Drosophila erecta and D. yakuba with respect to the D. melanogaster species complex has been a subject of controversy. All three possible groupings of the species have been reported in the past, though recent multi-gene studies suggest that D. erecta and D. yakuba are sister species. Using the whole genomes of each of these species as well as the four other fully sequenced species in the subgenus Sophophora, we set out to investigate the placement of D. erecta and D. yakuba in the D. melanogaster species group and to understand the cause of the past incongruence. Though we find that the phylogeny grouping D. erecta and D. yakuba together is the best supported, we also find widespread incongruence in nucleotide and amino acid substitutions, insertions and deletions, and gene trees. The time inferred to span the two key speciation events is short enough that under the coalescent model, the incongruence could be the result of incomplete lineage sorting. Consistent with the lineage-sorting hypothesis, substitutions supporting the same tree were spatially clustered. Support for the different trees was found to be linked to recombination such that adjacent genes support the same tree most often in regions of low recombination and substitutions supporting the same tree are most enriched roughly on the same scale as linkage disequilibrium, also consistent with lineage sorting. The incongruence was found to be statistically significant and robust to model and species choice. No systematic biases were found. We conclude that phylogenetic incongruence in the D. melanogaster species complex is the result, at least in part, of incomplete lineage sorting. Incomplete lineage sorting will likely cause phylogenetic incongruence in many comparative genomics datasets. Methods to infer the correct species tree, the history of every base in the genome, and comparative methods that control for and/or utilize this information will be valuable advancements for the field of comparative genomics.The phylogenetic relationship of the now fully sequenced species  Drosophila melanogaster species subgroup, a group of flies with three fully sequenced species in which phylogeny has been a source of controversy. Although the bulk of the data support a phylogeny with Drosophila melanogaster as an outgroup to sister species Drosophila erecta and Drosophila yakuba, large portions of their genes support alternative phylogenies. According to the authors, the most plausible explanation for these observations is that polymorphisms in the ancestral population were maintained during the two rapid speciation events that led to these species. Subsequent to speciation, polymorphisms were randomly fixed in each species, and in some cases non-sister species fixed the same ancestral polymorphisms, while sister species did not. In these cases the genes are correctly inferred to have conflicting phylogenies. The authors note that rapid speciation events will often lead to such conflict, which needs to be accounted for in evolutionary analyses.To take full advantage of the growing number of genome sequences from different organisms, it is necessary to understand the evolutionary relationships (phylogeny) between organisms. Unfortunately, phylogenies inferred from individual genes often conflict, reflecting either poor inferences or real variation in the history of genes. In this study, the authors examine relationships within the  Drosophila, the field of comparative genomics is now presented with the opportunity and challenge of understanding the function and history of every base in the model organism Drosophila melanogaster (Dmel). This process will hopefully result in the discovery of new biological phenomena and the development of new methodologies that will eventually help with the task of annotating other clades in the tree of life, particularly the human genome. Because most analyses of multiple genome sequences involve inferences about evolutionary history, they require an accurate description of the relationship of the species being analyzed.With the sequencing of 12 species from the genus Drosophila has been the subject of numerous studies, and the consensus from the literature suggests that the relationship of the 12 sequenced species is well resolved, with the exception of the species within the Dmel species subgroup and perhaps the placement of the Hawaiian species, D. grimshawi, and the virilis-repleta species, D. virilis and D. mojavenis [Dmel species group, the placement of D. erecta (Dere) and D. yakuba (Dyak) relative to the Dmel lineage has been the subject of numerous conflicting studies [Dmel, Dere, and Dyak, all three of the possible phylogenies ), which we shall refer to as tree 1, was supported by studies of polytene chromosome banding sequences [COI and COII mitochondrial genes [fru gene [Cu/Zn SOD gene [H3 gene family [Adh, Adhr, Gld, and ry [Adh, Amyrel, janA, janB, and Sod [,Dyak), which we shall refer to as tree 2, was supported by studies of an internal transcribed spacer region of ribosomal RNA genes [Amy gene [Adh gene [,Dere), which we shall refer to as tree 3, was supported by studies of protein electrophoresis [Adh gene [Amy gene [Dere and Dyak as sister taxa relative to the Dmel lineage. That Ko et al. found such strong support for tree 1, despite using the Adh gene, which on its own has been found to support the other two trees, suggests that the past incongruence was likely the result of sampling variance [The species history of the genus ojavenis . Within  studies ,6–15. Cologenies  have recequences , the COIal genes , mitochoal genes , the frufru gene , the Cu/SOD gene , the H3 e family , a concae family , a conca, and ry , and a c and Sod . The topNA genes , nucleotAdh gene ,21. The phoresis , mitochophoresis , the AdhAdh gene  and the variance ,23. Incovariance –28 that variance –31, as wvariance  and incovariance ,33–48.Dere and Dyak in the Dmel species subgroup, using the newly sequenced genomes in the genus Drosophila. Although we found that tree 1, placing Dere and Dyak as sister species, is the best-supported tree, we found genome-wide incongruence in substitutions, insertions/deletions (indels), and gene trees. We show that the branch separating the split of Dmel from the split of Dere and Dyak is sufficiently short that incomplete lineage sorting is a plausible explanation for the incongruence. We further show that the support for the three possible trees is nonrandomly distributed across the genome such that adjacent genes supporting the same tree are more likely in regions of low recombination, and substitutions supporting the same tree are most enriched roughly on the same scale as estimates of linkage disequilibrium, consistent with theoretical predictions under the coalescent [In this study, we set out to examine the possible causes of incongruence in this phylogeny and to investigate the placement of alescent . We testSophophora, we mapped Dmel gene annotations onto each unannotated genome. Dmel coding sequences  were mapped to potential orthologous regions in each species using TBLASTN, and GeneWise was used to build gene models based on the Dmel gene in each region. These GeneWise models were matched back to Dmel translations using BLASTP, and genes for which clear orthologs could be found were used in downstream analysis  for our initial analysis of the placement of Dere and Dyak within the Dmel species subgroup (we examine the effects of species choice on our results below). Dmel was chosen because the annotations were mapped from Dmel, and it is the primary model organism of the subgenus. D. simulans (Dsim) and D. sechellia (Dsec) were excluded from initial analysis because they were assumed to provide mostly redundant information to Dmel and they reduced the number of clear orthologs spanning the species by 2,544 genes, presumably because of lower sequence coverage and issues regarding the assembly of polymorphic reads in Dsim. Dana was chosen over D. pseudoobscura (Dpse) because it is the closest fully sequenced outgroup to the Dmel species subgroup. More than 9,000 genes  were found to have clear orthologs in all four of the chosen species. Of these seven subgenus Dere and Dyak together, was found to have the most support  methods ,54 to meThe median synonymous divergence trees for the sets of genes supporting each tree are: :0.0664,dana:1.3246) for tree 1, :0.0498,dyak:0.0757,dana:1.2871) for tree 2, and :0.0454,dere:0.0719,dana:1.3147) for tree 3 . The brat), where t is the time between speciation events in units of generations / 2Ne and Ne is the effective population size [t. In order to go from an estimate of the informative divergence to this probability, the substitutions per site per year, the ancestral generation time and the ancestral population size must be known. Synonymous substitutions per site per year has been estimated to be in the range of 1–2 × 10−8 in Drosophila [Dmel species subgroup is about ten and can be used as an estimate for the ancestral generation time [6 to 107, but this should be considered a poorly resolved parameter [5 to 1.9 × 106 years, or 9.5 × 106 to 1.9 × 107 generations. The range of values for t becomes 0.48 to 9.5, which produces probabilities for congruence in the range of 0.59 to 0.99995  = 1 − 2 / 3exp.Given that we observed incongruence in individual sites as well as for whole genes, we wanted to better understand the extent to which sites supporting the same tree are spatially correlated, with a particular interest in the compatibility of this structure with the incomplete lineage-sorting hypothesis. The above analysis of gene trees suggests that sites can be correlated out to the length of genes. To see if this correlation extends beyond individual genes we looked for blocks of adjacent genes supporting the same gene and tested for unusual block lengths. Using permutations of ML gene tree states to obtain significance, we found gene tree block lengths at expected frequencies, with the exception of an excess of long blocks supporting tree 3 in the range of 250 kb to 700 kb, three of which were highly significant (Dmel recombination rates [p < 0.1) as well for blocks for each specific tree, with tree 2 blocks showing the strongest correlation . These weak correlations suggest a minor role for recombination rates in determining the spatial structure of support for different trees across the genome; however, there are many reasons for why strong correlations would not be expected, including poorly conserved recombination rates across these species [If the blocks of genes supporting the same tree were the product of incomplete lineage sorting, then regions of low recombination ought to have larger blocks . Althougon rates . We foun species –69 and g species –72. NoneX2 test, p < 10−10).Using the whole-genome frequencies of informative amino acid and nucleotide substitutions supporting each tree, we looked to see if sites supporting the same tree are locally enriched across chromosomes . This is expected, as recombination is not thought to occur in the mitochondrial genome. While mitochondrial evolution differs from nuclear evolution in more ways than just recombination , the comThus far we have presented