Fusobacterium nucleatum genome sequence we have examined the evolutionary relationships of Fusobacterium genes by phylogenetic analysis and comparative genomics tools.The phylogenetic position and evolutionary relationships of Fusobacteria remain uncertain. Especially intriguing is their relatedness to low G+C Gram positive bacteria (Firmicutes) by ribosomal molecular phylogenies, but their possession of a typical gram negative outer membrane. Taking advantage of the recent completion of the Fusobacterium has a core genome of a very different nature to other bacterial lineages, and branches out at the base of Firmicutes. However, depending on the method used, 35–56% of Fusobacterium genes appear to have a xenologous origin from bacteroidetes, proteobacteria, spirochaetes and the Firmicutes themselves. A high number of hypothetical ORFs with unusual codon usage and short lengths were found and hypothesized to be remnants of transferred genes that were discarded. Some proteins and operons are also hypothesized to be of mixed ancestry. A large portion of the Gram-negative cell wall-related genes seems to have been transferred from proteobacteria.The data indicate that Many instances of similarity to other inhabitants of the dental plaque that have been sequenced were found. This suggests that the close physical contact found in this environment might facilitate horizontal gene transfer, supporting the idea of niche-specific gene pools. We hypothesize that at a point in time, probably associated to the rise of mammals, a strong selective pressure might have existed for a cell with a Clostridia-like metabolic apparatus but with the adhesive and immune camouflage features of Proteobacteria. Fusobacterium, together with some close relatives such as Leptotrichia, forms an ecologically and physiologically coherent group [Fusobacterium nucleatum has received particular attention being a key component of the human dental plaque that also has considerable pathogenic potential. In fact after Bacteroides, Fusobacterium is responsible for most human anaerobic infections, producing abscesses at different locations and aspiration pneumonia among other serious conditions [The genus nt group . They sent group . They arnt group ). The spnditions ,5.Bacteroides and other Gram negative anaerobes, their association became conflicting when, after the extensive gene sequencing carried out by the mid 80's, it became clear that Bacteroides showed a clear relationship to other aerobic Gram negatives such as Flavobacterium or Cytophaga [Fusobacterium appeared as a separate cluster only distantly associated to the low G+C Gram positives [Phylogenetically speaking the fusobacteria have become somewhat of a puzzle . Originaytophaga -9 while ositives ,11. Howeositives -14).Fusobacterium nucleatum genome [Clostridium species (low G+C Gram positives) genomic analysis showed also a strong proximity to Proteobacteria. Based on the ERGO chromosomal clustering tool, F. nucleatum had more "clusters" of genes with the same gene order in common with Escherichia coli than with Enterococcus or Staphylococcus, although less than with Clostridium or Bacillus [F. nucleatum thrives is an ecosystem with a large bacterial biodiversity. In a recent survey using 16S rDNA sequences from sub gingival plaque samples, 347 species or phylogroups were identified, and the best estimate of the total species diversity in the oral cavity is approximately 500 species [F. nucleatum interacts with a great deal of them, because it plays a crucial "bridge" role between early and late colonizers of the tooth surface [F. nucleatum interacts and aggregates  there is a great potential for HGT.The publication of the m genome  did not Bacillus . As expeBacillus . On the  surface  and form surface -21. BecaF. nucleatum, using a variety of bioinformatics tools, in an attempt to clarify the phylogenetic position of the Fusobacteria and the relative contributions of vertical descent and horizontal transfer in shaping the genome of this highly specialized organism. In addition, our study aims at providing material for further discussions on evolution of the gram-negative cell wall, and on the evolution of bacterial communities in micro-environments.We have reanalysed the fully sequenced genome of Fusobacterium with the cyanobacteria and the elongation factor tufA with the proteobacteria. Other cases are also unclear. DNA pol III is a complex holoenzyme formed by 10 subunits in E. coli [Thermotoga and Aquifex. The RNA-directed DNA polymerase and a RNA helicase seem related to archaeal counterparts. In Clostridium, for example, to which most informational genes of Fusobacterium have a best match, all subunits of both polymerases cluster clearly into the Firmicutes (data not shown). Summarizing, the fusobacteria appear as an independent taxon with remote relatedness to the Firmicutes. Although the phylogenetic signal for many genes was considerably weak, the rRNA genes and the ribosomal proteins were very congruent and therefore their trees  and Bacteroides thetaiotaomicron (the Gram-negative species to which it shows the highest number of homologs). Due to differences in mutational biases between the leading and lagging strands, it is common to find the GC skew value (G-C/G+C) with opposite signs on each replichore, the change in sign indicating the origin or terminus of DNA replication [F. nucleatum display a standard plot, with mainly positive values over the right replichore and negative values on the left replichore. Fusobacterium, however, does not show a clear pattern, with constant shifts in GC-skew values across the genome. This situation could be caused by horizontal gene transfer (HGT) incorporating xenologous sequences with a different GC-skew across the recipient chromosome, distorting a clear-cut plot. In favour of this, many GC skew oscillations coincide with clusters of putative xenologous origin . Although other related genomes are not available for comparison and the potential inversions could have happened prior to the split of both subspecies, the suggestion is that the oscillating GC-skew plot is not due to multiple inversions. Finally, the GC-skew plot of F. nucleatum could be partly due to multiple replication origins constantly shifting the values, but this situation has not been observed in any bacterial species.The observed GC-skew could also arise from chromosomal inversions . Howeveobserved ,31 and aobserved . Moreoveincentii  show an A sequence similarity search performed by BLASTP  against Fusobacterium chromosome and, in many cases, they are clustered  also hints at a secondary acquisition by HGT. Therefore, the interpretation of F. nucleatum as a gram-positive bacterium with gram-negative clothing [Looking into more detail at the gene clusters with a best hit outside Firmicutes it was found that many genes were involved in typical gram-negative features, mainly membrane-associated functions Table . For exaclostridiales and streptococci), which share the mouth and dental plaque ecosystem [F. nucleatum with most sequenced genomes, a weak phylogenetic signal may remain for many trees. The branching pattern in trees is also influenced by other variables like different rates of evolution for different genes, method of alignment or number of species included. Like in the sequence similarity method, the data presented are inferences based upon the data, and the limitations of each method should be kept in mind.BLAST analysis has the advantage of giving a closest similarity match for almost every gene. It is however a crude method that can give as the top sequence similarity hit a species that is not the closest from a phylogenetic point of view  and it ccosystem . The phyF. nucleatum protein-coding genes belonged to clusters of 2 or more genes that had some order conservation in other bacteria. From these, 35% had the same order as most Firmicutes  best sequence similarity match, respectively. The tree analysis confirms that the β chain is likely to have a proteobacterial origin. Interestingly, although the β chain is located in a proteobacterial cluster (at the edge of cluster 12), it is contiguous to the Firmicutes related α chain gene, separated by a very short spacer without a promoter. This exemplifies how selection may have put together two functionally related genes, presumably to ease cotranscription, even though their phylogenetic origin appears to be different.To explore the possibility that the chimeric nature of bacteria . An inteMethanosarcina acetivorans, to which it presents the highest sequence similarity, whereas the second and third operon appear to be a blend of genes with relatedness to firmicutes, proteobacteria, spirochaetes and Thermotoga. These genes are present in many Gram negatives including Helicobacter and other Proteobacteria [F. nucleatum, this time dispersed along the genome. Although one of the sets has best matches to Firmicutes species, another one appears to be of spirochaete source , whereas the other three are, according to sequence similarity, gene order and tree reconstruction a mixture of genes with archaeal, Firmicutes and proteobacterial origin. A third case can be seen in the three copies of a hemin transport system located away from each other and formed by a hemin receptor and the genes hmuT, hmuU and hmuV. Although the different taxonomical origin analysis methods are not always consistent, two of the hemin operons are probably of proteobacterial origin  and a potential transferred gene, whereas only 8 appeared between core genes. Since intergenic spacer regions are known to increase in length as a result of genomic rearrangements and pseudogene formation [-5) to one another and to other five long spacers scattered throughout the genome. In all cases except one, these long spacers are flanked by outer membrane proteins of Gram-negative origin, suggesting that they may represent remnants of old membrane-associated genes. A similar case is that of the long spacer located after the hemolysin activator protein precursor (FN1818), which shows high sequence similarity to a hemolysin activator located someplace else in the genome (and to another spacer and a short ORF with unknown function).An indication of massive gene transfer events comes from looking at intergenic spacer regions of ormation , many ofvincentii), spanning 450 sequences that represent 20% of the genome. On average, these orphan genes are extremely short , glycine permease, periplasmic-like proteins, etc.Another potential signature of ancient transfers subsequently erased is the high number of ORFs without a match in the complete, non-redundant NCBI database . As it is unlikely that all these short ORFs are highly expressed, we believe that this biased codon utilization is reflecting very divergent pseudogene fragments. Thus, the picture that emerges is that of massive gene transfer leaving many non-coding segments that are remnants of unnecessary genes and genomic rearrangements.In addition, some short ORFs appear to be degraded fragments of bigger genes. For example, there are 3 sequences with similarity to HIPA proteins, one of which is less than half the length of the other two. As it also has a very biased codon usage, it is likely that it represents a degraded remnant of this protein. The 3 copies of integrases scattered across the genome show another case. Two of them are around 900 bp long and have a normal codon usage. The third copy (FN0402) is only 177 bp long, is flanked by a long spacer and has a very skewed codon usage. In general, the codon usage of these orphans is very biased  points to HGT as the most likely origin of these genes. Even less apparent, our work suggests multiple episodes of gene transfer to or from phylogenetically-related bacteria, like certain Firmicutes species (such as the cariogenic bacterium S. mutans or some Clostridia), that might be confounded with vertically inherited traits.The genome of  example ). One poFusobacterium requires special consideration. Some type of Gram-negative cell wall seems to be the default phenotype in Bacteria  with Gram-negative cell wall structures [Fusobacterium does not show any primitive trait and its outer membrane and transport mechanisms show all the characteristics of any sophisticated Gram-negative cell wall. In addition, many of the outer membrane proteins are closest to specific taxa  and not equally dispersed among species with a Gram-negative cell wall. Thus, many of the genes involved