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A novel approach to identify epithelial defense mechanisms against normal luminal bacteria
A1054 AGAABSTRACTS • G4313 ANALYSIS OF ANTI-COLONIC AUTOANTIBODIES IN PRIMARY SCLEROSING CHOLANGITIS. J_A_AOdin 1, L Casciola-Rosen 2, A Rosen I. Departments of 1Medicine and 2Dermatology, Johns Hopkins School of Medicine, Baltimore, MD. Patients with primary sclerosing cholangitis (PSC) frequently have concomitant inflammatory bowel disease (IBD), especially ulcerative colitis (UC). Anti-colon and anti-portal tract antibodies as well as perinuclear antineutrophil cytoplasmic antibodies (p-ANCA) have been shown to be prevalent in the sera of patients with PSC suggesting an autoimmune pathogenesis. The identity of the colon target antigen(s) is unclear. Our goal was to further characterize the anti-colon antibodies in sera of patients with PSC and identify any clinical associations such as the presence of IBD. Methods: Sera from twenty-five patients with PSC (biopsy- and ERCPproven) were analyzed by Western blotting of lysates of Caco-2 cells (a human colon epithelial cell line) and HeLa cells (a human cervical epithelial cell line). Antigens recognized by the serum autoantibodies were detected with HRP-labeled goat anti-human IgG followed by enhanced chemiluminescence. Results: 28% of patients with PSC had high titer (up to 5000-fold dilution) IgG autoantibodies targeted against colon antigens migrating at 75 and 57 kDa. 72% of these patients had autoantibodies recognizing both antigens. Surprisingly these autoantibodies were present in patients with or without known IBD. The MW of these antigens differs from any previously reported in the sera of patients with PSC, These antigens did not co-migrate with the common primary biliary cirrhosis associated autoantigens and were not detected in lysates of HeLa cells. Additionally 8% of the sera recognized a 32 kDa antigen in both Caco-2 and HeLa cell lysates possibly representing anti-histone (HI) autoantibodies described previously in UC, though neither patient had known UC. Conclusion: High titer anti-colon IgG autoantibodies directed against novel antigens migrating at 75 and 57 kDa were detected by Western blotting in 28% of patients with PSC. Identification of these antigens may give insight into the pathogenesis of IBD in PSC. • G4314 A NOVEL APPROACH TO IDENTIFY EPITHELIAL DEFENSE MECHANISMS AGAINST NORMAL LUMINAL BACTERIA. H. O~awa. K.Fukushima, I. Sasaki, H.Naito, Y.Funayama, T.Masuko, K.Takahashi, S.Satoh, T.Ueno, A.Hashimoto, S.Matsuno. Tohoku University, Sendai, Japan. Background/Aims: Critical role of normal enteric flora for gut inflammation has been described in IL-2 knockout and HLA-B27 transgenic rodents. Since intestinal epithelial cells (IEC) represent a key barrier against bacteria, the identification of IEC gene products involved in this response may lead to a better understanding of mucosal defense under physiological and pathological conditions such as ulcerative colitis. The aim of this study was to investigate the small bowel and colon IEC gene expression induced by luminal bacteria. Methods: ICR mice were kept under specific pathogen-free (SPF) or sterile (germ free; GF) conditions. GF mice were fed chow contaminated with feces from SPF mice at 3 days, 7 days, 14 days before sacrifice at the age of 8 weeks. IEC were separately isolated from small bowel and colon from SPF, GF, and ex-GF mice (4 in each group). Patterns of IEC mRNA expression before and after bacterial reconstitution were examined by differential display. Cloning, sequencing, and northern blot analysis were performed to confirm the mRNA expression. Results: After bacterial reconstitution, acute self-limited inflammation developed in the colon but not the small bowel of ex-GF, with no evidence of residual chronicity. In 60 displays, we cloned 4 different bands differentially-expressed between small bowel and colon before and after reconstitution. One represented the cryptdin-related sequence 4C (CRS4C) which was strongly expressed only in small bowel IEC after but not before reconstitution. Another represented serum amyloid A1 (SAA1) mRNA which was strongly expressed after reconstitution only in colon IEC. The enhanced SAA1 mRNA expression in colon IEC was also detected in human mucosal inflammation, including ulcerative colitis. Conclusion: The genes detected by this reconstitution model seem to translate the response of IEC against normal bacterial flora. Since mice with normal genetic background are used, this model is ideally suited to study the response of small bowel and colon epithelium against enteric constituents in health and intestinal inflammation. • G4315 RABBIT COLONIC MUCINS INDUCE AUTOANTIBODIES AND MAY CAUSE ULCERATIVE COLITIS-LIKE MUCOSAL DAMAGE. S. Ohara, M. Igarashi*, S. Nakano*, T. Kubota*, F. Ogawa, K. Hotta. Dept, of Biochem. and * Int. Med., Sch. of Med., Kitasato Univ., Sagamihara228, JAPAN. Autoimmunity has been suggested in the etiology of ulcerative colitis. However, auto-antigens have not been identified so far. We have previously reported that the rat colonic mucins induce anti-colonic mucin antibodies in rats, and the mucin-immunized rats suffer from a colitis-like disorder. In the present study, we perform the same kind of experiment using rabbits. Methods: Japanese white rabbits were used. The purification of colonic mucins has been described as follows. Briefly, distal colonic tissue was homogenized in Tris-HC1 buffer (pH 7.2) containing 2 % Triton X-100 and 6
GASTROENTEROLOGYVol. 114, No. 4 M urea. Mucins were purified from the extract using gel-filtration and CsC1 equilibrium centrifugation. The rabbits were sensitized by subcutaneous injections of 1 mg of mucins emulsified with Freund's complete adjuvant. Two and three weeks later, their sensitivity was boosted with 0.5 mg mucins emulsified with Freund's incomplete adjuvant. Control rabbits were injected with only the adjuvant on the same time schedule. One week after the last immunization, sera were collected from the mucin-immunized rabbits and control rabbits. We performed ELISA and Westem blot analysis for detection of anti-mucin antibodies. A pathologic observation was performed using histochemical study. Results: ELISA and Western blot analysis showed that the immunized rabbits had anti-mucin antibodies. Macroscopically, we could not detect mucosal damage, but histochemical study showed infiltration of inflammatory cells and erosion at the colonic mucosa, Conclusion: Our results suggest that colonic mucins are one of the autoantigens of ulcerative colitis. • G4316 THE ROLE OF CD8÷ T CELLS FOR DEVELOPMENT OF COLITIS IN Gcti2-DEFICIENT MICE. L. Ohman*, U. Rudolph, L. Birnbaumer, G. R. Harriman. and E. H6rnauist* *University of G6teborg, Sweden and Baylor College of Medicine, Houston, TX. Mice with targeted deletion of the G protein subunit Gtxi2 develop an inflammatory bowel disease closely resembling ulcerative colitis. Histopathologically the Gcti2-/- mice show acute and chronic mucosal inflammation with ulcerations, crypt abscesses and loss of goblet cells. The inflammation is limited to the colonic mucosa with no skip areas, and the small intestines appear normal. Immunological characterization of the intestinal mucosa has revealed a local increase in memory CD4 ÷ T cells and proinflammatory Thl-type cytokines in Gtxi2-/- mice with colitis. We have previously shown that CD8 ÷ T cells exert a local suppression of mucosal immune responses in normal mice in that CD8-deficient mice exhibit a three-fold stronger gut mucosal immune response following oral immunization, but not systemically following parenteral immunization, as compared to wild type mice. To further elucidate the role of local suppression of mucosal immune responses in development of colitis in this animal model if IBD, Gcti2-/- mice were bred with CD8-/- mice to obtain Gcti2-deficient mice lacking CD8÷ T cells. Transmission of the Gai24- genotype on the CD8-/- background did not show a mendelian distribution: Significantly fewer Gai2-/- mice and correspondingly more Gcti2+/+ mice than expected were found. However, development of colitis was not found to be influenced by the absence of CD8÷ T cells. Thus, Gcti2-/-CD8-/- double knockout mice had to be sacrificed due to severe disease symptoms at a mean age of 15_+4 weeks, and Gai2-/CD8+/- and Geti2-/-CD8+/+ mice were sacrificed at a mean age of 16 ± 7 weeks. Flow cytometric analysis of the CD4 ÷ population in the mesenteric and caudal lymph nodes (draining the small intestine, cecum, plus ascending colon, and distal colon and rectum, respectively) of Gcti2-/-CD8-/- double knockout mice demonstrated an increased frequency of CD4÷ T cells expressing a memory phenotype, i.e. CD44hi,CD62L]°. These CD4÷ T lymphocytes also exhibited an increased expression of the mucosal homing receptors tx4137 and ctEI37, as compared to Gai2+/-CD8-/- mice. The overall frequency of CD4 ÷ T ceils was not increased in the Gai2-/-CD8-/- mice. However, the frequency of CD3+CD4-8- T ceils was increased in both locations. A corresponding decrease in the frequency of CD19 ÷ B lymphocytes was found both in the mesenteric and caudal lymph nodes. Still, the frequency of B lymphocytes expressing the co-stimulatory molecules B7.1 (CD80), and to a lesser extent B7.2 (CD87) was increased in both locations. Analysis of cytokine production revealed a marked increase in proinflammatory Thl-type cytokines in inflamed colons, but not small intestines of Gtxi2-/-CD8-/- mice, as compared to Gt~i2+I-CD8-1- mice. IFN-), and IL-113 levels were increased 11 and 49-fold, respectively. Low levels of IL-4 was detected, but the production was not different between the two strains. No spontaneous production of IL-2, IL-3 or IL-12 was seen. In conclusion, the absence of suppressive CD8÷ T cells in the intestinal mucosa does not aggravate colitis in Gcti2-deficient mice. • G4317 CLINICAL AND RADIOLOGICAL FEATURES SUGGESTIVE OF DISSEMINATED TUBERCULOSIS IN PATIENTS WITH HIVRELATED ABDOMINAL PAIN. EA O'Keefe*, R Wood*, A Van Zyl#. Departments of Medicine* and Radiology#, University of Cape Town, South Africa. Disseminated TB (TB) is the most common cause of abdominal pain in South African AIDS patients. It is important to identify simple diagnostic criteria that differentiate this condition from other causes of HIV-related abdominal pain as it responds well to antituberculous therapy and surgery should be avoided. METHODS: HIV patients presenting to an HIV-referral centre in Cape Town with abdominal pain and a CD4 count <200 were studied prospectively. TB was defined by positive mycobacterial cultures from blood, bone marrow or 2 other sites. The association between the predominant site of pain, fever, weight loss, diarrhoea, hepatomegaly, respiratory symptoms, abnormal chest radiograph (CXR), abdominal ultrasound (US) findings and the presence or absence of TB was assessed.
GASTROENTEROLOGYVol. 114, No. 4 M urea. Mucins were purified from the extract using gel-filtration and CsC1 equilibrium centrifugation. The rabbits were sensitized by subcutaneous injections of 1 mg of mucins emulsified with Freund's complete adjuvant. Two and three weeks later, their sensitivity was boosted with 0.5 mg mucins emulsified with Freund's incomplete adjuvant. Control rabbits were injected with only the adjuvant on the same time schedule. One week after the last immunization, sera were collected from the mucin-immunized rabbits and control rabbits. We performed ELISA and Westem blot analysis for detection of anti-mucin antibodies. A pathologic observation was performed using histochemical study. Results: ELISA and Western blot analysis showed that the immunized rabbits had anti-mucin antibodies. Macroscopically, we could not detect mucosal damage, but histochemical study showed infiltration of inflammatory cells and erosion at the colonic mucosa, Conclusion: Our results suggest that colonic mucins are one of the autoantigens of ulcerative colitis. • G4316 THE ROLE OF CD8÷ T CELLS FOR DEVELOPMENT OF COLITIS IN Gcti2-DEFICIENT MICE. L. Ohman*, U. Rudolph, L. Birnbaumer, G. R. Harriman. and E. H6rnauist* *University of G6teborg, Sweden and Baylor College of Medicine, Houston, TX. Mice with