results suggesting that incomplete lineage sorting is a plausible explanation for the observed incongruence. We next sought to rule out alternate explanations.Is the incongruence in gene trees unexpected given the strength of support for each inference? To address this question, we used the bootstrap  value, RWe next tested whether the incongruence is robust to model choice. An empirical study of model choice and accuracy by Ren et al found that codon-based models are able to recover both recent and deep divergences well, while nucleotide-based models are less efficient at deep divergences and amino acid–based models are less efficient at recent divergences . They alComparing congruence across models, simpler models seem to produce more congruence than more complex models . For eacSophophora: Dmel, Dsim, Dsec, Dere, Dyak, Dana, and Dpse. All 21 possible species combinations that include Dere and Dyak and at least one of Dmel, Dsim, and Dsec, as well as at least one of Dana and Dpse, were considered. The HKY model was used both because it was found to produce the most congruence in the original four species as well as because it is considerably more computationally efficient than the codon models. Across all species combinations, incongruence is consistently greater than expected from bootstrap values, suggesting that incongruence is not species choice dependent  contributes most to congruence, while Dsim (80.8%), Dsec (80.4%), and Dpse (79.7%) contribute roughly equally and Dmel (78.9%) actually contributes least to congruence. We note that the presence of Dmel in the most congruent species combination goes against this general trend, perhaps reflecting further complexities in the impact of species choice on congruence.Ranking species combinations by levels of congruence reveals that our original species choice produces the most congruence , suggestAlthough the incongruence appears to be robust to model and species choice, a much more stringent test is to look at incongruence in the partition of genes that consistently support the same tree across all models and across all species combinations . Of the Dmel, Dere, Dyak, and Dana species combination to look at congruence as a function of bootstrap value. As shown in X2 test, p < 10−3).To assess the statistical significance of the incongruence in the partition of genes consistent across all models and species combinations , we usedDmel, Dere, Dyak, and Dana species combination and the HKY model. Interestingly, each tree-specific concatenation supported its tree with 100% bootstrap support [To further test whether the statistical support from the incongruent genes is the result of consistent signal, as opposed to having hidden support  for tree support . Thus, tWe next looked at sequence and evolutionary properties of the genes supporting each tree to see if any clear biases could explain the incongruence. The properties we examined are sequence quality, gene length , base composition (GC content) across the species at each position in the codon, transition–transversion ratio (kappa), ratio of nonsynonymous to synonymous divergence (dN/dS), informative synonymous divergence (ISD), ratio of informative synonymous divergence to noninformative synonymous divergence (RINSD), and total synonymous divergence (TSD). The strongest and most consistent correlations with bootstrap value are for ISD and RINSD , which aDana  ,93,94 anDere and Dyak as sister species, relative to the Dmel lineage, in the Dmel species subgroup, using the fully sequenced genomes of seven species in the subgenus Sophophora. Although we did find that the best-supported phylogeny is that which places Dere and Dyak as sister species, we also found pervasive incongruence of substitutions, indels, and gene trees  would b complex ) and theubgroups , would aDrosophila, we believe our findings have important implications for comparative genomics as a whole. The idea that speciation events have occurred in rapid bursts throughout the tree of life [Although this study should prove quite valuable to the increasing numbers of comparative genomics researchers studying the genus  of life –102 is l of life ), but thWe envision three types of methods that will need to be developed to appropriately account for this kind of variation. The first are methods that can infer the most likely species tree using an entire genome in a single calculation, considering lineage sorting explicitly. The second are methods that can infer the most likely history of every base in every species, given the species tree. Last, comparative genomics methods that use phylogenies would need to be altered to control for and utilize the output from the second kind of method. Progress is being made in the first two categories ,104–113,Dmel release 4.2 genome, cDNA, and translation sequences were downloaded from Flybase (http://www.flybase.net). Prepublication assemblies for Dere and Dana , sequenced and assembled by Agencourt Bioscience (http://www.agencourt.com), and for Dsec , assembled and sequenced by the Broad Institute (http://www.broad.mit.edu), were downloaded from the Berkeley AAA website (http://rana.lbl.gov/drosophila). The prepublication assemblies for Dyak  and Dsim  were downloaded from the Washington University School of Medicine Genome Sequencing Center's website (ftp://genome.wustl.edu/pub). The Dpse v1.04 assembly was downloaded from Flybase. Assemblies can be found in http://ncbi.nlm.nih.gov/Traces/trace.cgi; species_code, “DROSOPHILA ERECTA,” “DROSOPHILA YAKUBA,” “DROSOPHILA ANANASSAE,” “DROSOPHILA SIMULANS,” “DROSOPHILA SECHELLIA,” “DROSOPHILA PSEUDOOBSCURA”).Dmel gene models onto the unannotated genome in a pairwise fashion using a modified reciprocal–BLAST approach [Dmel translation, we used the protein sequence as a NCBI TBLASTN [−3) against the scaffolds of the target assembly. (2) The scaffolds were ordered by the hit e-value reported by TBLASTN, and up to two regions were selected from the two best scaffolds and used as input to construct gene models using GeneWise. To improve the chance of constructing a complete gene model using GeneWise, the regions were selected by clustering high-scoring pairs on the scaffold such that every high-scoring pair within 100 kb of another high-scoring pair was included in the same region, and a buffer of 10 kb was included at the ends of the regions. (3) The predicted translations of the models reported by GeneWise were then used as BLASTP queries against a database of Dmel translations, with an e-value threshold of 1 × 10−3.Each of the sequence assemblies were annotated separately by mapping approach  to assigapproach ,118, to  TBLASTN  query  the rank of alternative translations from the gene corresponding to the starting Dmel translation, and (3) whether or not there were highly ranked hits to genes other than the gene corresponding to the starting Dmel translation. One-to-one orthology was assigned when the only top-ranked hits in the BLASTP results were translations from the gene corresponding to the starting Dmel translation. Hits that had e-values within one order of magnitude were considered equivalently ranked. For genes with more than one translation with clear orthologs in each species, the first historically annotated (translation with the lowest letter ID) was used to represent the gene.We then assigned orthology/paralogy relationships using a heuristic algorithm that takes into account (1) the rank of the starting cDNA and translation sequences can be found in Dmel and Dana together and Dere and Dyak together. Likewise, tree 2 grouped Dmel and Dere together and tree 3 grouped Dmel and Dyak together.Informative substitutions supporting each tree were counted across all cDNA and peptide alignments. Only single substitutions that split the four species into two groups of two were considered. Informative substitutions for tree 1 grouped Dana and one of the ingroup species. Such insertions, where the inserted sequence is the same in the two species containing it, provided strong, unambiguous characters.Informative indels supporting each tree were counted across all peptide alignments. Indels were classified as informative in the same way that substitutions were. Indels were further filtered to avoid artifacts from alignment errors. Only indels with five amino acids of perfect identity in flanking sequences, with no mono-, di-, or tri-amino acid repeats, were included. Insertions were inferred based on an absence in ,Dana); tree 2, ,Dyak,Dana); and tree 3, ,Dere,Dana) (see The Codeml program of the PAML package (version 3.14) ,120 was ana) see . Codeml Dmel, calculated by Hey and Kliman [R statistic, were downloaded. The average R in each block was calculated where a gene could be found in their set. The Pearson correlation of the average R within blocks and the length of blocks was calculated using the R statistics package [Based on the ML tree for each gene, the genome was divided up into blocks supporting each tree. A ten-gene sliding window was used to calculate a running average of the support for each tree along each chromosome. Each window was assigned a tree based on the most frequent genealogy in the window. Each gene was then reassigned a tree based on the most frequent tree of all the windows that contained it. This effectively allows the neighbors of a gene to influence its assignment, and near neighbors have more influence than far neighbors. Adjacent genes that support the same tree were combined together into blocks. To measure the significance of the size of the blocks, the labels for each gene in the genome were randomized 1,000 times and the blocks were recalculated for each replicate, using the windowing method described above. Recombination rates for a subset of genes in d Kliman  using th package .X2 significance of windows was calculated by comparing the observed frequencies of informative mutations supporting each tree with the genome averages of those frequencies.Informative substitutions in genes were used to look at the structure of support for the different trees across the genome independent of the likelihood inference. The counts of each type of informative substitution were calculated in 60 nonoverlapping 1-kb windows surrounding each informative substitution across all chromosomes. The frequency of each kind of informative substitution across the whole genome was used to calculate an expected count for each 1-kb window. In each window, the enrichment of informative substitutions supporting the same tree was calculated. The RELL bootstrap values  from 10,All models were run using the same settings as described above for F3×4 except where HKY (model = 4) or WAG+F (model = 3) was specified and where the gamma function was used .L is the likelihood of the model given the data, and N is the degrees of freedom [Akaike's information criterion (AIC) was calculated as AIC = −2 ln L + 2 N, where  freedom . Only coDmel, Dere, Dyak, and Dana species combination. The Spearman rank correlations were calculated using the R statistics package [The sequence quality in each species was calculated as the mean sequence quality score of the coding bases. Bootstrap value, length, GC content, transition–transversion