Helicobacter organisms play a role in human UC has been debated but not comprehensively investigated. The aim of this study was to develop a molecular approach to investigate the presence of Helicobacter organisms in adults with and without UC.Changes in bacterial populations termed “dysbiosis” are thought central to ulcerative colitis (UC) pathogenesis. In particular, the possibility that novel Helicobacter was developed. Oligonucleotide probes against the genus Helicobacter were designed and optimised alongside a validation of published H. pylori probes. A comprehensive evaluation of Helicobacter genus and H. pylori PCR primers was also undertaken. The combined approach was then assessed in a range of gastrointestinal samples prior to assessment of a UC cohort. Archival colonic samples were available from 106 individuals for FISH analysis (57 with UC and 49 non-IBD controls). A further 118 individuals were collected prospectively for dual FISH and PCR analysis (86 UC and 32 non-IBD controls). An additional 27 non-IBD controls were available for PCR analysis. All Helicobacter PCR-positive samples were sequenced. The association between Helicobacter and each study group was statistically analysed using the Pearson Chi Squared 2 tailed test. Helicobacter genus PCR positivity was significantly higher in UC than controls . Sequence analysis indicated enterohepatic Helicobacter species prevalence was significantly higher in the UC group compared to the control group . PCR and FISH results were concordant in 74 (67.9%) of subjects. The majority of discordant results were attributable to a higher positivity rate with FISH than PCR.A dual molecular approach to detect Helicobacter organisms warrant consideration as potential pathogenic entities in UC. Isolation of these organisms from colonic tissue is needed to enable interrogation of pathogenicity against established criteria. Ulcerative colitis (UC) is a chronic condition of the human colon which affects the superficial mucosal layer from the rectum and extending proximally for variable distances Helicobacter pylori was the causative agent underpinning gastric and duodenal ulceration and ultimately gastric cancer revolutionised our understanding of these conditions and resulted in a Nobel prize for Robin Warren and Barry Marshall. The tantalising possibility that a similar agent is responsible for IBD warrants consideration and exploration The discovery that Helicobacteraceae contains the genera Helicobacter and Wolinella. The Helicobacter genus can be split into two groups, gastric Helicobacter, describing those that preferentially colonise the stomach, and enterohepatic Helicobacter, which preferentially colonise the intestinal or hepatobilliary system (Helicobacter organisms have been cultured from both Cotton-top tamarin monkeys (Saguinus oedipus) and rhesus monkeys (Macaca mulatta) with colitic disease , whilst Helicobacter hepaticus and Helicobacter bilis have been shown to be capable of causing IBD-like disease in immunodeficient rodent models Helicobacter spp. on a background of host immunodeficiency can lead to colitis, and that “auto-immune” type reactions to commensal bacteria can be initiated by such organisms, would suggest the possibility of parallel mechanisms in humans resulting in IBD.The family y system . EnterohHelicobacter spp., from the negative studies of Bell and Grehan pylori Helicobacter (npH) in both IBD and controls Helicobacter species level have been limited. Unfortunately, no-one has successfully cultured non-pylori Helicobacter organisms from IBD tissue , H. canadensis (ATCC 700968), H. canis (ATCC 51402), H. cholecystus (ATCC 700242), H. cinaedi (CCUG 18818), H. felis (ATCC 49179), H. hepaticus (ATCC 51449), H. pullorum (NCTC 12824), H. pylori (ATCC 700392), Pseudomonas fluorescens , Listeria monocytogenes , Aeromonas caviae , Aeromonas sobria , Campylobacter jejuni , Proteus mirabilis (NCTC 3177), Enterobacter aerogenes (NCIMB 10102), Yersinia enterocolitica (NCIMB 2124), Bifidobacterium longum (NCIMB 8809), Bifidobacterium infantis (DSM 20088), Eubacterium rectale (NCIMB 14373), Roseburia intestinalis (DSM 14610), Bacteroides vulgatus (DSM 1447), Bacteroides thetaiotaomicron (NCTC 10582), Eubacterium hallii (DSM 17630), Enterococcus faecalis (NCIMB 13280), Pseudomonas aeruginosa (NCIMB 8626), Enterobacter cloacae (NCIMB 8556), Proteus vulgaris (NCTC 4175), Salmonella enteritidis (NCTC 12694), Salmonella poona (NCTC 4840), Salmonella typhimurium (NCIMB 13284), Escherichia coli (NCIMB 12210), Shigella sonnei (ATCC 25931), Staphylococcus epidermidis (NCIMB 8853), Bacillus subtilis (NCIB 8054), Bacillus cereus (ATCC 10876), Klebsiella pneumoniae (NCIMB 13281), Staphylococcus aureus (NCIMB 12702), Streptococcus gordonii (ATCC 35105), Faecalibacterium prausnitzii strain A2-165 (DSM 17677), Megasphaera elsdenii (ATCC 25940), Bifidobacterium adolescentis isolate L2-32, Lactococcus lactis strain MG1363, Enterococcus faecalis strain JH2-2, Ruminococcus albus strain SY3, Ruminococcus flavefaciens strain 17, Eubacterium cylindroides strain T2-87, Coprococcus spp L2-50, Methanobrevibacter smithii ATCC 35061, Acinetobacter baumanii, Lactobacillus acidophilus (ATCC 43561) and Streptococcus bovis strain Z6. Aerobic and microaerobic strains were grown at 37°C on Columbia agar with 10% horse blood. Anaerobic strains were grown at 37°C on M2GSC in-situ hybrisation (FISH) and PCR optimisation studies.A bacterial reference panel was used to screen a series of primer combinations. The bacterial strains used in this study included: Helicobacteraceae (genera Helicobacter and Wolinella), 8 Helicobacteraceae PCR primer pairs were assessed. The nested PCR combination of C05 and C97 H. pylori specific PCR, numerous H. pylori specific primer sets targeting the 16S rRNA gene were assessed with the most successful pairing being identified as 27f (5′-AGAGTTTGATCMTGGCTCAG-3′) 5′-CTGGAGAGACTAAGCCCTCC-3′) H. pylori and H. hepaticus-derived DNA were spiked into faecal samples which were previously analysed by FISH and PCR and found to be negative for Helicobacteraceae. Dilutions ranged from 500 pg to 0.05 pg of Helicobacter DNA with the detection level of 0.5 pg Helicobacter DNA (representing approximately 30 bacteria) consistently being achieved.Initial assessment used a universal 16S bacterial PCR described previously Helicobacteraceae , Sulfurovum (9 of 42) and Wolinella (4 of 5). The specificity of the 5 newly designed Helicobacteraceae probes along with four previously published H. pylori specific probes  including a panel of 9 Helicobacter type strains Helicobacteraceae probes, it was identified that the Helicobacteraceae probe S-G-Hel-1047-a-A-21 and the H. pylori specific probe Hp16S2 hybridized only to the respective target organisms but not to any of the other organisms tested. Both the Eub338 and Hp16S2 hybridised at 50°C and could be co-hybridised using discriminating fluorescent labels (Rhodamine red and Oregon green 488 respectively). S-G-Hel-1047-a-A-21 hybridised at 52°C.Five broad-specificity probes were designed to target the small subunit rRNA of the family teraceae . The newc probes  was testH. pylori status (50 positive and 50 negative). H. pylori status had been confirmed previously by CLO test and histology; however this was blinded to researchers until after molecular assessment. FISH was performed on archival paraffin tissue sections and PCR was performed on DNA extracted from fresh biopsies.In order to calculate the sensitivity and specificity of the dual molecular approach 100 gastric samples were selected on the basis of H. pylori coupled with the universal bacterial probe. Sections 2, 4 and 6 were used for assessing the presence of all Helicobacteraceae. Biopsy sections were deparaffinised using xylene and ethanol Biopsy blocks were cut to a thickness of 4 µm using a Leica RM2125RT rotary microtome with sections cut per block, and mounted on ChemMate capillary gap slides, 75 µm, . Following microtome sectioning and mounting of tissue, slides were dried vertically at room temperature and incubated overnight at 37°C to ensure that the tissue was adhered to the slide. Slides were then arranged by patient and block number and sections 1, 3 and 5 were used for assessing the presence of DNA extraction of mucosal biopsies was performed using the commercially available Qiagen QIAamp Mini kit (Qiagen Crawley UK) with the following amendments. Biopsy samples were kept frozen until the addition of ATL buffer before allowing biopsies to equilibrate to room temperature, an additional 10 µl of Proteinase K was added for an initial lysis period of 18 hours to ensure complete lysis of the biopsy material prior to DNA extraction. PCR was performed as described above with biopsy DNA initially subjected to universal bacterial PCR One hundred infectious diarrhoea samples were also collected for inclusion in the validation cohort. Samples were obtained from the Department of Medical Microbiology  and DNA was extracted using the Nucleon phytopure DNA extraction kit. PCR was performed as described above with faecal DNA initially subjected to universal bacterial PCR Paraffin embedded colonic specimens from a total of 106 patients were obtained from the Department of Pathology . Fifty-seven ulcerative colitis (UC) patients and forty-nine healthy controls (HC) were included. All UC patients were assessed during active disease and analysis was performed on all available colonic sites that were biopsied at the time of colonoscopy. The HC subjects comprised individuals who had undergone a colonoscopy in which colonic tissue was macroscopically normal, and subsequently confirmed as microscopically normal by histology. This cohort was examined exclusively by the FISH method outlined above. This sample cohort was not amenable to PCR methodologies.A total of 145 individuals were recruited for the prospective fresh biopsy study, 86 formed the ulcerative colitis (UC) cohort and were analysed alongside a cohort of 59 healthy controls (HC). Of the UC cohort 9 individuals were excluded, 3 could not undergo colonoscopy for clinical reasons and 6 had an alternative final diagnosis. The HC cohort comprised two groups. The first group (N = 32) were recruited specifically for this study and had biopsies collected for both FISH and PCR studies (as outlined above). The second group (N = 27) had been recruited previously and all 27 had biopsies collected from normal colon whilst undergoing polypectomy.Biopsies were collected during colonoscopy using standard endoscopic forceps (Boston Scientific Nanterre Cedex France). The colonic mucosa was rinsed with sterile water via the colonoscope to remove residual faecal material. Biopsies were immediately snap frozen in liquid nitrogen and then transferred to a −80°C freezer until used for DNA analysis. Additional biopsies were also sent for histopathology assessment and FISH analysis.de-novo) and 59 healthy controls. The entire UC group had biopsies collected for both fluorescent in-situ hybridisation (FISH) and PCR studies. Samples that generated