Title: Antibodies to CBir1 Flagellin Define a Unique Response That Is Associated Independently With Complicated Crohn’s Disease
Abstract: Background & Aims: Antibody responses to certain microbial antigens define heterogeneous groups of Crohn’s patients; multiple and high-level responses to these antigens are associated with aggressive clinical phenotypes. The flagellin, CBir1, identified by investigations in the C3H/HeJBir mouse model, has been identified as a dominant antigen capable of inducing colitis in mice and eliciting antibody responses in a subpopulation of patients with Crohn’s disease (CD). The aim of this study was to evaluate serum response to CBir1 flagellin in CD patients and to compare this response to responses defined previously to oligomannan (anti-Saccharomyces cerevisiae antibody), I2, OmpC, and neutrophil nuclear autoantigens (pANCA), and to determine anti-CBir1-associated phenotypes. Methods: A total of 484 sera from the Cedars Sinai Medical Center repository, previously typed for anti-Saccharomyces cerevisiae antibody, anti-I2, anti-OmpC, and pANCA were tested for anti-CBir1 by enzyme-linked immunosorbent assay, and results were assessed for clinical phenotype associations. Results: The presence and level of immunoglobulin G anti-CBir1 were associated with CD independently. Anti-CBir1 was present in all antibody subgroups and expression increased in parallel with increases in the number of antibody responses. pANCA+ CD patients were more reactive to CBir1 than were pANCA+ ulcerative colitis patients. Anti-CBir1 expression is associated independently with small-bowel, internal-penetrating, and fibrostenosing disease features. Conclusions: Serum responses to CBir1 independently identify a unique subset of patients with complicated CD. This bacterial antigen was identified in a murine model and has a similar pattern of aberrant reactivity in a subset of CD patients. Background & Aims: Antibody responses to certain microbial antigens define heterogeneous groups of Crohn’s patients; multiple and high-level responses to these antigens are associated with aggressive clinical phenotypes. The flagellin, CBir1, identified by investigations in the C3H/HeJBir mouse model, has been identified as a dominant antigen capable of inducing colitis in mice and eliciting antibody responses in a subpopulation of patients with Crohn’s disease (CD). The aim of this study was to evaluate serum response to CBir1 flagellin in CD patients and to compare this response to responses defined previously to oligomannan (anti-Saccharomyces cerevisiae antibody), I2, OmpC, and neutrophil nuclear autoantigens (pANCA), and to determine anti-CBir1-associated phenotypes. Methods: A total of 484 sera from the Cedars Sinai Medical Center repository, previously typed for anti-Saccharomyces cerevisiae antibody, anti-I2, anti-OmpC, and pANCA were tested for anti-CBir1 by enzyme-linked immunosorbent assay, and results were assessed for clinical phenotype associations. Results: The presence and level of immunoglobulin G anti-CBir1 were associated with CD independently. Anti-CBir1 was present in all antibody subgroups and expression increased in parallel with increases in the number of antibody responses. pANCA+ CD patients were more reactive to CBir1 than were pANCA+ ulcerative colitis patients. Anti-CBir1 expression is associated independently with small-bowel, internal-penetrating, and fibrostenosing disease features. Conclusions: Serum responses to CBir1 independently identify a unique subset of patients with complicated CD. This bacterial antigen was identified in a murine model and has a similar pattern of aberrant reactivity in a subset of CD patients. Immunologic responses to bacterial products are key to the induction of inflammatory bowel disease in humans and in experimental models. The relationship of these immune responses to the underlying genetic and clinical phenotypes is just beginning to emerge. Thus, among patients with Crohn’s disease (CD), immune responses to different microbial antigens may be related to different pathophysiologic mechanisms and may represent distinct genotypes and phenotypes. Investigations based on experimental models of CD have led to the current theory that chronic intestinal inflammation is the result of an aberrant immunologic response to commensal bacteria residing within the gut lumen.1Strober W. Fuss I.J. Blumberg R.S. The immunology of mucosal models of inflammation.Annu Rev Immunol. 