7.2) expression on B cells. This difference was observed between CpG-ODN vaccinated IFN-β^-^/- mice and PBS control IFN-β^-^/- mice as well as between CpG-ODN vaccinated IFN-β^-^/- and CpG-ODN vaccinated wild type mice (Figure [3](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Percent of expression of CD86/B7.2 on B cells in splenocytes of mice at day 17 after immunization and vaccination of ODN-CpG in IFN-β^-^/- mice (KO) and wild type litter-mates (WT). ::: ![](1465-9921-6-25-3) ::: CpG-ODN vaccination induces mild synovitis particularly in IFN-β-/- mice ------------------------------------------------------------------------ Mice did not show any clinical visually deformation. While surveying the capacity of CpG-ODN vaccination to induce IFN-β in different tissues, it was noticeable that there were pathological changes in joints of some mice. Thus, we stained the paws of mice (n = 3) with hematoxylin and erythrosine and evaluated the pathologic changes in joints. Data revealed mild synovitis and pannus formation in multiple joints of CpG-ODN vaccinated mice while no control mice had any pathologic changes. Furthermore, we discovered that mice lacking IFN-β were more affected than their wild type littermates (table [2](#T2){ref-type="table"} and figure [4](#F4){ref-type="fig"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Histopathologic evaluation of joints for arthritis changes. ::: Groups Vaccination ------------------ ----------------- ------------- CpG-ODN Control IFN-β^-^/- (n.1) ++ \- IFN-β^-^/- (n.2) ++ \- IFN-β^-^/- (n.3) \+ \- WT (n.1) ++ \- WT (n.2) \- \- WT (n.3) \- \- Hematoxylin-eosin staining of joints from four different groups of mice (IFN-β^-^/- and their WT littermates with CPG-ODN treatment or control) were analyzed. This revealed mild synovitis and pannus formation in 3/3 IFN-β-/- mice treated with CPG-ODN and 1/3 WT treated with CPG-ODN while no pathological changes were observed in these two non-treated groups. ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Illustration of joint synovitis after hematoxylin-eosin staining. A. It shows synovitis and pannus formation in IFN-β^-^/- mice treated with CPG-ODN. B. It shows no pathologic changes in a control treated IFN-β^-^/- mice. ::: ![](1465-9921-6-25-4) ::: Cell profile in airways after vaccination withCpG-ODN ----------------------------------------------------- The CpG-ODN vaccination reduced the number of cells in OVA-sensitized B10.RIII mice. However, the number of cells recovered in IFN-β^-^/- mice did not significantly change (table [3](#T3){ref-type="table"}). ODN vaccinated mice had a slight increase in numbers of eosinophils compared with non-treated WT. CpG-ODN therapy diminished the number of eosinophils in WT mice, while it was only partially effective in prevention of eosinophilia in IFN-β^-^/- mice with significant differences between the CpG-ODN treated and non-treated mice in WT and IFN-β^-^/- (table [3](#T3){ref-type="table"}). Similarly, vaccination with CpG-ODN showed an enhanced response of macrophages in IFN-β^-^/- mice compared to WT mice, but this macrophage response was similar in treated and non-treated WT mice. Lymphocyte and neutrophil response in airways of treated-IFN-β^-^/- mice was also significantly enhanced compared to WT mice. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Effects of vaccination with CpG-ODN (prevention study) on eosinophil and total BAL cell recruitment. ::: Treatment Genotype Total cells Eosinophils Monocytes Lymphocytes Neutrophils --------------- -------------- ----------------- ----------------- --------------- ----------------- ----------------- PBS B10.RIII 245 ± 43 3 ± 1 232 ± 20 5 ± 1 5 ± 1 IFN-β^-^/- 259 ± 14 3 ± 1 242 ± 33 6 ± 1 8 ± 2 OVA B10.RIII 622 ± 37\* 381 ± 43\* 144 ± 17 62 ± 3\* 35 ± 2\* IFN-β^-^/- 683 ± 66\* 427 ± 83\* 178 ± 22 55 ± 8\* 22 ± 4\* OVA+CpG B10.RIII 227 ± 18† 2.7 ± 2† 142 ± 19 67 ± 4 14 ± 1 IFN-β^-^/- 574 ± 32 52 ± 7 † ‡ 321 ± 39† ‡ 130 ± 38† ‡ 70 ± 22† ‡ Cell types quantified in BALF were eosinophils, macrophages, lymphocytes and neutrophils and are expressed as no. of cells × 10^3^/ml. n= 5/group, \*P\< 0.05 vs. untreated groups. † P\< 0.05 vs OVA-treated mice. ‡ P\< 0.05 vs WT mice treated with CpG-ODN. OVA-treated mice and control groups. ::: Inhibition of OVA-specific IgE in the prevention study (vaccination) by CpG-ODNs is independent of IFN-β -------------------------------------------------------------------------------------------------------- It has been shown that systemic administration of CpG-ODN do not inhibit established IgE response while vaccination inhibits IgE production\[[@B32]\], however the role of INF-β was not investigated. Here, we examined what the function of IFN-β was in prevention of OVA-specific IgE in CpG-ODN vaccine. We found that CpG-ODN vaccine resulted in inhibition of OVA-Specific IgE in both WT and IFN-β-/- mice (figure [5](#F5){ref-type="fig"}). IgG2a levels were similar in both WT (118 ± 15 μg/ml) and IFN-β-/- (135 ± 25 μg/ml) mice. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### OVA-specific IgE levels in the prevention study (vaccination). B10.RIII/WT (□) and IFN-β^-^/- (■) were sensitized to OVA by intraperitoneal injection either OVA alone or with CpG-ODN and subsequently challenged with OVA by intranasal drops on days 14and 16; control mice received PBS alone. Cells were harvested on day 17. n= 5/group, \*P\< 0.05 vs. OVA groups. † P\< 0.05 vs OVA-treated WT mice. ::: ![](1465-9921-6-25-5) ::: Allergen specific Th1 response as a result of CpG-ODN vaccination is partly impaired in the absence of IFN-β ------------------------------------------------------------------------------------------------------------ To address if splenocytes from WT and IFN-β^-^/- respond differently in vitro, cells from naïve mice were stimulated and cell proliferation was measured. Splenocytes from both groups, WT and IFN-β^-^/-, had the same proliferation levels after stimulation with concavalin A, CpG or culture media (figure [6A](#F6){ref-type="fig"}). However, cells from WT immunized mice vaccinated with CpG in vivohad more cell proliferation after restimulation with OVA than IFN-β^-^/- immunized and CpG vaccinated mice (figure [6B](#F6){ref-type="fig"}). Next we assessed whether OVA specific Th1 response, i.e. IFN-γ, IL-2 and IL-12, were affected by CpG-ODN vaccination plus OVA treatment in vivo. We found that IFN-γ, IL-12 and IL-2 were significantly lower in OVA-primed/OVA-challenge IFN-β-/- mice compared to WT mice (figure [6C](#F6){ref-type="fig"}). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Ex-vivoimmune response in the prevention study (vaccination). A. In vitrostimulation of splenocytes from naïve mice with con A and CpG does not show any difference between B10.RIII (□) and IFN-β-/- mice (■). B. In vitroproliferation of OVA restimulated T cells from in vivoCpG-vaccinated OVA-primed B10.RIII (□) and IFN-β-/- mice (■). Mice were primed and challenged as in Figure 2. In vitroproliferation after recall with OVA was weaker in IFN-β-/- mice (■) than B10.RIII mice (□). C. Th-1 cytokines from supernatants after in vitroproliferation of OVA restimulated T cells in OVA-primed/CpG-vaccinated mice. IFN-γ, IL-12 and IL-2 production in supernatants from cell cultures was higher in B10.RIII than in IFN-β-/- mice. n= 5/group \*P\< 0.05 vs. OVA-treated B10.RIII mice. ::: ![](1465-9921-6-25-6) ::: Discussion ========== Synthetic unmethylated CG dinucleotides within particular sequence context (CpG motifs) mimic bacterial DNA, and are responsible for the immunostimulatory activity of that \[[@B6]\]. CpG oligonucleotides have shown to produce a strong activation of B cells\[[@B33]\], NK cells \[[@B34]\], macrophages\[[@B35]\] and dendritic cells\[[@B36]\] by a direct mechanism. However CpG have also the ability to exert activation of T cells by an indirect mechanism through via IFN-α/β \[[@B37],[@B38]\]. Furthermore, CpG in mice results in production of inflammatory and antiinflammatory cytokines including IL-1, IL-2, IL-6, IL-18, TNF-α, type I IFN (IFN-α/β) and type II IFN (IFN-γ) \[[@B39]-[@B41]\]. Type I IFNs (IFN-α/β) have pleiomorphic effect on the immune system with activation of macrophages and stimulation of NK cells to produce IL-12, which in turn induces Th1 cell development\[[@B42]\]. Some of these immunostimulatory effects have been applied in animal models of several diseases including allergic disorders\[[@B8],[@B43]-[@B50]\]. It have been shown that therapies using oligonucleotides containing CpG have the ability of immunomodulation with a downregulation of elevated IgE and eosinophilic inflammation in the airways, both of which are orchestrated by cytokines elaborated by Th2 cells. However, systemic administration of CpG has been reported to increase side effects, owing in part to high dose of these oligonucleotides. Systemic immunization, even with adjuvants, induces robust adaptive immune responses at systemic sites but weak in the airways, while local immunization can elicit both systemic and mucosal responses \[[@B51]-[@B53]\]. In this report, we have demonstrated that concomitant intranasal administration of low doses of CpG and the offending antigen exerted significant reduction of total number of infiltrating cells, including eosinophils in BALF (table [1](#T1){ref-type="table"}). As mentioned before, CpG in mice results in production of several cytokines including type I IFN (IFN-α/β)\[[@B37],[@B38],[@B54]-[@B56]\] which have the ability to exert indirect activation of T cells \[[@B37],[@B38]\]. IFN-β treatment, used by either oral\[[@B18]\] or parenteral\[[@B17]\] via in mice, have shown to produce an inhibition of antigen-induced bronchial inflammation and airway hyperresponsiveness \[[@B17],[@B18]\] probably influenced by the inhibition of Th-2 airway eosinophilia by the suppressive effect on eosinopoiesis \[[@B57]\]. We have recently demonstrated that lung eosinophilic inflammatory response was exacerbated by the lack of IFN-β gene\[[@B19]\]. Even though it is believed that immunomodulatory effects of CpG-ODN may be mediated by type I IFNs \[[@B15]\], the relative role of IFN-β, a type I IFN, has not been defined. Here, we aimed to elucidate whether IFN-β have a key role in the anti-allergic effect of CpG motifs. Our results demonstrate that therapy with CpG-ODN prior to and after the allergen challenge resulted in significant reduction of total number of infiltrating cells, including eosinophils, in BALF in WT mice while CpG-ODN did show an enhanced response of macrophages, lymphocytes and neutrophils in airways of IFN-β-/- mice. These findings might be explained since CpG motifs in bacterial DNA can delay apoptosis of neutrophil granulocytes \[[@B58]\] and macrophages \[[@B59]\], indicating a possibility of inhibition of macrophage apoptosis by CpG and a difference of cellular responses downstream of different Toll-like receptors \[[@B59]\]. Another possibility might be that phosphorothioated ODNs used in our experiments might have been chemoattractants for primary macrophages\[[@B60]\] in the absence of IFN-β. This chemoattractant activity have been exposed as independent of CpG activity\[[@B60]\], since it has not been seen with phosphodiester CpG-ODNs. However, up to our knowledge this is the first reference about the influence of CpG on neutrophils. It has been shown that systemic administration of CpG-ODN do not inhibit established IgE response while vaccination inhibits IgE production\[[@B32]\]. We found that CpG-ODN vaccine resulted in inhibition of OVA-Specific IgE in both WT and IFN-β^-^/- mice (figure [5](#F5){ref-type="fig"}). These data underline that IFN-β is not required for the beneficial effect of CpG-ODN vaccine in a model of allergic inflammation. Vaccination with a single low dose of CpG-dinucleotide inhibited OVA-specific IgE production with subsequent upregulation of IgG2a in both groups. The success in inhibiting established IgE response is most likely due to the timing of the protocol where mice received CpG-ODN at the time of priming. This early intervention presumably prevents presence of IgE-plasma cells in the bone marrow as suggested earlier by Peng et al \[[@B32]\]. Production of the Th1 cytokine, IFN-γ, has been reported to be dependent on CpG-ODN-induced IFN-α/β as demonstrated by antibodies that block IFN-α/β\[[@B54]\]. Since, earlier reports target both IFN-α and β, it was unclear if one or both of these cytokines mediate the biological effects of CpG-ODN. In addition, we have recently reported that IFN-β knock out mice do not have any failing mounting a T~H~1 response, measured by IFN-γ production. In contrary, IFN-γ production was significantly elevated as a result of experimental autoimmune encephalomyelitis (EAE), a T~H~1-mediated disease model for multiple sclerosis. Consequently IFN-β knock out mice had more severe and chronic symptoms than their WT littermates