positive PCR results with Helicobacteraceae or H. pylori PCR primers were sequenced to confirm identity. Based on sequence analysis results, samples that were suspected of containing multiple Helicobacter sequences were cloned Biopsies were only collected for PCR based studies and so FISH analysis was not studied in this cohort. Therefore mucosal biopsies were obtained from 136 individuals, 77 with a clinical and histological diagnosis of UC .Helicobacteraceae and H. pylori specific probes along with the universal Eub338 probe. The same 50 samples tested positive by PCR for Helicobacteraceae, H. pylori specific and universal bacterial primer sets with a subset of results (N = 10) subjected to sequence analysis which confirmed the presence of H. pylori (sequence identities >99%). The molecular results were 100% concordant with the findings of clinical investigation indicating that the combined approach had a high diagnostic sensitivity and specificity.Fifty of the gastric biopsy samples demonstrated FISH positivity using both Helicobacter by FISH and both Helicobacteraceae and H. pylori specific PCR with sequencing confirming the presence of H. pylori. As non-pylori Helicobacter organisms have been isolated from diarrheal samples previously, we performed a series of spiking experiments to confirm that the diarrheal samples were not inhibiting PCR amplification. Ten samples which were negative for Helicobacteraceae by both FISH and PCR were spiked with H. pylori DNA (500 pg to 0.5 pg) prior to PCR amplification. All spiked samples yielded positive PCR and subsequent sequencing results  confirming that if present these organisms would have been detected.The combined molecular approach was then applied to 100 infectious diarrhoea samples. All infectious diarrhoea samples showed positivity with the universal bacterial FISH probe (Eub338) and the universal bacterial PCR. One diarrhoea sample was positive for Helicobacteraceae from the available pathology blocks of the right and left colon including rectum. Subjects were considered to be positive if appropriate fluorescent organisms were observed in at least 1 slide. All fluorescent in-situ hybridisations were assessed alongside H. pylori positive gastric biopsy reference slides and a selection of Helicobacter reference strains. All of the 106 subjects had bacteria detected with the universal bacterial probe. Of the 55 subjects positive for Helicobacteraceae, 29 were from the UC cohort and 26 were from the HC cohort. In both groups there was no statistically significant correlation with gender or age of the subjects and in the UC cohort the extent of disease did not correlate with the rate of positivity either as detailed in Helicobacter pylori with 13 of the Helicobacteraceae positive subjects from the HC cohort also having a positive result for Helicobacter pylori compared with only 3 of the UC cohort (p = 0.002). Assuming that these Helicobacter pylori positive results represent transported gastric Helicobacter pylori we consequently removed these from the Helicobacteraceae positive results, thus creating a new category of non-pylori Helicobacter positive organisms. By this approach there was also a statistically significant difference in the presence of non-pylori Helicobacter organisms between the UC and HC cohorts (p = 0.04). There was no correlation with age, gender or severity of disease as detailed in H. pylori organisms were entirely responsible for the genus positivity or if the H. pylori were cohabiting with other non-pylori Helicobacter. Indeed, it was also not possible to ascertain if more than one Helicobacteraceae species was present within a sample. Attempts were made to extract microbial DNA from the archival tissue but these were unsuccessful and therefore a prospectively collected cohort was established with samples taken for PCR based analyses and FISH analysis.The UC archival cohort  had a median age of 40 (range 15–82) at the time of colonoscopy and were classified as extensive (40%), left sided (37%) and proctitis (23%) according to the Montreal criteria Helicobacteraceae and 3 (2%) were PCR positive for both Helicobacter pylori and Helicobacter genus  with only Helicobacter pylori identified.The prospective UC cohort  had a median age of 42 (range 16–84) at the time of index colonoscopy and were classified as extensive (25%), left sided (62%), proctitis (13.0%). A total of 137 biopsy sites were analysed. Twenty one (27%) subjects had a single site analysed, all of which represented inflamed mucosa and 56 (73%) had more than one biopsy site assessed, of which 42 both had inflamed and uninvolved mucosa. The prospective HC cohort  had a median age of 63 (range 30–75) at the time of index colonoscopy. There was a statistically significant median age difference between the UC cohort and the control groups (Mann Whitney U test p<0.001). A single biopsy site (n = 59) was analysed from each control subject. All 136 subjects (77 UC and 59 controls) had colonic biopsies available for PCR analysis and all were positive for universal bacterial PCR indicating the presence of bacterial DNA within all samples. Of the 136 subjects, 43 (32%) were PCR positive for er genus . SubsequHelicobacter species and a Wollinella succinogenes with 6 subjects having more than one species identified  versus 11 of 59 (19%), p = 0.004). By analysing the sequences obtained and including only those Helicobacter species classified as “enterohepatic,” the prevalence was 29 of 77 (38%) in the UC group versus 2 of 59 (3%) in the controls (p<0.0001). There was also a negative association between the identification of gastric Helicobacter species in UC (2 of 77) versus controls  (= 0.007) . There wHelicobacteraceae PCR positivity was also considered. Of the 77 UC subjects, 32 had full bowel preparation prior to colonoscopy along with all 59 control subjects. Helicobacteraceae PCR positivity in subjects with bowel preparation was significantly higher in UC than controls . There was also a significant difference in Helicobacteraceae PCR positivity within the UC cohort based on bowel preparation 20 of 32 (63% full bowel preparation), 1 of 5 (20% phosphate enema preparation) vs 11 of 40 (28% no bowel preparation), Pearson Chi squared test p = 0.007). There was also no statistically significant association with antibiotic usage.The effect of bowel preparation on Helicobacteraceae positive whilst 1 was also H. pylori positive. In the 32 controls, 12 (38%) were Helicobacteraceae positive whilst 2 were also H. pylori positive (Helicobacteraceae positivity was significantly higher in the UC cohort (p<0.0001) but no negative association with Helicobacter pylori as seen in the archival FISH and prospective PCR studies. As in the other studies there was no correlation between the age, gender or extent of disease. Correlation between PCR and FISH results for these 109 subjects were examined which demonstrated concordance in 74 (68%) of subjects. The majority of discordant results were attributable to a higher positivity rate for Helicobacteraceae with FISH than PCR  of the 136 subjects, samples were available for both FISH and PCR based analyses. All samples analysed by FISH were Eub338 positive indicating the presence of bacteria. 62 of 77 (81%) UC were positive . The Helthan PCR .H. pylori status. A further validation cohort utilising diarrhoeal samples demonstrated limited Helicobacteraceae positivity. However spiking experiments on this cohort indicate that organisms would have been identified had they been present. These findings suggest that Helicobacteraceae are not a prominent causative agent in infectious diarrhoea in our setting although they have been isolated from diarrhoeal samples by other investigators Helicobacteraceae, rather than one based solely on either PCR or FISH, was that the combination of techniques allows visualisation of organisms in-situ and species-level identification from sequencing. PCR-only studies can be criticised based on the possibility that contaminant environmental DNA could bias results. In the gastrointestinal tract for instance, DNA could be transited to the colon in the faecal stream from foodstuffs. FISH addresses these concerns by allowing direct visualisation and localisation of organisms to the colonic mucosa. FISH-only studies however are limited by the constraints of designing species-specific probes and therefore they lack species-level sensitivity at times. By utilising both approaches Helicobacteraceae species present could be visualised and also identified. Based on the strength of these validation studies, we considered that this combined methodology was suitable for investigating Helicobacteraceae prevalence in UC colonic biopsies. A combined FISH/PCR approach has also been utilised to examine Helicobacteraceae prevalence in a small cohort of children with IBD (n = 12) (Crohn's disease n = 11), irritable bowel syndrome  and controls (n = 4) Helicobacteraceae were identified including Helicobacter ganmani, Wolinella succinogenes, H. hepaticus and H. pylori. Two further bands were identified as Helicobacter although equal sequence similarity was attributed to multiple species.During the development of our combined molecular approach, both the PCR and FISH techniques were highly sensitive and specific (100% each) when interrogating gastric biopsies with known Helicobacteraceae positivity are significantly higher in the colonic tissue of UC patients than in controls. When sequencing data is analysed and species identities attributed, in control patients, the species identified are almost exclusively gastric (namely H. pylori and H. mustelae (99–100% sequence similarity); comprising 9 of 11). This finding is in stark contrast to the Helicobacter sequences from the UC cohort where 32 patients had Helicobacteraceae species identified, although only 2 of these were attributed to H. pylori and curiously, H. mustelae was absent. Our study was not designed to obtain gastric Helicobacter species. As such it was not possible to confirm or refute the notion that Helicobacter species detected in the colon are truly colonising the mucus or merely transiting from the stomach. For the former, Helicobacter grown from both sites (stomach and colon) would be necessary in order to undertake a detailed strain comparison, which was not feasible within this study. As indicated in the results section there was also a statistically significant difference in age between the UC and control groups. The difference in age between the two groups is a result of the control group predominantly being recruited from a colorectal cancer screening programme whose lower age limit is 50 years.The results of our study show that rates of Helicobacter species. It might be anticipated that the wash-out effect of bowel preparation might reduce the positivity. This surprising result may be the result of an unidentified confounding factor or could be a surrogate disease severity marker. This was due to it being a clinical decision whether subjects received bowel preparation or not. As no bowel