in the construction of the Gram-negative outer membrane have probably been horizontally transferred. The extent of this transfer deserves further examination. If we assume that Fusobacterium evolved after the Gram-positive/negative divergence on the low-GC Gram-positive lineage, massive HGT is the most likely explanation for the formation of the outer membrane. On the other end of possible explanations, most genes of the outer membrane would already be present in the common ancestor of fusobacteria and Firmicutes, where a massive loss would be responsible for the differences observed today.The origin of the Gram-negative cell wall found in  example ), being ructures ,59. On tructures . It mighructures . HoweverThermotoga is paradigmatic, a hyperthermophilic bacteria that is assumed to have recruited genes from the archaeal hyperthermophiles to reach its unusual (for bacteria) thermotolerance. Here  there is not such an obvious explanation. F. nucleatum natural habitat seems to be the dental plaque of mammals, a rather unique and special environment that probably requires very special features to survive. Strong adhesion mechanisms, such as those found often in the Proteobacteria, probably represent an essential ability for survival in the early stages of plaque formation, particularly for non-motile cells. Also the mucose-associated immune system that prevails in the mouth of mammals could have acted as a strong selective pressure favoring the Gram-negative envelopes that are often less immunogenic and easier to disguise thanks to the LPS polysaccharide O chain [Fusobacterium and its interaction with the environment, such as peptide transport systems, cell adhesins and outer membrane components have probably been acquired by gene transfer. It is therefore not only the number of horizontal transfers but also their contribution to niche adaptation that makes the HGT mechanism of dramatic impact on genomes. It is interesting that some of these genes are shared by different organisms inhabiting the dental plaque. From an applied point of view, some of these highly transferred genes are likely to provide a critical advantage in the establishment and adaptation of the bacteria to their niche, and could be used as potential targets for antimicrobial agents.Recently, the idea of gene pools that are characteristic of certain environments has been advanced to explain the large number of common genes among groups of thermoacidophiles distantly related by ribosomal phylogeny . The pre O chain . Thus, iThe different rRNA and conserved protein data sets were analyzed with Bayesian methods using the program MrBAYES 3 . For theRickettsia prowazekii, Rickettsia conorii, Wigglesworthia brevipalpis, and Buchnera aphidicola, and with the addition of Bacteroides thetaiotaomicron and and Desulfovibrio vulgaris. An alignment was generated for each ribosomal gene, using the Clustalw software with default parameters [Fusobacterium nucleatum, S1 is absent/truncated in the Mollicutes subgroup of the low-GC gram-positives, L25, L30, L31, L32, L33 contained a high number of paralogs and/or were absent in several key species.The amino-acid sequences of ribosomal genes S1–S20 and L1–L35, excluding S1, S14, L24, L25, L30, L31, L32 and L33, were retrieved from the KEGG website from a total of 60 different bacteria. The bacteria chosen were all those represented in the KEGG ribosomal genes ortholog table , except rameters . When twrameters , excludirameters . Exclusirameters , L24 is Fusobacterium nucleatum subsp. nucleatum ATCC 25586 were retrieved from . Peptide sequence database of all non-redundant GenBank CDS translations + PDB + SwissProt + PIR was retrieved from . We performed an all against all BLASTP [Fusobacterium nucleatum subsp. nucleatum ATCC 255586 against peptide sequence database. We then recorded the top hit for each protein sequence with an E-value of 10-5, filtering the hits whose sequence identity and length was lower than 30 and 50%, respectively. We categorized all the hits into 8 categories as belonging to the CFB group, Firmicutes bacteria, α,β,γ-Proteobacteria, δ,ε-Proteobacteria, Spirochaetes, other Bacteria, Archaea and Eukaryotes/no hit. Hits to Firmicutes (the group to which Fusobacterium appear to be more closely-related) were refined by further BlastP analysis between F. nucleatum and the 31 sequenced bacteria available from this group. If the gene had a homolog in only one genus from all the available low-GC gram-positive species, it was considered a HGT event from/to this group. If it was present in more than one genus it was considered vertically inherited and consistent with the ribosomal phylogeny. There were 61 cases of genes found in more than one genera from a single subgroup of this taxon . These can be equally explained by HGT or by common descent and were conservatively assigned to the vertical inherited category.The protein sequences of l BLASTP  search oF. nucleatum gene, the protein sequences of up to 50 best blast hits with e-value lower than e-5 were retrieved (the hits were identified by the "Blast method" described above). All sequences were then automatically clustered with the Clustalw alignment tool with default parameters. A neighbor-joining tree with 1000 bootstrap replicates was generated from the resulting alignment, using Clustalw with default parameters. The trees were visualized with NJPLOT [F. nucleatum genes to a taxonomic group was done using the following criteria:For each h NJPLOT . In all F. nucleatum gene was determined to originate from the firmicutes if it was found in the tree most closely associated with at least 5 different species from that group, or with at least 3 species from 2 different subgroups . If the F. nucleatum gene branched at the base of the firmicutes, the gene was assigned as being consistent with phylogeny; otherwise, it was assigned as a potential horizontal gene transfer (HGT) from the firmicutes.A F. nucleatum genes with trees fulfilling this criteria were assigned as HGTs from proteobacteria. Note that a distinction was made between the grouping of the alpha-, beta-, and gamma- proteobacteria, and the grouping of the epsilon- and delta- proteobacteria whenever possible.Same as described above (low-GC gram-positives), the subgroups in this case were:alpha-, beta-, gamma-, gamma-entero-, delta-, and epsilon-proteobacteria. In the case of the proteobacteria, all F. nucleatum had to be closest to at least 3 species, and there had to be a clear association between the two groups, i.e. the branches were relatively short, and the tree topology did not resemble a "star phylogeny".To be assigned as originating from the archaeales, the F. nucleatum gene had to be found closest in the tree to at least 3 different species, there had to be a clear association  and there should have been no obvious evidence of gene transfer from the Fusobacteria. Evidence of transfer from the Fusobacteria would be when, apart from the association to some species of the high-GC gram positives , the tree placed the F. nucleatum gene in agreement with the accepted species phylogeny (just outside of the firmicutes).To be assigned to these groups, the F. nucleatum gene had to be found closest to at least 1 species from that group . A clear association was necessary, as well as no evidence of transfer from Fusobacteria (see above).The F. nucleatum gene had to be closest to at least 2 species from that group, or 1 species with a clear association, and no evidence of transfer from Fusobacteria.The F. nucleatum gene, it was then assigned as "unknown/not resolved".If the tree contained less than four hits other than eukaryotes and other Fusobacteria, the gene was not considered for further analysis. In cases where it was not possible to clearly associate a taxonomic group to the F. nucleatum and other genomes, all available amino-acid protein sequences sets for all replicons of all published bacterial and archaeal genomes at the time (may 1st 2004) were downloaded from the NCBI ftp website . All Orthologs of F. nucleatum genes  were detected between the two replicons (F. nucleatum and replicon X). Clusters of consecutive orthologs were found (consecutive orthologs are determined in terms of the numbered position in both F. nucleatum (exactly consecutive) and replicon X (possible gap of 2 genes in between the orthologs), and for each cluster, a score was assigned as follows:In order to identify clusters of at least two genes with conserved order between )*(1-(deletions/numberOfGenesInCluster)) *Mean(Identity%)*Mean(Length%)*numberOfGenesInCluster/10000F. nucleatum gene the orthologs that were part of clusters were ordered by their score, and an excel table was generated for manual investigation. If the gene order of a given gene cluster was not preserved in Firmicutes species but maintained in another procaryotic group, the genes were assigned as HGT from/to the group with the highest score (highest gene-order conservation). If the order was preserved in at least one species from two or more groups of low-GC gram-positives  the cluster was assumed to be ancestral to the divergence of fusobacteria and Firmicutes, and consistent with the ribosomal phylogeny. If gene order was preserved in one or more species from only one of the low-GC gram-positive groups the cluster was classified as HGT from low-GC gram-positive bacteria.Where "totalCost" was determined by the program derange2 ,72 with F. nucleatum genes by function was based on the TIGR Gene Attribute Annotation [Classical GC-skew plots were done using the formula (G-C)/(G+C) in 5000 bp windows, following Lobry's methods ,26. The notation .AM carried out the gene-order analysis of gene transfer, and the study of chimeric operons and short ORFs. FRV conceived the study, and FRV and AM drafted the manuscript. RP and BAL did the bioinformatics work. RP and AM carried out the BLAST analysis of gene transfer and made the figures, except the phylogenetic trees. BAL developed the phylogenetic and gene-order method and did the ribosomal proteins tree. DM did all Bayesian trees. All authors read and approved the final manuscript.Fusobacterium nucleatum based on phylogenetic analysis and comparative genomics. Supplementary material published as additional information in the manuscript Mira et al. 2004. Evolutionary relationships of The file contains three additional figures. It is available in pdf format and includes figure legends.Click here for file


Keratocyte loss by apoptosis following epithelial debridement is a well-recognized entity. In a study of corneal buttons obtained from patients of corneal ulcer undergoing therapeutic keratoplasty, we observed loss of keratocytes in the normal appearing corneal stroma, surrounding the zone of inflammation. Based on these observations, we hypothesized that the cell loss in the inflammatory free zone of corneal stroma is by apoptosis that could possibly be a non-specific host response, independent of the nature of infectious agent.Acanthamoeba keratitis. The corneal sections were reviewed for morphologic changes in the epithelium, stroma, type, degree and depth of inflammation, loss of keratocytes in the surrounding stroma . TUNEL positivity was evaluated in the corneal sections, both in the zone of inflammation as well as the surrounding stroma. A correlation was attempted between the keratocyte loss, histologic, microbiologic and clinical features.To test our hypothesis, in this study, we performed Terminal deoxyribonucleotidyl transferase-mediated d-Uridine 5" triphosphate Nick End Labelling (TUNEL) staining on 59 corneal buttons from patients diagnosed as bacterial, fungal, viral and Acanthamoeba (8) keratitis. The morphological changes in corneal tissues noted were: epithelial ulceration , destruction of Bowman's layer , mild to moderate  to severe inflammation . Morphologic evidence of disappearance or reduced number of keratocytic nuclei in the corneal stroma was noted in 49 (83%) cases; while the TUNEL positive brown cells were identified in all cases 53/54 (98%), including cases of fungal (19), bacterial (14), viral (13), and Acanthamoeba keratitis. TUNEL staining was located mostly in the deeper stroma and in few cases the peripheral stroma. TUNEL positivity was also noted with the polymorphonuclear infiltrates and in few epithelial cells  cases, more with viral infections .The corneal tissues were from 59 patients aged between 16 years and 85 years (mean 46 years) and included fungal (22), viral (15), bacterial (14) and We report apoptotic cell death of keratocytes in the corneal stroma in infectious keratitis, a phenomenon independent of type of infectious agent. The inflammatory cells in the zone of inflammation also show evidence of apoptotic cell death. It could be speculated that the infective process possibly triggers keratocyte loss of the surrounding stroma by apoptosis, which could possibly be a protective phenomenon. It also suggests that necrotic cell death and apoptotic cell deaths could occur simultaneously in infective conditions of the cornea. Acanthamoeba keratitis and had proposed that one of the mechanisms for this loss was apoptosis of keratocytes [Infections of the cornea are potentially blinding diseases and may be caused by fungi, bacteria, viruses and a few protozoans. Despite best efforts with early diagnosis and specific antimicrobial treatment about one-third of cases require surgical intervention . Those watocytes . We haveApoptosis is an active process orchestrated by a series of events starting from activation of genes, release of enzymes, specific morphologic changes and ultimately leading to "an environmental-friendly cell death" whereby the dead cells are removed without affecting the bystander cells -15. ThisThe corneal buttons removed during therapeutic keratoplasty for uncontrolled infections could possibly throw some light on these events. This study was designed to look for the evidence of apoptotic cell death of keratocytes, along with the morphological changes in corneal tissues in infective conditions.Acanthamoeba (8) keratitis in the study. The diagnosis of fungal, bacterial and Acanthamoeba keratitis was based on the microbiological investigations as described below. The etiologic diagnosis was consistent with histopathology of the corneal buttons excised during therapeutic penetrating keratoplasty. Clinical information was available in 43 cases from the ulcer database of our institute. The cases with missing clinical data on a specific parameter were excluded while calculating the statistical significance of that parameter. The clinical data was analyzed for duration of symptoms, size of ulcer, location, depth of infiltrates, presence of perforation, thinning, anterior chamber infiltration and duration of treatment.Corneal tissues from patients diagnosed as infectious keratitis undergoing therapeutic keratoplasty between 1998 and 2002 were reviewed. The corneal sections of those cases, in which the normal stroma surrounding the zone of inflammation and necrosis was visible, were included in the study. We included 59 patients diagnosed for fungal (22), viral (15), bacterial (14) and Acanthamoeba. Corneal scrapings were subjected to Grams, Giemsa and Calcofluor white stain for direct smear examination and culture on blood agar, chocolate agar, brain heart infusion broth, thioglycollate broth, Sabouraud's dextrose agar and non-nutrient agar with Escherichia coli overlay. Immunofluorescent assay for HSV-1 was done on cold acetone fixed corneal scraping smears using anti-rabbit HSV-1 polyclonal antibody and FITC conjugated swine antirabbit immunoglobulin . In addition, polymerase chain reaction using primers specific for HSV-1 glycoprotein D gene was done on DNA extracted from corneal scraping with commercially available DNA zol solution All patients had undergone microbiological investigations by processing of their corneal scrapings for bacteria, fungus, The excised corneal buttons of the 59 patients were subjected to routine histopathology processing. The paraffin embedded sections were stained with hematoxylin eosin, periodic acid Schiff's and special stains like Gomori's methenamine silver stain and Grams stains. The sections were studied for histologic changes viz. ulceration, inflammation, vascular channels, necrosis, keratocyte loss and other details. Inflammation was graded in a semi-quantitative way as mild, moderate or severe based on the density of the inflammatory cells and the visibility of the stromal collagen in the background. We looked for the absence or the reduced number of the keratocytic nuclei in the corneal stroma surrounding the zone of inflammation.Terminal deoxyribonucleotidyl transferase-mediated d-Uridine 5" triphosphate Nick End Labeling (TUNEL) staining was performed on all the 59 sections, as per the manufacture's instructions using In-Situ Cell Death Detection Kit, POD . In brief, the sections were de-paraffinized, re-hydrated and incubated with proteinase K (20 ug/ml) for 10 minutes at room temperature. After rinsing with phosphate buffered saline (PBS) the sections were blocked with 3% hydrogen peroxide in methanol for 10 minutes. The sections were then incubated in the permeabilization solution consisting of 0.1% triton × 100 in 0.1% sodium citrate for 20 minutes. After washing the sections were incubated with TUNEL reaction mix at 37°C in a humidified chamber for 2 hours followed by washing and incubation with POD converter for 2 hours, the sections were stained with diaminobenzidine and counter stained with hematoxylin and observed under the microscope. Sections from the normal corneal tissues obtained from the eyebank corneas were used as control slides. Statistical analysis was done using Chi-Square test and Fisher Exact test.Acanthamoeba keratitis. The mean age of the patients was 46 years (range 15–85 years) with male to female ratio of 1 (22): 1.7 (37). Based on microbiologic and histologic findings, the buttons were diagnosed as fungal keratitis in 22; bacterial in 14, viral in 15 and Acanthamoeba keratitis in 8 cases.We studied the excised corneal buttons of 59 patients who underwent therapeutic penetrating keratoplasty for fungal, bacterial, viral and The clinical features of 43 patients at the time of presentation are given in Table The histologic changes were observed in the sections of the excised corneal tissues are described in Table Acanthamoeba etiology. In addition, TUNEL positive cells were seen in the epithelium in 10 cases,  declare that they have no competing interests.This work was conceived and planned by Geeta K Vemuganti. Clinical and microbiological support was provided by Prashant Garg and Savitri Sharma respectively. The bench work and analysis was done by Kishore Reddy and Ghazala Iftekhar.The pre-publication history for this paper can be accessed here:


Some currently available birth weight for gestational age standards are customized but others are not. We carried out a study to provide empirical justification for customizing such standards by sex and for whites and blacks in the United States.We studied all male and female singleton live births and stillbirths  in the United States in 1997 and 1998. White and black singleton live births and stillbirths were also examined. Qualitative congruence between gestational age-specific growth restriction and perinatal mortality rates was used as the criterion for identifying the preferred standard.The fetuses at risk approach showed that males had higher perinatal mortality rates at all gestational ages compared with females. Gestational age-specific growth restriction rates based on a sex-specific standard were qualitatively consistent with gestational age-specific perinatal mortality rates among males and females. However, growth restriction patterns among males and females based on a unisex standard could not be reconciled with perinatal mortality patterns. Use of a single standard for whites and blacks resulted in gestational age-specific growth restriction rates that were qualitatively congruent with patterns of perinatal mortality, while use of separate race-specific standards led to growth restriction patterns that were incompatible with patterns of perinatal mortality.Qualitative congruence between growth restriction and perinatal mortality patterns provides an outcome-based justification for sex-specific birth weight for gestational age standards but not for the available race-specific standards for blacks and whites in the United States. Birth weight-specific perinatal mortality curves among male and female births intersect to produce a paradox: overall perinatal mortality rates and perinatal mortality rates at lower birth weights are relatively higher among male births, while at higher birth weights perinatal mortality rates are relatively higher among female births . This puIn this paper, we demonstrate the paradoxical crossover of birth weight-specific perinatal mortality curves among male and female births and show how this phenomenon is resolved using the fetuses at risk approach. We also explore issues related to fetal growth restriction among males and females using the same approach. This latter issue is particularly important from a conceptual and clinical standpoint because the current literature on birth weight for gestational age standards  is confusing. Some standards provide unisex reference values -24, seveWe used the fetuses at risk approach to contrast growth restriction and perinatal mortality rates among males and females in order to provide empirical justification for sex-specific (vs unisex) birth weight for gestational age standards. We also constructed and compared gestational age-specific growth restriction and perinatal mortality curves among whites vs blacks in order to evaluate currently available birth weight for gestational age standards (single standard vs separate standards for whites and blacks in the United States).We used data on all reported live births and stillbirths in the United States in 1997 and 1998 . Live births and infant death records for these years have been previously linked and gestational duration has been calculated based on the last menstrual period (LMP). Missing or inconsistent information on gestational age has been imputed or replaced in a small fraction (approximately 7 percent) of records by the National Center for Health Statistics . Gestational age was imputed from the month and year of the LMP when the exact LMP day was missing . LMP-basAnalyses were restricted to singleton live births and stillbirths ≥22 weeks gestational age and ≥500 g birth weight in order to eliminate potential problems arising from regional differences in birth registration. Male and females births were first contrasted in terms of their gestational age and birth weight distributions. Birth weights were categorized into 500 g intervals for this purpose . Birth weight-specific perinatal mortality rates, calculated within these birth weight categories, were computed as per convention by dividing the number of stillbirths and early neonatal (0 to 6 days) deaths in any birth weight category by the number of total births (stillbirths and live births) in that birth weight category. Similarly, gestational age-specific perinatal mortality rates among male and female births were contrasted, with rates computed by dividing perinatal deaths at any given gestation by the number of total births at that gestation.The numbers of fetuses at risk for stillbirth and early neonatal death at each gestation were then used to calculate a second set of perinatal mortality rates. Under this fetuses at risk formulation, the stillbirth rate at 28 weeks gestation was computed by dividing the number of stillbirths at 28 weeks by the number of live births and stillbirths at 28 or more completed weeks of gestation. This implies that fetuses who delivered at 29, 30, 31 and 32 or more weeks gestation were also at risk of stillbirth at 28 weeks -19,41-44th percentile cut-off from a birth weight for gestational age standard based on live births in the United States [th percentile values provided by this standard [th percentile of birth weight for gestational age  compared with appropriate-for-gestational age births at the 50th percentile of birth weight for gestational age . We also examined gestational age-specific growth restriction differences among males and females using rate ratios  and contrasted these with gestational age-specific differences in stillbirth and neonatal mortality rates . This was done to ascertain the relationship between patterns of growth restriction and patterns in the two components of perinatal mortality .We also examined gestational age-specific patterns of fetal growth restriction using the fetuses at risk approach ,17-19. Td States . Gestatistandard  to evaluComparisons of male and female gestational age-specific growth restriction and gestational age-specific perinatal mortality patterns were contrasted with similar comparisons