ratio, dN/dS, ISD, NSD, and TSD were taken directly from the PAML output for the ML tree from the original analysis using the F3×4 model and the  package .To examine the correlation of divergence with the proportion of sites supporting each tree in local areas across the genome we used 5-kb and 1-kb windows, overlapping by 2.5 kb and 0.5 kb, respectively. Using the synonymous site divergences reported by Codeml from the original analysis, we calculated the synonymous divergence per coding site in each window. We also calculated the proportion of sites supporting each tree in each window. Windows with no synonymous coding sites were excluded.Dataset S1(38 MB ZIP)Click here for additional data file.Dataset S2(42 MB ZIP)Click here for additional data file.Dataset S3(45 MB ZIP)Click here for additional data file.Dataset S4(51 MB ZIP)Click here for additional data file.Dataset S5(41 MB ZIP)Click here for additional data file.Dataset S6(61 MB ZIP)Click here for additional data file.Dataset S7(4.2 MB ZIP)Click here for additional data file.Dataset S8(2.7 MB ZIP)Click here for additional data file.Dataset S9(4.7 MB ZIP)Click here for additional data file.Dataset S10(3.0 MB ZIP)Click here for additional data file.Dataset S11(6.3 MB ZIP)Click here for additional data file.Dataset S12(4.0 MB ZIP)Click here for additional data file.Dataset S13(5.5 MB ZIP)Click here for additional data file.Dataset S14(3.5 MB ZIP)Click here for additional data file.Dataset S15(5.8 MB ZIP)Click here for additional data file.Dataset S16(3.8 MB ZIP)Click here for additional data file.Dataset S17(5.5 MB ZIP)Click here for additional data file.Dataset S18(3.5 MB ZIP)Click here for additional data file.Figure S1Dmel, Dere, Dyak, and Dana using the HKY model (A), the HKY+G model (B), the F3×4 model (C), the F3×4+G model (D), the WAG+F model (E), and the WAG+F+G model (F). Genes were binned by bootstrap value, and the proportion of genes supporting tree 1 (red line), tree 2 (green line), and tree 3 (purple line) were plotted. The expected congruence based on the bootstrap value in each bin (black solid line) demonstrates the excess incongruence.An excess of incongruence above what is expected by chance was observed for genes from (829 KB EPS)Click here for additional data file.Figure S2Dmel, Dsec, Dsim, Dere, Dyak, Dana, and Dpse (A), Dmel, Dsec, Dsim, Dere, Dyak, and Dana (B), Dmel, Dsec, Dsim, Dere, Dyak, and Dpse (C), Dmel, Dsec, Dere, Dyak, Dana, and Dpse (D), Dmel, Dsim, Dere, Dyak, Dana, and Dpse (E), Dsec, Dsim, Dere, Dyak, Dana, and Dpse (F), Dsec, Dere, Dyak, Dana, and Dpse (G), Dmel, Dsim, Dere, Dyak, and Dana (H), Dsim, Dere, Dyak, Dana, and Dpse (I), Dmel, Dsec, Dere, Dyak and Dana (J), Dsim, Dere, Dyak, and Dana (K), Dsec, Dsim, Dere, Dyak, and Dana (L), Dsec, Dere, Dyak, and Dana (M), Dmel, Dsec, Dere, Dyak, and Dpse (N), Dmel, Dsim, Dere, Dyak, and Dpse (O), Dmel, Dere, Dyak, and Dpse (P), Dsec, Dsim, Dere, Dyak, and Dpse (Q), Dsim, Dere, Dyak, and Dpse (R), Dsec, Dere, Dyak, and Dpse (S), and Dmel, Dere, Dyak, Dana, and Dpse (T). Genes were binned by bootstrap value, and the proportion of genes supporting tree 1 (red line), tree 2 (green line), and tree 3 (purple line) were plotted. The expected congruence based on the bootstrap value in each bin (black solid line) demonstrates the excess incongruence.An excess of incongruence above what is expected by chance was observed using the HKY model for genes from (68 KB PDF)Click here for additional data file.Figure S3Dmel, Dere, Dyak, and Dana species combination.Although the distributions of the RINSD for incongruent genes are biased toward lower values relative to congruent genes for the set of all genes (A), distributions are similar across trees for the set of consistent genes. Distributions were calculated using results from the original ML analysis using the F3×4 model and the (719 KB EPS)Click here for additional data file.Figure S4Dmel, Dere, Dyak, and Dana species combination.TSD is distributed similarly across consistent and inconsistent genes (A) as well as across trees for consistent genes (B), with a slight bias toward lower values for inconsistent genes and consistent genes supporting trees 2 and 3. Distributions were calculated using results from the original ML analysis using the F3×4 model and the (701 KB EPS)Click here for additional data file.Figure S5Dmel, Dere, Dyak, and Dana species combination.GC content is distributed nearly identically across species for first (A) and second (B) codon positions in all genes. Distributions were calculated using results from the original ML analysis using the F3×4 model and the (677 KB EPS)Click here for additional data file.Figure S6Dere (A), Dyak (B) and Dana (C). Distributions were calculated using results from the original ML analysis using the F3×4 model and the Dmel, Dere, Dyak, and Dana species combination.Mean sequencing quality scores for coding nucleotides in a gene are distributed nearly identically across trees in the set of all genes for (845 KB EPS)Click here for additional data file.Figure S7Dmel, Dere, Dyak, and Dana species combination.Transition–transversion ratios are similarly distributed across trees for the set of all genes. Distributions were calculated using results from the original ML analysis using the F3×4 model and the (657 KB EPS)Click here for additional data file.Figure S8Dmel, Dere, Dyak, and Dana species combination.dN/dS values are similarly distributed across trees for the set of all genes. Distributions were calculated using results from the original ML analysis using the F3×4 model and the (657 KB EPS)Click here for additional data file.Figure S9Dmel, Dere, Dyak, and Dana using RY coding and the F81 model. Genes were binned by bootstrap value, and the proportion of genes supporting tree 1 (red), tree 2 (green line), and tree 3 (purple line) were plotted. The expected congruence based on the bootstrap value in each bin (black solid line) demonstrates the excess incongruence.An excess of incongruence above what is expected by chance was observed for genes from (644 KB EPS)Click here for additional data file.Figure S1010 (observed / expected).Controlling for differences in GC content using RY coding, the enrichment of informative nucleotide substitutions near other substitutions that support the same phylogeny was found for all three trees and is on a scale roughly similar to estimates of linkage disequilibrium. At each informative site in the genome, the counts of informative sites supporting each of the three trees in 1-kb windows extending 30 kb up- and downstream were measured. For each type of informative site, the enrichment of the same type of informative site in each 1-kb window was calculated using the observed counts and the expected number of sites based on their genome-wide frequency. Enrichment is log(645 KB EPS)Click here for additional data file.Table S1(26 KB XLS)Click here for additional data file.


Allele frequencies reported from public databases or articles are mostly based on small sample sizes. Differences in genotype frequencies by age, race and sex have implications for studies designed to examine genetic susceptibility to disease.In a community-based cohort of 9,960 individuals, we compared the allele frequencies of 49 single nucleotide polymorphisms (SNPs) of genes involved in inflammatory pathways to the frequencies reported on public databases, and examined the genotypes frequencies by age and sex. The genes in which SNPs were analyzed include CCR2, CCR5, COX1, COX2, CRP, CSF1, CSF2, IFNG, IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL13, IL18, LTA, MPO, NOS2A, NOS3, PPARD, PPARG, PPARGC1 and TNF.Mean(SD) age was 53.2(15.5); 98% were Caucasians and 62% were women. Only 1 out of 33 SNPs differed from the SNP500Cancer database in allele frequency by >10% in Caucasians , whereas 12 SNPs differed by >10% (up to 50%) in African Americans (n = 105). Two out of 15 SNPs differed from the dbSNP database in allele frequencies by >10% in Caucasians, and 5 out of 15 SNPs differed by >10% in African Americans. Age was similar across most genotype groups. Genotype frequencies did not differ by sex except for TNF(rs1799724), IL2(rs2069762), IL10(rs1800890), PPARG(rs1801282), and CRP(rs1800947) with differences of less than 4%.When estimating the size of samples needed for a study, particularly if a reference sample is used, one should take into consideration the size and ethnicity of the reference sample. Larger sample size is needed for public databases that report allele frequencies in non-Caucasian populations. Chronic inflammation has been implicated in a wide variety of diseases, including cardiovascular disease, cancer, diabetes, neurological disorders, arthritis, among others -8. KnowlSeveral genes have been found to be involved in inflammatory responses. Examples are genes coding for interleukins (ILs) , tumor nWhen designing a study to investigate genetic susceptibility to diseases, information on the allele frequencies of single nucleotide polymorphisms (SNPs) in the source population is crucial for ensuring sufficient statistical power. To date, the estimation for allele or genotype frequencies of candidate genes has been primarily based on limited numbers of individuals. For example, the SNP500Cancer database, a useful resource often used for referencing sequences and allele frequencies of validated SNPs, were based on 102 anonymous individuals with self-described heritage  . On the Age and sex are often used as matching factors in epidemiological association studies because in most cases, they are associated with disease risk or survivorship. When genotype frequency is associated with age or sex, by matching on these factors in case-control studies, one would make the genotype frequency artificially similar between cases and controls. Under this circumstance, over-matching may occur.To assess how allele frequencies reported on public databases are commensurate with the allele frequencies in the general population, we compared the allele frequencies of selected SNPs in candidate genes involved in inflammatory pathways in a large, community-based population in Washington County, Maryland to the allele frequencies reported from SNP500Cancer database, and if unavailable from SNP500Cancer, the dbSNP database. In addition, we compared the genotype frequencies among age and sex groups to explore whether overmatching by age or sex in an association study of genetic polymorphisms and disease risk may be of concern. The candidate genes included were CCR2, CCR5, COX1, COX2, CRP, CSF1, CSF2, IFNG, IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL13, IL18, LTA, MPO, NOS2A, NOS3, PPARD, PPARG, PPARGC1 and TNF.Characteristics of the study population, the Odyssey and CLUE II subcohort, were presented in Table Among the 33 SNPs that were both reported by the present study and the SNP500 Cancer Database, the allele frequency of only 1 SNP differed by more than 10% in Caucasians, whereas the allele frequencies of 7 SNPs differed by 11–20%, 4 SNPs differed by 21–30%, and 1 SNP differed by 50% in African Americans , even a discrepancy in allele frequency between study samples and the samples used in the SNP500Cancer project is up