preparation was given when there was the clinical impression of severe disease. This suggests that Helicobacteraceae positivity is associated with less severe disease. However no association between Helicobacteraceae positivity and the Montreal classification of disease extent and severity was observed.Interestingly, bowel preparation appeared to increase the detection of Helicobacter species present within their colonic tissue. Two of the six had different Helicobacter sequences identified from the same biopsy whereas the other four had single Helicobacter species identified from biopsies taken from different regions of the colon. Mixed species were not identified in the control cohort although it should be acknowledged that only single colonic sites were investigated by biopsy. Nevertheless our findings suggest that more than one enterohepatic Helicobacter species can be present in the same human host although this does not appear to be the case for gastric species.Six of the UC patients appear to have multiple Helicobacter species identity cannot be firmly established by 16S sequencing and the 400 bp product of the nested PCR further compounded this by only allowing sequencing over this short segment. However there is confidence that the sequence belonged to the genus Helicobacter based on the sequenced product. For example, although our nested PCR technique amplified a hyper-variable region of the 16S rRNA Helicobacter genome which equated to an estimated average evolutionary diversity of 14 base pairs within the 9 species identified Helicobacter species without additional genotypic or phenotypic characterisation Helicobacter organisms were present that had identical sequences over the 400 bp 16S rDNA region analysed. It is possible that alternative identification approaches including denaturing gradient gel electrophoresis (DGGE) could have been used to address this potential issue. Where sequence analysis indicated that mixed species were present, the additional cloning and sequencing approach was undertaken which demonstrated the presence of multiple Helicobacter sequences within a few samples.It should be noted that allocation of species should not rely solely on 16S rRNA sequencing as comparison of these sequences can be misleading and does not always provide conclusive evidence for species level identification. Helicobacter species from the prospective cohort, however this was not successful. Helicobacter species are notoriously fastidious and although novel Helicobacter species have been isolated from human faeces, to date none have been isolated from colonic tissue. Examining both FISH and PCR analyses on a large number of samples (n = 109) revealed a correlation rate of ∼68%. In the majority of cases, discordant results showed that FISH was more likely to yield a positive result than PCR. This was not seen in our gastric validation cohort. The most likely explanation is that the number of H. pylori strains present in infected gastric tissue is higher than the corresponding number of Helicobacter species in the colon. These results suggest that the FISH technique is more sensitive but lacks the specificity to identify the Helicobacteraceae species. Regardless of these technical issues, both techniques demonstrate a statistically significant correlation between the presence of Helicobacteraceae species, particularly the enterohepatic Helicobacter species and the UC cohort.The use of nested PCR is known to increase the sensitivity of the PCR test; however it is also known that without strict use of appropriate control strategies including sequencing of positive results, it can lead to false positives. In the current study, all positive nested PCR findings were sequenced in order to eliminate the query of false positives due to nested PCR. Extensive attempts were made to isolate Helicobacteraceae but a single PCR was used for H. pylori. It is likely that a nested PCR for H. pylori would have been more sensitive in the colonic samples and would have increased the detection rate. This is a plausible explanation for at least 3 of the four samples which were shown by sequence analysis of the Helicobacteraceae PCR product to contain H. pylori despite H. pylori PCR results being negative. Nevertheless, in order to identify every Helicobacter that was detected, we chose to sequence every positive PCR reaction including those that were H. pylori positive in order to determine whether multiple species were present.The PCR methodology that we developed used a nested PCR approach for Attributing causation to putative pathogens has always been a difficult endeavour with the gold standard remaining fulfilment of Koch's postulates There must be a clear link between a pathogen and a disease,The pathogenic organism should be identified and characterised ,There should be positive evidence of the chain of infection Knowledge of a specific pathogen should assist the development of new diagnostic methods and treatmentWe would add that host factors, in particular genetic or immunological susceptibility should be considered, particularly in the context of IBD. We believe that our data adds considerable weight to fulfilling Swidsinski's second postulate and that the first has already been firmly established in animal models. Further work is required to address the third postulate which would clearly be aided by successful culture of these organisms from the colonic tissue of UC patients.H. mustelae in the colonic tissue of controls but not UC patients warrants further consideration. Since H. mustelae is a gastric organism  for H. pylori in human gastric disease. It may be that H. mustelae represents a less pathogenic organism in the human host which nonetheless confers the IBD protective benefits of H. pyloriHelicobacters our findings clearly demonstrate compelling molecular evidence for their presence in the human colon. Their presence in the human host is not well established compared to animal models however; there is no reason to suggest that they do not reside in the human intestinal tract. Clearly these hypotheses require further exploration.Finally, the presence of Table S1Bacterial Sequence Identification* of Helicobacteraceae positive samples.(DOC)Click here for additional data file.


Few prognostic biomarkers are approved for clinical use primarily because their initial performance cannot be repeated in independent datasets. We posited that robust biomarkers could be obtained by identifying deregulated biological processes shared among tumor types having a common etiology. We performed a gene set enrichment analysis in 20 publicly available gene expression datasets comprising 1968 patients having one of the three most common tobacco-related cancers  and identified cell cycle related genes as the most consistently prognostic class of biomarkers in bladder (BL) and lung adenocarcinoma (LUAD). We also found the prognostic value of 13 of 14 published BL and LUAD signatures were dependent on cell cycle related genes, supporting the importance of cell cycle related biomarkers for prognosis. Interestingly, no prognostic gene classes were identified in squamous cell lung carcinoma or head and neck squamous cell carcinoma. Next, a specific 31 gene cell cycle proliferation (CCP) signature, previously derived in prostate tumors was evaluated and found predictive of outcome in BL and LUAD cohorts in univariate and multivariate analyses. Specifically, CCP score significantly enhanced the predictive ability of multivariate models based on standard clinical variables for progression in BL patients and survival in LUAD patients in multiple cohorts. We then generated random CCP signatures of various sizes and found sets of 10–15 genes had robust performance in these BL and LUAD cohorts, a finding that was confirmed in an independent cohort. Our work characterizes the importance of cell cycle related genes in prognostic signatures for BL and LUAD patients and identifies a specific signature likely to survive additional validation. Gene expression profiling of human cancers has revolutionized our understanding of the disease and expedited the discovery of prognostic and predictive biomarkers Here we address this lack of robust prognostic biomarkers by postulating the existence of common cellular processes (modules) across multiple tumor types of common etiology, whose abnormal activity could be captured by a transcriptional signature. As our goal was to develop robust bladder cancer biomarkers, we selected additional cancer types with smoking as a well-defined major etiological factor. The identification of such a process would presumably allow for development of robust biomarkers that are prognostic across multiple patient cohorts and tumor types.An overview of our analysis is provided in %), lung adenocarcinoma , lung squamous cell carcinoma , and head and neck squamous cell carcinoma . All gene expression datasets used in this analysis are publicly available and can be downloaded from the Gene Expression Omnibus (GEO) Tables S1–S4 in File S2. References to the original study for each cohort are also provided in Tables S1–S4 in File S2. For more details see Supporting Materials and Methods in File S1.We analyzed 20 gene expression datasets comprising 1968 patients with bladder urothelial cell carcinoma  was always used if available (three BL cohorts). Overall survival (OS) was used if DSS was unavailable . Recurrence-free survival (RFS) was used if neither DSS nor OS were available (one LUAD and two HNSCC cohorts). The events for these endpoints are death from disease, death from any cause, and disease recurrence, for DSS, OS, and RFS, respectively. In one additional HNSCC cohort . Sample R code and output are provided in the form of a Sweave document that includes output from our analysis interweaved with corresponding R code (File S3). Additional R code is available upon request.The Database for Visualization and Annotated Discovery  in these gene lists. Cell cycle related modules were the most consistently enriched modules across BL and LUAD patient cohorts (for details see Supporting Results and Discussion in File S1). In contrast, LUSC and HNSCC patient cohorts did not have a common overrepresented pathway (for details see Supporting Results and Discussion in File S1).Data from 1968 patients were examined for the outcomes of tumor progression (where available) and survival  score (average expression of 31 cell cycle genes) that predicted time to recurrence or death in prostate cancer Overall, CCP score was significantly predictive (P<0.05) of progression and survival in all BL cohorts with these endpoints, and of survival in 5/8 LUAD cohorts, with high CCP scores associated with poor prognosis in all cases. Specifically, CCP score was predictive of progression in CNUH , Lindgren , and Dyrskjot  through forward stepwise selection of informative variables (P<0.05). Such a model selects a concise, ‘optimal’ set of variables and may select CCP score over standard clinical variables. Because clinical variables are readily available this final