according to maternal race. Specifically, live births and stillbirths ≥22 weeks of gestational age and ≥500 g birth weight in the United States in 1997 and 1998 were used to compare gestational age-specific growth restriction and perinatal mortality rates among whites vs blacks.Identification of SGA live births among blacks and whites was carried out using a single standard for both races  and alsoDifferences in rates were assessed using rate ratios and excess risks. Taylor series 95% confidence intervals were calculated on all rate ratios. All p values presented are two-sided. Sensitivity analyses were carried out to assess the potential effect of gestational age errors on patterns of growth restriction and perinatal mortality among males and females. Specifically, we reassessed growth restriction and mortality patterns among males and females after excluding all births for whom menstrual-based gestational age was either imputed or replaced by the clinical estimate of gestation.There were 3,905,694 singleton male births in the United States in 1997 and 1998 . The low birth weight  rate among male live births was 5.5%, and 10.5% of male live births were born preterm (<37 weeks). There were 3,723,153 female births in the United States during the same period and relative to males, female live births had a higher rate of low birth weight  but a lower rate of preterm birth . Males had a 14%  higher perinatal mortality than females; perinatal mortality rates among males and females were 6.78 and 5.95 per 1,000 total births, respectively.The gestational age distribution of male live births Figure  was 'shiGestational age-specific perinatal mortality rates calculated using the fetuses at risk approach showed that perinatal mortality rates increased with increasing gestational age Figure . Males hhigher growth restriction rate than females at the same gestational week (sex-specific standard) and this was qualitatively congruent with a 17 percent  higher perinatal death rate among males compared with females at 35 weeks gestation. On the other hand, when a unisex standard was used to identify growth restricted live births, males had a lower rate of growth restriction at all gestational ages and this was not qualitatively congruent with the higher gestational age-specific pattern of perinatal mortality among males .Figure s Figure . For insOverall growth restriction rates based on a sex-specific standard showed that rates were 3% (95% CI 2 to 3) higher among males. Stillbirth and early neonatal mortality differences (rate ratios) among male vs female births both favored females , despite a significantly higher perinatal mortality rate among blacks at 40 weeks gestation . On the other hand, rates of gestational age-specific growth restriction were qualitatively congruent with patterns of gestational age-specific perinatal mortality when growth restriction among blacks and whites was defined using a single birth weight for gestational age standard . Growth restriction (based on a single standard for both races) and perinatal mortality rates were substantially higher among births to black mothers as compared with births to white mothers at all gestational ages  for identifying fetal growth restriction at all gestational ages. Our findings challenge the former assumption and show that in fact fetal growth restriction rates are better viewed as increasing with advancing gestational age . This may be a consequence of the use of sex-specific birth weight for gestational age standards or sex-specific ultrasound-based fetal growth standards and, as mentioned, probably also reflects higher rates of suspected fetal compromise among pregnancies with male fetuses. Despite the marginally higher rates of labor induction among pregnancies with male fetuses, however, mortality differences persist. Research should be directed at ascertaining whether excess neonatal mortality among males can be successfully reduced through explicit recognition of male sex as a factor for altering the threshold for obstetric intervention.Although contemporary birth weight for gestational age standards have substantial face validity ,47,48, tOur study has limitations that are typical of studies that use large data bases. Errors in gestational age information are inevitable, although the magnitude of these errors is likely to be similar among male and female births. The overall rate of missing gestational age was low, however (0.9 percent among white live births and 0.8 percent among black live births). Our estimates of gestational age-specific fetal growth restriction rates are approximate. Ideally, estimation of the incidence of fetal growth restriction requires identification of fetal growth restriction on a longitudinal basis among continuing pregnancies . The altThe fetuses at risk approach resolves the paradox of intersecting perinatal mortality curves. Male births have higher rates of gestational age-specific perinatal mortality than female births. There is empirical justification for using sex-specific standards of birth weight for gestational age since gestational age-specific growth restriction patterns based on such standards correspond qualitatively with gestational age-specific perinatal mortality patterns. On the other hand, a single birth weight for gestational age standard for whites and blacks in the United States appears more appropriate than currently available race-specific standards since gestational age-specific growth restriction patterns among blacks and whites (based on a single standard) are qualitatively congruent with gestational age-specific patterns of perinatal mortality.The author(s) declare that they have no competing interests.KSJ proposed the study, carried out the analyses and drafted the manuscript. The results of the analyses were presented and discussed at a meeting of the Fetal and Infant Health Study Group of the Canadian Perinatal Surveillance System. All authors contributed to revising the manuscript for intellectual content. All authors read and approved the final version.The pre-publication history for this paper can be accessed here:


Biliary atresia (BA) is a neonatal cholestatic disease of unknown etiology. It is the leading cause of liver transplantation in children. Many similarities exist between BA and graft versus host disease suggesting engraftment of maternal cells during gestation could result in immune responses that lead to BA. The aim of this study was to determine the presence and extent of maternal microchimerism (MM) in the livers of infants with BA.Using fluorescent in situ hybridization (FISH), 11 male BA & 4 male neonatal hepatitis (NH) livers, which served as controls, were analyzed for X and Y-chromosomes. To further investigate MM in BA, 3 patients with BA, and their mothers, were HLA typed. Using immunohistochemical stains, the BA livers were examined for MM. Four additional BA livers underwent analysis by polymerase chain reaction (PCR) for evidence of MM.By FISH, 8 BA and 2 NH livers were interpretable. Seven of eight BA specimens showed evidence of MM. The number of maternal cells ranged from 2–4 maternal cells per biopsy slide. Neither NH specimen showed evidence of MM. In addition, immunohistochemical stains confirmed evidence of MM. Using PCR, a range of 1–142 copies of maternal DNA per 25,000 copies of patients DNA was found.Maternal microchimerism is present in the livers of patients with BA and may contribute to the pathogenesis of BA. Biliary atresia (BA) is a cholestatic disease of infancy characterized by the destruction of the biliary tree. -3 Both tMany hypotheses on the etiology of BA exist. Two leading hypothesis are that BA occurs as a result of ductal plate malformations occurring during development of the liver, or as a result of an immune mediated process triggered by yet to be determined stimulus.  As more + T – helper (Th) 1 cells. [Graft versus host disease (GVHD), seen after allogeneic hematopoietic stem cell transplantation, is an immune mediated process triggered by the transfer of alloimmune cells from a donor to a host. GVHD shares many similarities with BA. Both BA and GVHD are characterized by a lymphocytic infiltration around the portal triad and damage to the biliary ducts.  The pred1 cells. -14 As wi1 cells. Given the similarities between BA and GVHD, we hypothesized that a contributing etiology of BA could be an alloimmune reaction such as in GVHD triggered by maternal microchimerism. Maternal microchimerism occurs when a small number of maternal cells are transferred to the offspring during pregnancy. This is known to occur in up to 40% of normal pregnancies. ,16 In adThis study was performed with the approval of the Committee of Human Research at USCF (H9048-20247-01). Cases of BA were identified through a data search in the Anatomic Pathology CoPath system by diagnosis or during patient medical visits to the UCSF Division of Pediatric Gastroenterology, Hepatology, and Nutrition. An explanation of the study was given to each study candidate. Written consent was obtained from each study participant.We modified a previously reported method of FISH,  using X ®, Brown Deer, WI, USA).Patients with BA and their mothers were HLA typed. The HLA typing HLA-A, -B, and -DRB1 alleles of the child and mother were determined by sequence specific PCR (SSP)  were fixed in acetone for 10 minutes at 4°C then washed in PBS (5 minutes centrifugation × 3). Sections were blocked with Protein Block  for thirty minutes at room temperature. Sections were then incubated with anti-HLA-B14 mouse monoclonal antibody  then washed in PBS. Sections were incubated in goat anti-mouse conjugated with FITC for 30 minutes at room temperature. Sections were rewashed and then counterstained with DAPI. Slides were then examined using a fluorescent microscope.Using the HLA types of the patients with BA and their mothers, and a previously reported method of kPCR, evidence of maternal DNA was explored within the liver biopsy specimens of explanted BA livers.  The minoUsing FISH, we examined 11 male BA liver biopsies and 4 male NH liver biopsies. After initial digestion and staining of the liver specimens, we were able to interpret 8 of the BA and 2 NH liver biopsy specimens  has a bright signal for HLA-B14 while there is only background staining for HLA-B14 in specimen B (negative control). Specimen C, the test specimen, has isolated scattered bright signals suggesting maternal microchimerism.To further investigate our findings, three patients with BA and their mothers were HLA typed. Using the HLA marker B14, we examined the BA livers for evidence of maternal microchimerism using immunohistochemistry techniques. An HLA-B14 positive patient served as our positive control Figure . SpecimeFinally, using polymerase chain reaction, in conjunction with the HLA typing of patients and mothers, we were able to affirm that maternal microchimerism occurs in the BA livers. An additional four fresh frozen liver specimens of BA infants, obtained at the time of transplantation, were examined for maternal DNA. All the livers had evidence of maternal microchimerism, with the range of 1 to 142 copies of maternal DNA detected per 25000 copies of patients' DNA. Figure In all eleven of twelve patients with BA had evidence of maternal microchimerism by FISH or kinetic PCR, while neither of the two neonatal hepatitis patients had evidence of maternal microchimerism (p = 0.03).BA is a progressive cholestatic disease of infancy, which is characterized by a mild to moderate lymphocytic infiltration within the extra and intrahepatic bile ducts. It is plausible, that maternal microchimerism may play a role in the etio-pathogenesis of BA by causing an alloimmune reaction given the similarities between BA and GVHD. We have shown that maternal microchimerism is present within the livers of patients with BA.Maternal microchimerism occurs when maternal cells reside in the body of offspring. Maternal-fetal lymphocytic transfer is known to occur during pregnancy starting as early as the tenth week of gestation and continuing up to delivery.  The numbDisease has been shown to occur in mothers who have had fetal microchimerism, and children/adults who have had maternal microchimerism. Children who have immunodeficiencies are at greater risk for graft verses host disease owing to engraftment of maternal lymphocytes. In both SCID and DiGeorge syndrome, maternal microchimerism and GVHD have been well described. In a study by Susanne Müller et al., 121 patients with SCID were evaluated for maternal microchimerism using HLA typing. Maternal cells were found in 48 patients, with 19 patients showing signs of GVHD. GVHD manifested itself in the skin and in the liver.  