to 20% may not influence investigators' decision on including into a particular SNP into a study. However, for rarer alleles, sampling errors resulting in variations in allele frequencies estimates can have an impact on SNP selection. For example, we chose to study a SNP (rs1726803) of POLD1 gene and a SNP (rs6413413) of ADH2 gene that were reported on the SNP500Cancer database to have a minor allele frequency of 5%. After analyzing approximately 3,000 samples, we found no variation in the SNP allele frequency and stopped this genotyping analysis.Among the 49 SNPs examined, 10 SNPs did not follow the Hardy-Weinburg equilibrium. Genotyping is not 100% accurate and failures to call out genotypes might have been a reason for the H-W disequilibrium. On the other hand, the sample size is fairly large in this study, and the larger the sample size is, the easier for any discrepancy in the observed allele frequency from the expected frequency (according to an H-W equilibrium) to reach statistical significance.Although in the present study, the number of African American participants was limited (n = 105), it exceeded the size reported on the SNP500Cancer databases (n = 24). As expected, there were greater differences in the allele frequencies between the present study and the SNP500Cancer database for African Americans; allele frequencies significantly differed for 35% of the SNPs for which comparisons could be made. This finding raises concerns about the usefulness of the SNP500Cancer database as a reference for candidate gene selection for African Americans.We observed sex- or age-differences in genotype frequencies for some of the SNPs. Chance alone cannot be excluded from being a possible explanation for the statistically significant differences in genotype frequencies between age groups or sex groups, particularly because the differences in genotypes between age and sex groups were small in the present study. Replication is needed for testing the robustness of these findings.In conclusion, when estimating the size of samples needed for a study, particularly if a reference sample is used, one should take into consideration the size and the ethnicity of the reference sample. The greater differences in allele frequencies among African Americans between the present study and public databases indicate a need for basing public databases on a larger sample size for this ethnic group. The small differences in genotype frequencies by age or sex for some candidate genes may be explained by chance alone, and more published data are needed for replication.The study population consists of participants in two community-based cohorts, CLUE I and CLUE II, in Washington County, Maryland. Washington County has a slowly growing population. The majority (98%) of the Washington County residents is Caucasian; 1.5% are African Americans. CLUE I  was established in 1974 for the purposes of collecting brief questionnaire data and setting up a serum bank for research purposes. Participants were recruited at mobile trailers that were stationed in various locations in Washington County to offer opportunities for research participation. A total of 26,147 individuals participated in CLUE I. Of these, 23,951 were Washington County residents. Participation was better in women, in those who had more years of education and in the age group of 45–64 years.The CLUE II was a similar campaign conducted in 1989 in which plasma samples, buffy coat samples and information on participants' selected characteristics and diet were collected. A total of 32,896 individuals participated in CLUE II, and of these, 25,079 were Washington County residents. Both CLUE I and CLUE II cohorts included approximately 30% of the County residents. The racial distributions of the CLUE I and CLUE II cohorts were similar to that in the Washington County. CLUE II participation was slightly better among women, among those who had more years of school education, and among those who were at age of 45–70 years.The present study includes the Odyssey cohort and a CLUE II subcohort. The Odyssey cohort is composed of the 8,394 individuals who took part in both CLUE I and CLUE II. Within Odyssey, DNA was successfully extracted from the buffy coat samples of 8,307 individuals. The CLUE II subcohort  is an age- and sex-stratified random sample (10%) of the CLUE II participants who were residents of Washington County and its neighboring areas. DNA samples from this subcohort were successfully extracted from all buffy coat samples. Among these, 807 samples overlapped with the 8,307 Odyssey samples. Hence, 9960 samples are included in the present study.SNP data were rendered anonymous by in-house Anonymous Data Management software. The study identifiers were encrypted by modern cryptographic technologies that destroyed the link from genetic information back to identities, while anonymously allowing one-way linkage from identities to genetic information. Informed content to take part in the CLUE I and CLUE II research campaign was obtained from each participant. The protocol was approved by the Institutional Review Board of the Johns Hopkins Bloomberg School of Public Health.Candidate genes were selected based on the following criteria: (a) estimated allele frequencies of ≥ 5% in Caucasians in published literature or databases, (b) known or promising importance in the development of cancer, cardiovascular diseases, and/or longevity, (c) validated allele substitutions, and/or (d) functional changes linked to allele substitutions that have been published in the literature.At CLUE II enrollment, blood samples were collected into 20-ml heparinized Vacutainers . Samples were refrigerated at 4°C and most samples were centrifuged within 2 to 6 hours after blood collection and were never 24 hours later. Plasma aliquots from each participant were placed in two 5-ml Cryotubes  and were stored at -70°C. Buffy coat samples were stored in separate vials at -70°C until extraction. Barcoding was performed as part of the blood collection process. Labels were printed with the study numbers barcoded so that they could be scanned for accuracy in data entry and inventory maintenance.TaqMan assay.DNA was extracted from buffy coat by the alkaline lysis method . FollowiAssay reliability was assessed for the 17 SNPs analyzed by Celera, using 86 duplicate samples randomly inserted into the plates; the percent concordance was 82–95%. Two SNPs (rs 1800587 and rs 17561) were both analyzed by Celera and ABI. The percent concordance was 0.89 and 0.90, respectively.Characteristics of the Odyssey and the CLUE II subcohort study participants were summarized by the frequency distributions of categorical variables and by the means (SDs) of continuous variables. The genotype frequencies of each candidate gene were compared between the Odyssey and the CLUE II subcohort. For most SNPs, no differences in genotype frequencies were observed between the Odyssey and the subcohort. The only exceptions were LTA (rs3093543) for which a higher proportion of AC as compared to AA genotype was observed [OR = 1.18 ] and PPARGC1 (rs8192678) for which a higher proportion of AA as compared to GG genotype was observed [OR = 1.28 ] in the Odyssey compared to the subcohort. Subsequent analyses were performed on the combined Odyssey and subcohort data.2 tests for age categories by genotypes, was used to compare age as continuous variable across the genotype categories. Genotype frequencies were compared between men and women by χ2 test.Allele frequencies were examined separately in Caucasians and African Americans, and were compared to the frequencies reported from the SNP500Cancer database or if unavailable from SNP500Cancer, the dbSNP database. Statistically significant differences were reported if the allele frequencies from public databases fell beyond the 95% confidence intervals (95% CIs) of the allele frequencies among the CLUE population. The Hardy-Weinberg equilibrium was tested by a goodness-of-fit approach. Genotype frequencies by age groups of 10-year intervals were examined. Because age as a continuous variable provides more information, the One-Way Analysis Of Variance (ANOVA), rather than χIL: interleukins; TNFs: tumor necrosis factors; COX: cyclooxygenase; NOS: nitric oxide synthase; PPARs: peroxisome proliferator-activated receptors; CCR: chemokine receptors, MPO: myeloperoxidase; IFNG: interferon-gamma; CSF: colony-stimulating factors; LTA: lymphotoxin alpha; CRP: C-reactive protein.HYH conceived the study aims, participated in study design and statistical analysis, interpreted the data and drafted the manuscript. LT performed statistical analysis. PS participated in study design and interpreted the data. SCH participated in study coordination and data management. GWC helped to interpret the data. KJH participated in study design and interpreted the data. All authors read and approved the final manuscript.Table 2. Allele frequencies of selected SNPs in the CLUE II study and the SNP500Cancer database; description: List of allele frequencies of selected SNPs involved in inflammatory pathways observed on CLUE II study samples and on the SNP500 Cancer database, and tests for H-W equilibrium.Click here for fileTable 3. Genotype frequencies by age groups in Odyssey and CLUE II subcohort; description: Genotype distribution by age of 10 years intervals in the study samples.Click here for fileTable 4. Genotype frequencies by sex in Odyssey and CLUE II subcohort; description: Genotype frequencies in men and women of the combined Odyssey and CLUE II subcohort.Click here for file


Nitroimidazoles have been shown to be potent sensitisers of certain clinically active chemotherapeutic agents. This process of chemopotentiation has been shown to be hypoxia-mediated. The present studies evaluated whether increasing the level of hypoxia in the tumour tissue, by treatment with the vasoactive agent hydralazine, could modify the chemosensitising ability of nitroheterocyclics. Administering either misonidazole or RSU 1164 before, or hydralazine after, the chemotherapeutic agents melphalan, cyclophosphamide or the nitrosourea CCNU, increased the extent of cell kill in both the KHT sarcoma and RIF-1 tumour. However, even greater enhancements could be achieved when hydralazine was used in treatment protocols in which a nitroimidazole was combined with chemotherapy. For example, a 5.0 mg kg-1 dose of hydralazine given 30 min after melphalan, or a 2.5 mmol kg-1 dose of misonidazole administered 30 min before melphalan, increased, compared to melphalan alone, the resultant tumour cell kill by factors of approximately 1.9 and approximately 1.3, respectively. By comparison, when hydralazine was given after the melphalan plus misonidazole combination, treatment efficacy was enhanced approximately 3-fold compared to melphalan alone. Yet in contrast to the results of the tumour response studies, the inclusion of hydralazine did not increase the bone marrow toxicity associated with the chemotherapeutic agent when used alone or in conjunction with a nitroimidazole. The results, therefore, imply that the addition of hydralazine to the chemotherapy, or chemotherapy-sensitiser protocol, led to a therapeutic advantage.