2002; 20: 495-549Crossref PubMed Scopus (1147) Google Scholar, 2Blumberg R.S. Saubermann L.J. Strober W. Animal models of mucosal inflammation and their relation to human inflammatory bowel disease.Curr Opin Immunol. 1999; 11: 648-656Crossref PubMed Scopus (407) Google Scholar, 3De Winter H. Cheroutre H. Kronenberg M. Mucosal immunity and inflammation. II. The yin and yang of T cells in intestinal inflammation: pathogenic and protective roles in a mouse colitis model.Am J Physiol. 1999; 276: G1317-G1321PubMed Google Scholar In addition, there are several animal models of CD in which disease development and progression hinge on the presence of T-cell reactivity toward specific commensal bacteria.4Cong Y. Brandwein S.L. McCabe R.P. Lazenby A. Birkenmeier E.H. Sundberg J.P. Elson C.O. CD4+ T cells reactive to enteric bacterial antigens in spontaneously colitic C3H/HeJBir mice increased T helper cell type 1 response and ability to transfer disease.J Exp Med. 1998; 187: 855-864Crossref PubMed Scopus (333) Google Scholar, 5Cong Y. Weaver C.T. Lazenby A. Elson C.O. Colitis induced by enteric bacterial antigen-specific CD4+ T cells requires CD40-CD40 ligand interactions for a sustained increase in mucosal IL-12.J Immunol. 2000; 165: 2173-2182Crossref PubMed Scopus (79) Google Scholar, 6Cong Y. Weaver C.T. Lazenby A. Elson C.O. Bacterial-reactive T regulatory cells inhibit pathogenic immune responses to the enteric flora.J Immunol. 2002; 169: 6112-6119PubMed Google Scholar In the C3H/HeJBir mouse model, serologic responses toward enteric bacteria are present, indicating a seroreactivity to specific commensal microorganisms.7Brandwein S.L. McCabe R.P. Cong Y. Waites K.B. Ridwan B.U. Dean P.A. Ohkusa T. Birkenmeier E.H. Sundberg J.P. Elson C.O. Spontaneously colitic C3H/HeJBir mice demonstrate selective antibody reactivity to antigens of the enteric bacterial flora.J Immunol. 1997; 159: 44-52PubMed Google Scholar In addition, the mono-association of specific bacteria in the germ-free interleukin-10−/− mouse model results in colitis, but with diverse severity and location depending on the organism. This finding implies that similar genetic backgrounds may yield distinct disease manifestations in response to different microbial antigens.8Kim S.C. Tonkonogy S. Albright C. Sartor R.B. Regional and host specificity of colitis in mice monoassociated with different non-pathogenic bacteria.Gastroenterology. 2003; 124: A-485Google Scholar In human beings, specific associations between a particular bacterial species and the development of disease or its characteristics have not been established. However, supporting the role of bacteria in human disease, studies have shown that the fecal stream is critical to disease development and progression,9D’Haens G.R. Geboes K. Peeters M. Baert F. Penninckx F. Rutgeerts P. Early lesions of recurrent Crohn’s disease caused by infusion of intestinal contents in excluded ileum.Gastroenterology. 1998; 114: 262-267Abstract Full Text Full Text PDF PubMed Scopus (736) Google Scholar, 10Rutgeerts P. Goboes K. Peeters M. Hiele M. Penninckx F. Aerts R. Kerremans R. Vantrappen G. Effect of faecal stream diversion on recurrence of Crohn’s disease in the neoterminal ileum.Lancet. 1991; 338: 771-774Abstract PubMed Scopus (648) Google Scholar that antibiotics and probiotics have shown promise in therapeutic trials,11Prantera C. Kohn A. Mangiarotti R. Andreoli A. Luzi C. Antimycobacterial therapy in Crohn’s disease results of a controlled, double-blind trial with a multiple antibiotic regimen.Am J Gastroenterol. 1994; 89: 513-518PubMed Google Scholar, 12Prantera C. Zannoni F. Scribano M.L. Berto E. Andreoli A. Kohn A. Luzi C. An antibiotic regimen for the treatment of active Crohn’s disease a randomized, controlled clinical trial of metronidazole plus ciprofloxacin.Am J Gastroenterol. 1996; 91: 328-332PubMed Google Scholar, 13Prantera C. Kohn A. Zannoni F. Spimpolo N. Bonfa M. Metronidazole plus ciprofloxacin in the treatment of active, refractory Crohn’s disease results of an open study.J Clin Gastroenterol. 