There haThe histologic and immunologic similarities of BA and GVHD are striking. The site of damage in BA and GVHD after bone marrow transplantation is the same. In each, lymphocytes congregate around the bile ducts. The damage occurs to both the intra-and extrahepatic biliary tracts. In a mouse model of acute GVHD, Nonomura et al. showed that transfer of allogeneic cells set along minor HLA mismatch can cause damage to both the intra and extrahepatic biliary ducts. Interestingly, the timeframe in this mouse model of acute GVHD, in terms of lymphocytic infiltration and fibrosis, appear similar to that of BA.  Initiall+ T lymphocytes. The genes in BA livers showed differential lymphocytic function, with activation of osteopontin, a regulator of cell-mediated immunity in Th 1 cells, and interferon gamma. [+ T lymphocyte. Although a variety of complex mechanisms are involved in causing the end organ damage in GVHD, evidence exists showing similarities in effector mechanisms. In a SCID mouse model of acute GVHD, an increase in interferon-gamma secretion with a synchronized increase in activated Th cells was seen during acute GVHD. [The lymphocytic infiltrates in both BA and GVHD are predominantly CD4n gamma.  Similarlte GVHD.  The inflte GVHD. ,25,26Maternal microchimerism is not a rare occurrence in healthy individuals, but is usually not associated with disease. The high occurrence of maternal microchimerism in healthy individuals has been suggested to have a tolergenic effect that may contribute to long-term microchimerism. AdditionaMaternal microchimerism occurs in the livers of patients with BA. Given the previously well-described relationship between microchimerism and GVHD, and the similarities between BA and GVHD, these findings indicate a potential etio-pathogenisis for BA. Further investigations into the role of maternal microchimerism in BA are warranted.None declared.DLS conceived of the hypothesis and contributed to the design and coordination of the study; he also drafted the manuscript. PR and MBH participated in the design of the study and manuscript preparation. DK carried out the kPCR and HLA typing analyses. GM performed the FISH analyses. LBL carried out the kPCR and HLA typing analyses and participated in the study design. MOM participated on the study design and coordination, immunohistochemistry stains and the manuscript preparation.The pre-publication history for this paper can be accessed here:


The demonstration of tissue-bound immunoreactants by direct immunofluorescence microscopy (DIF) is a valuable parameter in the diagnosis of various autoimmune and immunecomplex-mediated skin diseases. For preservation of tissue-bound immunoreactants, biopsies are usually fresh-frozen in liquid nitrogen or transported in Michel's fixative. But even optimally preserved tissue specimens are no guarantee for the correct diagnosis by DIF, especially when weak to moderate IgG fluorescence of the epidermal basement membrane zone is involved. In such cases false negative results are easily obtained due to the relatively high dermal "background" fluorescence produced by polyclonal anti-human IgG fluorescein conjugates.In the present study we have compared the use of normal saline (0.9% NaCl) with liquid nitrogen and Michel's fixative as transport medium for skin biopsies. From 25 patients with an autoimmune skin disease  four matched skin biopsies were obtained and transported in either saline for 24 and 48 hours, liquid nitrogen, or Michel's fixative for 48 hours.Direct IF microscopy showed significant reduction of background fluorescence (p < 0.01) and relatively enhanced desired specific  staining in biopsies transported in saline. A conclusive or tentative IF diagnosis was reached in 92% after 24 h saline, 83% after 48 h saline, 68% after freezing in liquid nitrogen, and 62% after 48 h Michel's medium (n = 25).We conclude that transporting biopsies without freezing in normal saline for 24 hours is an adequate and attractive method for routine IF diagnosis in autoimmune and immune complex-mediated dermatoses. The superior results with saline incubation are explained by washing away of IgG background in dermis and epidermis. The demonstration of tissue-bound immunoreactants by direct immunofluorescence microscopy (DIF) is a valuable parameter in the diagnosis of various autoimmune skin diseases. ReliablThe present study was initiated by the unexpected finding of significant increase of the signal-to noise ratio in a skin biopsy submitted for DIF and accidentally kept overnight in normal saline. The biopsy, obtained from a patient suspect of pemphigoid, showed substantial reduction of IgG background fluorescence and relatively bright specific IgG fluorescence along the BMZ. This finding encouraged us to compare diagnostic results of DIF in matched skin biopsies using standard snap-freezing, Michel's fixative and normal saline.The 25 patients included in this study were selected on the basis of previously confirmed positive direct immunofluorescence (IF) in skin biopsies transported in liquid nitrogen. The final diagnosis was reached by clinical, routine laboratory, histological and direct IF findings. In case of bullous autoimmune diseases, serum samples were characterized by indirect IF on 1.0 M NaCl-split skin ,9, immun2 to minimize the risk of local variation of immunoreactants. The matched skin specimens from each patient were immediately placed in one of the following transport media: (a) liquid nitrogen, (b) Michel's fixative 48 hours with appropiate pH, (c) saline 24 hours and (d) saline 48 hours. We used 5 ml screw-capped polypropylene vials for transporting biopsies in Michel's fixative and saline.From each patient four skin specimens were obtained by punch biopsy (4 mm) using lidocaine as the local anaesthetic. The biopsies were taken from perilesional skin within an area of 2 cmBiopsies were placed in aluminum vials, snap-frozen in liquid nitrogen and stored at -80°C until further processing within two weeks for DIF (see below).1 Biopsy specimens were kept in 5 ml Michel's fixative for 48 hours (Mi48) at room temperature, followed by washing for 30 minutes in Michel's buffer solution. The specimens were then blotted on filter paper to remove excess moisture, and stored at -80°C until further processing.Michel's fixative and buffer solution were prepared monthly according to the original description.We used normal saline solution (0.9% NaCl in aqua dest.) without addition of calcium or magnesium. Skin specimens were kept in 5 ml saline solution for 24 hours (S24) and 48 hours (S48) at room temperature. Preliminary experiments with saline time of 6 hours did not result in improvement of the signal-to noise ratio. The specimens were then blotted on filter paper and stored at -80°C until further processing for DIF.2 antibodies against human IgG, IgA and IgM , and rabbit antibodies against human C3c and fibrinogen/fibrin . Proper conjugate dilutions were made in phosphate-buffered saline  supplemented with bovine serum albumin 1%. After washing in 1000 ml PBS for 30 min, the sections were coverslipped under fresh PBS/glycerol (50% v/v). The slide preparations were kept at 4°C until microscopic examination within two days.For comparative purposes, matched skin specimens of each patient were processed for direct IF microscopy at the same occasion. Cryosections of 4 μm thickness were mounted on polysine™ glass slides, air-dried for 30 min before a fan, and encircled with a hydrophobic emulsion . The sections were then stained for 30 min in a moist chamber at room temperature, using fluorescein (FITC)-labeled, Fc-specific goat F(ab')12;13.: (a) epidermal in vivo ANA, (b) epidermal intercellular spaces, (c) basement membrane zone , and (d) blood vessel walls. In addition, the background staining (USS plus NSS) of the upper dermis was scored. All sections were read blindly by the same experienced observer (MCJMdJ). The diagnosis made by direct IF was regarded as conclusive if the DSS score of the relevant target structure was at least weak but definite (Table). The diagnosis was regarded as tentative in case of weak staining that was not consistently distributed at the relevant target structure. A case was regarded as non-diagnostic if target structures showed negative to doubtful DSS scores.The sections were examined with a Leica DMRA microscope  for selective incident light fluorescence using a xenon arc (XBO 75W) as light source and PL Apo ×40/0.80 dry objective. The fluorescent staining was graded as follows: - (negative), ± (doubtful), + (weak), ++ (moderate), +++ (bright). The desired specific staining (DSS) of the following target structures was scoredStatistical analysis of IgG and IgA background fluorescence in matched biopsies was done by the McNemar test.A total of 95 biopsies from 25 patients were examined; three biopsies were missing, and two biopsies proved unsuitable at cryosectioning. Cutting of 4 μm cryosections was easiest with biopsies transported in saline and hardest with biopsies transported in Michel's fixative.Biopsies kept in saline for 24 and 48 hours showed statistically significant reduction of background fluorescence in the dermis, especially with IgG, as compared with fresh-frozen and fixed biopsies p < 0.01). After 24 hours, this resulted in enhanced signal-to noise ratios and accordingly more easy detection of immunoreactants at target structures, in particular the epidermal basement membrane zone (BMZ) and subepidermal blood vessel walls . We know that our visual perception is sensitive for light contrast and not for intensity . The staA disadvantage of saline is the limited time of transport 24 h) for consistently reliable results. If biopsies are kept longer than 24 hours in saline, decreased fluorescence of tissue-bound immunoreactants may be encountered, although we have observed bright specific fluorescence of the BMZ in biopsies kept in saline for at least 5 days. Michel's fixative seems to have a similar limitation: Skeete and Black found that biopsies stored in this fixative should be received within 1 day of biopsy for consistently reliable results [ h for coin vivo ANAs, possibly due to extraction or degradation of nuclear antigens. Another disadvantage of saline might be, from a histopathological point of view, morphological disturbances such as hydropic degeneration [The second disadvantage of saline is the loss of at least some epidermal neration , and splneration . That isneration . Neitherneration ,6,20. Honeration .Besides the diagnostic benefit, the preference would also go to the use of saline because it is a ready available, inexpensive, and convenient transport medium, and it certainly improves cutting properties of skin biopsies compared to biopsies fixed in Michels' medium. Saline can be used as standard medium at room temperature for express postal delivery of IF biopsies to the laboratory, if delivery is guaranteed within 24 hours. Transport of biopsies by express postal delivery or in-house airtube post is much more cost-effective than courier delivery necessary for biopsies transported in liquid nitrogen. If it is expected to tide over a longer period we advise to store the biopsy in saline for 24 hours and then to place it in Michel's fixative for further transport.Practically, place the biopsy in a screw-capped 5 ml polypropylene tube filled to the top with saline. The saline does not have to be sterile. If the specimen arrives in the laboratory the same day after biopsy, we advise to keep it overnight in saline at room temperature, followed by snap-freezing and  storage at -80°C the next day.The use of normal saline offers an attractive alternative to liquid nitrogen and Michel's fixative for diagnostic IF in autoimmune and immune complex-mediated dermatoses, but is only consistently reliable if the specimens are received within 24 hours after biopsy.None declared.BMZ: basement membrane zoneBP : bullous pemphigoidDH: dermatitis herpetiformisDIF: direct immunofluorescence microscopyDSS: desired specific stainingEBA: epidermolysis bullosa acquisitaIF: immunofluorescence microscopyLAD: linear IgA dermatosisLE : lupus erythematosusMMP: mucous membrane pemphigoidNSS: non-specific stainingPF : pemphigus foliaceusPV : pemphigus