model may not be cost-effective currently but may be so in settings where cancers are staged and graded according to their molecular pathology. Finally, we evaluated if CCP score increased the prognostic value of a best available model that included clinical variables that are readily available in current settings. Such an increase suggests that CCP score has clinical utility. For more details of the methodology see Supporting Materials and Methods in File S1.We next evaluated whether CCP score contributes independent prognostic information. We performed a multivariate analysis and included clinically relevant variables such as age, gender, and grade in the BL and LUAD cohorts where CCP score was prognostic in the univariate analyses. For each cohort we also developed a best multivariate model , and was the only significant variable in the multivariate analysis. When survival was the endpoint, CCP score was the most consistently significant variable in the multivariate analysis (P<0.05), along with stage, which were each significant in three cohorts. CCP score was selected for the final model in two of these cohorts (Table S7 in File S4). In patients with LUAD, CCP score was the most consistently significant variable in the multivariate analysis and also the most frequently selected variable in the final model (Table S8 in File S4). In the best available models, addition of CCP score led to an increase in prognostic ability (P<0.05) in two BL, one BL, and three LUAD cohorts when progression, survival, and survival were the endpoints, respectively (Tables S9–S10 in File S5). These results suggest that CCP score may be superior than standard clinical variables in both BL and LUAD cancers and that CCP score may have immediate clinical utility for predicting progression in BL patients and survival in LUAD patients. For more details see Supporting Result and Discussion in File S1. Consistent with the gene set enrichment analysis above, CCP score was not prognostic in patients with squamous tumors (LUSC and HNSCC) .CCP score was selected for the  and Figure S1), we hypothesized that the performance of previously defined BL progression and LUAD survival signatures would be dependent on cell cycle related genes. BL survival signatures were not examined because a previous study found that their performance was poor in independent datasets Supporting Materials and Methods in File S1) that attenuates the predictive capability of signature genes that are correlated with CCP score Because modules most consistently associated with progression in BL patients and survival in LUAD patients were cell cycle related  and evaluated for progression in the BL cohorts and survival in the LUAD cohorts. A heatmap indicates each signature's prognostic value and whether its predictive ability is lost when adjusted for CCP score  (see Supporting Results and Discussion in File S1 for details). To independently confirm this finding and to evaluate smaller refined signatures we analyzed 4 “robust” signatures  (Table S12 in File S8) which include genes predictive of outcome in multiple cohorts .Because CCP signature genes are highly correlated Supporting Materials and Methods in File S1). All scores were predictive (P<0.05) in univariate and multivariate analyses which included CAPRA-S The CCP-4, -7, -10, and -12 signatures were provided to Myriad Genetics  for “blinded” evaluation, i.e., without indication of our hypothesis regarding the relationship between signature size and prognostic value. The predictive ability of the signature scores for biochemical recurrence in prostate cancer was evaluated in 353 patients receiving radical prostatectomy http://cancergenome.nih.gov). CCP score was calculated as before  using the normalized RNA counts  and downloaded from The Cancer Genome Atlas (TCGA) . First, it suggests that in BL and LUAD cancers, prognostic biomarkers will not validate across multiple cohorts unless they are (directly or indirectly) associated with cell cycle Figure 4et. al. found that in breast cancer the prognostic value of AURKA expression was comparable to multi-gene models. AURKA is one of the cell cycle genes we examined but was not as robust as CCP score, being predictive (P<0.05) in only 5/13 BL and LUAD cohorts . In general, redundancy obtained from multiple genes may overcome the variable quality of RNA derived from fixed tissue, which can cause individual gene assays to fail, thus ensuring robust analytical results. While we found that CCP-12 predicted outcome in an independent cohort additional steps such as defining thresholds that would separate patients into high and low risk groups and prospective evaluation in independent cohorts using predefined endpoints are required before this panel is ready for clinical use.Our work also tackles an important but seldom addressed issue in biomarker development, the impact of signature size on predictive ability. Cost effectiveness and feasibility of assessing multiple genes in a small quantity of biopsy tissue weigh against the need for large enough signatures that overcome technical variability from the assay and biological variability from tumor sample heterogeneity. Our results indicate the prognostic performance of cell cycle genes plateaus at 10–15. In contrast, Haibe-Kains File S1Supporting Materials, Results and Discussion.(DOCX)Click here for additional data file.File S2Summary of bladder cancer (Table S1), lung adenocarcinoma (Table S2), lung squamous cell carcinoma (Table S3), and head and neck squamous cell carcinoma (Table S4) patient cohorts.(PDF)Click here for additional data file.File S3Sweave document containing sample R code and output.(PDF)Click here for additional data file.File S4Multivariate analyses of progression in bladder cancer (Table S6), survival in bladder cancer (Table S7), and survival in lung adenocarcinoma (Table S8).(PDF)Click here for additional data file.File S5Comparison of prognostic power of CCP score and best available clinical variables in bladder (Table S9) and lung adenocarcinoma (Table S10).(PDF)Click here for additional data file.File S6Prognostic modules associated with outcome in tobacco-related cancers (Table S5) (Excel XLSX file). In each cohort, over-represented Gene Ontology (GO) terms and KEGG pathways were identified from lists of genes significantly predictive of disease outcome  using the DAVID gene annotation enrichment analysis toolkit. Consistently prognostic modules were identified by ranking all modules first by the number of cohorts with significant results (FDR<20%) and then by average p-value. There were no significantly over-represented prognostic modules in HNSCC patient cohorts at FDR<20%.(XLS)Click here for additional data file.File S7Prognostic bladder progression and lung adenocarcinoma survival gene signatures used in the adjustment analysis (Table S11) (Excel XLSX file).(XLS)Click here for additional data file.File S8CCP genes in five refined signatures (Table S12). Signatures are denoted by the number of CCP genes.(DOCX)Click here for additional data file.File S9Prognostic modules associated with bladder progression and lung adenocarcinoma survival signatures (Table S13) (Excel XLSX file). Over-represented Gene Ontology (GO) terms and KEGG pathways were identified for each signature using the DAVID gene annotation enrichment analysis toolkit. Modules are ranked first by the number of signatures with significant results (FDR<20%) and then by average p-value.(XLSX)Click here for additional data file.Figure S1Prognostic modules associated with progression in bladder cancer. In each cohort, over-represented Gene Ontology (GO) terms and KEGG pathways were identified from lists of genes significantly predictive of progression (P<0.01) using the DAVID gene annotation enrichment analysis toolkit. Consistently prognostic modules were identified by ranking all modules first by the number of cohorts with significant results (FDR<20%) and then by average p-value. Each subfigure includes ten modules: the most consistently prognostic modules and the ‘top hit’ for each cohort, marked by an asterisk (*), which is defined as the module with the lowest false discovery rate (FDR) in that cohort that has an FDR<20% in multiple cohorts. A, over-represented GO terms associated with progression in bladder cancer. B, over-represented KEGG pathways associated with progression in bladder cancer.(TIF)Click here for additional data file.Figure S2Prognostic value of CCP score in squamous cell lung cancers and head and neck squamous cell carcinomas.A, prognostic value of CCP score in squamous cell lung carcinomas (SCLC). Kaplan-Meier (KM) curves were generated for overall survival (OS) in the Bild (N = 53) and Takeuchi (N = 35) cohorts and for recurrence-free survival (RFS) in the Son (N = 76) cohort. KM curves were generated for patients with CCP scores at the lower (green), middle (blue), and upper (red) 33% and the log rank P-value of the continuous CCP score is reported. B, prognostic value of CCP score in head and neck squamous cell carcinomas (HNSCC). The Colo (N = 81) cohort did not include clinical follow-up time and so we evaluated the ability of CCP score to discriminate between node negative (N0) and node positive (N+) patients or between patients with non-recurrent (NR) and recurrent (R) tumors. The Pavon cohort (N = 63) did not include any clinical endpoints and so we evaluated the ability of CCP score to discriminate between N0 and N+ patients. In the Cohen cohort (N = 44), KM curves were generated for RFS. Abbreviations: HR, hazard ratio, corresponding to 1-unit increase in CCP score.(TIF)Click here for additional data file.Figure S3Relationship between prognostic value of CCP score and signature size based on proportion of significant signatures. Up to 10,000 gene signatures of sizes 1, 2, 4, …30, 31 were generated as described in Supporting Materials and Methods in File S1. Solid lines indicate proportion of signatures at each size that predicted survival (P<0.05) and are colored according to A, bladder patient cohort and B, lung adenocarcinoma cohort. Vertical dotted lines correspond to number of CCP genes (of 31) profiled in each cohort and are colored according to cohort.(TIF)Click here for additional data file.Figure S4Relationship between prognostic value of CCP score and signature size based on p-values. Up to 10,000 gene signatures of sizes 1, 2, 4, …30, 31 were generated as described in Supporting Materials and Methods in File S1. Boxplots of log10 p-values of signature scores for each signature size are plotted in A, bladder patient cohorts and B, lung adenocarcinoma cohorts. The blue horizontal line corresponds to a p-value of 0.05.(TIF)Click here for additional data file.Figure S5Prognostic value of CCP signature genes in A, bladder cancer and B, lung adenocarcinoma cohorts. In each cohort a gene is either significantly predictive of outcome , not significantly predictive of outcome , or was not profiled (white box) in each cohort. * indicates CCP score  is prognostic (P<0.05).(TIF)Click here for additional data file.