1994; 19: 79-80Crossref PubMed Scopus (41) Google Scholar, 14Rutgeerts P. Hiele M. Geboes K. Peeters M. Penninckx F. Aerts R. Kerremans R. Controlled trial of metronidazole treatment for prevention of Crohn’s recurrence after ileal resection.Gastroenterology. 1995; 108: 1617-1621Abstract Full Text PDF PubMed Scopus (706) Google Scholar, 15Guslandi M. Mezzi G. Sorghi M. Testoni P.A. Saccharomyces boulardii in maintenance treatment of Crohn’s disease.Dig Dis Sci. 2000; 45: 1462-1464Crossref PubMed Scopus (618) Google Scholar and that the disease predominantly affects areas with bacterial stasis (ileum and cecum). Immune responses to commensal enteric organisms have been investigated in CD. Duchmann et al16Duchmann R. May E. Heike M. Knolle P. Neurath M. Meyer zum Buschenfelde K.H. T cell specificity and cross reactivity towards enterobacteria, bacteroides, bifidobacterium, and antigens from resident intestinal flora in humans.Gut. 1999; 44: 812-818Crossref PubMed Scopus (234) Google Scholar showed that CD patients have a reactivity to hundreds of bacterial antigens created from sonication of multiple bacterial species. We have shown that CD patients have antibodies to specific bacterial antigens and that patients can be clustered into 4 groups depending on their antibody response patterns.17Landers C.J. Cohavy O. Misra R. Yang H. Lin Y.C. Braun J. Targan S.R. Selected loss of tolerance evidenced by Crohn’s disease-associated immune responses to auto- and microbial antigens.Gastroenterology. 2002; 123: 689-699Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar These clusters are as follows: (1) antibody responses against oligomannan (anti-Saccharomyces cerevisiae [ASCA]); (2) antibody responses to both Escherichia coli outer membrane protein C (anti-OmpC) and a CD-related protein from Pseudomonas fluorescens (anti-CD-related bacterial sequence [I2]); (3) antibody responses to nuclear antigens (perinuclear antineutrophil cytoplasmic antibody [pANCA]); or (4) low or no serologic response to any of the tested antigens. These distinct antibody response patterns may indicate unique pathophysiologic mechanisms in the progression of this complicated disease. In addition, phenotypic associations with specific serologic response patterns have been discovered.17Landers C.J. Cohavy O. Misra R. Yang H. Lin Y.C. Braun J. Targan S.R. Selected loss of tolerance evidenced by Crohn’s disease-associated immune responses to auto- and microbial antigens.Gastroenterology. 2002; 123: 689-699Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 18Vasiliauskas E.A. Plevy S.E. Landers C.J. Binder S.W. Ferguson D.M. Yang H. Rotter J.I. Vidrich A. Targan S.R. Perinuclear antineutrophil cytoplasmic antibodies in patients with Crohn’s disease define a clinical subgroup.Gastroenterology. 1996; 110: 1810-1819Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 19Vasiliauskas E.A. Kam L.Y. Karp L.C. Gaiennie J. Yang H. Targan S.R. Marker antibody expression stratifies Crohn’s disease into immunologically homogeneous subgroups with distinct clinical characteristics.Gut. 2000; 47: 487-496Crossref PubMed Scopus (294) Google Scholar, 20Mow W.S. Vasiliauskas E.A. Lin Y.C. Fleshner P.R. Papadakis K.A. Taylor K.D. Landers C.J. Abreu-Martin M.T. Rotter J.I. Yang H. Targan S.R. Association of antibody responses to microbial antigens and complications of small bowel Crohn’s disease.Gastroenterology. 2004; 126: 414-424Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar Recently, we used serologic expression cloning to identify an immunodominant antigen, CBir1 flagellin, to which strong B-cell and CD4+ T-cell responses occur in colitic mice. Transfer of CBir1-specific CD4+ Th1 T cells to C3H/SCID mice generated a severe colitis dependent on endogenous expression of CBir1 flagellin in the cecum and colon. These findings prove that CBir1 flagellin is an immunodominant antigen of the enteric microbial flora. Of note, approximately 50% of patients with CD had serum reactivity to CBir1, whereas patients with ulcerative colitis (UC), patients with other inflammatory gastrointestinal diseases, and control subjects had little or no reactivity to this flagellin.21Lodes M.J. Cong Y. Elson C.O. Mohamath R. Landers C.J. Targan S.R. Fort M. Hershberg R.M. Bacterial flagellin is a dominant antigen in Crohn disease.J Clin Invest. 