vulgarisUSS: undesired specific stainingRV carried out the studies, participated in the design and data analysis and drafted the manuscript. MdJ participated in patient selection and carried out the immunofluorescence microscopy and imaging. HM performed the immunofluorescence technical work up. MW participated in material harvesting. HP participated in patient diagnosis. MJ conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Wolbachia infection in D. bifasciata in Hokkaido island of Japan, in contrast to the presence of infection on the proximal island of Honshu, was associated with failure of the infection to function properly on the Hokkaido genetic background.Selfish genetic elements that distort the sex ratio are found widely. Notwithstanding the number of records of sex ratio distorters, their incidence is poorly understood. Two factors can prevent a sex ratio distorter from invading: inability of the sex ratio distorter to function (failure of mechanism or transmission), and lack of drive if they do function (inappropriate ecology for invasion). There has been no test to date on factors causing variation in the incidence of sex ratio distorting cytoplasmic bacteria. We therefore examined whether absence of the male-killing D. bifasciata may occur at different densities in Hokkaido and Honshu populations, giving some credence to the idea that ecological differentiation could be important.The male-killer both transmitted and functioned well following introgression to each of 24 independent isofemale inbred lines carrying Hokkaido genetic backgrounds. This was maintained even under stringent conditions of temperature. We therefore reject the hypothesis that absence of infection is due to its inability to kill males and transmit on the Hokkaido genetic background. Further trap data indicates that The absence of the infection from the Hokkaido population is not caused by failure of the male-killer to function on the Hokkaido genetic background. Selfish genetic elements that distort the sex ratio of their host are known widely in arthropods . DespiteDrosophila pseudoobscura, frequency declines at both high latitude and high altitude. This is not associated with resistance to the action of the driver (no resistance is known), and variation in rates of multiple mating is suspected as a cause [D. subobscura, X drive is present in North Africa and absent in Europe. Here, absence of X drive is associated with the less efficient function of X drive on the European genetic background [Factors causing variation in prevalence/incidence over space may be either ecological or associated with differences in host genetic constitution. For instance, in the case of X chromosome meiotic drive in  a cause . In contckground .Drosophila prosaltans, for instance, there is intra-population host genetic variation in refractoriness to male-killer action/transmission [et al. [This study pertains to the factors affecting the incidence of male-killing bacteria. These bacteria pass from a female to her progeny, and kill any males they enter. Male-killers are common in insects, but an appreciation of the factors underlying their incidence is lacking . In the smission . Thus, ismission . For ins [et al.  have demWolbachia in Drosophila bifasciata in Japan. Drosophila bifasciata feeds on sap fluxes in deciduous forests, and sampling across 10 populations within Honshu island in Japan revealed a relatively constant frequency of infection, with between 5 and 7% of females infected with the male-killer [Wolbachia still present on Honshu to this day [In this paper, we examine the factors that could cause incidence variation for the male-killing e-killer . The malthis day .D. bifasciata populations [In contrast to collections from Honshu, past surveys across Hokkaido, the North island of Japan, indicated flies in this area are not infected with the male-killer, despite the relative proximity of the sites to the infected populations in the Northern most sites in Honshu. In total, 559 flies from six locations within Hokkaido were tested, with no evidence of sex ratio distortion in any case . We can ulations . Second,D. bifasciata ecology between Hokkaido and Honshu islands. Our results indicated that the Hokkaido genetic background supported the transmission and action of the male-killer even under stringent conditions, ruling out genetic differentiation as a cause of the absence of the male-killer from Hokkaido. We did observe higher capture rates of D. bifasciata in the island of Hokkaido, and future work should therefore be focussed on the degree to which ecological differences affecting the drive of the infection dictates incidence in this species.We tested whether the absence of infection in Hokkaido was associated with an effect of host genotype on the efficiency of male-killer transmission or strength of male-killing ability. Beyond this, we examined whether trap collection data were consistent with difference in Twenty-eight female flies were collected from the field in Hokkaido. Of these, 4 failed to produce progeny. The remaining 24 all produced a normal sex ratio, consistent with continued absence of the male-killing trait in Hokkaido.Wolbachia from Honshu by introgression of the infection onto the Hokkaido genetic background. The progeny from the 24 Hokkaido females were maintained as isofemale inbred lines for three generations to capture genetic variation within them. The male-killing Wolbachia from Honshu was then placed onto each inbred background via crossing infected females from Honshu to males from each of the Hokkaido lines, with subsequent generations being maintained through further crossing to males from the appropriate Hokkaido line.We tested whether the Hokkaido genetic background supported the male-killing The sex ratio produced following introgression of the infection to the Hokkaido genetic background was female biased and penetrance of the male-killing phenotype was perfect in the first generation. A few males appeared sporadically in six of the 24 lines in one or more subsequent generations, with highest frequency in lines 15 and 25  than in the two samples from the Northern Honshu populations  and the population from Mid Honshu (Kiyosumi). Increased capture rate in Hokkaido was also reflected in an increase in the proportion of all drosophilids sampled that were bifasciata . This high efficiency was maintained even at the threshold for complete male-killing in Honshu, 23.5 C . Thus, the male-killer is proficient at being transmitted and killing males on the Hokkaido background even under relatively stressful environmental conditions. Further, we continue to maintain the infection on the Hokkaido background (we are now at generation 15) without any loss of the infection and without appearance of males. Thus, it is not tenable to argue that the hosts themselves are not genetically suitable for the function of male-killer, at least on an ecological timescale. This situation contrasts with the case of meiotic drive in the related fly D. subobscura, where absence of drive in Europe was associated with refractoriness to the action of the sex ratio distorting element.Previous study has shown that male-killing bifasciata captured at lower rate in the populations from Honshu than in Hokkaido. Thus, ecological heterogeneity is a possible source of the incidence pattern. There are three parameters in male-killer dynamics that may be environmentally influenced. First, the advantage to male-killing may not be as strong in Hokkaido populations. Second, the cost of infection to female flies may be higher in Hokkaido than in Honshu. Third, the transmission efficiency may alter, mediated via elevated temperature, or possibly by reduced overwinter temperature.In the absence of variation in the ability of the male-killer to function on the different genetic backgrounds, the question arises as to the features that cause infection to be absent from the Hokkaido population. Our study did reveal differences in trap collection rates between the populations of Hokkaido and those of Northern Honshu, with In our view, the latter factor is unlikely to be driving the observed pattern. It is notable that the survey of Ikeda revealed the infection to be present in Northern Honshu, but not in Southern Hokkaido. Given these two areas are geographically and climatically very close, temperature differences have poor explanatory power. Explanations based on temperature are also weak because this species exhibits a degree of homeostasis in temperature, moving to elevated altitudes to avoid excess temperature.bifasciata observed in Hokkaido translates into many females laying eggs in a single sap flux, male death will not greatly increase the survival of infected females over uninfected, and infection will decline in frequency. The second factor that may cause infection to decline in frequency is if costs of infection are higher in Hokkaido than in Honshu. This factor can be ecologically contingent. Ikeda demonstrated that the relative fitness of infected flies compared to uninfected flies was lower under high densities in the laboratory [This leaves us with two hypotheses to explain absence of infection in Hokkaido. The first is that there is a weaker advantage to male-killing in Hokkaido than on Honshu, such that there is insufficient drive to maintain the bacterium or permit its spread. The advantage of male-killing to the bacterium depends upon the number of females ovipositing in a single patch . If the boratory . Thus, iWolbachia from Hokkaido.Aside from these possibilities, which we consider most likely, other ecological discontinuities between Hokkaido and Honshu deserve investigation. Some symbionts, for instance, give the host protection against parasitoids , and thuWolbachia to function on different host genetic backgrounds that drives presence or absence of infection in the D. bifasciata- male-killing Wolbachia system. We have demonstrated that despite being absent from Hokkaido, Wolbachia can both be maintained and express male-killing on the Hokkaido host genetic background. We observe that the two populations show differences in trap capture rates, and argue that either ecologically contingent benefits or ecologically contingent costs of infection may explain presence and absence of infection in this species, and that future research be focussed at this level.It is not variation in the ability of D. bifasciata were collected on the campus of Hokkaido University, Sapporo , Japan, in May 2003. These were then brought into the laboratory, where they were maintained individually in vials at 21°C on a modified cornmeal-agar diet . These female were checked for the presence of the male-killing trait through scoring of the sex ratio, and maintained by sib-sib inbreeding  for three generations to make inbred isofemale lines. In total, 24 isofemale inbred lines were created (4 lines went extinct), which were maintained thenceforth by simple tossing every three weeks.Twenty eight wild female Wolbachia infected virgin females extracted from a culture derived from Honshu island, Japan . This pished in ) with liTemperature is known to affect the stability of the male-killing trait, and previous study demonstrated that an upper threshold of 23.5 C existed for stable maintenance of the infection on the Honshu genetic background . We testDrosophila communities. Drosophila communities were sampled in 4 deciduous forests in Southern Hokkaido, 2 sites in Northern Honshu, and one site in Mid Honshu, in early-mid October over a number of years between 1973 and 1984 (Figure D. bifasciata is a typical forest-canopy dweller, sampling from the forest canopy is essential for estimating its population density. These traps were cleared 7 or 10 days after setting, and the capture rate of bifasciata per trap per day calculated at each locality to represent the density of this fly in this region. All drosophilid flies were identified, and the proportion of flies caught that were bifasciata recorded.Evidence of differences in fly density can be derived from sampling the ZV helped with the design of the crossing scheme, conducted the crosses involved, and performed the analysis of these crosses. MT designed and conducted the field sampling and scored trap collections. GH conceived the project, organised collection, helped with design of the crossing scheme, and wrote the paper. All authors read and commented on drafts of the manuscript, and approved the final manuscript.