Primary cultures represent an invaluable tool to set up functional experimental conditions; however, creation of tissue cultures from solid tumors is troublesome and often unproductive. Several features can affect the success rate of primary cultures, including technical issues from pre-analytical procedures employed in surgical theaters and pathology laboratories. We have recently introduced a new method of collection, transfer, and preservation of surgical specimens that requires immediate vacuum sealing of excised specimens at surgical theaters, followed by time-controlled transferring at 4°C to the pathology laboratory. Here we investigate the feasibility and performance of short-term primary cell cultures derived from vacuum packed and cooled (VPAC) preserved tissues. Tissue fragments were sampled from 52 surgical specimens of tumors larger than 2 cm for which surgical and VPAC times (the latter corresponding to cold ischemia time) were recorded. Cell viability was determined by trypan blue dye-exclusion assay and hematoxylin and eosin and immunohistochemical stainings were performed to appreciate morphological and immunophenotypical features of cultured cells. Cell viability showed a range of 84–100% in 44 out of 52 (85%) VPAC preserved tissues. Length of both surgical and VPAC times affected cell viability: the critical surgical time was set around 1 hour and 30 minutes, while cells preserved a good viability when kept for about 24 hours of vacuum at 4°C. Cells were maintained in culture for at least three passages. Immunocytochemistry confirmed the phenotype of distinct populations, that is, expression of cytokeratins in epithelioid cells and of vimentin in spindle cells. Our results suggest that VPAC preserved tissues may represent a reliable source for creation of primary cell cultures and that a careful monitoring of surgical and cold ischemia times fosters a good performance of primary tissue cultures. Primary cultures represent an invaluable tool to set up functional experimental conditions that are instrumental to demonstrate biological mechanisms directly on human-derived tumor cells, however, creation of tissue cultures from solid tumors is troublesome and often unproductive particularly when dealing with primary cultures of carcinomas. Major biological issues are related to a relatively slow doubling time of epithelial cancer cells and to a rapid overgrowth with fibroblasts In this study we tested the feasibility of setting up primary cell cultures derived from vacuum packed and cooled (VPAC) preserved tissues and to evaluate their performance and success rates.Tissue samples for cell cultures were collected in RPMI 1640 serum free medium, supplemented with 1% penicillin-streptomycin-fungizone.The basal media used for cell culture was a mixture of DMEM (Dulbecco’s Modified Eagle Medium) and F12 in 1∶1 proportion. For preparation of complete media, 10% fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin-streptomycin-fungizone were added to the basal media mixture. The complete media was supplemented with 10 ng/mL human epidermal growth factor (EGF), 5 µg/mL insulin and 400 ng/mL hydrocortisone. For breast cancer samples, the complete media contained also 5 ng/mL 17-beta-estradiol and 500 ng/mL progesterone.For enzymatic cell dissociation Collagenase Type IV was used while trypsin-EDTA was used for passaging. All reagents were from Sigma-Aldrich, St Louis, MO, USA.All materials used in this experiment were sterile to prevent contamination.www.milestonemedsrl.com), according to a procedure originally reported by our group The study was approved by the ethic institutional review board for “Biobanking and use of human tissue for experimental studies” of the Pathology Services of the Azienda Ospedaliera Città della Salute e della Scienza di Torino. Written informed consent was obtained from all patients for their tissue to be used in research. Following excision in the surgical theater, surgical specimens were immediately placed into beta-ray sterilized plastic bags and vacuum sealed using the TissueSAFE machine  in order to evaluate the decrease of temperature by using a digital thermometer with stainless steel sensor probe at different conditions of storage .2 at 37°C. Culture medium was changed first at the time of cell attachment and, subsequently, three times a week. Cell growth was monitored daily in flasks with an EVOS inverted microscope  during the first week. The morphology of the cell population (spindle or mixed spindle-epithelioid) was recorded.The study was conducted on surgical specimens of tumors larger than 2 cm, leading to a cohort of 52 surgical samples, including 13 colorectal carcinomas, 6 lung carcinomas, 27 breast carcinomas, 2 adrenocortical adenomas, 3 gastric carcinomas and 1 thyroid carcinoma  to carry out Hematoxylin and Eosin (H&E) staining and immunocytochemical (ICC) reactions. Each time cells grown at confluence were split was considered a new passage.Viability of cells was determined by trypan blue dye (0.4% in PBS)-exclusion assay. Aliquots of cell suspension were incubated with trypan blue solution (1∶1) for 5 min. Finally cells were transferred to the Burker chamber and counted by light microscope. Dead cells were defined as those stained with the dye. The percentage of living cells was calculated by the relationship between the number of viable cells and the total number of cells counted. Aliquots of cells were cryopreserved into sterile cryo-tubes in 1.5 mL freezing medium (FBS containing 10% DMSO). Tubes were kept at −80°C (for a maximum of 6 weeks) or, following overnight cooling, transferred into liquid nitrogen (for longer storage periods).To evaluate the proportion of stroma and tumor cells in the sample collected for primary cultures we examined the H&E histological slide obtained from sampling of a parallel tumor area. To evaluate morphology of cultures, cells were grown on coverslips  and washed in phosphate-buffered saline (PBS 1X) for 5 minutes, fixed in 4% neutral-buffered formalin for 10 minutes and dehydrated through a series of alcohols up to absolute alcohol and stained with H&E staining.If the culture showed a mixed population we tried to characterize the cells using specific organ related markers. ICC was performed using an automated slide processing platform . The following primary antibodies were used: mouse anti-pancytokeratin  for 4 minutes), anti-vimentin  for 20 minutes), anticytokeratin-19 , anti-cytokeratin-14 , anti-cytokeratin-7  and anti-TTF1 .Results were analyzed by contingency tables using the Fisher’s exact test. Paired Student’s T-test was used for continuous variables. A p value <0.05 was considered statistically significant.VPAC produced a more rapid decrease of temperature as compared to non-VPAC procedures, while vacuum sealing at room temperature did not significantly affect temperature decrease . StatistThen, we analyzed the influence of VPAC time on cell viability in more in details. Cell growth was not feasible in 10.7% of specimens with VPAC time ≤24 hours, in 15.8% of tissues with 24< hours ≤48 VPAC time and in 40% of samples with >48 hours VPAC time  . Howeverversus tumor, as derived from histological slides, seemed not to affect the morphology of cell cultures. In 12 out of 44 viable cultures (27%) the cell population had uniform spindle morphology since the first passage  starts when the specimen is excised and ends with incision of tissue and placement in a suitable tissue fixative Another issue that should be considered is the optimal temperature to guarantee cell viability. In transplantation pathology it has been shown that a rapid induction of hypothermia at 0–4°C by perfusion of organ with specific solutions better preserves organ viability In conclusion, VPAC represented a reliable and reproducible tissue handling protocol for creation of primary cell cultures. Our results also showed how a careful monitoring of surgical and cold ischemia times fostered a good performance of primary tissue cultures. Further studies are needed to more carefully define key parameters governing the reliability of the VPAC method. Nonetheless, the environmentally safe VPAC collection, preservation and storage of surgical specimens already represented a helpful strategy to bridge diagnostic and experimental pathology, offering a new tool for biobanking and improved generation of primary cultures as clinically relevant models of neoplastic lesions.


Batrachochytrium dendrobatidis (Bd), to high depth and characterized rapidly changing features of its genome that we believe hold the key to the worldwide success of this organism. Our analyses show three processes that contribute to the generation of de novo diversity. Firstly, we show that the majority of wild isolates manifest chromosomal copy number variation that changes over short timescales. Secondly, we show that cryptic recombination occurs within all lineages of Bd, leading to large regions of the genome being in linkage equilibrium, and is preferentially associated with classes of genes of known importance for virulence in other pathosystems. Finally, we show that these classes of genes are under directional selection, and that this has predominantly targeted the Global Panzootic Lineage (BdGPL). Our analyses show that Bd manifests an unusually dynamic genome that may have been shaped by its association with the amphibian host. The rates of variation that we document likely explain the high levels of phenotypic variability that have been reported for Bd, and suggests that the dynamic genome of this pathogen has contributed to its success across multiple biomes and host-species.Pathogenic fungi constitute a growing threat to both plant and animal species on a global scale. Despite a clonal mode of reproduction dominating the population genetic structure of many fungi, putatively asexual species are known to adapt rapidly when confronted by efforts to control their growth and transmission. However, the mechanisms by which adaptive diversity is generated across a clonal background are often poorly understood. We sequenced a global panel of the emergent amphibian pathogen,  Batrachochytrium dendrobatidis (Bd). Firstly, we identified widespread chromosome copy number variation (CCNV) across our lineages, with individual isolates harboring between 2 to 5 copies of each chromosome and rapid rates of CCNV occurring in culture. In addition, by using in vitro divergence of replicate lines of Bd, we showed that changes in ploidy can occur within as few as 40 generations. Secondly, we identified uneven rates of recombination across the genomes and lineages, revealing hot spots in known classes of virulence factors. Finally we identified significant evidence of diversifying selection across the secretome of Bd, and showed that selection also targets putative virulence factors. These findings add to our knowledge of genome-dynamicity and modes of evolution manifested by eukaryote microbial pathogens, and may explain the varied phenotypic responses observed in Bd.Pathogenic fungi constitute a growing threat to both plant and animal species on a global scale. However, many features of the fungal genome that enable them to successfully adapt to infect diverse hosts and ecological niches remain cryptic, especially for newly evolved emerging lineages. In this paper, we report three novel features of genome diversity linked to pathogenicity in the emerging amphibian pathogen,  In particular, it is increasingly apparent that pathogenic fungi manifest highly plastic genome architecture in the form of variable numbers of individual chromosomes, known as chromosomal copy-number variation (CCNV) or aneuploidy. This feature has been identified across the fungal phylum Ascomycota, ranging from Batrachochytrium dendrobatidis (Bd), which has so far been identified in over 50 countries worldwide and infecting over 500 species of amphibians http://www.bd-maps.net). One of the most enigmatic aspects of Bd's population genetic structure has been the low levels of genetic variation identified between globally distributed isolates. However, recent studies have shown the existence of up to five separate lineages BdGPL) against that of a separate, distantly related  lineage that appears to have originated in South Africa (named BdCAPE), using SOLiD sequencing. BdGPL was found to harbour evidence of