The loss of cartilage volume was also slightly associated with less knee pain. No association was found with other Western Ontario McMaster Osteoarthritis Index (WOMAC) scores, joint space width, or urine biomarker levels. Meniscal damage and bone edema are closely associated with more cartilage volume loss. These data confirm the significant advantage of qMRI for reliably measuring knee structural changes at as early as 12 months, and for identifying risk factors associated with OA progression.The objective of this study was to further explore the cartilage volume changes in knee osteoarthritis (OA) over time using quantitative magnetic resonance imaging (qMRI). These were correlated with demographic, clinical, and radiological data to better identify the disease risk features. We selected 107 patients from a large trial  evaluating the effect of a bisphosphonate on OA knees. The MRI acquisitions of the knee were done at baseline, 12, and 24 months. Cartilage volume from the global, medial, and lateral compartments was quantified. The changes were contrasted with clinical data and other MRI anatomical features. Knee OA cartilage volume losses were statistically significant compared to baseline values: -3.7 ± 3.0% for global cartilage and -5.5 ± 4.3% for the medial compartment at 12 months, and -5.7 ± 4.4% and -8.3 ± 6.5%, respectively, at 24 months. Three different populations were identified according to cartilage volume loss: fast , intermediate , and slow  progressors. The predictors of fast progressors were the presence of severe meniscal extrusion ( With the aging of the world population, osteoarthritis (OA) is becoming an increasingly common cause of disability ,2 DiarthMagnetic resonance imaging (MRI) allows for the precise visualization of joint structures such as cartilage, bone, synovium, ligaments, and menisci, as well as their pathological changes. Our group ,6, amongA large clinical trial assessing the effects of a bisphosphonate on knee OA structural changes was recently completed. A subset of 110 of these patients underwent MRI in addition to the clinical standardized radiograph and biomarker evaluations, as per the study protocol. In this longitudinal study, we assessed this larger cohort of OA patients and identified risk factors for greater disease progression. The cartilage volume changes contrasted with the clinical, radiological, and biomarker data. We felt it was necessary to confirm our previous results ,14 and pA subset of 110 patients was selected from 1,232 patients enrolled in a large clinical trial evaluating the impact of a bisphosphonate on knee OA. This specific subset of patients was recruited from the outpatient Rheumatology Clinic at the University of Montreal Hospital Centre (CHUM), Notre-Dame Hospital, and from the Rheumatic Disease Centre of Montreal, both in Montreal, Quebec, Canada. Both male and female patients were eligible for the study if they were between 40 and 80 years old, fulfilled the American College of Rheumatology criteria for knee OA , and hadPatients were excluded if they had chondrocalcinosis or an acute or chronic infection (including tuberculosis); if their OA of the knee was secondary to other conditions, including inflammation, sepsis, metabolic abnormalities, and trauma; or if they displayed any contraindication to the use of MRI. Further exclusion factors included patients' history of past or present gastrointestinal ulceration, their receipt of an intra-articular corticoid injection in the study knee within the six months prior to the outset of the study, as well as their classification as radiological grade IV on the Kellgren-Lawrence scale for the study knee or severe (class IV) functional disability. In patients in whom both knees were symptomatic, we chose the most symptomatic knee for the investigation. Patients were permitted to receive simple analgesics or NSAIDs, the regimens of which could be changed according to the preference of the rheumatologist and the clinical course of the patient. Such regimens, as well as any changes to them, were closely monitored and noted. Because of its potential to promote OA cartilage degeneration, the use of indomethacin was not permitted . A centrPatients underwent clinical evaluation at baseline and every 6 months thereafter until 24 months. They were first evaluated on the basis of the Western Ontario McMaster Osteoarthritis Index (WOMAC), a tri-dimensional self-administered questionnaire that probes pain (5 items), stiffness (2 items), and physical function 17 items) [ items 1. In addiThe JSW of the target knees was evaluated at baseline and at 12 and 24 months of follow-up, at the narrowest point in the medial tibio-femoral compartment according to the published protocol . This prHigh-resolution, three-dimensional MRI was obtained for each OA patient at baseline and at 12 and 24 months using the commercially available Magneton Vision 1.5 Tesla machine with a dedicated knee coil , as previously described  These ex3) and in percentage values for the entire knee  and for each of the knee compartments , respectively.The change in cartilage volume over time was calculated compared to baseline in absolute values  and presence or absence of one severe edema lesion (grade 2 only), regardless of the presence of additional smaller lesions.Reliability of our scoring system for meniscal and bone changes was excellent. The intra- and inter-reader correlation coefficient ranged from 0.86 to 0.96 for the meniscal tear, 0.85 to 0.92 for the meniscal extrusion and 0.88 to 0.93 for the bone marrow edema. Kappa statistics ranged from 0.79 to 0.89 for the meniscal changes and 0.78 to 0.87 for the bone marrow edema (data not shown).Level of urinary C-terminal cross-linking telopeptide of collagen type II (U-CTX-II), a biological marker of collagen type II degradation, was measured by a specific ELISA . Early mt test.All of the data  were systematically entered into a computerized database using a blinded double-entry procedure, after which descriptive statistics for patient characteristics were tabulated. The primary variable of interest for this publication was the change in cartilage volume over time for the entire knee  and for each of the knee compartments , respectively. The cartilage volume losses are presented as percentage losses compared to baseline (mean ± standard deviation) and statistical relevance assessed by a one-sample t tests, chi-squares, or the McNear exact test were performed to assess statistical significance. Multivariate linear analyses were used to assess predictors of cartilage volume loss independently from potential confounders like age, gender and BMI. Further analyses were done by dividing the cohort by quartiles of cartilage volume loss in which the first quartile demonstrated greater cartilage volume loss. Finally, the relationship between cartilage volume loss and the change in JSW was explored at 24 months using the Spearman correlation test. All statistical analyses were done using Statistica, version 6 packages . All tests were two-sided, and a p value = 0.05 was considered statistically significant. Analyses were not corrected for multiple comparisons.A K-means cluster analysis, a non-parametrical statistical method, was used to identify subgroups of disease progression based on cartilage volume loss at 24 months. These subgroups were further analyzed to contrast their baseline demographic, clinical, radiological, and biomarker features, and presented as mean ± standard deviation. Non-parametric Wilcoxon one-sample tests, one-sample and two-sample Student 2, the duration of knee OA was 8.9 ± 7.2 years, 91.4% were taking analgesics, and 72% were using NSAIDs, and these patients were exhibiting disease activity scores in the mild to moderate range according to the WOMAC , the Patient Global , the SF-36 (38.1 ± 9.5), and the Kellgren-Lawrence score . The mean JSW measure at baseline was 2.88 ± 0.64 mm. These baseline characteristics of the 110 subjects were very similar according to age, gender, BMI and baseline WOMAC pain, stiffness and function values to the 1,232 subjects enrolled in the large clinical trial. The patients represent all those enrolled in Montreal for the bisphosphonate study and all had MRI. There was no effect of any bisphosphonate treatment group on the rate of knee OA progression as measured by either cartilage volume per MRI or the JSW loss at two years (data not shown). We felt, therefore, that all the patients may be considered as a unique group and analyzed as such.A subgroup of 110 patients was assessed with quantitative MRI (qMRI); three patients were lost to follow-up early in the study. At baseline, the cohort was largely in line with the demographic and disease characteristics of a general OA population: the mean age was 62.4 ± 7.5 years, 64% of subjects were female, subjects had an average BMI of 30.6 ± 4.3 kg/mp < 0.0001, one-sample t test). At 24 months, further cartilage volume loss was evident in the global (-5.7 ± 4.4%), medial (-8.3 ± 6.5%), and lateral compartments (-3.5 ± 3.8%), which were all statistically significant when compared to baseline (p < 0.0001). The lower rate seen for the lateral compartment may be explained by the selection of this population according to the inclusion/exclusion criteria; patients with isolated lateral compartment knee OA as defined with standing knee X-rays were excluded. The results of the femoral and tibial compartments demonstrated similar and statistically significant results and are presented in Table At 12 months  compared to 48 patients with an intermediate rate of cartilage loss , and 48 patients with a slow loss rate . These three groups now defined as slow, intermediate or fast are labeled as such throughout the text. Among the three groups, a greater relative cartilage volume loss was observed in the medial compartment of the same patients identified as fast and intermediate groups at 24 months with -21.5 ± 0.1% and -9.9 ± 0.1% loss, respectively (p < 0.0001). In contrast, very little progression (-3.2 ± 0.6%) was found in the medial compartment of the slow progressors.With the use of cluster analysis, we identified three different subgroups of disease progression Figure  at 24 moTable p < 0.0001; ANOVA for the three groups). This was present in 73% of the fast compared to 19% of the slow progressors for the extrusion, and in 72% and 23%, respectively, for the tear .For the meniscal changes, in absolute numbers, 85 patients had a meniscal medial tear and/or extrusion, 55 had a lateral tear and/or extrusion, with some patients having both menisci compartments damaged. Only six patients had an intact meniscus. The severe medial meniscal extrusion and the severe medial tear Table  at baselp < 0.05, Chi-squared fast versus slow; and p = 0.03, ANOVA for the three groups).The presence of bone edema Table  in the tNo association of the presence of bone edema with clinical symptoms was found (data not shown). Analyses on the other compartments, medial, lateral, femoral, and tibial were also performed and did not provide new information (data not shown).p > 0.25 (Spearman correlation coefficient) were found for all the variables compared to cartilage volume loss (data not shown). For the JSW changes at 24 months, some weak correlations were demonstrated for changes at 24 months with WOMAC pain score and function score changes .The evaluation of the clinical course of all 107 OA patients revealed no significant correlation between the changes in cartilage volume and the changes in clinical variables such as the patients' and physicians' global assessments, the pain, stiffness, and function dimensions of the WOMAC, and the physical components of the SF-36. Values of r < 0.2 and As the medial compartment is the most closely related to the radiological changes, the results presented subsequently focus on this compartment. Multilinear regression analysis was used to investigate the association of medial cartilage volume loss at 24 months using baseline demographics, with clinical and imaging data as predictors controlling at the same time potential confounders such as age, gender, and BMI Table  and 6. Tp = 0.04). No additional association with the change of clinical parameters was found.Bone edema also showed an independent association with the cartilage loss, which was statistically significant using the stepwise method. The clinical variables, the SF-36 physical components, and the WOMAC total score also demonstrated some independent predictive values. Surprisingly, baseline medial cartilage volume was also independently associated with medial cartilage loss at 24 months. Additional multivariate analyses comparing changes in the clinical variables at 24 months and the medial compartment cartilage loss were also done Table . These dp < 0.001; data not shown). At 24 months, the changes in the cartilage volume in the medial compartments  and the JSW changes at 24 months , despite further characterization of the cohort into quartiles vis-à-vis the MRI data. However, the first quartile of cartilage loss represents a group in which JSW changes were also significant  .The presence of severe meniscal extrusion (score = 2), found in 38 patients at baseline, was associated with a smaller baseline JSW (2.45 ± 0.53 mm) compared to a greater JSW (3.09 ± 0.53 mm) in the absence of such an extrusion . Moreover, the baseline level of U-CTX-II did not predict cartilage volume loss over time.The levels of U-CTX-II, a marker of cartilage degradation, were compared with the changes in JSW and with the cartilage volume changes at 12 and 24 months. No correlations were seen, either between the level of the biomarker and the loss of cartilage volume assessed by qMRI , or between the level of the biomarker and the changes in JSW. The r values for both ranged from -0.06 to 0.10; no Very few studies have examined the quantitative changes in cartilage volume over time in a symptomatic knee OA population. Through the use of MRI in a longitudinal study of 107 subjects with symptomatic OA of the knee, we demonstrate a significant global cartilage volume loss at as early as 12 months. The changes in values are in line with those from a pilot study , and areThe present study further reinforces the heterogeneity of the OA patient population that previous clinical trials have described ,29 AccorQuestions remain regarding patients with slow disease progression. It is unlikely that this process reflects merely the normal aging process, as these patients experienced pain and loss of function and met the American College of Rheumatology criteria for knee OA. We hypothesize that the slow progressors might constitute a subgroup of patients in a quiescent phase of the disease.3 of global cartilage volume with a standard deviation of 459 mm3 was detected at 24 months, a change that was smaller than the mean. In comparison, for the same cohort, a JSW loss of 0.16 mm with a standard deviation of 0.49 mm, roughly three times greater than the mean change, was detected at 24 months. Therefore, the 'effect size', defined as the mean change divided by its standard deviation, of qMRI assessments appears superior to that of JSW assessments.Many consider the measurement of the change in the minimal JSW of standardized knee radiographs to be the best available methodology for evaluating the anatomical progression of OA ,32-34. Tp = 0.03) when using a multivariate analysis approach, which was, however, not demonstrated by direct correlation. The patients' analgesic and NSAID washout period prior to the clinical evaluation likely reinforces the explanation of this phenomenon. Our findings are in accordance with Hunter and colleagues [In this study, we found some association between the extent of cartilage volume loss and changes in pain at 24 months .This high level of meniscal damage (78%) may appear unusual. Our patients were selected based on inclusion/exclusion criteria for which the presence of radiological knee OA on the medial compartment plus the presence of JSW between 2 and 4 mm was necessary. It is probable that these patients may represent a more advanced disease state and that a meniscal lesion, a structure that greatly influences the JSW assessment, could almost be a prerequisite to obtain a JSW of 2 to 4 mm on standing X-rays. To further reinforce this hypothesis, our previous study on 32 subjects  showed aCicuttini and colleagues  suggesteOur study also demonstrated the association between cartilage volume loss and the presence of bone edema. Felson and colleagues (( have alrThe lack of correlation between the U-CTX-II levels and the cartilage volume loss found in the present study contradicts other studies ,27,42,43This study, like any other, has its limitations. Our cohort is representative of the average patient population with typical knee OA that is seen at a rheumatology clinic. This study is of notable confirmatory value in terms of the results obtained from a smaller number of patients . HoweverOne may also question the potential partiality of the non-blinding of the cartilage when meniscal or bone assessment was done. However, as the radiologist evaluation was performed completely separately from the assessment of cartilage volume, it is unlikely that the grading of meniscal damage was biased by the concomitant visualization of the cartilage. We also acknowledge that the FISP sequence may not be the optimal MR sequence for identifying all the meniscal and bone lesions. It has sufficient contrast, however, to identify significant lesions, especially edema, as demonstrated in this previous work, and this acquisition has the unique advantage of being able to assess simultaneously the cartilage, menisci and bone.Our study confirms the feasibility of the long-term longitudinal follow-up of cartilage volume changes over time in a large cohort of patients with OA. Significant knee cartilage volume loss was detected as early as 12 months in this study and as early as six months in our previous study , thus imBMI = body mass index; DMOAD = disease modifying osteoarthritis drug; JSW = joint space width; MRI = magnetic resonance imaging; NSAID = nonsteroidal anti-inflammatory drug; OA = osteoarthritis; qMRI = quantitative MRI; U-CTX-II = urinary C-terminal crosslinking telopeptide of collagen type II; WOMAC = Western Ontario McMaster Osteoarthritis Index.J-P Raynauld, J Martel-Pelletier, MJ Berthiaume, G Beaudoin, D Choquette, B Haraoui, H Tannenbaum and J-P Pelletier received financial support from Procter & Gamble Pharmaceuticals. J Martel-Pelletier and J-P Pelletier are consultants for Procter & Gamble Pharmaceuticals. JM Meyer, JF Beary and GA Cline are employees of Procter & Gamble Pharmaceuticals.JPR, JMP, MJB, GB, JMM, JFB, GAC, and JPP contributed to the study design. DC, BH, HT, and JMP were responsible for the acquisition of data. JPR, JMP, JMM, JFB, GAC, and JPP analyzed and interpreted the data. JPR, JMP and JPP prepared the manuscript. JPR and GAC performed the statistical analysis. All authors read and approved the final manuscript.