2004; 113: 1296-1306Crossref PubMed Scopus (659) Google Scholar This study determined the relationship of serum reactivity to CBir1 and the previously defined responses to oligomannan (ASCA), OmpC, I2, and pANCA in patients with CD and defined distinct clinical phenotypes. Results show that antibodies to CBir1 are associated independently with CD, have no correlation to levels of previously defined antibodies, are expressed in ASCA− and pANCA+ CD patients, and are associated independently with aspects of complicated CD. Serum samples from 484 patients (40 normal controls, 21 disease controls, 50 UC patients, and 373 CD patients) were selected from the serum archive of the Cedars-Sinai Inflammatory Bowel Disease Research Center. All research-related activities were approved by the Cedars-Sinai Medical Center institutional review board. The diagnosis for each patient was based on standard endoscopic, histologic, and radiographic features. The normal control group is a collection of environmental controls that contain sera from individuals with no symptoms/signs of disease (ie, spouses of patients). Disease controls include sera from patients with presumed infectious enteritis (stool culture negative for specific pathogens), blastocystis, celiac disease, collagenous colitis, irritable bowel syndrome, radiation proctitis, and acute schistosomiasis. For UC, groups chosen were pANCA− (n = 25, seronegative) and pANCA+ (n = 25, pANCA enzyme-linked immunosorbent assay unit [EU] > 45, no other antibody reactivity present). For CD, 2 cohorts were chosen: cohort 121Lodes M.J. Cong Y. Elson C.O. Mohamath R. Landers C.J. Targan S.R. Fort M. Hershberg R.M. Bacterial flagellin is a dominant antigen in Crohn disease.J Clin Invest. 2004; 113: 1296-1306Crossref PubMed Scopus (659) Google Scholar (n = 100) comprised patients with select antibody expression to test CBir-1’s specificity for CD and its relationship with other CD-associated antibodies; cohort 2 (n = 303) was unbiased, previously well clinically, and serologically characterized,20Mow W.S. Vasiliauskas E.A. Lin Y.C. Fleshner P.R. Papadakis K.A. Taylor K.D. Landers C.J. Abreu-Martin M.T. Rotter J.I. Yang H. Targan S.R. Association of antibody responses to microbial antigens and complications of small bowel Crohn’s disease.Gastroenterology. 2004; 126: 414-424Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar with an overlap of 30 patients between the two. Within cohort 1, groups chosen were seronegative (n = 40), ASCA+ (n = 15, immunoglobulin (Ig)G ASCA EU > 40, IgA ASCA EU > 45, and no other antibody reactivity present), I2+ (n = 15, anti-I2 EU > 40, and no other antibody reactivity present), I2+/OmpC+ (n = 15, OmpC EU > 30, and anti-I2 reactivity present), I2+/OmpC+/ASCA+ (n = 15, anti-I2, anti-OmpC, IgG, and IgA ASCA all positive, but no ANCA reactivity allowed), and pANCA+ (n = 25, ANCA EU > 35, no other antibody reactivity present). Cohort 2 was used for determining antibody groups as well as for phenotype analysis by using definitions of clinical subgroups previously reported.20Mow W.S. Vasiliauskas E.A. Lin Y.C. Fleshner P.R. Papadakis K.A. Taylor K.D. Landers C.J. Abreu-Martin M.T. Rotter J.I. Yang H. Targan S.R. Association of antibody responses to microbial antigens and complications of small bowel Crohn’s disease.Gastroenterology. 2004; 126: 414-424Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar Serum samples from 44 CD patients diagnosed as described earlier were analyzed for changes in antibody expression. Twenty of these patients were under treatment with infliximab and had experienced a CD activity index change of at least 70 (mean = Δ181) at time points at least 4 months apart with serum drawn at both times. The sera from the other 24 patients were drawn at the time of surgery and once at least 6 months after surgery. Enzyme-linked immunosorbent assay analysis of anti-CBir1 was performed as previously described21Lodes M.J. Cong Y. Elson C.O. Mohamath R. Landers C.J. Targan S.R. Fort M. Hershberg R.M. Bacterial flagellin is a dominant antigen in Crohn disease.J Clin Invest. 