historical recombination, manifested as patchily distributed heterozygosity, and phylogenetic incongruency across small spatial scales that we hypothesised has resulted from ongoing recombination Bd clearly has a more dynamic genome than a purely clonal, mitotic mode of reproduction would suggest. Here, we describe a new global panel of isolates that were subjected to high-depth Illumina sequencing in order to better understand cryptic genomic features that are associated with the rapid ascendancy of this pathogen.A contemporary EID that is gaining substantial notoriety is the aquatic chytrid fungus Bd with a geographical distribution spanning five continents to a high depth  of the five suspected lineages of BdBdGPL and BdCAPE's known geographic range (BdGPL into Switzerland and Ethiopia and BdCAPE into France). Across the Bd JEL423 genome, 96% was covered by at least four reads in every isolate. Additionally, 65% of the total identified variant sites  were called as either reference or polymorphic in all 22 isolates amounting to >10X the number of ‘covered in all’ polymorphic loci previously found using the ABI SOLiD 3 platform We sequenced 22 isolates of 52–195X;  using th52–195X; , which w52–195X; , which w52–195X; . These pBd and affecting nine of the largest fifteen supercontigs   had significant p-values showing between 1–3 chromosomes in lower ploidy levels relative to the remaining bi-allelic genome. Over these lower-ploidy chromosomes we observed greater numbers of tri-alleles than bi-alleles and no decrease in heterozygous base-calls (both of which should occur if these chromosomes were haploid). We therefore conclude that these four isolates have tetraploid genomes with the identified losses in read-depth corresponding to chromosomes that have lost a single copy and are now trisomic.Thirteen iploidy) , and sixBdCH, which were passaged for 40 generations with and without exposure to skin antimicrobial peptides collected from the water frog Pelophylax esculentus. In these culture lines, the ancestral putatively triploid isolate (BdCH ACON) differentially lost and gained copies of supercontig IV and V respectively when passaged without selection (BdCH CON2A), and gained a copy of supercontig V following treatment with antimicrobial peptide (BdCH APEP), which resulted in a significant reduction in mean growth inhibition  and AUL (FR)) showed at least one haplotype that included an inferred crossover  and BdCH ACON (CH) were separated. Crossovers were identified in every major chromosome, and predominantly identified in intergenic regions (143 Mb−1 compared with 57 Mb−1 for coding regions and 65 Mb−1 for introns)  , S13.BdCAPE and BdCH  compared with 0–0.2% in BdGPL. This was a surprising finding given that the three BdCH isolates were separated by only 40 passages in the lab and were derived from a single isolate that had been relatively recently isolated in 2007. This suggests two hypotheses: Either in vitro passage under selective conditions promotes rapid recombination, or our isolate of BdCH is descended from a population of Bd that is more recombinogenic than BdGPL. To further study the amount of recombination within lineages and between isolates, we extracted haplotypes that were phased across all of the isolates within a given lineage and contained at least two alleles per loci  for pairwise comparisons of each lineage across window lengths of 1.4 Kb and 10 Kb , introns and intergenic regions (−1 fewer heterozygous positions in the introns compared with the CDS for isolate MG1). This overall pattern can be explained through selection purging deleterious mutations from the CDS. In addition, we found homozygous polymorphisms to be highly supported in all lineages in terms of uniquely mapped reads, whilst un-phased bi-allelic heterozygous positions had a smaller total proportion in the divergent lineages compared with BdGPL, suggesting some heterozygous positions may be miscalled due to paralogs.To identify genes that are present in the reference sequence and absent in our panel of isolates (presence/absence polymorphism), we examined the read-depth across each of the genes. Only five genes were identified from our panel , includi regions . In everWe categorised each of the mutations within the CDS into synonymous and non-synonymous mutations . SNPs wedS), non-synonymous substitution (dN) and omega (dN/dS = ω) for every gene in every isolate and compared values by grouping isolates into their lineages , suggesting positive or diversifying selection. Although no clear pattern could be distinguished within BdGPL  of the genes showed a signature of positive selection compared with only 23% for each of the other two lineages. This finding suggests that BdGPL has been undergoing greater levels of positive selection than either BdCAPE or BdCH, despite the low numbers of sites under selection owing to the high levels of relatedness within this lineage.By analysing each of these 1,450 genes with ω≥1using branch site models (BSM) in PAML along each of the three lineages of tegories . Nine getegories , includiBd's pathogenesis to the presence of a number of putative virulence factors that include proteases and chitin-binding proteins Bd since its divergence from free-living saprobes such as Homolaphlyctis polyrhizaBd to bind to keratinized host cells and to subsequently enter the host cells Bd has only been inferred owing to their homology to host-translocated proteins of known virulence in oomycetes dN/dS (ϖ) ratios when compared against classes of genes that do not contain a signal peptide. This shows that these gene families are evolving most rapidly in Bd, and that gene-products that interact with the amphibian host are undergoing diversifying (or reduced purifying) selection when compared with those gene-products that remain intracellular. Our findings suggest that Bd has had an evolutionary association with amphibians that predates the radiation of the lineages that we have characterised here, and is further evidence that this chytrid has an obligate rather than an opportunistic association with its amphibian hosts.Recent studies have attributed aspects of Bd isolates from the level of SNPs up to heterogeneity in ploidy amongst genomes and amongst chromosomes within a single genome. Individuals from all three lineages harboured CCNV along with predominantly or even entirely diploid, triploid and tetraploid genomes. Recent research by Rosenblum et al.Bd recovered largely from infected amphibians in the Americas, including a single haploid chromosome in isolate BdGPL JEL289. This variation may itself, reflect only part of the full diversity in Bd,pathogensas +2/+3 shifts in ploidy, whole genomes in tetraploid, or chromosomes in pentaploid or greater, may occur and await discovery. Chromosomal genotype was shown to be highly plastic as significant changes in CCNV occurred in as few as 40 generations in culture. It is not known whether other chytrid species also undergo CCNV, or if this is a unique feature of Bd and hence may be intrinsic to its parasitic mode of life. Currently, CCNV is known to occur in a variety of protist microbial pathogens, including fungi, however it is currently not known whether this genomic-feature is specific to a parasitic life-style, or is a more general feature of eukaryote microbes; identifying the ubiquity of CCNV or otherwise across nonpathogenic species will therefore be of great interest. Further, the manner in which the plasticity of CCNV in Bd affects patterns of global transcription and hence the phenotype of each isolate also remains to be studied. However, it is clear from research on yeast, Candida and Cryptococcus, that CCNV significantly contributes to generating altered transcriptomic profiles, phenotypic diversity and rates of adaptive evolution even in the face of quantifiable costs; understanding the relationship between CCNV and Bd-phenotype will therefore likely be key to understanding its patterns of evolution at both micro- and macro-scales.By mapping read-depth and SNPs across these genomes, we discovered that widespread genomic variation occurs within and amongst via mitotic processes within compatible genomes. In order to study recombination amongst our isolates, we determined the phase of our reads and constructed haplotypes that were suitable for traditional population genetic tests. This showed that, whilst the majority of the genomes from all three lineages manifest widespread linkage disequilibrium, recombination could still be detected across each chromosome and in all genomes. Crossovers (measured both as the proportion of SNPs that change phase and the numbers of haplotypes failing Hudson's four-gamete test) were found to occur much more frequently within the BdCAPE and BdCH lineages compared to BdGPL, and these two lineages accordingly manifest lower average linkage disequilibrium. All of the BdCH genomes that we sequenced stem from a single isolate collected in 2007. This suggests that either the high rates seen here have accrued since the isolate was taken into culture (suggesting a very rapid rate of in vitro recombination), or that we are characterising recombination events that occurred prior to the isolation of BdCH and are segregating as a consequence of the multiple-ploidy nature of Bd. In support of the latter hypothesis, comparisons between population-level data for BdGPL and BdCAPE show that BdGPL is far less recombinogenic and has been undergoing a largely clonal expansion since its emergence, consistent with previous observations made by James et al.BdGPL population is derived from a less recombinogenic ancestor than either BdCH or BdCAPE, that contemporary recombination is not occurring at a rapid rate and, where it occurs, is the result of a selfing rather than outcrossing events.Whilst differing numbers of individual chromosomes presents a potential barrier to the standard model of meiosis, homologous recombination may still be occurring BdGPL compared against BdCAPE or BdCH does not support the notion that BdGPL is an outbred hybrid lineage as previously proposed Bd lineage found in Brazil (BdBrazil) along with an isolate that is a likely BdGPL/BdBrazil recombinant Bd retains the ability to outcross, despite having a primarily clonal genome and life cycle. However, values of FST across our dataset show no introgression between the three lineages; this demonstrates that they have remained largely separate since their divergence and suggests that outcrossing between lineages of Bd is rare or, if it has occurs, remains spatially restricted. Further broad-scale collections of isolates and extension of our comparative-population genomic analyses will allow the assignment of more accurate rates of introgression across evolutionary timescales.The discovery of a lower proportion of variable sites across haplotypes in addition to the lower proportion of heterozygous positions in Bd's genome. Within the oomycete genus Phytophthora, CRNs manifest diverse carboxy-terminal domains and high rates of homologous recombination targeted to the conserved HVLVXXP motif, suggesting that the mosaic domains of CRNs are being shuffled by recombination Bd CRNs have been shown to be highly expressed on host tissue in vitroBd lack a secretion signal, their expression, accumulation of genetic variation in terms of recombination and ϖ values, and similarities with oomycete CRNs strongly suggest that a number of these CRN-like genes are functional in Bd. However, whether they contribute directly or indirectly to the virulence of Bd remains to be determined.We show that rates of recombination are uneven across the genome, with CRN-like genes enriched for crossovers, suggests that either CRN-like genes might have features that favour recombination or that recombinants of these genes have a fitness advantage and are thus more likely to reach fixation than recombinants at other locations in the genome. CRNs were also enriched for non-synonymous polymorphisms, are characterised by a signal of directional selection, and are amongst the most polymorphic genes in Bd in terms of CCNV, ubiquitous and potentially targeted recombination, and natural selection, points to an ability to generate diversity without the necessity of an obligate sexual stage. Our study has uncovered high levels of genotypic plasticity that are likely to cause widespread phenotypic plasticity even