with more extensive CNS inflammation and higher demyelination \[[@B23]\]. Thus, here we aimed to investigate the profile of OVA-specific Th1 cytokines after CpG-ODN vaccination in the absence of IFN-β. We found a clear reduction in Th1 response (IL-2 and IFN-γ) in IFN-β knock out mice vaccinated with CpG-ODN which was in agreement with earlier reports\[[@B55]\]. As Th1-promoting activity of CpG-ODN is controlled by IL-12\[[@B12]\], we measured the levels of IL-12 and found that production was elevated in the CpG-ODN WT group. We also found that its induction is partially under the influence of IFN-β triggered by synthetic CpG sequences. Since IFN-γ is almost undetectable in non-treated mice, at least under the conditions used in this study, the results also suggest that CpG is capable of inducing IFN-β in substantial amounts to trigger IFN-γ production. Our findings of Th1 mediated response in systemic immune response were moreover supported by the fact that IFN-γ production was also defective in the inflammatory organ measured in BALF. Moreover, our results also provide evidence that IFN-β is an important cofactor for IFN-γ production through induction of IL-12 pathway as it has been suggested by Sun et al\[[@B37]\] While, it is crucial to underline that IFN-β-/- mice do not have a general defect on mounting a Th1 immune response\[[@B23]\] therefore it is more likely that the defect in inducing a proper Th1 response in IFN-β-/- mice is due to malfunctioning IL-12 and IFN-γ induction through TLR9 pathway as a result of CPG-ODN vaccination. This might also explain the lower proliferative response of OVA-specific Th1 cells in IFN-β-/- mice reported here. Once more, it should be mentioned that IFN-β-/- mice are capable of inducing significantly higher OVA-specific T cell proliferation of Th2 character \[[@B19]\] which might also partly contribute to suppression of a more profound Th1 response. It has been reported that CpG-ODNs do not directly stimulate T cells, but by inducing production of IFN-γ from APCs, thus activating T cells to express CD69 and B7.2\[[@B9],[@B37]\], while their proliferative responses are reduced\[[@B37]\]. It was also shown that CpG stimulate T cells by inducing APCs to synthesize IFN-I, which then act directly on T cells via IFNAR\[[@B37]\]. In addition, it has been suggested that production of type I IFNs by APCs is through increased availability of costimulatory signals on activated DC\[[@B37],[@B36]\]. It has also been reported that stimulation with CpG motifs induces the changes in surface molecules of APCs\[[@B25],[@B55],[@B37]\]. However, the reduced OVA-specific Th1 response in IFN-β-/- mice is less likely to be mediated by lack of upregulation of costimulatory molecules on APCs as we have previously reported that these mice have upregulated B7.1/2 on APCs\[[@B19]\]. After treatment with CPG-ODN we made an interesting observation that the mice developed a mild synovitis, which to our knowledge is the first report of mucosal administration of CPG-ODN causing joint modification. Synovitis is one of the phenotype features of the experimental murine animal models of autoimmune arthritis, such as collagen-induced arthritis (CIA), which is an extensive investigated model of human rheumatoid arthritis. This model can be elicited in susceptible strains by immunization with type II collagen (CII), the major protein of articular cartilage. Assessment of disease includes visual/clinical evaluation of arthritis severity, measurement of humoral and cellular immune responses, including CII-specific antibody titers and T cell responses to CII. In these models, joints are histologically scored for the changes of inflammation including synovitis and periarticular, pannus formation, cartilage damage with marginal erosions or diffuse changes, and bone damage including resorption and periosteal proliferation\[[@B31]\]. It is known that unmethylated CpG-ODN are responsible for induction of arthritis triggered by bacterial DNA\[[@B11],[@B61]-[@B63]\] that supports our data. Our finding that mucosal administration of CpG-ODN causes mild synovitis points out a potential hazardous side effect when using CpG-ODN as a treatment. In summary, we have demonstrated that the CpG-ODNs can partly prevent the development of eosinophilic airway inflammation and allergen specific IgE response in the absence of IFN-β, while Th1 response is defective. In addition, these results demonstrate that mucosal administration of CpG-ODN before allergen exposure could be a less harmful form of active immunotherapy in allergic diseases without impeding systemic immune responses as earlier suggested \[[@B8],[@B51]\]. However, due to the potential of hazardous side effects, meticulous caution must be undertaken prior to considering it as a therapy in allergic asthma. Abbreviations ============= APC: Antigen presenting cells; CpG, cytosine-guanine motifs; ODNs, oligodeoxynucleotides; DAB, 3.3 diamino benzidine tetrahydrochlorhide; BALF, bronchoalveolar lavage fluid; CREPA, (cyanide-resistant eosinophil peroxidase activity); IFNAR, type I IFN receptor; APC, antigen-presenting cells; DC, dendritic cells. Authors\' contributions ======================= VM conceived of the study, participated in its design and coordination, performed the experiments and drafted the manuscript. AT carried out the analysis of flow cytometry, prepared histological samples of joints and performed the clinical and histological analysis of joints for arthritis. AT and IT generated crossing of IFN-β ko mice to B10.RIII strain of mice, genotyped, backcrossed and maintained the IFN- β-/- mouse line. VN participated in the design and coordination of the study. SI-N participated in the direction of the study, performed histological analysis of joints, as well as writing and preparing the manuscript. All authors read and approved the final manuscript. Acknowledgements ================ We thank Sandy Liedholm, Isabelle Bohlin, Rebecka Ljungqvist and Carlos Palestro for taking excellent care of the animals and Emma Mondoc and Margareta Svejme for help with histological analysis. This work has been supported by grants from The Swedish Foundation for Health Care Sciences and Allergy Research, The Crafoord Foundations, The Edvard Welander Foundation, King Gustaf V\'s 80-year Foundation, Fundación SEAIC and Tore Nilsson\'s Foundation for Medical Research.	PubMed Central	2024-06-05T03:55:54.194804	2005-3-5	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555575/", "journal": "Respir Res. 2005 Mar 5; 6(1):25", "authors": [ { "first": "Victor", "last": "Matheu" }, { "first": "Alexandra", "last": "Treschow" }, { "first": "Ingrid", "last": "Teige" }, { "first": "Vaidrius", "last": "Navikas" }, { "first": "Shohreh", "last": "Issazadeh-Navikas" } ] }
PMC555576	Introduction ============ Diabetes is a complex and multifarious group of disorders characterized by hyperglycemia that has reached epidemic proportions in the present century. Infection is a leading cause of morbidity and mortality among the diabetic population \[[@B1]\]. Diabetes is associated with vascular and renal dysfunction characterized by hypertension, dyslipidemia, microalbuminuria, macroalbuminuria and glomerular mesangial expansion \[[@B2],[@B3]\]. Mitogen-activated protein kinases (MAPKs) are implicated in the etiology of diabetes \[[@B4],[@B5]\]. MAPKs are serine-threonine protein kinases involved in cell survival, proliferation and apoptosis \[[@B6]\]. Three MAPK subfamilies have been well characterized: extracellular signal regulated kinase 1 and 2 (ERK1/2), c-jun N-terminal kinases (JNK) and p38-MAPK \[[@B7]\]. ERK1/2 is involved in the growth response of cell while p38-MAPK and JNK are associated with cellular response to stress \[[@B7]\], inflammation \[[@B8]\] and vasoactive mediators such as endothelin-1 (ET-1) \[[@B9]\]. Also p38-MAPK activation stimulates inducible nitric oxide synthase (iNOS) expression in serum-deprived RAW 264.7 cells \[[@B10]\]. These observations suggest that signaling mechanisms can regulate various vasoactive molecules and vice-versa. However it is still not known if progression of diabetes produces a time dependent activation of p38-MAPK in vascular blood vessels and kidneys. We speculate a differential regulation of p38-MAPK and ERK1/2 in thoracic aorta and kidneys depending upon the duration and severity of diabetes. We have earlier demonstrated that diabetes during coronary artery bypass grafting, and chronic peritoneal sepsis produced an imbalance in the myocardial and systemic ET-1 and nitric oxide (NO) profiles \[[@B11],[@B12]\]. However, the profile of ET-1 during progression of diabetes is unclear. Diabetes, both type 1and type 2, is associated with decreased NO bioavailability \[[@B13],[@B14]\]. Conflicting reports (i.e. increased, unchanged and decreased) exist regarding the state of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) during diabetes \[[@B15],[@B16]\]. We have earlier reported that elevation of ET-1 and NO mechanisms, either systemically or locally in the myocardium, correlated well with the development of myocardial dysfunction during sepsis \[[@B11]\] and make the heart susceptible to myocardial injury \[[@B17]\]. We anticipate that the duration of hyperglycemia would differentially modulate systemic and renal ET-1 and NO mechanisms. We hypothesize that progression of diabetes \[mild (14-days) to moderate (28-days)\] would produce renal and vascular dysfunction that correlate with altered p38-MAPK phosphorylation in kidneys and thoracic aorta. Therefore, the specific aims of the study are to characterize the progression of STZ-induced renal and vascular dysfunction at 14- and 28-days; and to examine if the progression of STZ-induced diabetes would alter the biosynthesis and activation of ET-1, NO and the phosphorylation of p38-MAPK and ERK1/2 in thoracic aorta, kidney cortex and kidney medulla. Materials and methods ===================== Male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 350--400 g were used in the study. The rats were acclimatized to the laboratory conditions for at least 7 days following their arrival. All animal experiments were conducted in compliance with humane animal care standards outlined in the NIH Guide for the Care and Use of Experimental Animals, and were approved by the Institutional Animal Care and Use Committee of North Dakota State University. Experimental Protocol --------------------- All animals were age-matched at the onset of the study. The rats were randomly divided into three groups: control, 14-days and 28-days diabetic rats (n = 6 for each group). ### Induction of diabetes Diabetes was produced by a single tail vein injection of streptozotocin (STZ; 60 mg/kg, I.V.) \[[@B15]\]. Diabetes was confirmed by blood glucose estimation (Glucometer Elite^®^, Bayer Corporation, IN) 2 days after STZ-injection. Diabetes was confirmed if blood glucose \> 200 mg/dL. The animals were not given insulin supplementation. Food intake and water intake were examined everyday after STZ-injection. The non-diabetic control rats received an injection of 1 mL/kg saline. 14-days and 28-days post-STZ administration recordings of systemic hemodynamics, carotid blood flow, and renal arterial blood were done. Arterial blood was collected in plastic tubes containing EDTA (1 mg/mL) and heparin (5 units/mL) to determine the plasma concentration of ET-1, NO by-products (NOx) and creatinine. The animals were euthanitized by pentobarbital (100 mg/kg, I.P.), the thoracic aorta and kidney were harvested. The concentration of ET-1, NOx, creatinine and protein expression of preproET-1, iNOS, eNOS, phosphorylated p38-MAPK (pp38-MAPK), total p38-MAPK, phosphorylated ERK, total ERK were determined in the thoracic aorta, kidney cortex and kidney medulla obtained from each animal. Surgical protocol ----------------- Separate group of animals (N = 6) were used for hemodynamic study. On the day of experiment, the rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (Nembutal^®^, Abbott; 50 mg/kg). ### Common carotid arterial blood flow measurement (acute) Under pentobarbital (50 mg/kg I.P.) anesthesia, rats were placed in dorsal recumbency and through a midline cervical incision, the left common carotid artery was identified and carefully separated from adhering connective material. The carotid artery was cleared from the vagal nerve and a Transonic^®^flow probe (0.3 mm 1RB, Transonic Systems Inc., Ithaca, NY) was placed carefully around the artery. The probe was manually positioned so that the artery was centered within its window and then probe was held in position. Through a sterile 10 CC syringe loaded with sterile K-Y brand lubricating jelly (Johnson & Johnson, Arlington, TX), the acoustical window within the flow probe was filled while avoiding any air bubble. ### Renal arterial blood flow measurement (acute) Under pentobarbital anesthesia, rats were place in dorsal recumbency, and through a midline abdominal skin incision the left renal artery was carefully separated from renal vein and a 0.5 VB Transonic^®^flow probe was placed around it. The probe was manually positioned so that the artery is centered within the window and the probe was held in position. The acoustical window within the flow probe was filled with K-Y brand lubricating jelly (Johnson & Johnson, Arlington, TX). Renal and carotid artery blood flow was measured using Transonic^®^flow meter T206 attached to MP100 system of Biopac Systems Inc., CA via analog to digital conversion. The MP100 system was calibrated with minimum and maximum flow capacity of the individual probe connected to the flow meter. The sampling of data was carried out at 1000 Hz and recorded to a dedicated computer using AcqKnowledge™ software. Biochemical estimations ----------------------- ### Determination of the concentration of plasma creatinine The concentration of creatinine was determined in plasma using creatinine liquid reagents (end-point, colorimetric, DIAZYME). The blood samples immediately after collection were spun down at 5000 rpm for 10 minutes. The plasma was then decanted and stored at -20°C until the time of creatinine determination using manufacturer\'s instruction. Plasma concentration of creatinine (mg.dL^-1^.Kg^-1^) normalized to individual rat body weight was calculated. ### Determination of the plasma concentration of ET-1 The concentration of ET-1 was determined in plasma. The blood samples were collected in plastic tubes containing EDTA (1 mg/mL) before euthanasia. The sample was centrifuged at 3,000 × g for 15 min at 4°C and plasma was separated and assayed for ET-1. Plasma was acidified adding an equal volume of 20% acetic acid. ET-1 like material was extracted from plasma \[[@B11]\] using C-18 SEP-Columns (Peninsula labs, CA). The recovery of ET from plasma was approximately 87%. Immunoassay (IA) was performed using EIA kit for ET-1 (R and D systems, Minneapolis, MN). The assay was performed in microtiter plates coated with a rat antibody to human ET-1. Diluted anti-ET-1 HRP conjugate (100 μL) (ET-1 conjugated to horseradish peroxidase) was added in each well. Standards (0.25 pg -- 65 pg ET-1), parameter control (24.5 ET-1 pg/ml) or sample extract (100 μL, each) were added. The plates were covered with plate sealers and incubated for 1 hr at room temperature. The contents of each well were aspirated and washed using wash buffer provided with the kit. After the last wash, contents of each well were decanted and tetramethylbenzidine (100 μL) was added. After 30 min, stop solution (100 μL) containing 1 N HCl was added. Within 30 min of addition of the stop solution, the optical density of each well was measured using a micro plate reader at 450 (OD 450) and 620 (OD 620) nm separately. A standard curve was created and the concentration of ET-1 of each sample calculated and expressed as pg/ml of plasma. ### Determination of the concentration of nitric oxide byproducts (NOx) The concentration of endogenous NO~X~(nitrate + nitrite) was determined in plasma, thoracic aorta, kidney cortex and medulla. Blood (500 μL) was collected and 40 μL of heparin was added to each sample to prevent clotting. The blood sample was then decanted and stored at -20°C until the time of nitric oxide byproducts, (NO~2~+ NO~3~) NOx, determination. To determine the NOx level of thoracic aorta, kidney cortex and kidney medulla, those tissue samples were harvested from each experimental rat and immediately homogenized with cold phosphate buffered saline (PBS) on ice, which inhibited the activity of NOS ex vivo. The homogenate was centrifuged (3000 × g, 5 min) and the supernatant was collected. The supernatant obtained from tissues and plasma was passed through a 1.2 μm multiscreen filter plate. Plasma NOx and tissue NOx concentration was determined by using Greiss reaction \[[@B11],[@B15]\]. 6 μL of plasma was mixed with 44 μl distilled H~2~O, 20 μl 0.31 M phosphate buffer (pH 7.5) and 10 μL each of 0.86 mM NADPH (Sigma), 0.11 mM flavenidinine dinucleotide and 1.0 U/mL of nitrate reductase. NO~3~was converted to NO~2~by nitrate reductase (Boehriger Mannheim). Unknown tissue samples were run in duplicate. The samples were allowed to incubate for 1 hr at room temperature in the dark. Two hundred microliters of Greiss reagent \[1:1 mixture of 1% sulfanilamide in 5% H~3~PO~3~and 0.1% N-(1-naphthyl) ethyl-enediamine\] were added to each well and the plates were incubated for an additional 10 min at room temperature. Absorbance was measured at 540 nm using a plate reader and converted to NOx concentration using a nitrate standard curve and expressed as μM in plasma and μmoles/g protein in tissue. Protein in the supernatant obtained from each sample was determined using standard Lowry method. Immunoblot Analysis ------------------- PreproET-1, iNOS, eNOS, total and phosphorylated p38-MAPK and ERK protein expressions of thoracic aorta, kidney cortex and kidney medulla were determined using standard SDS-PAGE and immunoblot technique. Briefly, thoracic aorta, kidney cortex and kidney medulla tissues were homogenized in lysis buffer and centrifuged as described by Pollack et al. \[[@B16]\]. The supernatants, at a final protein content of 25 μl, were loaded to the gels using a 2:1 laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue and 710 mM β-mercaptoethanol). The prepared samples were electrophoresed on 10% denaturing sodium dodecyl sulfate (SDS) polyacrylamide gels. The proteins were transferred electrophoretically onto polyvinylidene difluoride (PVDF) membrane (Gelman Sciences, Pierce, Rockford, IL). Non-specific binding sites on the membrane were blocked overnight at 4°C with 5% nonfat dry milk in Tris-buffered saline containing Tween 20 (TBST, 20 mM Tris-HCl, 150 mM NaCl, 0.2% Tween 20, pH 7.4). The membranes were then probed with the primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hr at room temperature. The primary antibodies are highly specific against the proteins studies and had no cross-reactivity with related members. After five washings in TBST, the membranes were incubated with the secondary antibody (Sigma, St. Louis, MO) for 1 hr at room temperature. Finally membranes were washed three times with TBST. The specific proteins were detected by enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia Biotech). The blots were analyzed using Un-Scan-It™ software to estimate the density of the blots in pixels. Uniform loading was assessed by β-actin (Sigma) protein expression. While analyzing the data for immunoblot analysis, the beta-actin blots for each gel were analyzed first to confirm equal protein loading. Only after confirming that there was equal protein loading in the wells, as evidenced by no significant difference in pixel values of beta-actin blots, the bands for individual proteins were analyzed. Statistical Analyses -------------------- All the data were expressed as mean ± SEM. One-way ANOVA was performed to analyze the hemodynamic and biochemical data using SPSS software. Following a significant F value, a post hoc Students Newman Keuls test was performed for inter- and intra-group comparisons. Statistical significance was realized at p ≤ 0.05 to approve the null hypothesis for individual parameters. Results ======= General characteristics of the animals -------------------------------------- All control rats were freely moving in their individual cages through out the study. Although diabetic rats appeared to be lethargic and displayed restricted movements, there was no sign of infection or motor disorder in any of the rats studied. The food intake and water intake of 14-days and 28-days diabetes groups were significantly increased as compared to control group (Fig [1](#F1){ref-type="fig"}). The average body weight change, blood glucose and kidney weight/ 100 g body weight in all groups are summarized in Table [1](#T1){ref-type="table"}. The body weight change (%) was significantly decreased in 14-days and 28-days diabetes groups as compared to their age-matched control group. All STZ-treated rats were diabetic with mean blood glucose around 480 mg/dL. Blood glucose was significantly elevated in 14-days diabetes and 28-days diabetes groups as compared to their age-matched control group. Kidney weight normalized to body weight was significantly greater in diabetic (14-days and 28-days) rats than in control rats. Plasma creatinine concentration was increased 3-fold in 14-days diabetes group and 2-fold in 28-days diabetes group as compared to control group (Table [1](#T1){ref-type="table"}). Diabetes (14- and 28-days) produced no significant change in mean arterial pressure but caused a significant decrease in heart rate (Table [1](#T1){ref-type="table"}). Effect of STZ-induced diabetes on aortic and renal blood flow ------------------------------------------------------------- Control groups exhibited aortic and renal blood flow 10.8 ± 0.3 mL/min and 5.4 ± 0.1 mL/min, respectively. Induction of diabetes produced a significant decrease in renal (Fig [2A](#F2){ref-type="fig"}) and carotid blood flow (Fig [2B](#F2){ref-type="fig"}) at both 14-days and 28-days as compared to control group. Effect of STZ-induced diabetes on the concentration of ET-1 in plasma and expression of preproET-1 in thoracic aorta, kidney cortex and medulla ----------------------------------------------------------------------------------------------------------------------------------------------- To determine the effect of duration of diabetes (14-days to 28-days) on aortic and renal ET-1 biosynthesis we determined the concentration of ET-1 in plasma and expression of ET-1 precursor, preproET-1 in thoracic aorta, kidney cortex and kidney medulla. A significant elevation in plasma ET-1 concentration was observed in 28-days diabetes as compared to 14-days diabetes and control groups (Fig [3A](#F3){ref-type="fig"}). We did not find any significant change in the plasma concentration of ET-1 in 14-days diabetes group as compared to control group. The expression of preproET-1 was significantly increased in thoracic aorta in 14-days and 28-days diabetes groups as compared to control group (Fig [3B](#F3){ref-type="fig"}). We also found a significant increase in the expression of preproET-1 in kidney cortex in 14-days diabetes groups as compared to control group. A significant increase in the protein expression of preproET-1 of kidney cortex was observed in 28-days diabetes group as compared to 14-days diabetes and control groups (Fig [3C](#F3){ref-type="fig"}). Induction of diabetes did not produce any change in the expression of preproET-1 in kidney medulla at 14-days and 28-days diabetes groups as compared to control group (Fig [3D](#F3){ref-type="fig"}). Effect of STZ-induced diabetes on the concentration of NOx and expression of eNOS and iNOS ------------------------------------------------------------------------------------------ A significant increase in the concentration of NOx in plasma of 28-days diabetes group as compared to the control and 14-days diabetes groups was observed (Fig [4A](#F4){ref-type="fig"}). The NOx level was also elevated in thoracic aorta of 28-days diabetes group as compared to the control and 14-days diabetes groups (Fig [4B](#F4){ref-type="fig"}). In kidney cortex, we also found that NOx level was significantly increased in 14-days and 28-days diabetes groups as compared to control group (Fig [4C](#F4){ref-type="fig"}). But we did not find any significant change in kidney medulla of 14-days and 28-days diabetes groups (Fig [4D](#F4){ref-type="fig"}). A significant up-regulation of eNOS and iNOS in thoracic aorta in 14-days and 28-days diabetes groups was obtained as compared to control group (Fig [5A1--A3](#F5){ref-type="fig"}). Also in kidney cortex in 28-days diabetes, eNOS and iNOS were significantly increased as compared to 14-days diabetes and control groups. Protein expression of iNOS was also significantly increased in kidney cortex in 14-days diabetes group as compared to control group (Fig [5B1--B3](#F5){ref-type="fig"}). In kidney medulla we did not find any significant change in eNOS and iNOS in 14-days and 28-days diabetes groups as compared to control group (Fig [5C1--C3](#F5){ref-type="fig"}). Effect of STZ-induced diabetes on the expression of total and phosphorylated p38-MAPK and ERK1/2 in thoracic aorta, kidney cortex and medulla --------------------------------------------------------------------------------------------------------------------------------------------- Total p38-MAPK and phosphorylated p38-MAPK protein expressions in thoracic aorta were significantly elevated in 14-days and 28-days diabetes groups as compared to control group (Fig [6A1--A3](#F6){ref-type="fig"}). Expression of phosphorylated p38-MAPK was significantly increased in kidney cortex obtained from 28-days diabetes group as compared to 14-days diabetes and control groups. Total p38-MAPK was not altered in kidney cortex in 14-days and 28-days diabetes groups as compared to control group (Fig [6B1--B3](#F6){ref-type="fig"}). In kidney medulla of 14-days and 28-days diabetes groups we did not observe any significant change in total and phosphorylated p38-MAPK (Fig [6C1--C3](#F6){ref-type="fig"}). A significant decrease in the expression of phosphorylated ERK 1/2 of thoracic aorta in 28-days diabetes group but not in 14-days diabetes group was observed as compared to control group. We found that total ERK 2 but not total ERK 1 was significantly increased in thoracic aorta in 14-days diabetes groups as compared to control group. There was no change in the expression of total ERK 1 in 14-days and 28-days diabetes groups and ERK 2 in 28-days diabetes group as compared to control group (Fig [7A1--A3](#F7){ref-type="fig"}). In kidney cortex phosphorylated ERK 1/2 protein expression was significantly elevated in 28-days diabetes group as compared to 14-days diabetes and control groups. In kidney cortex, phosphorylated ERK 1/2 was significantly elevated in 14-days diabetes group as compared to control group. Total ERK 1/2 was not altered in kidney cortex in 14-days and 28-days diabetes groups (Fig [7B1--B3](#F7){ref-type="fig"}). In addition, in kidney medulla we did not observe any significant change in total and phosphorylated ERK 1/2 in 14-days and 28-days diabetes group as compared to control group (Fig [7C1--C3](#F7){ref-type="fig"}). Discussion ========== STZ has long been used as a drug of choice to induce type 2diabetes in various animal models. This well-established model is characterized by insulin deficiency associated with insulin resistance \[[@B18]\]. It was reported that a single intravenous injection of STZ (55 mg/kg) could cause increased plasma glucose levels, decrease in body weight and 17% mortality in rats \[[@B18]\]. In the present study, too, we have observed a mortality of 20% in 14-days diabetes group and 26% in 28-days diabetes group. STZ-treated rats, post 48-h, were confirmed to be hyperglycemic, lost body weight 10% and 13% in 14-days and 28-days diabetes groups respectively. Kavalali et al. \[[@B19]\] found that food and water intake amount was higher in diabetic groups than the control group. In our study too, we observed that diabetic rats had an increase in the food intake and water intake following 14-days and 28-days of STZ-administration. These observations suggested that single intravenous injections of STZ (60 mg/kg) produced a reproducible and consistent model of diabetes in our laboratory conditions. Renal hypertrophy can be detected as early as one day after the onset of diabetes and seen regularly post 60-hr of single STZ injection \[[@B20]\]. It has been reported that diabetes-induced renal hypertrophy produces increased dimensions of renal cells along with increased kidney weight \[[@B21]\]. In the present study, we observed an elevated kidney weight corrected by body weight in diabetic (14-days and 28-days) rats suggesting that STZ-induced diabetes produced renal hypertrophy. We also observed that both 14- and 28-days diabetic rats exhibited reduced renal blood flow along with 3-2 fold increase in plasma creatinine concentration. Umerani and Goyal \[[@B22]\] demonstrated an increase in serum creatinine as an indicator of deteriorated renal function in diabetic rats. Similar to our results, Itoh and coworkers \[[@B23]\] also demonstrated that serum creatinine levels in control group and diabetes group varied from 0.8 to 1.4 mg/dl respectively. Thus the results obtained in our study provide evidence for diabetes-induced renal dysfunction in the rat. ET-1, a potent vasoconstrictor peptide, has been implicated in diabetes and cardiovascular disorders. Elevated, unchanged and attenuated plasma ET-1 levels have been reported during diabetes \[[@B24]-[@B27]\]. Although the reason for such a variation in findings appears difficult to fathom, the discrepancies in data may be attributed to differences in species of animals, duration of hyperglycemia, dose of STZ administered etc. In the present study, we observed decreased aortic and renal blood flow in both 14-days and 28-days diabetics rats. However elevation of ET-1 and NOx in plasma was seen only in 28-days diabetic rats group but not in 14 days-diabetes group. Taken together, these observations suggest that STZ-induced hyperglycemia produced alterations in the systemic and regional blood flow, which could be due to altered systemic levels of ET-1 and NO. Makino et al. \[[@B27]\] demonstrated upregulated preproET-1 mRNA in the aorta from STZ-induced diabetic rats. They suggested that increased release of ET-1 from the aorta contributes to enhanced plasma level of ET-1 seen in diabetic rats. Also ET-1 concentration has been shown to increase in kidneys \[[@B28]\] following diabetes. Since ET-1 exists not only in the vascular endothelial cells but also in the mesangial cells or tubular cells, this increase in ET-1 could be attributed to increased biosynthesis of ET-1 in the renal cells \[[@B28]-[@B30]\]. In the present study, we also observed an elevated expression of preproET-1 proteins in aorta and kidney cortex but not in kidney medulla at 14-days and 28-days following diabetes induction. These findings suggest that during early diabetes an upregulation of ET-1 biosynthesis in vascular (thoracic aorta) and locally at the organ level (kidney cortex) could be responsible for vascular and renal dysfunction seen during diabetes. Although the mechanisms of ET-1 elevation during diabetes are relatively unknown, several research groups speculated that this increase could be due to an abnormal production by the affected endothelium \[[@B31]\] or lack of suppression of ET-1 release secondary to attenuated endothelium-derived relaxing factor production \[[@B32]\]. Hirata et al. demonstrated that ET-1 via binding to ET~B~receptors produces activation of NOS \[[@B33]\] and generates NO. In the present study, elevated concentration of NOx in plasma, thoracic aorta and kidney cortex but not in medulla was observed in 28-days diabetes group. Stockklauser-Farber et al. \[[@B7]\] demonstrated an increased myocardial NOS (iNOS and eNOS) activity that reached maximal values after 4 wk and 6 wk diabetes. We speculated that along with elevated NO production, activation of NOS isoforms may play a prominent role in the pathophysiology of nephropathy at different phases of STZ-induced diabetes \[[@B34],[@B35]\]. Increased expression of eNOS in afferent arterioles and glomeruli was found by Sugimoto et al.\[[@B36]\]. In a previous study, these authors demonstrated an enhanced renal expression of iNOS 5 days post diabetes that was sustained for 20 days, while eNOS and nNOS were not altered \[[@B37]\]. We also observed that the progression of diabetes from 14-days to 28-days upregulated iNOS and eNOS in thoracic aorta and kidney cortex while decreasing aortic and renal blood flow that correlated well with systemic and local increase in NOx levels in thoracic aorta. This suggests that NO stimulation in thoracic aorta and kidney cortex occurs with increased duration of hyperglycemia in STZ-induced diabetic rats. Since in the present study both ET-1 and NOS activation exhibit a similar course in 28-days diabetic rats, we speculate that NOS stimulation could be due to activation of ET~B~receptors via elevated ET-1 mechanisms. However, further studies will be required to strengthen this speculation. Signaling mechanisms in diabetes -------------------------------- Hyperglycemia has been shown to phosphorylate ERK1/2 in rat glomerular and mesangial cells \[[@B38]\] and p38-MAPK in vascular smooth muscle cells and aorta in derived from diabetic rats \[[@B39]\]. Pearson et al. \[[@B40]\] and Tian et al. \[[@B41]\] demonstrated that ET-1 stimulation of mesangial cell (MC) proliferation involves several pathways, among which MAPK figures prominently. In the present study, we observed that 14-days diabetes up-regulated phosphorylated p38-MAPK but not ERK1/2 in thoracic aorta. We also observed down-regulation of phosphorylated ERK 1/2 in thoracic aorta post 28-days diabetes. In kidney cortex although p38-MAPK was not altered post 14-days diabetic group, ERK1/2 is elevated. This suggests that ERK1/2 phosphorylation predominate during 14-days diabetes in kidney cortex but not in thoracic aorta, while both p38-MAPK and ERK1/2 remain unaffected in kidney medulla. Both in vivoand in vitroresults suggest that ERK and p38MAPK may be involved in high-glucose-induced cellular hypertrophy \[[@B42]\]. In the present study, we observed that ERK1/2 precedes p38-MAPK phosphorylation depending upon the progression of diabetes from 14-days to 28-days. We observed a profound increase in plasma NOx levels and iNOS expression with a corresponding increase in p38-MAPK activation in kidney cortex and thoracic aorta during 28-days diabetes. We propose that p38-MAPK activation could be an important signaling mechanism that causes iNOS activation as was reported by Liu et al. \[[@B14]\]. We speculate that activated p38-MAPK and iNOS mechanisms outweigh ERK1/2 mechanisms in kidney cortex and thoracic aorta during moderate diabetes. A similar finding, though not directly related to the present study, was shown by Purves et al. \[[@B43]\] where they demonstrated using cultured sensory neurons that co-treatment with high glucose and oxidative stress results in an additive effect on p38-MAPK phosphorylation without affecting ERK1/2 activation. The findings of the present study suggested that phosphorylation of p38-MAPK and not ERK1/2 was associated with iNOS activation and renal and vascular dysfunction following 28-days of STZ-induced hyperglycemia. The results obtained in the present study characterize the STZ (60 mg/kg, I.V.)-induced diabetic rat model in our laboratory. The marked characteristics of diabetic rat model include weight loss, increase food and water intake, and bradycardia. STZ-induced diabetes, both at 14-days and 28-days, produced renal dysfunction and vascular dysfunction that correlated well with levels of ET-1 and NOx and expression of preproET-1 and NOS proteins in kidney cortex and thoracic aorta. The data obtained in the present study demonstrate that progression of diabetes from 14-days to 28-days caused a factorial increase in p38-MAPK phosphorylation along with NOS upregulation in kidney cortex and thoracic aorta. The study provides evidence that diabetes produces vascular and renal dysfunction with a profound effect on signaling mechanisms at later stage of diabetes. However, more studies will be required to further delineate the inherent link or interaction between p38-MAPK upregulation, ET-1 and NO mechanisms and development of renal and vascular dysfunction during diabetes. Acknowledgements ================ First two authors contributed equally in this study. This work was supported by Beginning Grant-in Aid Award from American Heart Association Northland Affiliate, Academic Research Enhancement Award from National Institutes of Health-National Heart, Lung and Blood Institute grant No. HL 066016 and NDEPSCOR, National Science Foundation (ACS), Presidential Fellowship (AG) and fellowship to SB was provided from the funds obtained from National Institutes of Health-National Center for Research Resources 1 P20RR15566. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Food intake (gm/day) (A) and water intake (mg/day) (B) in control. 