2004; 113: 1296-1306Crossref PubMed Scopus (659) Google Scholar but using the NH2-terminal fragment of CBir1 (147aa) without knowledge of diagnosis or other serology results. Briefly, enzyme-linked immunosorbent assay plates were coated overnight with 100 ng/well of CBir1, then blocked with 1% bovine serum albumin in phosphate-buffered saline for 2 hours. Plates were washed and serum was added at a 1:200 dilution in 1% bovine serum albumin-phosphate-buffered saline for a 30-minute incubation. After washing, horseradish-peroxidase-conjugated anti-human IgG at a 1:10,000 dilution was added and incubated for 30 minutes. After another wash, the plates were incubated with tetramethylbenzidine substrate for 15 minutes. The reaction was stopped with 1 N sulfuric acid and read at 450 nm. Positive was defined as the mean ± 2 SD of the healthy controls. For cohort 2 and the longitudinal cohorts and phenotype cohorts, this assay was modified to be more similar to the ANCA, OmpC, and I2 protocols: alkaline phosphatase was substituted as the secondary conjugate and incubated for 1 hour followed by paranitrophenyl phosphate as substrate for 30 minutes. Differences between disease groups were tested with nonparametric (Wilcoxon signed-rank) statistics for quantitative levels. To determine the associations between antibody responses (positivity) toward microbial antigens, autoantigens, and disease phenotype characteristics, univariate analyses using χ2 tests were performed. The Cochran-Armitage test for trend was used to test if there was a linear trend in the proportion of patients with positive anti-CBir-1 expression as the number of antibody responses increased. A P value (P trend) of .05 or less suggests that the linear trend was statistically significant. A stratified Cochran-Mantel-Haenszel test was used to determine the association between anti-CBir1 and disease phenotypes. Multivariate analysis with logistic regression modeling also was performed to determine the primary associations among qualitative serologic responses with disease phenotypes. All statistic tests were performed using statistical analysis software (Version 8.02; SAS Institute, Inc, Cary, NC). Serologic expression cloning of a cecal bacterial antigen phage library identified the flagellin, CBir1, as an immunodominant antigen recognized by colitic mice21Lodes M.J. Cong Y. Elson C.O. Mohamath R. Landers C.J. Targan S.R. Fort M. Hershberg R.M. Bacterial flagellin is a dominant antigen in Crohn disease.J Clin Invest. 2004; 113: 1296-1306Crossref PubMed Scopus (659) Google Scholar and by approximately half of patients with CD. We used serum from 2 separate cohorts to investigate subgroups of CD patients. Cohort 1 consisted of sera from 100 CD patients selected on the basis of antibody profile. Newly tested sera from a group of 303 unselected patients that were studied and reported on by Mow et al20Mow W.S. Vasiliauskas E.A. Lin Y.C. Fleshner P.R. Papadakis K.A. Taylor K.D. Landers C.J. Abreu-Martin M.T. Rotter J.I. Yang H. Targan S.R. Association of antibody responses to microbial antigens and complications of small bowel Crohn’s disease.Gastroenterology. 2004; 126: 414-424Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar comprised cohort 2. For antigen, we used the amino domain of CBir1 flagellin because most of the IgG reactivity was to this region of the molecule. In addition, this form of CBir1 had a lower baseline reactivity (data not shown) among inflammatory controls, patients with UC or CD, and healthy control subjects, compared with the full-length construct used previously.21Lodes M.J. Cong Y. Elson C.O. Mohamath R. Landers C.J. Targan S.R. Fort M. Hershberg R.M. Bacterial flagellin is a dominant antigen in Crohn disease.J Clin Invest. 