without the need to invoke outcrossing. These large and small-scale changes are therefore likely to contribute to rapid evolutionary rates in the face of an effective host response. Such ‘genomic instability’ may explain the diverse phenotypic responses observed in BdOur demonstration of multiple hierarchies of cryptic genomic variation in Batrachochytrium dendrobatidis (Bd) strain JEL423 was downloaded from http://www.broadinstitute.org/ (GenBank project accession number AATT00000000). The feature file for JEL423 had all but the longest splice variants removed for each gene leaving 8794/8819 genes. We aligned our reads to the genome sequence using Burrows-Wheeler Aligner (BWA) v0.5.9 −05 e-value cut-off) to the non-redundant BLAST database, SignalP3.0 Full details are given in t-tests (with a cut-off of p<5−10) on the mean depths across the largest supercontig (supercontig 1) against each subsequent supercontig for each isolate (t-tests.Chromosome copy number variation (CCNV) was identified using changes in both depth of coverage and percent of reads specifying two most frequent alleles at any locus. To quantify these changes, we first performed  isolate . Next, w isolate  for each isolate . Using aA), detecting linkage disequilibrium for a given set of haplotypes if VD>L (Lold). We also calculated rBarD values and performed 4 gamete tests between every combination of loci in a haplotype (FST) according to the equations given in Multilocus 1.3 To detect recombination, we identified haplotypes using reads that overlapped two or more bi-allelic heterozygous positions. Haplotypes from each isolate were then compared to haplotypes in other isolates. We also calculated the Index of association  was subcultured into control (ACON) and peptide-treated (APEP) culture flasks containing 10 ml 1% tryptone media supplemented with 1% penicillin-streptomycin (Sigma) to reduce the risk of bacterial contamination. Cultures were incubated at 18°C and passaged every 4–5 d by scraping the side of the flask and transferring 1 ml into 9 ml fresh media. Peptide-treatment included addition to the media of 80 µg ml-1 skin defense peptides collected from Pelophylax esculentus (n = 15 combined) according to Daum et al. (2012) 50, or the concentration at which growth of Bd was inhibited by 50%.For Figure S1Bd isolate JEL423 were aligned to a modified JEL423 reference sequence. Additionally, simulated reads from a heterozygous reference sequence were made to the depths of the Illumina and SOLiD datasets. Single Nucleotide Polymorphisms (SNPs) and heterozygous positions were then called and the False Discovery Rates (FDR) ascertained. The SNP-caller BiSCaP v0.11 was tested using default settings, and SAM/BCFTools with VCFUtils was tested for its ability to call SNPs using its default settings. SNPs were also filtered for those found without first modifying the reference sequence (f = filtered). (A) 1 nt/Kb simulated SNPs or heterozygous positions  within the coding region (CDS) (B) 1 nt/100 nt simulated SNPs or heterozygous positions  within the CDS region. The new Illumina data was able to recover >95% of true positive SNPs and >80% true positive heterozygous positions using BiSCaP v0.11, outperforming the previous lower-depth SOLiD sequences.The previous SOLiD reads (5) and the new Illumina paired end reads of (PNG)Click here for additional data file.Figure S2A) The overlap of homozygous SNPs varied between 3% and 97% (B) The overlap of heterozygous positions varied between 3% and 75%.The percent of ECVA polymorphic sites shared between each of the 22 isolates. Greater overlap (≥30%) highlighted in red. ((PNG)Click here for additional data file.Figure S3BdGPL, BdCAPE and BdCH shown in red blue and green respectively). (A) A tree from 275 Kb ECVA polymorphic positions identified from Illumina sequencing. (B) A tree from 36 Kb ECVA polymorphic positions from Illumina and SOLiD sequencing. (C) A tree from 218 Kb EVCA homozygous positions identified from Illumina sequencing. (D) A tree from 8 Kb EVCA homozygous positions identified from Illumina and SOLiD sequencing.Phylogenetic trees were made using the UPGMA algorithm in PAUP from ECVA polymorphic positions identified in the nuclear genomes demonstrating three divergent lineages ((PNG)Click here for additional data file.Figure S4Bd nuclear genomes was identified using allele-frequencies and mean read depths across each chromosome normalised to the alignment depth for each isolate. Many BdGPL isolates can be seen to include more copies of chromosome 2 and 3, while the 3 BdCH and 3 of the 5 BdCAPE isolates have fewer copies of chromosome 9 and 11. Fewer copies of chromosome 9, 11 and 16 appear to be found in many of the isolates.CCNV in the (PNG)Click here for additional data file.Figure S5t-tests for the mean depth of read coverage across each chromosome against chromosome 1 revealed significant p-values demonstrating uneven chromosome copy number. Stringent cut-offs for ploidy differences relative to the largest chromosome (Chr. 1) of each isolate were chosen: p<5−10. Chromosomes with p-values below this cut-off, with a mean depth that is greater than chromosome 1 are highlighted in blue, while those with a mean depth lower than chromosome 1 are shown in green. All 308 chromosomal p-values (excluding chr1) are shown in the bottom plot ordered from smallest to greatest.(PNG)Click here for additional data file.Figure S6Bd. The most common allele is shown in black and the second most common allele is shown in blue. Bins were used to summarise the expected peaks for odd, even and odd numbers of chromosomes and shown in red . Individual chromosomes with a predominantly bi-allelic value are shown with a blue border, and those with a predominant tri-allelic value are shown with a black border.The percent of reads specifying the two most frequent alleles per chromosome using 2 representative isolates from each lineage of (PNG)Click here for additional data file.Figure S7Bd nuclear genomes showing homozygous SNPs minus heterozygous positions. Predominance of homozygous SNPs is shown in red and predominance of heterozygous positions in shown in blue. Windows across BdGPL isolates demonstrate highly uneven distribution of heterozygosity attributed to recombination whereas polymorphisms are more evenly spread across the genomes of BdCAPE and BdCH isolates.Sliding non-overlapping windows of 10 Kb across the 22 (PNG)Click here for additional data file.Figure S8A) All reads over all phased positions. A 90% cut-off was used to filter ambiguous phased positions or those with an excess of mismatches as shown by the red line. (B) Positions that agreed 90–100% for a single phase are shown as a percent of all reads.Heterozygous positions had their phase determined using overlapping reads. Reads from each isolate are shown as a separate black line on the graphs. Only bi-allelic polymorphisms were compared for phasing. Predominantly, overlapping reads agreed with a single bi-allelic phase. ((PNG)Click here for additional data file.Figure S9Illustrations of how phased haplotypes were extracted from the alignment. Heterozygous positions that did not pass the minimum depth or percent phased cut-offs, along with examples of pairwise crossovers and outcomes for a four-gamete test between three isolates.(PNG)Click here for additional data file.Figure S10A) Total numbers of matching phased heterozygous positions in same phase (Kb) (B) Percent of matching phase positions from the total number of shared phased positions.Pairwise comparisons for shared phased heterozygous positions. ((PNG)Click here for additional data file.Figure S11A) Total numbers of crossovers identified. (B) Percent of crossovers from the total number of shared phased positions.Phased heterozygous positions demonstrating crossovers were identified between every isolate. ((PNG)Click here for additional data file.Figure S12BdGPL subset (s.s.) 1 consists of isolates VC1, AP15 and JEL423. Subset 2 consists of subset 1, ETH4 and MODS27.Lengths of haplotypes (in nucleotides) that included at least two alleles per loci in every isolate of a given group, and were therefore suitable for population genetic analysis. (PNG)Click here for additional data file.Figure S13Bd, suggesting recombination is not confined to small or large chromosomes, or the ends of any given chromosome. The same is seen with rBarD values.Intra-lineage heterozygote's, the percent of heterozygote's that were phased (PP), the percent of PP's that demonstrated a crossover (XO) and the RbarD were plotted using non-overlapping windows across the genome (length 10 Kb). Both phased positions and crossovers were found across each of the chromosomes in each of the lineages of (PNG)Click here for additional data file.Figure S14FST) was calculated for each pairwise lineage across window lengths of 1.4 Kb (A) and 10 Kb (B). All three lineages are differentiated from one another across each chromosome, with some intra-chromosomal variation. Notably, the stretch of rDNA located at the start of chromosome 14 appears to have a reduced genetic distance between each of the three lineages of Bd. BdGPL subset (ss) 1 consists of isolates VC1, AP15 and JEL423. Subset 2 consists of subset 1, ETH4 and MODS27.The Fixation Index ((PNG)Click here for additional data file.Figure S15The total numbers of crossovers found within genes demonstrated variation between gene families. All crossovers were compared against total number of heterozygous and phased positions, transcript length and tribe size. Proteases and chitin recognition proteins had a greater number of crossovers than would be expected by random over their combined number of phased positions.(PNG)Click here for additional data file.Figure S16Crossovers at unique locations  occurred differentially across gene families. NR crossovers were compared against total number of heterozygous and phased positions, transcript length and tribe size. Proteases and chitin recognition proteins had a greater number of crossovers than would be expected by random over their combined number of phased positions.(PNG)Click here for additional data file.Figure S17dN) vs synonymous mutation per synonymous site (dS) from alignments to Bd JEL423 for each of the gene families for all isolates belonging to the BdGPL. The line designates the ω value (dN/dS), whereby everything above the line has ω>1 and represents genes undergoing the greatest levels of variation.The ratio of non-synonymous mutation per non-synonymous site ((PNG)Click here for additional data file.Figure S18dN) vs synonymous mutation per synonymous site (dS) from alignments to Bd JEL423 for each of the gene families for all isolates belonging to the three lineages. The lines designate the ω value (dN/dS), whereby everything above the line has ω>1 and represents genes undergoing the greatest levels of variation. Summaries of ω values for all genes in each of the three lineages are shown in the final three plots.The ratio of non-synonymous mutation per non-synonymous site ((PNG)Click here for additional data file.Figure S19A Venn diagram showing the total number of genes undergoing positive selection according to the Branch site model (BSM), where genes had 2D′>8.1887. The nine genes were identified in all three lineages were four uncharacterised (secreted) with transcript ID's 05565, 02533, 00379, 06783 and five uncharacterised (non-secreted) with transcript ID's 03962, 07794, 05877, 02935, 08088.(PNG)Click here for additional data file.Table S1Bd nuclear genomes relative to Bd JEL423 using BiSCaP v0.11 with default settings. (A) Tallies of each category of loci found in each separate isolate. (B) The percent of uniquely mapped reads over each type of category of loci. Bi-allelic heterozygous positions had a reduced percent of uniquely mapped reads in the 2 divergent lineages of Bd, which may result from structural variants. Additionally, 72.48% of the homozygous SNPs and heterozygous positions were phased, which came from reads >86% uniquely mapped to the genome in any given isolate.Polymorphisms and reference bases were identified in 22 (PNG)Click here for additional data file.Table S2The two most common allele frequencies over each base of each chromosome were determined by percent of read agreement with the reference base. Using 1000 Bootstrap replicates of these values, we recorded how often 47–53% reads agreeing with an allele predominated over 30–36% or 63–69% reads agreeing with an allele. Shown in white are chromosomes with >95% of replicates showing a predominantly bi-allele signature (even-ploidies). Chromosomes with <5% bootstrap support for an even number of chromosomes therefore had a high support for unbalanced allele frequencies (odd-ploidies), and shown in blue. Chromosomes not fulfilling these criteria are shown in green and considered ambiguous.(PNG)Click here for additional data file.Table S3t-tests respectively. For t-tests, all genes with <2 NRPP (the minimum required for a crossover) were excluded. Although both CRN-like and uncharacterised (secreted) were enriched for crossovers at unique loci (non-redundant), only CRN-like (between lineages) were enriched for XO/NRPP.Genes were tested for enrichment in non-redundant  crossovers (XO) and NR XO/NR phased position (NRPP) compared to the values for all genes using Hypergeometric tests and (PNG)Click here for additional data file.Table S4BdGPL subset (s.s.) 1 consisted of isolates VC1, AP15 and JEL423. Subset 2 consisted of subset 1, ETH4 and MODS27.Haplotypes over coding sequence that failed the four-gamete test were predominantly from coding-regions. Haplotypes overlapping a number of genes were included in the counts for each gene . After accounting for these extra counts, an additional 1,162 haplotypes were still found to come from coding regions compared with those from intergenic or intron regions. However, no gene group had a clear enrichment for haplotypes that failed the four-gamete test. (PNG)Click here for additional data file.Table S5BdGPL JEL423 were identified amongst BdCAPE and BdCH isolates, whilst none were identified amongst BdGPL isolates.Only five presence absence (PA) polymorphisms relative to (PNG)Click here for additional data file.Table S6Bd nuclear genomes. The total numbers of each variant-type are followed by their numbers per kilobase of genomic region in parentheses. For heterozygous positions, the affect on the transcript (synonymous/non-synonymous) was determined using the alternative allele. Where two alternative alleles to the reference sequence were found (infrequently), the first present within the VCF was chosen. With the exception of the reference strain Bd JEL423, the ratios of non-synonymous to synonymous changes were between 1.12–2.00 and 1.22–2.13 for homozygous and heterozygous positions respectively.Homozygous (A) and bi-allelic heterozygous (B) polymorphisms were found in the coding and non-coding regions of the (PNG)Click here for additional data file.Table S7ABC transporters, Chitin associated genes and CRN-like genes were tested for enrichment in homozygous SNPs. The total number, average and standard deviation of non-redundant homozygous SNPs for each gene family were calculated for all isolates, and lineage specific isolates. A Hypergeometric test was used to identify significant enrichment for variants where P<0.01 (*), P<0.001 (**) and P<0.0001 (***).(PNG)Click here for additional data file.Table S8Proteases, uncharacterized secreted genes and uncharacterized genes were tested for enrichment in homozygous SNPs. The total number, average and standard deviation of non-redundant homozygous SNPs for each gene family were calculated for all isolates, and lineage specific isolates. A Hypergeometric test was used to identify significant enrichment for variants where P<0.01 (*), P<0.001 (**) and P<0.0001 (***).(PNG)Click here for additional data file.Table S9Secreted and CRN-like genes are significantly enriched for heterozygous positions at unique loci. The total number, average and standard deviation of non-redundant heterozygous and phased positions for each gene family were calculated for all isolates and lineage specific isolates. A Hypergeometric test was used to identify significant enrichment for heterozygosity where P<0.01 (*), P<0.001 (**) and P<0.0001 (***).(PNG)Click here for additional data file.Table S10dS), non-synonymous substitution (dN) and omega (dN/dS = ω) for every gene in every isolate.The average rates of synonymous substitution ((PNG)Click here for additional data file.Table S11A) The total numbers of genes, the numbers of genes with ω≥1 among all isolates, and how many of those genes had 2D′≥11.4076 (1% significance after Bonferroni correction) and 11.4076>2D′>8.1887 (5% significance after Bonferroni correction). The final column shows the percent of genes with 2D′>8.1887 from those with ω≥1. (B) For each lineage, the numbers of genes with ω≥1 and those that also had 2D′>8.1887. Following both of these columns are the results from a hypergeometric test for enrichment. For the genes with ω≥1, the test is for enrichment from the entire set of genes, whilst for the genes with 2D′>8.1887, the test is for enrichment from just the genes with ω≥1. (C) Overlap of genes with 2D′>8.1887.Number and category of genes with ω≥1  that also were found to have undergone positive selection using the Branch Site Models in codeml. ((PNG)Click here for additional data file.Text S1Supplemental Materials and Methods. Full details of methods and analysis described in this manuscript.(DOC)Click here for additional data file.


Since multiple ELR+ CXC chemokines act on both receptors—CXCR1 and CXCR2—a pharmacologic agent blocking both receptors seems to be advantageous. So far, several CXCR1/2 antagonists have been developed and have been tested successfully in experimental studies. A newly designed CXCR1 and CXCR2 antagonist can be orally administered and was for the first time found efficient in humans. This review highlights the role of CXCR2 in acute lung injury and discusses its potential as a therapeutic target.In pulmonary inflammation, recruitment of circulating polymorphonuclear leukocytes is essential for host defense and initiates the following specific immune response. One pathological hallmark of acute lung injury and acute respiratory distress syndrome is the uncontrolled transmigration of neutrophils into the lung interstitium and alveolar space. Thereby, the extravasation of leukocytes from the vascular system into the tissue is induced by chemokines that are released from the site of inflammation. The most relevant chemokine receptors of neutrophils are CXC chemokine receptor (CXCR) 1 and CXCR2. CXCR2 is of particular interest since several studies implicate a pivotal role of this receptor in development and promotion of numerous inflammatory disorders. CXCR2 gets activated by ELR As an essential component of the unspecific immune defense, leukocytes migrate from the blood into inflammatory tissue. Uncontrolled, excessive infiltration of leukocytes into the tissue leads to a destruction of organ structure and is a main characteristic of acute and chronic inflammatory disorders like atherosclerosis, reperfusion injury or acute lung injury (ALI) .ALI and its more severe form acute respiratory distress syndrome (ARDS) are still life-threatening syndromes primarily seen on intensive care units with a mortality rate of 40% in approximately 200,000 adult patients per year in the USA . Only veIn pulmonary inflammation, recruitment of circulating polymorphonuclear leukocytes (PMNs) is essential for defense and reducing the bacterial burden in the alveolar space—and initiates the following specific immune response. If PMN migration into the lung is impaired, the immune response is severely disturbed . PMN migThe extravasation of leukocytes is controlled by chemokines, which are released at the site of inflammation and induce chemotaxis. Chemokines are small, soluble peptides and interact with cells through specific chemokine receptors. Besides chemotaxis, chemokines can activate integrins that mediate leukocyte adherence on endothelial cells. The existence of the multiplicity of chemokines and their specific receptors enable selective trafficking of different immune cells under normal and inflammatory conditions –12. So fNeutrophils are an essential component of the innate immune system and are the first group of cells that migrate to sites of infection. CXC chemokine receptor (CXCR) 1 and CXCR2 are the major chemokine receptors of neutrophils –16. CXCRLike most chemokine receptors, CXCR2 is a G protein-coupled receptor assembled by seven transmembrane domains and connected with heterotrimeric G proteins. CXCR2 (and CXCR1) is expressed by granulocytes, especially neutrophils, but also by eosinophils, mast cells, T lymphocytes and basophils , 39. CXC+ chemokines (glutamic acid-leucine-arginine containing). Generally, ELR+ chemokines have angiogenic activity [+ chemokines are potent neutrophil chemoattractants and activators and, when administered exogenously, induce neutrophil mobilization from the bone marrow into the circulation [CXCR1 and CXCR2 share 78% of their amino acid sequence and get activated by ELRactivity  and theractivity . ELR+ chculation –54.+ chemokines: CXCL1, CXCL2, CXCL3, CXCL6, and CXCL8 , and CXCL 8, have been identified as the primary chemokine receptor system mediating neutrophil recruitment in various models of acute and chronic inflammation [The murine CXCL1 and CXCL2/3, functional homologues of human growth-related oncogenes . Ozone induces a transient increase of 20% in sputum neutrophils . Pharmac+ CXC chemokines act on both receptors—CXCR1 and CXCR2—, a pharmacologic agent blocking both receptors seems to be desirable [Since multiple ELResirable ; even thesirable . In a reesirable .Gernez et al. summarized that CXCR2 inhibitors are likely to affect all four pools of neutrophils . These pGenerally, G protein blockers block only around 50% of activity of the corresponding receptor. Our group showed that heterozygote mice for CXCR2 still had a significant reduction on PMN migration in the interstitium of the lung and alveolar space  and therReparixin (formerly known as repertaxin), a non-competitive allosteric inhibitor of CXCR1 and CXCR2 with a 400-fold higher efficacy in inhibiting CXCR1 activity than CXCR2, has been described [escribed . Reparixescribed –37 and oescribed . Prophylescribed  K11R/G31P improved the course of LPS-induced lung inflammation in pigs as shown by decreased neutrophil migration into the lung (approximately 86%) and reduced TNFα (approximately 70%) and IL-1 (approximately 83%) levels in the BAL [ELR-CXC chemokine antagonist  the BAL . CXCL8. Currently, there is no data available on the effects of this drug in ALI.GSK1325756, a potent, competitive CXCR2 receptor antagonist developed for the treatment of chronic obstructive pulmonary disease (COPD) is currently investigated with the question how the pharmacokinetic is affected by age, gender and food  .y humans . SB-6569Pseudomonas aeruginosa [Nocardia asteroides [Aspergillus fumigatus [Streptococcus pneumonia [In certain inflammatory diseases with special pathogens, a functional CXCR2 is required for neutrophil recruitment and pathogen elimination. CXCR2 knockout mice or antagonism of the receptor led to massive bacterial/fungal outgrowth in ruginosa , Nocarditeroides , Aspergiumigatus , and Strneumonia —infectioTherefore, further studies on blocking CXCR2 under different disease conditions are needed to understand its mode of action and to find out interactions with other receptors. This attempt is quite difficult since typically, upon activation of ligands, chemokine receptors are quickly desensitized and internalized and therefore have only around 30 seconds for signal transmitting .However, there is one recently discovered example of formerly unknown interactions: receptor oligomerization can lead to a novel signal cascade besides the normal signal pattern. One group found a hetero-oligomerization of the human DOP opioid and CXCR2 receptors . TherebyIn summery, CXCR2 plays a pivotal role in ALI with regard to neutrophil recruitment from the bone marrow, the circulating pool, neutrophils adherent to the endothelium, and the tissue pool. Pharmacologic blocking strategies appear promising in many disease conditions, and in several experimental studies blocking CXCR2 has been shown to attenuate tissue damage und disease progress. Pharmacologic blocking agents are even orally available and offer promising results in humans, but further studies with a larger cohort are needed. There are further investigations on this receptor necessary about its functions and interactions in relation to other receptors.