Wolbachia and microfilaria loads, antigenemia, filarial dance sign (FDS), dilation of supratesticular lymphatic vessels, and plasma levels of lymphangiogenic factors VEGFR-3]). Lymphedema patients were additionally monitored for stage (grade) of lymphedema and the circumferences of affected legs. Wolbachia load, microfilaremia, antigenemia, and frequency of FDS were significantly reduced in microfilaremic patients up to 24 mo in the doxycycline group compared to the placebo group. The mean dilation of supratesticular lymphatic vessels in doxycycline-treated patients was reduced significantly at 24 mo, whereas there was no improvement in the placebo group. Preceding clinical improvement, at 12 mo, the mean plasma levels of VEGF-C and sVEGFR-3 decreased significantly in the doxycycline-treated patients to a level close to that of endemic normal values, whereas there was no significant reduction in the placebo patients. The extent of disease in lymphedema patients significantly improved following doxycycline, with the mean stage of lymphedema in the doxycycline-treated patients being significantly lower compared to placebo patients 12 mo after treatment. The reduction in the stages manifested as better skin texture, a reduction of deep folds, and fewer deep skin folds. In conclusion, a 6-wk regimen of antifilarial treatment with doxycycline against W. bancrofti showed a strong macrofilaricidal activity and reduction in plasma levels of VEGF-C/sVEGFR-3, the latter being associated with amelioration of supratesticular dilated lymphatic vessels and with an improvement of pathology in lymphatic filariasis patients.Lymphatic filariasis is a disease of considerable socioeconomic burden in the tropics. Presently used antifilarial drugs are able to strongly reduce transmission and will thus ultimately lower the burden of morbidity associated with the infection, however, a chemotherapeutic principle that directly induces a halt or improvement in the progression of the morbidity in already infected individuals would constitute a major lead. In search of such a more-effective drug to complement the existing ones, in an area endemic for bancroftian filariasis in Ghana, 33 microfilaremic and 18 lymphedema patients took part in a double-blind, placebo-controlled trial of a 6-wk regimen of 200 mg/day doxycycline. Four months after doxycycline treatment, all patients received 150–200 μg/kg ivermectin and 400 mg albendazole. Patients were monitored for  Lymphatic filariasis, caused by filarial worms, is transmitted by mosquitoes. The infection leads to pathology such as edema of the legs (lymphedema) or the scrotum (hydrocele). About 120 million people are estimated to be infected, and 1.2 billion are at risk of infection. The currently used drugs  to treat the disease are able to kill most of the larval stage (microfilariae) in the blood but have either no (ivermectin) or partial (DEC) effect on the adult worms that cause the pathology. They also do not sufficiently halt the progression of pathology, such as, lymph vessel dilation, hydrocele, and lymphedema.In search of a more effective drug capable of killing the adult worms, and also of halting the progression of the disease in already infected individuals, the authors recruited, in an endemic area in Ghana, 33 people who were infected with the worm and microfilaremic but had not yet developed the disease, as well as 18 lymphedema patients, and treated them with either 200 mg/d doxycycline or matching placebo for 6 wk. The findings presented here reveal that doxycycline is able to kill the adult worms, improves lymphatic vessel dilation, and ameliorates the conditions of lymphedema patients significantly. This suggests that doxycycline can be used to treat lymphatic filarial infections and pathology, making doxycycline the first drug already approved for human use and considered for use as an adjunct to current control programs, which in addition to parasite control achieves improvement of the quality of life of persons with pathology. Bancroftian filariasis is a mosquito-transmitted parasitic disease of humans characterized by lymphangitis, hydrocele, lymphedema, and elephantiasis, and is one of the most common causes of global disability . The disThe antifilarial drugs currently used, diethylcarbamazine (DEC) and ivermectin, are predominantly active against microfilariae (MF), with DEC showing partial activity against adult worms . Mass drWolbachia found in most filarial species, including W. bancrofti [Wolbachia organisms suggest that these bacteria are important both as chemotherapeutic targets and disease-causing organisms [Wolbachia was shown to mediate neutrophil infiltration and stromal haze when a worm extract including Wolbachia antigens was injected into the eyes of mice [Thus, a drug with stronger macrofilaricidal activity than reported for DEC, which simultaneously has ameliorating effects on lymphatic vessels, could considerably improve the prospect of developing a treatment for lymphatic pathology. One aspect of the biology of filarial nematodes that could be exploited in the effort to advance the elimination program is the presence of the endosymbiotic ancrofti . Recent rganisms . In a la of mice –17.The events that lead to the development of chronic pathology in LF are not fully understood, but the immune responses of the human host to the parasites are believed to play a significant role in determining the pathological manifestations of disease in infected individuals –21.The lymphatic vascular system is important for immune surveillance, tissue fluid homeostasis, and fat absorption ,23. Pertkeratin 14 transgenic promoter [Recent studies on the molecular mechanisms controlling the lymphatic vessels have shown that vascular endothelial growth factors C (VEGF-C) and VEGF-D specifically control lymphangiogenesis in humans ,28 by acpromoter . In thispromoter . These Kpromoter .Wolbachia–derived molecules from Brugia spp. induced proinflammatory cytokines, including TNF and IL-1B [Brugia and Onchocerca volvulus adult and microfilarial worms were also found to stimulate human peripheral mononuclear cells in vitro, resulting in the production of TNF, IL-1, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-10 [Acanthocheilonema viteae, a filarial species naturally devoid of Wolbachia, and, importantly, with O. volvulus extracts from patients that had been treated with doxycycline to deplete Wolbachia from the worms [Wolbachia are the major stimulating principle for proinflammatory cytokines such as TNF. From this it can be further hypothesized that exposure of host cells to Wolbachia from worms  may induce the production of lymphangiogenic factors such as VEGF-C by endothelial cells in LF patients.Studies in animal models have shown that nd IL-1B . Solublend IL-10 ,40. Thishe worms . Thus itWolbachia from both MF and adult filarial worms of W. bancrofti and O. volvulus [Wolbachia depletion by doxycycline was associated with a reduction of pretreatment elevated levels of proinflammatory cytokines such as TNF from plasma [Wolbachia by doxycycline may ameliorate filarial pathology through down-regulation of proinflammatory cytokines and VEGF-C/VEGFR-3. However, no studies to date have analyzed Wolbachia-induced secretion of VEGF-C and its receptor on lymphatic vessel dilation among human populations with lymphatic filarial disease.Treatment with doxycycline for 8 wk is well tolerated in humans and has shown macrofilaricidal activity . Doxycycvolvulus ,44. Of im plasma . TogetheWolbachia in filarial worms on the levels of VEGF-C and its soluble receptor VEGFR-3 and on dilation of the supratesticular lymphatic vessels in microfilaremic patients; and (3) the effect of doxycycline treatment on clinical manifestations associated with filarial lymphedema. To address these aims, we recruited microfilaremic and lymphedema patients in an endemic area in Ghana and treated them with 200 mg/d doxycycline for 6 wk. Lymphedema patients were followed up at 4 and 12 mo, whereas microfilaremic patients were re-examined at 4, 12, and 24 mo after the start of doxycycline treatment. We report that 6 wk of doxycycline had a strong macrofilaricidal activity; and targeting the endosymbiotic Wolbachia in filarial worms resulted in the reduction of plasma levels of VEGF-C/sVEGFR-3, which were associated with amelioration of dilated supratesticular lymphatic vessels and improvement of lymphedema in lymphatic filariasis patients.The aims of this study were to assess (1) a 6-wk course of doxycycline for macrofilaricidal activity; (2) the effect of targeting the endosymbiotic We treated a total 95 patients with bancroftian filariasis (76 microfilaremic and 19 lymphedema) with doxycycline or matching placebo for 6 wk. The treatment was well tolerated, with eight doxycycline and ten placebo patients experiencing adverse reactions, which in no case required a treatment stop. The adverse reactions arose on days 2 and 3 after commencement of treatment in both groups. The adverse reactions experienced by the eight doxycycline-treated patients included headache, dizziness, diarrhea, and itching skin. No adverse event went beyond 3 d or required more intervention than application of paracetamol tablets, oral dehydration salt, and ointment. The adverse reactions experienced by the ten placebo patients included headache, diarrhea, and painful scrotum. Again no event lasted for more than 3 d, except for one microfilaremic patient who had a painful scrotum for 6 d. In this group, the side effects were also treated with paracetamol tablets and oral dehydration salt. There was no significant difference between the doxycycline and placebo groups.Thirty-three (17 doxycycline and 16 placebo) microfilaremic patients and eighteen lymphedema patients (8 doxycycline and 10 placebo) were present at all time points , and theWolbachia level, microfilaremia, and antigenemia from baseline and at follow-up time points. In all the analyses, only participants present at all time points (17 doxycycline and 16 placebo) were included. Following doxycycline treatment, Wolbachia levels in MF were reduced by 95% (p < 0.0001) in the doxycycline group, but there was no significant change in the placebo group (p = 0.2588).p = 0.2741) whereas microfilaria levels had actually increased in the placebo group (p = 0.0017). Both doxycycline and placebo patients were given albendazole and ivermectin after the 4 mo re-examination to clear microfilaremia. At 12 mo post therapy, microfilaremia was virtually absent in the doxycycline group except in one of the 17 (6%) patients, who had a very low MF count. At 24-mo follow-up, 18% of doxycycline-treated patients that were re-examined had microfilaremia. In contrast, 50% of placebo patients became microfilaremic at 12 mo, and the number increased to 63% at 24 mo. The reduction of MF in the doxycycline group compared to placebo group was significant at the 12 mo (p = 0.0029) and 24 mo (p = 0.0211) follow-up time points. It must be mentioned that all patients continued to live in the endemic area; given a 5-y average life-span of adult worms and constant worm loads without treatment interference, new infections are expected to occur at a rate of about 20% per year.Microfilaremia was only slightly, though not significantly, reduced 4 mo after doxycycline treatment compared to pretreatment levels in the doxycycline group . However, the difference became significant at 24 mo post therapy (p = 0.0382), with median CFA levels in the doxycycline group being only 6% of pretreatment (p = 0.3271); however, the difference became significant at 12 and 24 mo after doxycycline treatment  levels in plasma, and presence or absence of worm nests in ultrasound examinations, were used to assess macrofilaricidal activity of doxycycline. CFA did not differ significantly between the two groups before and at 12 mo after treatment (reatment . Before p = 0.0404) in the doxycycline group but not in the placebo group (p = 0.2168). Of the nine doxycycline-treated patients, seven (78%) patients showed a reduction of the lymphatic vessels in comparison to 1/8 (13%) patients in the placebo group  showed that 17 male patients (nine doxycycline and eight placebo-treated) had supratesticular lymphatic vessel dilation. p = 0.0221, chi-square test).In support of the hypothesis that doxycycline can ameliorate lymphatic vessel alteration, more patients in the placebo group developed a hydrocele during the observation period compared to the doxycycline group. Thus, one patient in the doxycycline-treated group developed a clinical hydrocele whereas in another patient, the hydrocele disappeared. In contrast, in the placebo-treated group, five patients developed hydrocele . This increase of hydrocele in the placebo group was significant when compared to the pretreatment status  were significantly elevated in microfilaremic patients . No correlation was found between VEGF-C and lymphatic dilation, but a strong trend  was noticed between sVEGFR-3 and lymphatic vessel dilation. There was also a positive association between levels of VEGF-C and antigenemia  and between levels of sVEGFR-3 and antigenemia , but no associations between microfilaremia and levels of VEGF-C (p = 0.8864) and sVEGFR-3 (p = 0.7720) were found.Using pretreatment samples, we found a positive correlation between VEGF-C and sVEGFR-3 , whereas there was no significant difference in the placebo-treated group .There were reductions in the antigenemia levels in both doxycycline and placebo patients with lymphedema at 12 mo, but no significant difference in the reduction was obtained in any of the groups  at this p = 0.0051, Wilcoxon rank test) in the lymphedema stage of the doxycycline-treated patients at 12 mo post therapy compared to pretreatment, but not in the placebo group). The reduction in the stage manifested as better skin integrity (fewer “knobs”), reduction of deep and shallow skin folds, and fewer entry lesions of the affected legs (not shown). No change in the mean circumference of the affected legs in both doxycycline and placebo groups at 12 mo post therapy was observed.p = 0.0002, and p = 0.0012, respectively) compared to endemic controls. Interestingly, the plasma levels of sVEGFR-3 were significantly higher in lymphedema patients than in microfilaremic patients without lymphedema  in sVEGFR-3 levels between pretreatment and 12 mo.Mean plasma concentrations of VEGF-C and sVEGFR-3  and 4 weThe past decade has seen major advances that have changed LF from a neglected disease into a disease now accepted as potentially eradicable. The main reason is the identification of ivermectin, DEC, and albendazole, as effective antifilarial agents.While DEC has been shown to have some macrofilaricidal effect, it is still believed that a more potent macrofilaricide will add to efforts to achieve LF elimination, halting also the progression of the clinical manifestations induced largely by the adult worm . This paW. bancrofti, shorter than what we published earlier [Wolbachia endosymbionts and showed a strong macrofilaricidal activity, as evidenced by reduction of antigenemia by 94% and the absence of adult worms from the scrotal region in 89% of male participants by USG 24 mo after treatment. The second and fully novel advance is the reduction of lymphangiogenetic factors following doxycycline, and the reduction of lymphatic vessel dilation and improvement of lymphedema.Two major advances have been observed in this study. Firstly, a regimen of 6 wk of doxycycline against  earlier , depleteWolbachia load in doxycycline-treated patients 4 mo after treatment, and this reduction was sustained throughout the 24-mo follow-up period. The fact that there was an apparent, but not significant, increase in Wolbachia loads in three MF-positive doxycycline patients at 24 mo is probably due to new infections, which is likely to occur in this area of ongoing transmission, theoretically at a yearly rate of 20% of the total worm load, given that the average worm life span is about 5 y and that there is a rather stable adult worm load in adult patients. New infections with concurrent rise in Wolbachia levels following doxycycline treatment have already been documented for doxycycline treatment of onchocerciasis, where old, doxycycline-treated and thus Wolbachia depleted, female worms were located in onchocercomas next to young, nulliparous worms that were full with Wolbachia [Wolbachia depletion on embryogenesis and loss of microfilariae from host blood through natural attrition, as recorded in onchocerciasis [We recorded a 95% reduction of olbachia . Due to olbachia . The loserciasis ,53 and lerciasis ,44.Even though antigenemia levels were significantly reduced by 94% in doxycycline-treated microfilaremic as compared to 48% in placebo patients, antigen units remained elevated at 24 mo in some doxycycline patients, though USG results showed the absence of worm nests. Before doxycycline treatment, all 33 (17 doxycycline and 16 placebo) patients had antigenemia levels above 32,000 units . However, at 24 mo, 11/17 (65%) of doxycycline-treated patients had antigen units below 32,000, compared to only 4/16 (25%) of placebo patients. Given that USG demonstrated absence of worm nests in most doxycycline-treated patients analyzed, our data suggest that antigenemia is cleared from the blood slowly, but progressively, after the death of the adult worms, as suggested by others . The lown = 16); however, even if a significant difference had been detected with higher numbers, the extent of the reduction would not have increased. Thus, our findings rather support other reports that ivermectin plus albendazole has no significant macrofilaricidal effect [r = 0.754, p < 0.0001). This is consistent with another study [We cannot exclude that the partial reduction in antigenemia observed in the placebo patients could be due, to some extent, to a partial macrofilaricidal effect of ivermectin plus albendazole treatment. USG results did show 20% loss of worm nests at 24 mo; however, this reduction did not reach statistical significance. The lack of significance could be due to the rather small number of placebo patients  kits  in a cohort of healthy European individuals is 225  pg/ml. The levels of endemic normals in the present study was somewhat higher, with a mean of 1,851 pg/ml ; it is not clear whether this reflects genetic differences between people of European descent and Africans, or a possible exposure of these persons to stimuli of the lymphangiogenetic system other than lymphatic filariasis. However, the levels of VEGF-C in microfilaremic and lymphedema patients were significantly increased over those of endemic normals before treatment. This finding, and more so the yet significantly elevated levels of sVEGFR-3 in lymphedema patients in comparison to microfilaremic patients , raises the question about the biological significance. Despite a vast literature on VEGF-C or sVEGFR3 expression in tissue (quantitative PCR and immunohistology), few data on plasma exist for these two factors, which are the major axis specific for lymphangiogenesis. What can be concluded from the available data are the following: (1) serum levels of VEGF-C are usually 10-fold higher than plasma levels, because of release from platelets during coagulation; this also means that plasma levels would be the more reliable marker since there is no (dominant) interference by platelet-derived VEGF-C ; and (2)A report on VEGF-C levels in cervical cancer patients provides levels in serum, not plasma, and is therefore not directly comparable to our data (see the constraints above); nevertheless, in that report, after anticancer therapy the levels decreased to the level of controls, accompanied by concomitant improvement in the conditions of the patients , as alsoUnfortunately no data exist as yet with regard to plasma sVEGFR3 levels and their biological significance for tumors. However, the more than 3-fold elevation in lymphedema patients is not considered to be biologically meaningless.The degree of lymphatic dilation caused by filarial worms is considered an indirect measurement of the altered lymphatic function . One stuWolbachia from adult filarial worms in the lymphatic vessels. There are now valid data from human studies that Wolbachia stimulate proinflammatory cytokines such as TNF, IL-1B, IL-6, and nitric oxide [Wolbachia could affect lymphatic vessels via VEGF-C and its receptor sVEGFR-3. It is therefore conceivable that any therapeutic intervention that causes reduction of VEGF-C–inducing proinflammatory cytokines such as TNF, IL-1B [The mechanism of action of doxycycline on the reduction of VEGF-C/sVEGFR-3 and its ameliorating effect on lymphatic vessel is not yet fully clarified. However, this may be associated with depletion of ic oxide , which aic oxide . This raF, IL-1B , etc. woIn this regard, it is remarkable that there was a significant increase in the prevalence of hydroceles in placebo as compared to doxycycline treated patients in our study.Doxycycline treatment significantly reduced the stage of disease in lymphedema patients, whereas the stage was not significantly altered in placebo patients, who, rather, showed a trend towards deterioration. The improvement in the stage in doxycycline patients