2004; 113: 1296-1306Crossref PubMed Scopus (659) Google Scholar As shown in Figure 1, 50% of CD patients from cohort 1 had serologic responses to this CBir1 construct, as compared with very low numbers and low levels of response among inflammatory controls, patients with UC, or healthy control subjects. Among the control subjects who did respond to CBir1, the level of response was much lower than that of the patients with CD. In cohort 2, 55% (167 of 303) of sera were positive for antibodies to CBir1. Approximately half of CD patients, whether selected serologically or not, are reactive to CBir1. As we had determined with the previously defined CD-related antigens (I2/OmpC, oligomannan),17Landers C.J. Cohavy O. Misra R. Yang H. Lin Y.C. Braun J. Targan S.R. Selected loss of tolerance evidenced by Crohn’s disease-associated immune responses to auto- and microbial antigens.Gastroenterology. 2002; 123: 689-699Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 20Mow W.S. Vasiliauskas E.A. Lin Y.C. Fleshner P.R. Papadakis K.A. Taylor K.D. Landers C.J. Abreu-Martin M.T. Rotter J.I. Yang H. Targan S.R. Association of antibody responses to microbial antigens and complications of small bowel Crohn’s disease.Gastroenterology. 2004; 126: 414-424Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar we sought to determine whether the level of anti-CBir1 expression changed in association with disease activity. We collected serum samples from medically resistant patients with CD who were undergoing surgical removal of active disease. Samples were taken again 6 months postoperatively and analyzed for differences in response. In general, there was very little change before and 6 months after surgery when patients were in clinical and endoscopic remission (Figure 2A). We then performed the same analysis before, and 4 months after, treatment of CD with infliximab (Figure 2B, C). Among patients who achieved complete remission as evidenced by mucosal changes and healing, similar stability in anti-CBir1 expression is seen (Figure 2B, C). These findings are consistent with antibody responses to other microbial antigens.17Landers C.J. Cohavy O. Misra R. Yang H. Lin Y.C. Braun J. Targan S.R. Selected loss of tolerance evidenced by Crohn’s disease-associated immune responses to auto- and microbial antigens.Gastroenterology. 2002; 123: 689-699Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 20Mow W.S. Vasiliauskas E.A. Lin Y.C. Fleshner P.R. Papadakis K.A. Taylor K.D. Landers C.J. Abreu-Martin M.T. Rotter J.I. Yang H. Targan S.R. Association of antibody responses to microbial antigens and complications of small bowel Crohn’s disease.Gastroenterology. 2004; 126: 414-424Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar To determine the relationship of expression and level of anti-CBir1 expression to the previously defined antibodies to microbial antigens (anti-I2, anti-OmpC, ASCA), we used multiple logistic regression analysis with cohort 1 and we found that anti-CBir1 relates independently to CD when controlled for anti-I2, anti-OmpC, and ASCA (P < .001). In addition, there is no relationship between the level of response to CBir1 and any 1 of the other 4 antibodies (Figure 3A–D). Thus, reactivity to CBir1 defines another potentially pathophysiologically distinct subgroup of CD. As previously described by Landers et al,17Landers C.J. Cohavy O. Misra R. Yang H. Lin Y.C. Braun J. Targan S.R. Selected loss of tolerance evidenced by Crohn’s disease-associated immune responses to auto- and microbial antigens.Gastroenterology. 2002; 123: 689-699Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar we have defined homogeneous groups of CD patients based on selective antibody responses to specific microbial antigens and associated clinical features. Having shown that anti-CBir1 expression is an independent marker, we wanted to know how this response relates to previously defined serologic groupings in given patients. Thus far, the largest subgroup of CD has been stratified based on the expression of ASCA. Among patients selected for study based on their antibody profiles (cohort 1), anti-CBir1 is expressed in both ASCA− (46%) and ASCA+ (60%) CD patients (Figure 4). Anti-CBir1 also is expressed by patients who do not react to ASCA, OmpC, I2, or ANCA (40%), as well as those who express ASCA exclusively (33%) and anti-I2 exclusively (40%) (Figure 5). Anti-CBir1 expression and magnitude increases among patients reactive to both I2 and OmpC (60%), and increases more among those patients reactive to I2, OmpC, and oligomannan (87%) (Figure 5). These results were confirmed in cohort 2, in which we found