targeted deletion of the G protein subunit Gtxi2 develop an inflammatory bowel disease closely resembling ulcerative colitis. Histopathologically the Gcti2-/- mice show acute and chronic mucosal inflammation with ulcerations, crypt abscesses and loss of goblet cells. The inflammation is limited to the colonic mucosa with no skip areas, and the small intestines appear normal. Immunological characterization of the intestinal mucosa has revealed a local increase in memory CD4 ÷ T cells and proinflammatory Thl-type cytokines in Gtxi2-/- mice with colitis. We have previously shown that CD8 ÷ T cells exert a local suppression of mucosal immune responses in normal mice in that CD8-deficient mice exhibit a three-fold stronger gut mucosal immune response following oral immunization, but not systemically following parenteral immunization, as compared to wild type mice. To further elucidate the role of local suppression of mucosal immune responses in development of colitis in this animal model if IBD, Gcti2-/- mice were bred with CD8-/- mice to obtain Gcti2-deficient mice lacking CD8÷ T cells. Transmission of the Gai24- genotype on the CD8-/- background did not show a mendelian distribution: Significantly fewer Gai2-/- mice and correspondingly more Gcti2+/+ mice than expected were found. However, development of colitis was not found to be influenced by the absence of CD8÷ T cells. Thus, Gcti2-/-CD8-/- double knockout mice had to be sacrificed due to severe disease symptoms at a mean age of 15_+4 weeks, and Gai2-/CD8+/- and Geti2-/-CD8+/+ mice were sacrificed at a mean age of 16 ± 7 weeks. Flow cytometric analysis of the CD4 ÷ population in the mesenteric and caudal lymph nodes (draining the small intestine, cecum, plus ascending colon, and distal colon and rectum, respectively) of Gcti2-/-CD8-/- double knockout mice demonstrated an increased frequency of CD4÷ T cells expressing a memory phenotype, i.e. CD44hi,CD62L]°. These CD4÷ T lymphocytes also exhibited an increased expression of the mucosal homing receptors tx4137 and ctEI37, as compared to Gai2+/-CD8-/- mice. The overall frequency of CD4 ÷ T ceils was not increased in the Gai2-/-CD8-/- mice. However, the frequency of CD3+CD4-8- T ceils was increased in both locations. A corresponding decrease in the frequency of CD19 ÷ B lymphocytes was found both in the mesenteric and caudal lymph nodes. Still, the frequency of B lymphocytes expressing the co-stimulatory molecules B7.1 (CD80), and to a lesser extent B7.2 (CD87) was increased in both locations. Analysis of cytokine production revealed a marked increase in proinflammatory Thl-type cytokines in inflamed colons, but not small intestines of Gtxi2-/-CD8-/- mice, as compared to Gt~i2+I-CD8-1- mice. IFN-), and IL-113 levels were increased 11 and 49-fold, respectively. Low levels of IL-4 was detected, but the production was not different between the two strains. No spontaneous production of IL-2, IL-3 or IL-12 was seen. In conclusion, the absence of suppressive CD8÷ T cells in the intestinal mucosa does not aggravate colitis in Gcti2-deficient mice. • G4317 CLINICAL AND RADIOLOGICAL FEATURES SUGGESTIVE OF DISSEMINATED TUBERCULOSIS IN PATIENTS WITH HIVRELATED ABDOMINAL PAIN. EA O'Keefe*, R Wood*, A Van Zyl#. Departments of Medicine* and Radiology#, University of Cape Town, South Africa. Disseminated TB (TB) is the most common cause of abdominal pain in South African AIDS patients. It is important to identify simple diagnostic criteria that differentiate this condition from other causes of HIV-related abdominal pain as it responds well to antituberculous therapy and surgery should be avoided. METHODS: HIV patients presenting to an HIV-referral centre in Cape Town with abdominal pain and a CD4 count <200 were studied prospectively. TB was defined by positive mycobacterial cultures from blood, bone marrow or 2 other sites. The association between the predominant site of pain, fever, weight loss, diarrhoea, hepatomegaly, respiratory symptoms, abnormal chest radiograph (CXR), abdominal ultrasound (US) findings and the presence or absence of TB was assessed.