14-days and 28-days STZ-induced diabetes groups (N = 6 in each group); \* p ≤ 0.05 Vs control group. ::: ![](1475-2840-4-3-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Renal blood flow (mL/min) (A) and carotid blood flow (mL/min) (B) in control, 14-days and 28-days STZ-induced diabetes groups (N = 6 in each group); \* p ≤ 0.05 Vs control group. ::: ![](1475-2840-4-3-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### The concentration of plasma ET-1 (pg.mL^-1^) in control, 14-days and 28-days STZ-induced diabetes groups (A) (N = 6 in each group). Effect of duration (14-days and 28-days) of STZ-induced diabetes on the expression of preproET-1 protein in thoracic aorta (B), in kidney cortex (C) and in kidney medulla (D) and % change in densitometric units of preproET-1 (N = 3 in each group); \* p ≤ 0.05 Vs control group, \# p ≤ 0.05 Vs 14-days diab group. ::: ![](1475-2840-4-3-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Effect of STZ-induced diabetes on the plasma (A), thoracic aorta (B), kidney cortex (C) and kidney medulla (D) concentration of nitric oxide byproducts (NOx) in control, 14-days and 28-days diabetic rats. (N = 6 in each group); \* p ≤ 0.05 Vs control group, \# p ≤ 0.05 Vs 14-days diab group. ::: ![](1475-2840-4-3-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Effect of STZ-induced diabetes on the protein expression of iNOS and eNOS in thoracic aorta obtained from control, 14-days and 28-days diabetic rats. Representative protein expression of inducible- and endothelial-NOS (A1), % change in densitometric units of eNOS (A2) and iNOS (A3) in aorta, eNOS (B2) and iNOS (B3) in kidney cortex and eNOS (C2) and iNOS (C3) in kidney medulla. (N = 3 in each group); \* p ≤ 0.05 Vs control group, \# p ≤ 0.05 Vs 14-days diab group. ::: ![](1475-2840-4-3-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Effect of STZ-induced diabetes on the protein expression of total and phosphorylated p38-MAPK in thoracic aorta obtained from control, 14-days and 28-days diabetic rats. Representative protein expression of phospho- and total- p38-MAPK (A1, B1 and C1), % changes in densitometric units of phospho-p38-MAPK (A2, B2 and C2) and total-p38-MAPK (A3, B3 and C3) in aorta, kidney cortex and kidney medulla respectively. (N = 3 in each group); \* p ≤ 0.05 Vs control group, \# p ≤ 0.05 Vs 14-days diab group. ::: ![](1475-2840-4-3-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Effect of STZ-induced diabetes on the protein expression of total and phosphorylated ERK 1/2 in thoracic aorta obtained from control, 14-days and 28-days diabetic rats. Representative protein expression of phospho- and total-ERK (A1, B1 and C1), % changes in densitometric units of p-ERK (A2, B2 and C2) and Total-ERK (A3, B3 and C3) in aorta, kidney cortex and kidney medulla respectively. (N = 3 in each group); \* p ≤ 0.05 Vs control group, \# p ≤ 0.05 Vs 14-days diab group. ::: ![](1475-2840-4-3-7) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effect of STZ-induced diabetes on percent body weight (BW) change, blood glucose levels (mg.dL^-1^), mean arterial pressure (mmHg), heart rate (beats per min), kidney weight/100 g BW, and plasma creatinine concentration (g.dL^-1^.kg^-1^BW) at 14-days (Diab, 14-days) and 28-days (Diab, 28-days). (N = 6 in each group) \* p ≤ 0.05 Vs control group. ::: Parameter Control Diab (14-days) Diab (28-days) -------------------------------------- ------------- -------------------- -------------------- BW change (%) 10 ± 3 -10 ± 3\* -13 ± 2\* Blood glucose (mg.dL^-1^) 84 ± 2 480 ± 3\* 486 ± 11\* Mean arterial pressure (mmHg) 130 ± 3 120 ± 6 117 ± 2 HR (Beats per min) 419 ± 5 326 ± 7\* 339 ± 10\* Kidney weight/100 g BW 0.72 ± 0.03 1.1 ± 0.05\* 1.0 ± 0.03\* Plasma creatinine (mg.dL^-1^.kg^-1^) 2.4 ± 0.5 6.4 ± 3.5 4.5 ± 3.5 :::	PubMed Central	2024-06-05T03:55:54.197552	2005-3-5	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555576/", "journal": "Cardiovasc Diabetol. 2005 Mar 5; 4:3", "authors": [ { "first": "Hongmei", "last": "Chen" }, { "first": "Sachin", "last": "Brahmbhatt" }, { "first": "Akanksha", "last": "Gupta" }, { "first": "Avadhesh C", "last": "Sharma" } ] }
PMC555577	Background ========== The complex anatomy of coronary vessels has made the investigation of coronary flow and hemodynamics one of the most difficult and challenging studies until today. Leonardo Da Vinci (generally called as \"man ahead of time\"), developed insightful sketches (some 550 years ago) of the anatomy of the heart and its blood vessels, as shown in Figure [1](#F1){ref-type="fig"}. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Leonardo da Vinci\'s sketches showing the distribution of the heart and blood vessels. ::: ![](1475-925X-4-14-1) ::: The major cause of death in both the developed and developing countries is cardiovascular disease. The study of coronary arterial circulation is important, because it is crucial in maintaining heart perfusion and function. The process of arteriosclerosis involves the formation of atherosclerotic plaques in the coronary tree. With time, this results in stenosis of the blood vessel and in turn decreases coronary flow. When it cannot match the oxygen demand of the myocardium, it results in ischemia and thereafter in an infarct. In order to overcome this problem of diminished perfusion of the affected myocardium, CABG is one of the treatment options that are being used to improve coronary perfusion. In this procedure, new routes around narrowed and blocked arteries are constructed with both arterial and venous conduits, allowing sufficient blood flow to be delivered to the ischemic heart muscles. In spite of CABG being an effective surgical technique to revascularize the myocardium, 20--50% of bypass grafts fail due to the formation of intimal hyperplasia \[[@B1]-[@B3]\]. Previous studies have reported anastomotic intimal hyperplasia thickening to be associated with several factors, such as hemodynamic factors \[[@B4]\], compliance mismatch between graft material and host artery \[[@B5]\], biomaterials incompatibility \[[@B6]\]. For considering the influence of hemodynamic factors on graft patency, it is important to replicate the in-vivo geometry of the entire bypass conduit. In other words an arterial bypass flow model must be anatomically accurate, so as to capture the essential hemodynamics of the true geometry \[[@B7]\]. Relation between wall shear stress and intimal hyperplasia ---------------------------------------------------------- Flow regimes in end-to-side anastomosis, provide pertinent information concerning wall shear stress gradient which affects intimal hyperplasia thickening. The three- dimensional experimental studies of flow in an end-to-side anastomosis by Ojha et al \[[@B8]\] have revealed a relation between low and fluctuating wall shear stress and intimal hyperplasia. For the distal end-to-side anastomosis model, Hughes and How \[[@B9]\] have shown that intimal hyperplasia occurs in regions of flow separation at the toe and the heel, and that flow-stagnation is observed on the floor of the anastomosis. Additionally, animal model studies, conducted by Bassiouny et al \[[@B10]\], provide correlation of regions of low wall shear stress with areas of intimal hyperplasia. Using CFD for wall shear stress determination --------------------------------------------- Although wall shear stress distribution is a major factor in the onset of coronary diseases it cannot be measured directly and is hence calculated from velocity profiles. Even though these velocity profiles can be obtained in vivo using magnetic resonance (MR) or color-flow Doppler ultrasound (CDU), there are some limitations to measurements due to the small dimensions of the arteries. Thus the alternative to measure the wall shear stress is to use CFD to simulate flow in CABG geometry and thereafter compute the wall shear stress. Anastomosis geometry -------------------- Unfortunately, most of the modeling work in graft-flow is limited to only a part of the total bypass conduit geometry, namely the anastomosis site. As regards to the anastomosis geometry, Song MH et al \[[@B11]\] developed a Y-Figure anastomotic model for proximal arterial stenosis (at angles ranging from 10° to 30°), in order to analyse the three-dimensional simulation of coronary artery bypass grafting. In this work, in the end-to-side anastomosis model, all the vessels were adopted to be of the same diameter. The simulation results suggest that the more acute the angle of anastomosis, the smaller is the energy loss. Even three-dimensional CFD simulations have been performed on end-to-side anstomosis of a stenosed coronary bypass, by Bertolotti and Deplano \[[@B12]\]. Herein, the anastomosis geometry was based on the assumption that the graft and the host vessel are of the same diameter, with the graft inclined at 45° to the host vessel. The inputs into the anastomotic domain were flow-rates from the graft and from the occluded proximal artery. The flow features were compared for different flow rates and distance of the anastomosis from the site of occlusion. It was concluded that the risk of intimal hyperplasia could be minimized if the anastomosis was sutured at a sufficient distance of length. Proximal artery flow conditions ------------------------------- An important factor is the effect of proximal artery flow condition on the hemodynamics at the distal end-to-side anastomosis. This effect has been analysed by Kute and Vorp \[[@B13]\], using CFD through the idealized geometry model consisting of equal diameter vessels with the graft inclined at 30°. The boundary conditions for the anastomotic model involved blunt velocity profiles into the graft and from the proximal artery, with a fixed pressure at the distal artery outlet. The velocity vectors for all the proximal arterial flow conditions exhibited skewing towards the floor of the artery, and their distribution varied with the flow conditions in the proximal artery. Graft flow determination ------------------------ The combined use of imaging techniques and CFD may be necessary for meaningful clinical studies. With the availability of imaging techniques like coronary angiography, magnetic resonance angiogram (MRA), computed tomography (CT), several researchers have analysed the flow in realistic bypass graft models. There have also been some studies combining experimental measurements and CFD analyses \[[@B14]-[@B17]\]. Doppler analysis is generally preferred since it is safe, accurate and rapid when compared to angiography. Even though angiography certainly gives a definite answer about graft patency, there is more risk involved and it is time consuming and requires the assistance of a cardiologist. Doppler methods have been used by Bertolotti et al \[[@B18]\] and Lin et al \[[@B19]\] for graft flow analysis during minimally invasive CABG. The functioning of the graft has also been analysed through methods such as intraoperative angiography, probing of the anastomosis, electromagnetic flow meter, and the transit time flow measurement \[[@B20]-[@B22]\]. Limitations of works carried out -------------------------------- It is clear from literature review that an important step towards realistic flow simulations concerns the generation of appropriate coronary vasculature geometry (involving the entire flow domain from the aorta to the perfused artery) with physiological boundary conditions. Unfortunately, a lot of work in these areas has been carried out on subsections of this bypass flow domain \[[@B8],[@B9],[@B11]-[@B13],[@B15],[@B16],[@B18]\], and that too involving idealized geometry of the anastomosis section. Therefore in our work, we have addressed this drawback by studying the flow characteristics (by means of a three-dimensional CABG model) in the complete bypass domain under representative physiological conditions. We have thereby analysed realistic physiological simulations, and highlighted the resulting flow patterns and wall shear stress that are deemed to play a major role in intimal hyperplasia. Methods ======= Geometrical Model ----------------- The bypass models simulating the flow field of the anastomosis in the right and left aorto-saphenous bypass grafts are illustrated in Figures [2a](#F2){ref-type="fig"} and [2b](#F2){ref-type="fig"} respectively. The ascending aorta is modeled to be of length 80 mm with diameter 25 mm. The normal coronary artery is modeled to be of circular cross-section. The 100% occluded coronary artery is modeled as a straight tube, with dimensions of length 45 mm and diameter 2 mm. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Geometry (plane view) and dimensions (in mm) of the bypass models of (a) The aorto-right coronary artery bypass model. (b) The aorto-left coronary artery bypass model. (PSCA-Perfused Segment of the Coronary Artery; OSCA-Occuluded Segment of the Coronary Artery; T-Toe; H-Heel) ::: ![](1475-925X-4-14-2) ::: In Figures [2a](#F2){ref-type="fig"} and [2b](#F2){ref-type="fig"}, the venous graft is shown to have a non-uniform circular cross-section, larger than that of the coronary artery. The intersection between the graft and the coronary vessel has an elliptical shape, which is due to the deformation of the larger diameter graft due to its suturing to the smaller diameter coronary vessel surface. The dimensions are provided by our cardiac surgeon joint-author (TYS) and the model has been constructed with this data. Model Assumptions, Data Input, and Boundary Conditions ------------------------------------------------------ The blood is assumed to be incompressible, with a Newtonian behaviour having dynamic viscosity (μ) of 0.00408 Pa and a density (ρ) of 1050 kg/m^3^. The blood vessel walls are assumed to be rigid and impermeable. For a quasi-steady, three-dimensional and laminar flow, the Navier-Stokes equations (for mass and momentum conservation) governing fluid motion is written as follows: ![](1475-925X-4-14-i1.gif) ![](1475-925X-4-14-i2.gif) where ![](1475-925X-4-14-i3.gif) denotes the velocity vector in three dimensions. The distributions of velocity and wall shear stress are obtained by computationally solving the above equations subject to the boundary conditions given below. In accordance with equation (2), the flow field is automatically updated during each time-interval, by adopting the time-varying input data of the aorta (Figures [3a](#F3){ref-type="fig"} &[3b](#F3){ref-type="fig"}), the left coronary artery (Figure [3c](#F3){ref-type="fig"}) and the right coronary artery (Figure [3d](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### \(a) Flow-rate waveform at the inlet of aorta. (b) Flow-rate waveform of the ascending aorta, reverse flow seen due to 4% of the stroke volume that goes into the ascending aorta. (c) Flow-rate waveform at the left coronary artery, derived from the flow velocity waveform obtained using intravascular Doppler ultrasonic flow technique. (d) Flow-rate waveform imposed at the right coronary artery, measured using a Doppler flow meter catheter. ::: ![](1475-925X-4-14-3) ::: ### Systolic phase This flow is obtained by prescribing a uniform velocity (across the cross-section) at the inlet to the aorta, from the left ventricle (LV). The velocity magnitude is computed from the physiologically representative stroke volume over the ejection period, based on the flow wave form \[[@B23]\] shown in Figure [3a](#F3){ref-type="fig"}. Through out this period, the left ventricle contracts and squeezing the coronaries, which are embedded in the cardiac muscle. This allows very little amount of blood to flow into the coronary arteries. During systole, the inputs to the model consist of (i) monitored time-varying flow-rate waveform at the inlet of the aorta, (depicted in Figure [3a](#F3){ref-type="fig"}), adopted from Ganong \[[@B23]\], as well as (ii) monitored time-varying flow-rate waveform at the left coronary artery(depicted in Figure [3c](#F3){ref-type="fig"}) obtained from in-vivo intravascular Doppler ultrasonic technique \[[@B24]\], and (iii) monitored time-varying flow-rate waveform imposed at the exit of the right coronary artery (depicted in Figure [3d](#F3){ref-type="fig"}) obtained by means of a Doppler flow-meter catheter \[[@B25]\]. ### Diastolic phase At the start of diastole, there exists a small amount of back-flow into the left ventricle through the aortic inlet (Figure [3a](#F3){ref-type="fig"}). For the rest of the diastolic phase, the aortic valve remains closed. In all these cases, blood flows back from the ascending aorta into the coronary arteries. Herein, we take into account that a major portion (second/third) of the backflow into the ascending aorta, (flow waveform displayed in Figure [3b](#F3){ref-type="fig"}) goes into the left coronary artery (Figure [3c](#F3){ref-type="fig"}), allowing the remainder to flow into the right coronary vessel (Figure [3d](#F3){ref-type="fig"}). This yields the blood volume flowing into the left-coronary vessel (during a cardiac cycle) to be 2.67% of the stroke volume, with the remaining 1.33% of the stroke volume going into the right coronary artery. From these specified flow-rates at the left coronary artery, and at the right coronary artery, the backflow from the ascending aorta into the aortic root domain is calculated. Hence the data input to the model consists of (i) the calculated uniform velocity at the ascending aorta, and (ii) the flow conditions at the left and right coronaries obtained from the monitored time-varying input flow-rate waveforms (Figures [3c](#F3){ref-type="fig"} &[3d](#F3){ref-type="fig"}). Fluid Dynamics Simulation Setup ------------------------------- The fluid dynamics simulations are performed by using a control-volume-based technique, implemented in the computational fluid dynamics (CFD) code Fluent \[[@B26]\]. The computation procedure of the commercial code consists of (i) construction of the geometry using a pre-processor, Gambit \[[@B27]\], (ii) meshing the computation domain, (iii) assigning boundary conditions in terms of velocities and flow-rate weightings, (iv) assigning fluid properties, and (v) the solution algorithm. The geometry of the aorto-coronary bypass graft modelsis constructed in Gambit, using the dimensions provided by our cardiac-surgeon joint-author (TYS). The elements employed to mesh the computational domain consisted primarily of regular structured hexahedral elements as well as wedge elements wherever necessary. In order to carry out the mesh sensitivity analysis, numerical simulations were carried out by varying the number of mesh elements in the computational domain. Initially, the domain was discretized into 120974 hex/wedge elements. The accuracy of the simulation results was then improved by employing a finer mesh that contained 419765 elements. This number was further increased to 623138, but resulted in no significant improvement in the results. Thus to maintain a balance between the computational cost and the numerical accuracy, we concluded (based on the mesh independence test) that the appropriate number of elements for our study is 419765. In the solution algorithm used by Fluent, the governing equations are solved sequentially. Because the governing equations are non-linear (and coupled), several iterations of the solution loop need to be performed before a converging solution is obtained. Using this approach, the resultant algebraic equations for the dependent variables in each control volume are solved sequentially by a point implicit (Gauss Seidel) linear equation solver, in conjunction with an algebraic multi-grid (AMG) method. The governing equations are solved iteratively until convergence of all flow variables is achieved. The solutions of all the flow variables are deemed to have converged once their residuals computed from two successive iterations are below the set desired convergence criteria of 10^-3^. The study was also carried out by setting the convergence criteria as 10^-4^; this did not alter the results obtained earlier. Results ======= For these numerical simulations we have incorporated the unsteady flow character by dividing the cardiac cycle into a number of small time interval, and analyzing for steady flow within these time intervals. To observe the velocity distribution features of the entire flow field, the computed velocity vectors are illustrated in the plane of symmetry at two different instants of the cardiac cycle. Simulated flow field for the aorto-right coronary bypass (Figures [4](#F4){ref-type="fig"} and [5](#F5){ref-type="fig"}, for t = 0.13 sec and t = 0.7 sec respectively) ----------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: {#F4 .fig} Figure 4 ::: {.caption} ###### \(a) Velocity vectors computed at t = 0.13 sec are depicted on the centre plane of the aorto-right coronary bypass model. (b) Enlarged view of the velocity vectors inside aorta. At the start of ejection, blood from the aortic inlet flows into the ascending aorta. The blood flow at a distance of 10 mm from the entrance of the aorta behaves almost like an inviscid flow. Very little amount of flow enters the coronaries; this may be due to the high pressure in the myocardium. (c) Parabolic profiles of the velocity vectors are observed inside the graft. Slight skewing of the flow profiles is seen due to the influence of the graft curvature. (d) A close view of the recirculation region in the artery-graft junction. Major portion of the flow exiting from the graft moves towards the distal portion of the right coronary vessel. (e) The flow pattern shows slight skewing towards the floor of the artery. With increasing distance skewing disappears thus shifting the maximum velocity magnitude 0.37 m/s to the centreline of the host artery. (f) A wide variation in the wall shear stress is observed at the distal anastomotic region. The weak recirculation zone at the proximal portion of the bypassed vessel results in negligible wall shear stress with the peak wall shear stress magnitude, 5.75 Pa appearing at the floor of the artery. ::: ![](1475-925X-4-14-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### \(a) Velocity vectors computed at t = 0.7 sec are depicted on the centre plane of the aorto-right coronary bypass model. (b) At the mid-diastolic instant, the aortic valve is fully closed and thus back flow from the ascending aorta enters both the coronary vessels, the left coronary vessel and the bypassed right coronary vessel. (c) Parabolic profiles of the velocity vectors are seen inside the graft. Maximum perfusion of the graft occurs at mid-diastole. (d) Maximum flow velocity approaching the graft exit is around 0.95 m/s. The flow exiting the graft with a higher velocity results in a stronger impingement on the floor of the artery. The strong recirculation region seen at the proximal portion of the distal anstomotic region forces the flow to move towards the right coronary artery exit. (e) The inner wall of the host artery exhibits significant skewing of the velocity profiles. The maximum flow velocity magnitude, 2.04 m/s, seen close to the floor of the artery is shifted to the centre line of the vessel with increasing axial distance. (f) Maximum perfusion occurs at the mid-diastolic instant, t = 0.7 sec. This gives rise to maximum wall shear stress with the peak magnitude being 72.22 Pa. ::: ![](1475-925X-4-14-5) ::: ### (a) Flow field in the aorto-right coronary bypass model at the onset of ejection, t = 0.13 sec At the onset of ejection, at t = 0.13 sec, Figure [4a](#F4){ref-type="fig"} depicts the distribution of velocity-vectors in the flow field. The flow-velocity distributions in different sections of the flow field are depicted in Figures [4b--4e](#F4){ref-type="fig"}. The uniform velocity at the inlet to the aorta is 0.035 m/s. At a distance of 10 mm from the entrance, the computed flow field appears to be like inviscid flow. There is hardly any flow entering the graft at the start of systole as shown in Figure [4c](#F4){ref-type="fig"}. Figures ([4d](#F4){ref-type="fig"} &[4e](#F4){ref-type="fig"}) depict the flow-velocity distribution in the anastomosis domain. The flow pattern in the graft anastomed to the aorta at a distance of around 40 mm from the aorta entrance is parabolic, with very slight skewing. The forward flow coming from the graft into the host artery shows a small region of recirculation at the heel of the anatomosis domain (i.e. where the occluded (proximal) bypassed coronary artery connects to the anastomosis domain) as indicated in Figure [4d](#F4){ref-type="fig"}. The flow coming into the perfused right coronary vessel gets fully developed, with a peak velocity of 0.37 m/s, as depicted in Figure [4e](#F4){ref-type="fig"}. The computed wall shear stress (product of velocity gradient at the wall and the viscosity of fluid) is depicted in Figure [4f](#F4){ref-type="fig"} as (i) a low wall shear-stress region in the heel section of the anastomosis where there is flow-stagnation along the floor of the artery and (ii) a high wall shear-stress at the toe of the anastomosis (associated with the disturbed flow patterns, shown in Figure [4d](#F4){ref-type="fig"}) which is prone to intimal hyperplasia. ### (b) Flow field in the aorto-right coronary bypass model at the mid-diastolic instant, t = 0.7 sec At the mid-diastolic instant (at t = 0.7 sec), when the aortic valve is closed, Figure [5a](#F5){ref-type="fig"} illustrates the velocity vector plots. Some amount of the backflow coming from the ascending aorta enters into the graft, and the rest goes into the left coronary vessel as shown in Figure [5b](#F5){ref-type="fig"}. Along the graft, the peak velocity is skewed towards the outer wall. Initially the flow is seen to follow the outer wall, and then this slowly turns to the centre as demonstrated in Figure [5c](#F5){ref-type="fig"}. In the anastomosis domain, a strong region of recirculation is observed near the occluded end of the artery, which forces the flow to move into the perfused right coronary (distal) artery as indicated in Figure [5d](#F5){ref-type="fig"}. The flow in the perfused distal artery exhibits significant skewing of the velocity profiles towards the inner wall of the perfused (distal) artery, with a peak magnitude of 2.04 m/s as seen in Figure [5e](#F5){ref-type="fig"}. The flow pattern variations give us insight into the wall-shear distribution. The high velocity gradients in the anastomosis give rise to large spatial variations in the resulting wall shear stress. The largest value of wall shear stress is seen in Figure [5f](#F5){ref-type="fig"}, near the toe of the graft-artery junction. Simulated flow field for the aorto-left coronary bypass (Figures [6](#F6){ref-type="fig"} and [7](#F7){ref-type="fig"}, for t = 0.13 sec and t = 0.7 sec respectively) ---------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: {#F6 .fig} Figure 6 ::: {.caption} ###### \(a) Velocity vectors computed at t = 0.13 sec are depicted on the centre plane of the aorto-left coronary bypass model. (b) The aortic valve is opened and blood flows from the left ventricle down into the aorta. The flow inside the aorta is almost like an inviscid flow. A small amount of the flow enters the right coronary vessel and the remaining moves towards the