anti-CBir1 expression in 46% (66 of 144) of ASCA−, 64% (102 of 159) of ASCA+, and 38% (23 of 61) of seronegative CD patients. The frequency of anti-CBir1 expression increases as the number of positive antibody responses increases in patients with 0, 1, 2, and 3 antigens (Figure 6, P < .001). Thus, in both cohorts 1 and 2, anti-CBir1 expression is highest in sera from patients who react to all 3 other antigens, but also is found along with any other combination of 1, 2, or 3 antibody responses.Figure 5Anti-CBir1 is found in all CD serologic subtypes, but is most prevalent in I2+/OmpC+/ASCA+ patients. The level of antibody response in defined subsets of CD to CBir1 flagellin is shown. Subsets are negative for all antibodies other than those listed. The gray area indicates the negative range as defined by 2 SD above the mean of the normal controls; lines indicate the median level for each group. The percentage of positive samples for each group is shown.View Large Image Figure ViewerDownload (PPT)Figure 6The frequency of anti-CBir1 expression increases with multiple microbial antibody expression. Frequency of anti-CBir1 expression in patients with no other microbial antibodies and those expressing 1, 2, or 3 other microbial antibodies (P trend < .0005).View Large Image Figure ViewerDownload (PPT) A small percentage of CD patients are solely pANCA+. pANCA is associated with UC, and in CD, pANCA marks for left-sided disease with UC-like features.18Vasiliauskas E.A. Plevy S.E. Landers C.J. Binder S.W. Ferguson D.M. Yang H. Rotter J.I. Vidrich A. Targan S.R. Perinuclear antineutrophil cytoplasmic antibodies in patients with Crohn’s disease define a clinical subgroup.Gastroenterology. 1996; 110: 1810-1819Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 19Vasiliauskas E.A. Kam L.Y. Karp L.C. Gaiennie J. Yang H. Targan S.R. Marker antibody expression stratifies Crohn’s disease into immunologically homogeneous subgroups with distinct clinical characteristics.Gut. 2000; 47: 487-496Crossref PubMed Scopus (294) Google Scholar, 22Esters N. Vermeire S. Joossens S. Noman M. Louis E. Belaiche J. De Vos M. Van Gossum A. Pescatore P. Fiasse R. Pelckmans P. Reynaert H. Poulain D. Bossuyt X. Rutgeerts P. Serological markers for prediction of response to anti-tumor necrosis factor treatment in Crohn’s disease.Am J Gastroenterol. 2002; 97: 1458-1462Crossref PubMed Scopus (7) Google Scholar, 23Peeters M. Joossens S. Vermeire S. Vlietinck R. Bossuyt X. Rutgeerts P. Diagnostic value of anti-Saccharomyces cerevisiae and antineutrophil cytoplasmic autoantibodies in inflammatory bowel disease.Am J Gastroenterol. 2001; 96: 730-734Crossref PubMed Google Scholar pANCA does not differentiate between UC and UC-like CD. We sought to determine whether anti-CBir1 expression had any bearing on this subgroup. Of the pANCA+ patients with CD, 40%–44% (cohort 2 and cohort 1, respectively) expressed anti CBir1 and none of the other antibodies vs only 4% in pANCA+ UC (Figure 7). This difference stratifies another subgroup of CD with a potential pathophysiologically unique disease mechanism. We previously determined that antibody responses to the microbial antigens OmpC, I2, oligomannan, and neutrophil nuclear antigen(s) are associated with anatomic location and disease expression.18Vasiliauskas E.A. Plevy S.E. Landers C.J. Binder S.W. Ferguson D.M. Yang H. Rotter J.I. Vidrich A. Targan S.R. Perinuclear antineutrophil cytoplasmic antibodies in patients with Crohn’s disease define a clinical subgroup.Gastroenterology. 1996; 110: 1810-1819Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 19Vasiliauskas E.A. Kam L.Y. Karp L.C. Gaiennie J. Yang H. Targan S.R. Marker antibody expression stratifies Crohn’s disease into immunologically homogeneous subgroups with distinct clinical characteristics.Gut. 2000; 47: 487-496Crossref PubMed Scopus (294) Google Scholar, 20Mow W.S. Vasiliauskas E.A. Lin Y.C. Fleshner P.R. Papadakis K.A. Taylor K.D. Landers C.J. Abreu-Martin M.T. Rotter J.I. Yang H. Targan S.R. Association of antibody responses to microbial antigens and complications of small bowel Crohn’s disease.Gastroenterology. 2004; 126: 414-424Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 24Arnott I.D.R. Lan