manifested as better skin texture, fewer deep folds, and also fewer entry lesions of the affected legs (unpublished data). This suggests that doxycycline can be used to improve the clinical manifestations of lymphedema due to filariasis. A reduction of the number of adenolymphangitis (ADL) attacks that was noted in both, doxycycline and placebo patients (unpublished data) could be attributed to the foot-care hygiene training given to all the lymphedema patients, and with an attempt by patients to demonstrate best compliance. This is consistent with other reports ,10,69,70Clinical manifestations of lymphedema do not only hasten the progression of lymphedema to elephantiasis , but alsWolbachia endosymbionts, by exogenous bacteria, or by both [The current treatment of lymphedema, which mainly relies on foot hygiene, is suboptimal and still empirical. This is because it is not clear at present to which extent ADL attacks are caused by either filarial worms, including  by both ,75.Wolbachia endosymbionts, has also been tested and reported to be beneficial in reducing only the incidence of ADL attacks [Wolbachia endosymbionts and reduced lymphangiogenic factors along with a reversion of lymph vessel dilation. In order for doxycycline to exert its ameliorating activity by targeting Wolbachia, it is mandatory that the targets, i.e., adult worms or at least incoming L3/4 larvae, are present in the host's body. Indeed, all our patients were either CFA-positive  [Several chemotherapeutic agents have been tested, but so far, only 5,6 benzo-alpha-pyrone (coumarin) showed some encouraging results . However attacks , but the attacks . In contatients) , or theyatients) . StudiesLymphedema patients have a high level of suffering, and it can be well expected that they would use doxycycline for 6 wk without the need of a directly observed treatment (DOT). This is so all the more since the current mass treatment does not improve lymphedema per se. Hence, doxycycline treatment will have a good chance to be the first chemotherapeutic approach to address lymphatic pathology.Wolbachia upon being killed, or  by skin commensals such as streptococci [Wolbachia nor exogenous species), and therefore has no effect on proinflammatory cytokines that regulate this VEGF family, although it does partially reduce adult worm levels.In conclusion, our data are in agreement with the hypothesis that progression of infection to lymphedema may be due to overexpression of lymphangiogenic factors such as VEGF-C and more so, sVEGF-R3, first due to the presence or the death of adult filarial worms in the lymphatic vessels and later (in the CFA-negative phase of the disease) either by the incoming larval stages of the parasite releasing ptococci ,79,80 thptococci  in whichptococci , which dp = 0.0024, Although both microfilaremic and lymphedema patients had elevated VEGF-C and sVEGFR-3 levels, those of sVEGFR-3 were yet significantly higher in lymphedema patients compared to non-lymphedema patients that are microfilaremic , Kumasi, Ghana, as well as by the ethics committee of the University of Liverpool which acted as a control body since this work formed part of a European study network funded by the European Commission. The study conformed to the principles of the Helsinki Declaration of 1975 (as revised in 1983 and 2000). The trial registration number is ISRCTN 14757.Individuals enrolled from the neighboring villages of Adjan, Domunli, and Akonu in the Western Region of Ghana took part in the study. No other human filarial species were endemic in these villages. A total of 76  microfilaremic A and 19 Randomization of patients was carried out using computer-generated random number software (StatView). Blinding was assured by the exclusion of persons involved in randomization or tablet packaging in any clinical or laboratory assessment.Participants received 2 × 100 mg capsules of doxycycline  or matching placebo supplied by the manufacturer daily for a total of 6 wk. Treatment was done and monitored by a trial clinician in the form of daily observed treatment (DOT). Four months after the start of treatment, all participants received a standard oral dose of 400 mg albendazole  and 150–200 μg/kg ivermectin .Male participants were examined using a portable ultrasound machine  equipped with a 7.5 MHZ linear transducer as described previously . BrieflyFor a quick screening in the night, the microfilarial load was determined by microscopic examination of finger-prick blood samples as published . SubsequWolbachia content was quantified by real-time PCR of the W. bancrofti Wolbachia–ftsZ gene, derived from 500–1,000 microfilariae using a Rotor-Gene 3000  at pretreatment and 4, 12, and 24 mo after treatment. Details of the technique are given in reference [W. bancrofti Wolbachia–ftsZ. The products were quantified by comparing with a standard curve of the plasmid containing the W. bancrofti Wolbachia–ftsZ fragment.eference . BrieflyW. bancrofti antigen was measured with the TropBio ELISA test kit . The manufacturer's protocol was followed except that the samples were diluted (1:20 ratio) with the diluent [For determination of circulating filarial antigenemia ,  diluent  before pThe circumference of both affected and normal legs were measured with measuring tape at five different points. Measurements were taken at 10 cm from the large toe, and 12 cm, 20 cm, 30 cm and 50 cm from the sole of the foot as described in reference , and theGrading or staging of lymphedema was performed along the guidelines of “Basic Lymphedema Management”  see Fig.Stage 1 = swelling that is reversible overnight, Stage 2 = swelling that is not reversible overnight, Stage 3 = shallow skin folds (ankle), Stage 4 = presence of knobs (lumps or protrusions), Stage 5 = deep skin folds plus knobs, Stage 6 = deep skin folds plus knobs and mossy lesions, and Stage 7 = parameters mentioned above plus patient is unable to perform routine daily activities.Before the treatment began, all lymphedema patients were taught about hygienic care and physical exercises for the affected legs, and importantly, daily cleansing of affected legs with soap and water, and keeping the affected leg dry between washes. All the lymphedema patients were given soap, towels, and plastic bowls for washing the legs. They were visited every 6 mo in their villages. At each visit, the patients were asked through a questionnaire the number of attacks experienced.B. malayi males were recovered 6 mo post infection from Mastomys coucha. Parasites were washed and stored at 4 °C for 4 h. They were then placed in canine musculature, frozen, and 7-μm sections were prepared and transferred onto glass slides. Quality was controlled by using a defined internal control serum (titer 1:320). For the IIFA test, 100 μl of plasma was inactivated at 56 °C for 30 min, and diluted 1:10 in PBS (pH 7.2). Seven-fold serial dilutions to 1:1,280 were made from the initial 1:10 screening dilution. A 10-μl volume of each of the diluted samples was added to separate wells on slides containing the B. malayi antigens, and incubated at 37 °C in a humid chamber for 45 min. After the incubation, the IIFA slides were washed four times for 20 min (3× in 0.1M PBS and 1× in deionized water). A fluorescein-labeled anti-total human immunoglobulin  diluted 1:90 in PBS was applied to each well and incubated for 45 min at 37 °C in a humid chamber. The slides were washed four times for 20 min (3× in 0.1M PBS and 1× in deionized water). Excess water was removed and the slides dried, covered with glycerol , and cover slides. Wells were observed with a immunofluorescence microscope . Fluorescence of either the complete worm section including mesenchym and cuticle or only mesenchym was defined as positive. Fluorescence of the mesenchym without cuticle was regarded as non-specific. A titer of nore than 1:80 (including cuticle fluorescence) was regarded as indicative for filarial infection. A titer equal to or less than 1:20 was regarded as negative. Positive and negative controls were included in the test. This test is validated in regular intervals within a German network of laboratories for quality control.Filarial antibodies were measured from all the lymphedema patients before and 12 mo after treatment using the indirect immunofluorescence antibody (IIFA) test. For antigen preparation, adult In all patients, the plasma concentrations of VEGF-C and sVEGFR-3 were measured using Quantikine immunoassay ELISA kits according to the manufacturer's instructions (R&D Systems). After stopping the reaction, plates were read at 450 nm and 540 nm with a microplate reader . Twenty-three endemic normals, i.e., residents of the same endemic area with no evidence of infection confirmed by the lack of mircofilaraemia and lack of circulating Og4C3 filarial antigen, despite exposure to infective larvae borne by mosquitoes, were included as controls.Wolbachia loads in worm tissue and microfilaremia were summarized as geometric means (GM). Differences in GM at baseline and subsequent follow-ups were analyzed using the Wilcoxon signed rank and Mann-Whitney U tests. Changes in the degree of antigenemia were calculated as percentages from baseline and analyzed between treatment groups at subsequent follow-up time points by the Mann-Whitney U test. Proportions of MF-positive and MF-negative individuals as well as individuals showing the fFDS before and after treatment were compared using Fisher' exact tests. Plasma levels of the VEGF molecules were expressed as mean ± standard deviations (SD), and differences in the levels of the VEGF molecules between endemic normals, microfilaremic (MF-positive), and lymphedema patients were analyzed using analysis of variance (ANOVA) with the Bonferroni/Dunn post hoc test. Differences in the levels of the VEGFs within the treatment groups before and 12 mo after treatment as well as changes in lymphatic vessel dilation, stages of lymphedema, and the size of affected limbs of lymphedema before and after treatment were assessed using paired t-tests. Positive relationships between the disease states and the VEGF molecules were assessed by the Spearman' rank correlation test and simple regression analysis (coefficient of determination indicated as r). A two-tailed p-value lower than 0.05 was considered significant. Independence of data was tested using multivariate analysis. All analyses were done using StatView version X for Mac OS 9.5.http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the gene and gene products discussed in this paper are: soluble VEGFR-3 (NM_002020); VEGF-C (CAA63907); and Wuchereria bancrofti Wolbachia–ftsZ gene (AF081198).The GenBank (