ascending aorta. An additional flow into the ascending aorta is due to that coming from the left coronary vessel. (c) There is a change in flow direction in the graft. The reverse flow from the left coronary vessel enters the graft. Parabolic profiles of the velocity vectors are seen inside the graft. (d) Very little amount of flow moves towards the proximal portion of the left coronary vessel as it is 100 % occluded. A small region of weak recirculation is seen close to the occluded site. (e) At the start of ejection, there is a reverse flow that comes from the left coronary artery. This flow arises due to the myocardium which is subjected to the intra-myocardial pressure during early systole. As blood flows through the host artery the flow becomes fully developed, this feature no longer exists once the flow reaches the junction. (f) The low velocity gradients in the distal anstomotic junction exhibit low wall shear stress distribution. The maximum wall shear stress magnitude is 1 Pa. ::: ![](1475-925X-4-14-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### \(a) Velocity vectors computed at t = 0.7 sec are depicted on the centre plane of the aorto-left coronary bypass model. (b) The aortic valve remains fully closed at mid-diastole and the flow from the ascending aorta enters both the coronary vessels with a high velocity. (c) Significant skewing of the velocity profiles seen at the outer wall of the graft with a high velocity indicating that good perfusion occurs during mid-diastole. (d) A small amount of flow exiting from the graft moves towards the proximal portion of the bypassed left coronary vessel. As this region is 100% occluded the flow reverses thus giving rise to a recirculation region. (e) Maximum perfusion of the bypassed left coronary vessel occurs during mid-diastole. The peak velocity magnitude is 1.84 m/s seen close to the floor of the host artery. (f) The low velocity components in the vicinity of the occluded region show negligible wall shear stress while elevated shear stresses with a magnitude of 59.43 Pa in the junction region where most of the flow moves towards the distal portion of the left coronary artery. ::: ![](1475-925X-4-14-7) ::: ### (c) Flow field in the aorto-left coronary bypass model at the onset of ejection, t = 0.13 sec The distribution of velocity-vectors in the flow field at the onset of ejection, t = 0.13 sec is shown in Figure [6a](#F6){ref-type="fig"}. The aortic valve opens, and blood flows into the aorta with an entrance uniform velocity of magnitude 0.043 m/s, with some fluid entering the right coronary vessel. The flow inside the aorta behaves like inviscid flow as can be observed in Figure [6b](#F6){ref-type="fig"}. During early ejection, as the left coronary vessel is embedded in the myocardium, the myocardium is subject to intramyocardial pressure which causes some reverse flow. The effect of LV contraction on the graft flow is incorporated by adopting the measured flow profile in the left coronary vessel at a point prior to its anastomosis with graft. In this way, we simulate the reversal of flow in the graft at the onset of ejection. A parabolic velocity profile of the back-flow in the graft is seen in Figure [6c](#F6){ref-type="fig"}. Most of the fluid flows into the graft; a small amount goes towards the heel of the anastomosis, which results in a weak recirculation region at the occluded region of the bypassed left coronary vessel, as demonstrated in Figure [6d](#F6){ref-type="fig"}. The back-flow in the distal portion of the left coronary vessel (coming into the anastomosis domain) gets fully developed as it approaches the anastomosis domain, with a maximum velocity of magnitude 0.074 m/s seen along the centreline of the vessel as shown in Figure [6e](#F6){ref-type="fig"}. The low velocity gradients in the distal anastomotic junction result in low wall shear stress, with maximum magnitude of 1 Pa, as shown in Figure [6f](#F6){ref-type="fig"}. ### (d) Flow field in the aorto-left coronary bypass model at the mid-diastolic instant, t = 0.7 sec The velocity vectors plots at mid-diastole are displayed in Figure [7a](#F7){ref-type="fig"}. The aortic valve remains closed, and the flow entering the coronaries is caused by the back flow from the ascending aorta as shown in Figure [7b](#F7){ref-type="fig"}. Most of the fluid enters the graft, exhibiting significant skewing with high velocity magnitude, resulting in maximum perfusion of the host artery as depicted in Figure [7c](#F7){ref-type="fig"}. In the anastomosis domain, a strong recirculation region is seen in the heel region of the anastomosis at the distal end of the bypassed vessel, Figure [7d](#F7){ref-type="fig"}. Maximum perfusion occurs during the mid-diastole phase. The velocity distribution in the perfused host artery segment shows significant skewing towards the floor of the artery (Figure [7e](#F7){ref-type="fig"}), with a peak velocity magnitude of 1.84 m/s. In the anastomosis heel region (in the occluded arterial segment) there is negligible shear stress. However, elevated shear stress (of magnitude 59.43 Pa) is seen in the anastomotic toe region (as shown in Figure [7f](#F7){ref-type="fig"}), where most of the flow moves towards the distal segment of the host coronary vessel. Discussion ========== Model Geometry -------------- Our model differs from other models in the following ways: (i) both the proximal and distal anastomotic regions are included, while other works \[[@B8],[@B9],[@B11]-[@B13],[@B15],[@B16],[@B18]\] have emphasized the distal anastomotic site alone, (ii) the dimensions taken up are close to true dimensions at surgery, unlike the idealized geometries cited in previous investigations\[[@B11]-[@B13],[@B18]\] that had all vessels of equal diameters, (iii) the anastomosis geometry is also more realistic compared to the idealized junction angles adopted in previous works, and (iv) our model incorporates the varying cross-section of the graft, that alters gradually along its length from a circular to an elliptical shape in order to fit the smaller artery at the distal anastomosis (thus attempting to reproduce the same geometry as obtained with the surgical procedure). Flow patterns ------------- Our results for the aorto-right coronary bypass model and the aorto-left coronary bypass model at two different instants of the cardiac cycle, clearly reveal the following features: (i) at the onset of ejection, in the aorto-right coronary bypass model, very little flow enters the graft with a velocity magnitude around 0.0495 m/s; the maximum flow velocity inside the graft is around 0.2 m/s; (ii) at the mid-diastolic instant, the flow profile in the graft is skewed towards the outer wall, with the peak velocity increasing as it travels downstream; close to the graft exit, the maximum flow velocity attained is around 1 m/s; (iii) at the onset of ejection, in the aorto-left coronary bypass model, there is a backflow from the left coronary artery into the graft; the peak velocity of flow at the entrance to the graft is 0.0788 m/s, and very little perfusion is given to the host artery; (iv) at the mid-diastolic instant, the graft perfusion is maximum, with peak velocity magnitude of 1.1 m/s. Features -------- Our study confirms that blood flow through the coronary artery bypass graft primarily occurs only during the diastolic phase of the cardiac cycle. This is in agreement with the physiological observation of coronary blood flow. There is however some difference between the flow patterns in the right and left coronary graft at the onset of ejection, with some backflow from the left coronary artery into the bypass graft which is not obtained in the case of the right coronary arterial bypass. The phenomenon (of reversal of flow during systole) can be explained by the predominant intra-cardiac course of the left coronary artery system. This aspect is also an original feature of our work. Lastly, our study has also shown (i) a low wall shear-stress region near the heel region of the anastomosis domain, and (ii) a high wall shear-stress in the toe region of the anastomosis domain, making it prone to intimal hyperplasia. This may have some clinical significance. We should take this into consideration in designing a coronary anastomotic device, so as to minimize biomechanical injuries to the coronary arterial wall. In doing so, we can alleviate or retard the development of intimal hyperplasia, which is the Achilles heel of the effectiveness of coronary artery surgery with saphenous vein. Conclusion ========== The computed results have revealed that (i) maximum perfusion of the occluded artery occurs during mid-diastole, and (ii) the maximum wall shear stress variation was observed around the toe of the anastomotic region. According to our cardiac surgeon joint author (TYS), this preliminary result can enable the clinicians to have a better understanding of vein graft disease, and hopefully we can offer a solution to alleviate or delay the occurrence of vein graft disease. Authors\' contributions ======================= MS carried out the computational fluid dynamic studies and drafted the manuscript. DNG and LPC guided the study, helped in interpretation of results and critically reviewed the manuscript. YST provided the surgical aspect of the study, and the dimensions for the model. All authors have read and approved the final manuscript.	PubMed Central	2024-06-05T03:55:54.200303	2005-3-4	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555577/", "journal": "Biomed Eng Online. 2005 Mar 4; 4:14", "authors": [ { "first": "Meena", "last": "Sankaranarayanan" }, { "first": "Leok Poh", "last": "Chua" }, { "first": "Dhanjoo N", "last": "Ghista" }, { "first": "Yong Seng", "last": "Tan" } ] }
PMC555578	Background ========== The mechanical properties of canine trabecular bone in the femoral head are important for a better understanding of normal biomechanics of the bone and are needed for assessing changes occurring under pathological conditions like osteoarthritis of the hip, osteonecrosis or Legg-Calvé-Perthes disease of the femoral head. Particularly the elastic moduli are important for finite element modeling of the proximal femur. Several methods have been used in the literature for identifying the elastic modulus of bone, such as mechanical testing \[[@B1]-[@B4]\], combinations of micro-computed tomography and finite element modeling \[[@B4]-[@B11]\], and ultrasonography\[[@B12]\] including acoustic microscopy \[[@B13]\]. Some studies have investigated canine bone \[[@B13]-[@B19]\], but to our knowledge no publication has presented any details about directional elastic moduli of canine femoral heads including degrees of anisotropy. Neither is it clear whether assuming isotropy on the tissue level is a justified simplification for trabecular bone in canine femoral heads as Kabel et al.\[[@B9]\] reported in a study of whale bone specimens. Therefore, in this study we determined Young\'s moduli of trabecular bone obtained from healthy canine femoral heads by ultrasonography. We then calculated degrees of anisotropy and used statistical testing in order to estimate whether assuming isotropy of the trabecular tissue might be a valid simplification. Methods ======= Eight dogs (weight 30--63 kg) were selected that had been euthanized for several medical reasons. From each dog both femora were obtained and were examined by a veterinarian for signs of metastatic malignant disease, Legg-Calvé-Perthes disease, osteoarthritis of the hip or bone necrosis. An x-ray of the whole femur was obtained with the femoral head and the intertrochanteric region placed directly on the film, and the main pressure and tensile trajectories were marked on the image. The bones were kept moist, wrapped in plastic bags and stored at -21°C. Each bone was placed on the x-ray image and the direction of tension and pressure trajectories were marked on the bone according to the x-ray template. An orthogonal coordinate system was defined (Fig. [2](#F2){ref-type="fig"}). The positive x-axis was oriented along the main pressure trajectories and the y-axis was aligned with the main tension trajectories. One cubic specimen of 10 × 10 × 10 mm was cut (Fig. [1](#F1){ref-type="fig"}) from each frozen femoral head using a precision bone saw (Exakt Makro 310 CP, EXAKT Apparatebau, Norderstedt, Germany). The edges of the cubes were cut parallel to the x, y and z axes of the coordinate system. The cubic specimens were weighed using a laboratory scale (Acculab ALC-110.4, Acculab Europe, Göttingen, Germany). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Schematic representation of femoral head and neck. The main tensile and compressive trajectories and the orientation of the cubic specimen and coordinate system are shown. ::: ![](1475-925X-4-17-2) ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Cubic specimen cut from one canine femoral head. ::: ![](1475-925X-4-17-1) ::: For sonographic testing, a specially designed device with a custom-made ultrasound transducer (Institute of Materials Science, University of Hannover) was used. An ultrasound frequency of over 2 MHz was chosen for measuring the material properties of trabecular bone tissue\[[@B20]\]. The ultrasound frequency was adjusted so that a clear signal could be detected by the ultrasound receiver. A frequency of 100 MHz was chosen because ultrasound signals remained undetectable when using lower frequencies even with maximum power. The cubic specimens were placed in a container after thawing and immersed in standard Ringer\'s solution at room temperature. An ultrasound receiver was placed at the surface of the cube opposite the transducer which was also directly touching the specimen surface, and the runtime through the bone material of each bone cube (n = 16) was recorded ten times in all three orthogonal directions. The edge lengths of each cube were measured using the digital image analysis system IMAGE C^®^(IMTRONIC GmbH, Berlin, Germany), and the specimen volumes were calculated. Specimen densities were determined by equation (1): ![](1475-925X-4-17-i1.gif) where ρ is specimen density, m is specimen mass and V is the specimen volume The ultrasound wave runtimes were processed by excluding the minimum and maximum results of the ten subsequent measurements, and the average of the remaining results was calculated. The transmission velocity was calculated by equation (2) and the elastic modulus was determined using equation (3). ![](1475-925X-4-17-i2.gif) where c~long~is the transmission velocity, s denotes the edge length of the specimen and equals the distance between ultrasound transmitter and receiver which are placed in direct contact with the opposing specimen surfaces, t~1~and t~0~the time at reception and sending of the ultrasound wave, respectively. ![](1475-925X-4-17-i3.gif) where E~x,y,z~is Young\'s modulus along x, y and z axes and ρ is the specimen density calculated from equation (1). C~long~is the ultrasound velocity calculated from specimen edge length and transmission time (see equation (2)) Mean density and mean Young\'s moduli of the specimens and standard deviations were determined using the statistical software package SPSS 12.0 (SPSS, Chicago, USA). Strong correlations between density and elastic moduli could be expected from equation (3), nonetheless Pearson correlations were calculated for confirmation. The directional elastic moduli were checked for significant differences using one-way ANOVA (analysis of variance). The degrees of anisotropy \[[@B4],[@B21]\](E~x~/E~z;~E~x~/E~y~; E~z~/E~y~) were calculated and subsequently checked for a significant relationship with specimen density using Pearson correlations. Results ======= The edge lengths of the bone specimens varied by ± 1 % (± 0.1 mm). Elastic moduli in the bone specimens ranging from 6.3 to 14.3 GPa were found. The elastic moduli in X, Y and Z direction were 11.2 ± 0.4 GPa, 10.5 ± 2.1 GPa and 10.5 ± 1.8 GPa, respectively. Minimum, maximum, mean values and standard deviations of bone sample density and directional Young\'s moduli are listed in Table [1](#T1){ref-type="table"}. Pearson correlations between density and directional Young\'s moduli (E~x~, E~y~and E~z~) were significant as could be expected from equation (3) (p \< 0.005). The degrees of anisotropy ranged from 0.82 to 1.59 and were significantly correlated with specimen density (Table [2](#T2){ref-type="table"}, Figure [3](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Degrees of anisotropy in dependence of specimen density. Correlation coefficients are listed in the symbol legend. ::: ![](1475-925X-4-17-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Minimum, maximum and mean values for sample densities and directional Young\'s moduli Moduli are arranged according to testing direction (along x, y and z axes, see fig. 2). ::: Minimum Maximum Mean Standard deviation ------------------------- ------------- ------------- ---------- ------------------------ Density \[g/cm^3^\] 1.19 1.51 1.40 0.09 E~X~\[MPa\] 10600 11760 11217 376 E~Y~\[MPa\] 6283 14285 10459 2071 E~Z~\[MPa\] 6832 13097 10506 1839 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Degrees of anisotropy The minimum and maximum degrees of anisotropy (range), the mean values, standard deviations (SD) and the correlation coefficients (r) indicating correlation of the respective degree of anisotropy with specimen density ρ. p designates the significance probability (p-value). ::: Range Mean SD r p --------------- ------------ ---------- -------- ------- ------- E~x~/E~z~ 0.89--1.59 1.10 0.18 -0,93 0.000 E~z~/E~y~ 0.92--1.11 1.01 0.05 -0,58 0.009 E~x~/E~y~ 0.82--1.73 1.11 0.22 -0,96 0.000 ::: Discussion ========== This study presents detailed data about mechanical properties of canine femoral trabecular bone tissue and degrees of anisotropy. Despite the strengths of our work, some limitations have to be noted. The correct calculation of the sample volume depends on exactly cubic specimens, but deviations might occur due to errors in the sawing technique. We found a maximum variation of edge length of ± 0.1 mm (1%) in our specimens so this error appears to be negligible. The apparent densities of the specimens were calculated by weighing whole samples and measuring sample volumes instead of calculating the densities of ashed samples\[[@B2],[@B19]\] or cleaning the bone marrow out of the specimens using water jets prior to measurement. According to Rho \[[@B20]\] ultrasonic waves at frequencies of \>2 MHz travel along the trabecular material and allow calculation of the elasticity of the trabecular bone material rather than the elasticity of whole specimens which is investigated by conventional compression testing. Considering this statement, in our study using a 100 MHz ultrasound transducer the application of apparent densities rather than the densities of the trabecular bone material might lead to an conspicuous underestimation of the elastic modulus (equation (3)) because it is expected that the density of the trabecular bone tissue is higher than the density of whole specimens. However, Kang et al. \[[@B22]\] measured densities of cylindrical trabecular bone specimens from canine femoral heads and reported a mean density of 1.17 ± 17 g/cm^3^for whole bone cylinders after cleaning and 0.65 ± 0.09 g/cm^3^for ashed samples which is much lower than our results. The lower densities might be caused by the specimen volume that was used by the authors: they geometrically measured the volume of the cylindrical bone specimens so that a considerable intertrabecular volume is included and bone tissue densities are underestimated according to equation (1). Hence the results of Kang\'s publication can not be used for comparison or correction of our density data. It is noticeable that in our study we found a small relative standard deviation (SD/mean) in apparent densities and Young\'s modulus in x direction (6.6 % and 3.3 %) although we used femora from a heterogeneous selection of different breeds. However, the relative SD in y and z directions was computed to be 19.5 % and 17.5 %, respectively. It is not clear whether the broader range of elastic moduli in these directions reflects real differences or whether it might be caused by a less accurate positioning of the saw when cutting the specimens from the femoral heads. The significant relationship between the specimen density ρ and directional elastic moduli found in our study was to be expected because Young\'s modulus was calculated from ρ. This significant relationship was also described in a study using compression testing of bone cubes from human donors (0.74 \<= r \<= 0.84; p \< 0.001) \[[@B2]\]. No significant differences were found between directional elastic moduli (p = 0.34). This result could support the concept of an \"effective\" isotropic elastic tissue modulus as described by Kabel et al.\[[@B9]\]. Several works have been published concerning elastic moduli of canine or human bone. Studies that investigated the apparent elastic modulus of human bone specimens using mechanical testing and finite element models found much lower elastic moduli\[[@B4],[@B21]\] than we did in our work. Kang et al. \[[@B22]\] reported elastic moduli of trabecular specimens from canine femoral heads of 428 ± 237 MPa which is also much lower than what we observed in our study; their results were obtained by conventional compression testing. Several other studies have been carried out investigating elastic moduli of canine trabecular bone specimens\[[@B14],[@B16],[@B19]\]. Those results are not comparable with our data because the authors measured elastic moduli of whole specimens rather than Young\'s moduli on the tissue level. Additionally, Odgaard et al. reported that conventional compression testing underestimates Young\'s modulus by about 20%\[[@B23]\]. Keaveny et al. \[[@B24]\] found a percentage difference in modulus when using platens compression testing of up to 86%. They recommend using the endcap technique for obtaining more accurate data, which however restricts testing to one direction. Jacobs et al. \[[@B25]\] investigated porous samples made from bone cement. Using finite element modeling and mechanical testing, they found that the mean error when using parallel platen compression testing was 8 or 15% depending on FE mesh size and was reduced to 2 or 0.5% with the endcap technique. Only one study investigating elastic moduli of canine trabecular bone tissue is available to our knowledge: Jorgensen and Kundu \[[@B13]\] used a 1 GHz acoustic microscope for examining a trabecular strut obtained from a canine distal femur. They computed a mean Young\'s modulus of 19.9 ± 2.5 GPa which is higher than our results; this could be caused by a different trabecular structure and higher bone volume fraction in the distal femur. The authors state that anisotropy was clearly detected at micrometer level, but no further quantification is given. More studies are available reporting mechanical properties of human trabecular bone. Rho \[[@B20]\] found an elastic modulus of 14.9 ± 1.7 GPa in trabecular bone specimens from human tibiae using a 2.25 MHz transducer. This modulus is higher than in our data but within the same order of magnitude. Ashman and Rho \[[@B26]\] measured elastic moduli from three human trabecular bone specimens using an ultrasonic technique and found a mean elastic modulus of 13.0 GPa which is about 30% higher than in our study of canine bone. This observation is supported by Kuhn\'s \[[@B19]\] assertion that elastic moduli are higher in human trabecular bone than in canine bone. Our results are further supported by Zysset et al. \[[@B27]\] who used a nanoindentation technique and found average elastic moduli in trabecular lamellae of human femoral necks of 11.4 ± 5.6 GPa. Conclusion ========== Our study provides detailed data about elastic moduli and degrees of anisotropy of canine femoral bone tissue. No significant differences between directional Young\'s moduli were found indicating that the concept of an effective isotropy of trabecular bone in canine femoral heads might be a justified simplification. The degrees of anisotropy were highly correlated with specimen densities. The results of elastic moduli are comparable to similar studies of canine and human trabecular bone tissue \[[@B13],[@B20],[@B26],[@B27]\]. Authors\' contributions ======================= TP carried out the statistical analysis of the results, took part in the detailed design of the study and prepared the manuscript. AB designed the study, prepared the bone specimens, conducted the ultrasound measurements and calculated the elastic moduli. UV and AML obtained the canine femora, examined the bone for exclusion criteria and prepared the bone specimens. BAB, IN and HW designed the study concept from a technical and medical perspective, respectively, and corrected the manuscript. Acknowledgements ================ We wish to thank the Institute of Materials Science, University of Hannover, for providing the experimental setup of the ultrasound tests. This study was supported by a grant from Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 599, project D6). We also thank Dr. Hoy, Biometrical Institute, Hannover Medical School for his advice concerning statistical testing.	PubMed Central	2024-06-05T03:55:54.203568	2005-3-17	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555578/", "journal": "Biomed Eng Online. 2005 Mar 17; 4:17", "authors": [ { "first": "Thomas", "last": "Pressel" }, { "first": "Anas", "last": "Bouguecha" }, { "first": "Ute", "last": "Vogt" }, { "first": "Andrea", "last": "Meyer-Lindenberg" }, { "first": "Bernd-Arno", "last": "Behrens" }, { "first": "Ingo", "last": "Nolte" }, { "first": "Henning", "last": "Windhagen" } ] }

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