Mycoplasma hominis, the genes encoding the surface-localized membrane complex P60/P80 are in an operon with a gene encoding a cytoplasmic, nucleotide-binding protein with a characteristic Histidine triad motif (HinT). HinT is found in both procaryotes and eukaryotes and known to hydrolyze adenosine nucleotides in eukaryotes. Immuno-precipitation and BIACore analysis revealed an interaction between HinT and the P80 domain of the membrane complex. As the membrane anchored P80 carries an N-terminal uncleaved signal peptide we have proposed that the N-terminus extends into the cytoplasm and interacts with the cytosolic HinT.Mycoplasmas are cell wall-less bacteria which encode a minimal set of proteins. In E. coli and differing in the N-terminal region revealed that mutation of the +1 position of the mature protein (Asn to Pro) which is important for signal peptidase I recognition resulted in reduced P80 secretion. All other P80 variants were released into the supernatant, in general as a 74 kDa protein encompassing the helical part of P80. Incubation of M. hominis cells in phosphate buffered saline supplemented with divalent cations revealed that the release of mycoplasma proteins into the supernatant was inhibited by high concentrations of calciumions.Further characterization of P80 suggested that the 4.7 kDa signal peptide is protected from cleavage only in the membrane bound form. We found several proteins were released into the supernatant of a logarithmic phase mycoplasma culture, including P80, which was reduced in size by 10 kDa. Western blot analysis of recombinant P80 mutants expressed in M. hominis implies a two-step process. In general the P80 protein is transported across the membrane and remains complexed to P60, surface-exposed and membrane anchored via the uncleaved signal sequence. Loss of the 4.7 kDa signal peptide seems to be a pre-requisite for P80 secretion, which is followed by a proteolytic process leading to a helical 74 kDa product. We propose that this novel form of two-step secretion is one of the solutions to a life with a reduced gene set.Our model for secretion of the P80 protein of  Listeria, Yersinia and even some of the mycoplasmas are able to invade the host cells [The contact of a pathogenic bacterium with its eukaryotic host provokes a multitude of reactions. A prerequisite for successful infection with the host is the cytadhesion of the bacterium generally mediated by surface localized proteins . Besidesst cells -4. An inst cells -7.The large majority of exported proteins possess an N-terminal signal sequence . Most siMethanococcus jannaschii to 37% in the mollicute Mycoplasma pneumoniae [Bacillus subtilis found that only 50% of the secreted proteins were detected by genomic prediction, indicating that proteomic analyses of secreted proteins (the secretome) are necessary for a comprehensive definition of all secreted proteins [In the last few years, computer programs such as PSORT-II, PSORT-B, ExProt and SignalP have been developed to facilitate the identification of putative secreted proteins -18. Compeumoniae . Analysiproteins .M. hyopneumoniae. P102 is encoded as a precursor protein carrying a type I signal sequence and is found exclusively in the extra-cellular milieu suggesting cleavage by signal peptidase I. The expression of secreted P102 is coupled to that of the surface-exposed cilium adhesin P97, which seems to represent a new variant of processed surface antigens. P97 is derived from a 126 kDa precursor protein by cleavage at amino acid residue (aa) 195. The cleaved 22 kDa N-terminal fragment, which carries an uncleaved type I signal sequence is found embedded in the membrane, in the cytoplasm and in a soluble form in the supernatant, whereas the mature P97, proposed to be membrane bound, is the target of complex proteolytic cleavage, which leads to the subsequent release of some fragments into the supernatant [M. fermentans is a further example of a surface-localized protein undergoing proteolysis after reaching its target. Site-specific cleavage leads to the generation of the membrane bound immune-stimulatory lipopeptide MALP-2 and the release of the remainder (the RF fragment) in a soluble form into the supernatant [Only a handful of mollicute genomes have been decoded, but no analyses of their secretomes have been conducted. A secreted protein, probably processed by the classical mechanism described above, has been characterized in the swine pathogen ernatant ,21. The M. hominis, which is mainly found as a commensal in the urogenital tract, but has also been associated with human urogenital tract infections [In fections ,24, two fections . Recentlfections . We havefections .The data in this paper suggest that the signal peptidase I recognition site is protected in the membrane bound form of P80, but is accessible as the first step in the release of the helical part of P80 into the cell culture supernatant, a process which is accompanied by a decrease in size of 10 kDa.E. coli, the TGA codons were mutated to TGG to allow expression. However, even though we mutated all TGA codons to TGG in the hitA gene, which encodes P80, we were not able to express and purify P80 peptides with an intact N-terminus. While P80-2, the C-terminal region of P80 from AA 320 to AA 713, was stably expressed in E. coli, P80-NT, the initial 21AA of the N-terminus, and P80-1, the N-terminal portion of P80 from AA 1 to AA 326, were degraded at the N-terminus during purification  and as type I signal peptidases share the unusual feature of being resistant to general inhibitors of the four other peptidase classes  we specuE. coli signal peptidase I, we characterized different mutants of the recombinant P80 protein according to their processing in E. coli. Using PCR, we constructed clones expressing the mature P80 protein without the 44 AA signal peptide (ΔSP) and an 89 AA N-terminal truncated P80 protein, which corresponds to the proposed helical, surface-localized part of P80 (Helic.). As the absence of proline at the +1 position of the mature protein is consistent with the observation that the SPase I of E.coli was inhibited by recombinant precursor proteins having a proline residue at this position [N/P) at the (+1) position in the mature protein expecting a reduced or complete inhibition of signal peptide cleavage  mutant, while the sizes of secreted P80 variants corresponded to that of the helical variant  and P80-(Helic.), the two variants lacking signal sequences as translocation signals, this transport may be mediated by an alternative, Sec-independent export mechanism.In order to test the hypothesis that the degradation of rP80 is mediated by position , we genee Figure . Westernt Figure . Small aE. coli is signal peptidase I-mediated and therefore we investigated the protection of the signal peptidase I recognition site of P80 in Mycoplasma hominis [The data suggest that the recombinant P80 processing in  hominis . TherefoAs might be expected the stationary phase culture contained several proteins that might be released from lysed cells, including P50, P60, OppA and cytoplasmic proteins such as P55 Figure . HoweverInterestingly, the P80 protein was the only one of the proteins analyzed that had a decreased size in the supernatant. To exclude the possibility that the reduction in size was simply a result of the large amount of albumin in the culture medium, we purified P80 from the supernatant of a culture in mid logarithmic growth using affinity chromatography. In addition we analyzed P60 as a control. As shown in Figure As the activity of proteases often depends on the presence of divalent cations, we examined the secretion of mycoplasma proteins in the presence or absence of divalent cations. Mycoplasma cells were incubated for 1 h at 37°C in phosphate buffered saline (PBS) supplemented with up to 5 mM magnesium or calcium chloride, or chelating agents as EDTA and EGTA. The addition of divalent cations did not increase the processing of P80 (data not shown). As the presence of 5 mM calcium ions resulted in a complete disappearence of P80 from the supernatant we examined the release of mycoplasma proteins from cells following stepwise addition of calcium ions at concentrations of 0 to 5 mM Figure . Silver Bacillus subtilis [The cytoplasmic proteins that were detected in the supernatant were not released from lysed cells as the titres of the cultures remained the same throughout the experiment (data not shown). These findings are in accordance with those of Antelmann and coworkers, who found several cytoplasmic proteins, such as elongation factor G (EF-G) and arginase (RocF) as to be components of the secretome of subtilis .2inhibited a release of most mycoplasma proteins from the cells  anchored in the membrane and exposed on the surface, and is also released into the extra-cellular environment. As P80 and P60 are concomitantly expressed and both complexed in the membrane fraction it is likely that the soluble P80 variant is derived from the membrane bound precursor. Interestingly, the findings from the heterologous expression of different P80 mutants in E. coli suggest that release of P80 is initiated by cleavage of the N-terminal type I signal sequence, followed by amino-terminal proteolysis by an unknown mechanism, leading to a stable P80 peptide with a predicted helical structure in the supernatant.The data presented here indicate that the membrane protein P80 of M. genitalium, the smallest known self-sufficient organism, lacks a gene for the type I signal peptidase [Mollicutes, despite the fact that several proteins with type I-signal sequences had been identified [Mycoplasma gallisepticum (MGA_1091) and M. pulmonis (MYPU_6300) [As the genome of eptidase  it was ientified ,25. HowePU_6300) ,32.M. hyopneumoniae in the extra-cellular matrix suggests that the intrinsic N-terminal type I signal peptide is immediately cleaved after translocation of the precursor through the membrane, as would be expected from current knowledge of the classical protein secretion pathway, the precursor proteins P80 and P120' of M. hominis are attached to the membrane without being processed or released into the supernatant [The release of mycoplasmal proteins into the extra-cellular milieu may follow different pathways to those described previously for other bacteria. While the exclusive detection of P102 of ernatant ,25,26.M. fermentans that leads to the generation of the immune-stimulatory lipopeptide MALP-2 and showed that the residual RF peptide was soluble and was released into the supernatant [M. hyopneumoniae appears to be quite complex. The precursor protein P126, which carries a type I signal sequence with a cleavage site between AA 31 and AA 32, is predominantly cleaved between AA 194 and AA 195, suggesting that the SP-I signature may be essential only for the translocation of the whole precursor protein across the membrane. The cleavage products P22 and P97 both remain closely associated with the membrane. While P97 is generally subjected to further site-directed proteolysis, leading to the release of peptides into the extra-cellular milieu, further processing of P22 occurs to be strain dependent [The later release of a surface-localized peptide is described here for the first time. Davis and Wise described site-specific proteolysis of the lipid-anchored MALP-404 of ernatant . Processependent .Mycoplasma hominis, and are considered to be required for the establishment of an infection at these sites. Recently in Legionella pneumophila a type II protein secretion system was characterized as a virulence factor linked to an intracellular infection [Release of proteins into the surroundings of a mycoplasma cell should lead to an immediate alteration of the cell surface architecture and, as most membrane proteins are targets for the host immune response ,34, may nfection .M. pulmonis  the gene of which is followed by P60 (MYPU_0070) and HinT (MYPU_0080) gene homologues, and a rhoptry protein of Plasmodium yoelii yoelii (gi23481286). Rhoptry proteins are released during host cell invasion by apicomplexans [P80 has significant similarity with two protein sequences, a hypothetical protein of mplexans .2+-binding calmodulin [2+ ions – may activate the signal peptidase induced processing of the membrane bound P80 protein. This would lead to a soluble P80-helix and the membrane anchored signal peptide. After loss of the transmembrane spanning C-terminal amino acids or due to a leakage of the whole signal sequence (as described above) the signal peptide could reach the cytoplasm where an interaction with HinT may be a further step in modulating or promoting cellular processes such as growth or activation of gene expression, a process we are currently investigating.As P80 interacts with HinT, a cytoplasmic protein found in all kingdoms, which in eukaryotes hydrolyses AMP derivatives and in yeasts functions as a regulator of an RNA polymerase II domain , a furthlmodulin , or are lmodulin , where tlmodulin . Our resMycoplasma hominis. This is, to our knowledge, the first description of secreted protein with a type I signal sequence that is stably embedded in the membrane as precursor protein and, as we propose as a model of secretion, is subsequently released from the membrane into the supernatant after signal peptidase I cleavage. As in other mycoplasmas several proteins have been shown to either possess an uncleaved signal sequence or to be further processed after anchoring in the membrane, this may be a common phenomenon in mycoplasmas. Because of their minimal coding capacity, mycoplasmas may have evolved a strategy to use secretory proteins in a dual role, as surface localized proteins in the membrane for cell surface architecture, and, in response to a change in the environment, as a soluble protein released from the cell surface.The data presented here clearly demonstrate that P80 is a secreted antigen of Mycoplasma hominis strain FBG was amplified by PCR [Helic., AA 90–713), amplifying a fragment encoding the mature P80 polypeptide , and mutating the asparagine codon at position +1 of the mature protein to a proline codon (N/P).The P80 protein encoding region (ACC Z29068) of d by PCR  using old by PCR . To facid by PCR , restricRcaI/BglII restricted amplicon in frame into the NcoI/BglII restricted plasmid pQE60 and inserted the BamHI/BglII restricted DHFR-fragment of pQE40 in the BglII site of the plasmid pQE60-P80 NT. Plasmids were propagated in DH5αF' for constitutive expression [Amplicons were cut at the restriction sites within the primers and ligated in-frame into the expression vector pQE30 . To express the P80 N-terminus (AA 1–21) as a fusion protein with dihydrofolate reductase and a poly His-tag, we ligated the respective pression .g, 30 min, 4°C). In the secretion assay, the pelleted cells and supernatant, which was concentrated by lyophilizing, were subjected to Western blot analysis. The recombinant peptides P80-NT and P80-2 were purified by Ni-NTA chelation according to the manufacturer's protocol, while rP80 and P80-1 were purified with the Sepharose-coated P80-specific antibodies LF8 and NB12 as described by Henrich et al. [Cells from a mid-logarithmic phase culture in LB broth  containing ampicillin (100 μg/ml) were harvested by centrifugation . The integrity of the different plasmid sequences was confirmed by analysis on an ABI sequencer using the method of Sanger .Mycoplasma hominis strain FBG was cultivated in PPLO broth supplemented with arginine from frozen stocks of a mid-logarithmic phase broth culture as described previously [g, 10 min, 4°C), and the cell pellets and supernatants were subjected to Western blot analysis. Alternatively, cell pellets derived by low speed centrifugation  were re-suspended in phosphate-buffered saline  containing 5.0, 2.5, 1.25, 0.63, 0.31 or 0.00 mM CaCl2 and incubated at 37°C for 60 min. Soluble proteins were then separated from insoluble material by centrifugation , and all samples analyzed by Silver staining [eviously . The titeviously . For prostaining  and WestProteins were separated in 9.5% polyacrylamide gels , transfeM. hominis culture as part of her thesis. MH carried out the immunoassays and completed the secretion assays. BH carried out the molecular genetic studies, participated in the design of the study and drafted the manuscript. All authors have read and approved of the final manuscript.RH began the characterization of antigens released into the supernatant of a


Cancer of unknown primary remains a mallignancy of elusive biology and grim prognosis that lacks effective therapeutic options. We investigated angiogenesis in cancer of unknown primary to expand our knowledge on the biology of these tumors and identify potential therapeutic targets.Paraffin embedded archival material from 81 patients diagnosed with CUP was used. Tumor histology was adenocarcinoma (77%), undifferentiated carcinoma (18%) and squamous cell carcinoma (5%). The tissue expression of CD34, VEGF and TSP-1 was assessed immunohistochemically by use of specific monoclonal antibodies and was analyzed against clinicopathological data.VEGF expression was detected in all cases and was strong in 83%. Stromal expression of TSP-1 was seen in 80% of cases and was strong in 20%. The expression of both proteins was not associated with any clinical or pathological parameters. Tumor MVD was higher in tumors classified as unfavorable compared to more favorable and was positively associated with VEGF and negatively with TSP-1.Angiogenesis is very active and expression of VEGF is almost universal in cancers of unknown primary. These findings support the clinical investigation of VEGF targeted therapy in this clinical setting. Cancer of unknown primary (CUP) is a unique clinical entity that accounts for an approximately 3% of human cancers. PatientAngiogenesis, the formation of new vessels, is essential for tumor growth and the development of metastases. It evolves though a complex multifactor process that involves interaction of pro-angiogenic and anti-angiogenic signals from tumor, endothelial and stromal cells. The angiogenic activity is reflected in the development of novel microvessels in tumor tissue that is quantified by the intratumoral microvessel density (MVD). Among several molecules implicated, Vascular Endothelial Growth Factor (VEGF) and Thrombospondin-1 (TSP-1) appear to be most relevant. Much evidence indicates that VEGF is a key activator of angiogenesis,5 and TSWe were prompted to investigate angiogenesis in unknown primary cancer in an attempt to enrich our understanding of the biology of these tumors. We studied by immunohistochemistry the tissue expression of VEGF and TSP-1 in CUP and correlated with MVD and clinicopathological parameters. In a recently published study vascular endothelial growth factor, and CD34, factors were not found to be of prognostic value in adenocarcinoma of unknown primary.A total of 81 patients diagnosed with CUP and treated in three University Medical Oncology Settings  between January 1997 and December 2002 were selected on the basis of availability of archival tumor tissues and accessibility to medical notes. Pathology diagnosis was reviewed by two pathologists blinded to written pathology report and representative paraffin blocks were selected for immunohistochemistry.Eligible cases categorized into unfavorable and more favorable subgroups tables  and 2. Pp = 0.04) and a longer survival, 11.5 vs 8.5 months (p = 0.01).Systemic chemotherapy was given in 64 patients (78%); four patients with cerebral metastases received whole brain irradiation. Chemotherapy was consisted of a platinum based combination. Objective response to chemotherapy was observed in 34 patients (53%) while one patient with brain metastases responded to radiotherapy. Median survival for all patients was 10.5 months  method. In brief, tissue sections were deparaffinised in xylene and dehydrated. They were immersed in citrate buffer  and subjected to microwave twice for 15 min. Subsequently, all sections were treated for 30 min with 0,3% hydrogen peroxide in methanol to quench endogenous peroxidase activity. Mouse monoclonal antibodies directed against human CD34 antigen  in dilution 1/50 β) VEGF Ab-3  in dilution 1/50 and c) thrombospondin  in dilution1/50 were used. Positive control slides were included in all cases. All dilutions were made in TBS-1% BSA solution and were followed by overnight incubation.The assessment of immunostaining was made by two experienced pathologists using light microscope. Tumor specimens too small to provide sufficient sections for all the immunoassaying procedures were disregarded from the study.Staining of endothelial cells for CD34 was used to evaluate the MVD. Any CD34 positive endothelial cell clusters clearly separated from each other were considered as single countable microvessels. A lumen was not required to identify a vessel. Larger vessels with muscular walls were excluded from counting. In each sample three areas of most prominent vascular density (hot spots) were identified at ×40 power field and microvessel counting was done under ×400 magnification. Counting was performed by two independent observers blinded to clinical information. The median count was used to make distinction between low and high MVD.Immunoreactivity for VEGF was observed in stromal and epithelial cells. Only staining of tumor cells was considered for analysis. To evaluate the expression of VEGF protein, we devised a combined score that corresponds to the sum of staining intensity  and percentile quadrants of positive cells . The maximum score was 6. Score 2 was regarded to represent weak expression, score 3 intermediate and score 4–6 strong expression.Staining for TSP-1 was only considered in regard to extracellular matrix. The expression of TSP-1 was characterized according to the extent and the intensity of staining classified as negative, +1: mild, focal, +2: intermediate, multifocal, +3: strong, diffuse reactivity.Staining results were analyzed against clinical subgroup, histological differentiation, response to treatment and survival. The association between MVD and clinical subgroup, histological differentiation and response to treatment was assessed by an unpaired t test. A Fisher's exact test was used to determine associations between VEGF and TSP-1 and the clinical subgroup, histological differentiation and response to treatment. Spearman non parametric correlation test was used for associations between MVD, VEGF and TSP-1.Survival was calculated by Kaplan-Meier method and comparison of survival curves was performed by the log-rank test. For statistical significance a two-tailed p value was considered. The Graphpad Instat version 3.05  and Prism version 4  software programs were used for statistical analysis and graphing.Demographics of studied cases are depicted in Tables 2    declare that they have no competing interests.NP conceived, coordinated and designed the study, interpreted the data and drafted the manuscript; VK, EB, designed and carried out the study, performed the statistical analysis, interpreted the data and drafted the manuscript; VMM carried out the pathological and immunohistochemical study, interpreted data and drafted the manuscript; ET, EK carried out the pathological and immunohistochemical study, interpreted data and drafted the manuscript; HK, GF, TF participated in designing the study, acquisition and interpretation of data and revising critically the manuscript. All of the authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here: