- Email: [email protected]
HLA genes are important phenotype determining genes in the peripheral arthropathies of inflammatory bowel disease (IBD)
A1056 AGA ABSTRACTS • G4321
GENES ARE IMPORTANT PHENOTYPE DETERMINING GENES IN THE PERIPHERAL ARTHROPATHIES OF INFLAMMATORY BOWEL DISEASE (IBD). TR Orchard. S Thiyagaraja, KI Welsh, BP Wordsworth, DP Jewell Gastroenterology Unit, Radcliffe Infirmary, Oxford, UK. B a c k g r o u n d - The detection of phenotype determining genes as opposed to disease susceptibility genes requires precise phenotypic characterisation of patients. Peripheral arthropathies in IBD are well recognised and are often classified with the HLA-B27 related spondyloarthropathies. However, previous small studies of HLA status in IBD have failed to show such an association in peripheral disease. We have described a clinical classification which divides these arthropathies into 2 types. Type 1 is a large joint acute oligoarthropathy associated with relapse of the IBD, whilst Type 2 is a persistent polyarthropathy which runs a course independent of the IBD. Both are seronegative and non-erosive. We report the results of HLA studies in these patients. M e t h o d s - DNA from 30 patients in each group underwent HLA genotyping using a PCR based phototyping method. Results from each patient group were compared with 603 Oxfordshire controls (with corrections for multiple comparisons) and then inter-group comparisons were made in the light of existing hypotheses. Results - Type 1 arthropathy was associated with HLA-DR103 in 40% vs 3% (p<0.0001, RR12.1), B35 in 33% vs 15%(p=0.01, RR2.2) and B27 in 27% vs 7% (p=0.001, RR4.0). Type 2 was associated with HLA-B44 in 63% vs 31%(p=0.01, RR2A). These associations were also significant when the two types of arthropathy were compared (DR103 40% vs 0% p=0.0001, B35 33% vs 7% p=0.02, RR5,0, B27 27% vs 3% p=0.03, RR8.0, B44 63% vs 13% p<0.0001, RR12.1). Conclusion - These data suggest that our clinical classification describes immunogenetically distinct entities and that HLA genes are important in determining the clinical phenotype of joint disease in IBD. Type 1 arthropathy is clinically and immunogeneticaily similar to the HLA-B27 related arthritides, whereas Type 2 may have a different aetiopathogenesis. HLA
• G4322 ILEOCAECAL RESECTION IS ASSOCIATED WITH A DECREASED INCIDENCE OF ARTICULAR COMPLICATIONS IN CROHN'S DISEASE. TR Orchard, DP Jewell, Gastroenterology Unit, Radcliffe Infirmary, Oxford UK. B a c k g r o u n d - Studies in HLA-B27 transgenic rats have demonstrated that the bacterial population in the ileocaecal region is important in the initiation and perpetuation of inflammation at distant sites. Diversion of the faecal stream away from this region abrogates the distant inflammation without affecting the colitis. To assess the role of the ileecaecal region in arthropathy in Crohn's disease we compared the incidence of arthropathy in patients pre- and postileocaecal resection (ICR) with patients who had never undergone surgery. Methods - Data on length of follow up, dates and nature of surgery and presence of Type 1 and Type 2 peripheral arthropathy and ankylosing spondylitis were obtained by case note review and patient interview of all patients attending the Oxford inflammatory bowel disease (IBD) clinic. The incidence of arthropathy pre-and post-surgery was compared for patients who had undergone ICR with patients who had never undergone surgery. Results were expressed in number of years of patient follow up per joint complication. Periods of remission induced by surgery were not counted as years of follow up. Results - 217 patients who had never had surgery and 194 who had undergone ICR were identified. Median length of follow-up was 10.7 years and 10.2 years respectively. In the non-surgery group there was 1 articular complication per 76 years of patient follow up compared to 80 years of patient follow up in the surgical patients pre-surgery. Post- ICR the incidence of articular complications was 1 per 656 years of patient follow up. Conclusion - The incidence of articular complications is significantly lower after ICR even after correction for surgically induced remission. One explanation for these findings may be that the change in intestinal flora after ICR reduces the risk of inflammation at extraintestinal sites. G4323
AN ANIMAL MODEL OF H. PYLORI-POSITIVE CHRONIC ACTIVE GASTRITIS USING NEONATAL THYMECTOMIZED MICE. C. Oshima K.Okazaki, K. Uchida, Y.Matsushima, T.Chiba. Department of Gastroenterology, Hepatology, and Endoscopy, Kyoto University, Kyoto, Japan. Aim: The immunological interaction of the host with H.pylori seems to play an important role in the pathophysiology of H.pylori-infected gastric diseases, However, it is still unknown whether autoimmune mechanism may be involved in H.pylori-infected chronic gastritis or not. To clarify it, we have established an animal model using neonatal thymectomized mice having autoimmune gastritis, similar to type A human gastritis. Methods and Materials: Ten Balb/c mice in each group were thymectomized on the 3rd day after birth (3dTx). To confirm whether they have autoimmune gastritis or not, serum levels of autoantibody against parietal cells were measured by ELISA system. At the period of 6-8 weeks old, mice were orally
GASTROENTEROLOGY Vol. 114, No. 4
infected with l0 s of H.pylori and sacrificed after 2, 4, and 6 month. Three groups (normal mice with or without H.pylori, and 3dTx-mice without H.pylori) were served as controls. Antibodies against parietal cells and H.pylori were serially measured. We studied histological findings of the stomach, and the subsets and intracellular cytokines (interferon-'/and IL-4) of infiltrating lymphocytes by immuno-histochemistry and flow cytometic analysis. Gene expressions of interferon-'/and IL-4 were also analyzed by RT-PCR. Results: In each group of H.pylori-infected 3dTx-mice, histological findings showed chronic active gastritis with lymph follicles in the gastric mucosa. Four months after H.pylori infection, formation of germinal center occurred in one of ten mice. Flow cytometric analysis of infiltrating lymphocytes showed 30, 3, and 6% as B cells, CD4, and CD8 positive cells, respectively. Paneth or goblet-like cells, suggesting intestinal metaplasia of the gastric mucosa, were found in some cases. Expression of interferon-'/ was identified in all of H.pylori-infected 3dTx-mice, but IL-4 was equivocal. The population of lymphocytas by flow cytometric analysis, staining intracellular cytokines, were consistent with the data of RT-PCR. On the other hand, infiltrating cells were few in the control mice. Serum levels of autoantibody were not correlated with the severity of gastritis. Conclusion: Autoimmune mechanism and predominant Thl type response may be involved in chronic active gasttitis in H.pylori-infected 3dTx Balb/c mice. G4324
AUTOIMMUNITY INDUCES A SEVERE RESPONSE TO HELICOBACTER FELIS IN NEONATALLY THYMECTOMIZED MICE. C. Oshima. K. Okazaki, Y. Matsushima, T. Chiba Department of Gastroenterology, Hepatology, and Endoscopy, Kyoto University, Kyoto, Japan. Aim: In humans, atrophic gastritis is classified as two types, type A and B. We have reported that neonatally thymectomized (nTx) mice have autoimmune gastritis (AIG), similar to human type A gastritis. On the other hand, it has been suggested that Helicobacter pylori is involved in type B chronic gastritis. Helicobacter fells (H.f) colonizes in the normal Balb/c mouse. In this study, we investigated influence of autoimmunity on pathophysiology, and host immune response to H.felis, using nTx mice. Material and methods: Ten Balb/c mice in each group were thymectomized on the 3rd day after birth. To confirm whether they have AIG or not, serum levels of autoantibody against parietal cells were measured by ELISA system. Group 1 (AIG (+), H.f (+)) and Group 2 (AIG (-), H.f (+)) mice were orally infected with 10s of H. fells at 8 weeks old. They were sacrificed at one and three months, and histopathological findings were compared with those of the control groups, Group3 (AIG (+), H.f (-)) and Group4 (AIG (-), H.f (-)). Antibodies to parietal cells and H.f were serially measured. Results: In Group1, both the corpus and antrai gastric mucosa showed more severe inflamation than that in other three groups. Serum levels of antibodies to parietal cells and H.fin Groupl were higher than those in other three groups. Conclusion: These data suggested that autoimmune mechanism may be involved in more severe gastritis with H.f positive nTx Baib/c mice. G4325
ALTERNATION OF DECAY-ACCELERATING FACTOR EXPRESSION ON GUINEA PIG GASTRIC MUCOSA AFTER ISCHEMIA REPERFUSION. T.Oshima, T.Joh, K.Seno, M.Ikai, M.Sasaki, H.Katanka, Y.Yokoyama, M.Nonaka*, N.Okada*, H.Okada*, T. Tada**, M.Itoh. 1st Dept. Int. Med., *Dept. of Molecular Biology and **Pathology. Nagoya City University Medical School, Nagoya, Japan. The complement regulatory protein, decay-accelerating factor (DAF), protects host tissues from damage due to autologous complement activation, We have previously shown that expression of human gastric mucosai DAF was markedly enhanced relative to the degree of inflammation (Gastroenterol 112: A 1082, 1997). However, expression of DAF in gastric mucosa has not been well investigated using experimental models. We recently identified 6 isoforms of guinea pig (GP) DAF (transmembrane types; TM-a, TM-ab, TM-abc, and glycosylphosphatidylinositol anchored types; GPI-a, GPI-ab, GPI-abc). In this study, we investigated the changes in DAF gene and protein expression following GP gastric injury induced by ischemia reperfusion (I/R). Methods: Male Hartley guinea pigs (250-300g) were subjected to an overnight fast and anesthetized. Gastric ischemia was induced by clamping the left gastric and epiploic arteries for 30 min. Prior to sacrifice, stomachs were removed 0, 6, 12, 24 hr, 3 and 7 days after I/R. Total RNA was extracted from half of each sample collected. Polyadenylated RNA was isolated as mRNA, and the amount of DAF mRNA was evaluated by Northern blot analysis. The other half of each sample was fixed in acetone, and immunohistochemical staining was performed with a specific monoclonal antibody to GP DAF (J. Immunol. 154:6103). Expression of DAF in gastric mucosa was semiquantitatively evaluated by scoring using 3 grades and data was then statistically analyzed, Expression of DAF isoform mRNA was assessed by RT-PCR with isoform specific primers. Results: Mucosai erosion was observed from 6 hr to 3 days after I/R (p<0,05), but was not detected at 7 days. Expression of DAF mRNA was constitutively demonstrated in normal GP gastric mucosa and was most intense 6 hr after I/R, however, it was detected at normal levels at 24 hr, 3 and 7 days. Expression of DAF protein was not significant from 0 to 12 hr after I/R, but it
GENES ARE IMPORTANT PHENOTYPE DETERMINING GENES IN THE PERIPHERAL ARTHROPATHIES OF INFLAMMATORY BOWEL DISEASE (IBD). TR Orchard. S Thiyagaraja, KI Welsh, BP Wordsworth, DP Jewell Gastroenterology Unit, Radcliffe Infirmary, Oxford, UK. B a c k g r o u n d - The detection of phenotype determining genes as opposed to disease susceptibility genes requires precise phenotypic characterisation of patients. Peripheral arthropathies in IBD are well recognised and are often classified with the HLA-B27 related spondyloarthropathies. However, previous small studies of HLA status in IBD have failed to show such an association in peripheral disease. We have described a clinical classification which divides these arthropathies into 2 types. Type 1 is a large joint acute oligoarthropathy associated with relapse of the IBD, whilst Type 2 is a persistent polyarthropathy which runs a course independent of the IBD. Both are seronegative and non-erosive. We report the results of HLA studies in these patients. M e t h o d s - DNA from 30 patients in each group underwent HLA genotyping using a PCR based phototyping method. Results from each patient group were compared with 603 Oxfordshire controls (with corrections for multiple comparisons) and then inter-group comparisons were made in the light of existing hypotheses. Results - Type 1 arthropathy was associated with HLA-DR103 in 40% vs 3% (p<0.0001, RR12.1), B35 in 33% vs 15%(p=0.01, RR2.2) and B27 in 27% vs 7% (p=0.001, RR4.0). Type 2 was associated with HLA-B44 in 63% vs 31%(p=0.01, RR2A). These associations were also significant when the two types of arthropathy were compared (DR103 40% vs 0% p=0.0001, B35 33% vs 7% p=0.02, RR5,0, B27 27% vs 3% p=0.03, RR8.0, B44 63% vs 13% p<0.0001, RR12.1). Conclusion - These data suggest that our clinical classification describes immunogenetically distinct entities and that HLA genes are important in determining the clinical phenotype of joint disease in IBD. Type 1 arthropathy is clinically and immunogeneticaily similar to the HLA-B27 related arthritides, whereas Type 2 may have a different aetiopathogenesis. HLA
• G4322 ILEOCAECAL RESECTION IS ASSOCIATED WITH A DECREASED INCIDENCE OF ARTICULAR COMPLICATIONS IN CROHN'S DISEASE. TR Orchard, DP Jewell, Gastroenterology Unit, Radcliffe Infirmary, Oxford UK. B a c k g r o u n d - Studies in HLA-B27 transgenic rats have demonstrated that the bacterial population in the ileocaecal region is important in the initiation and perpetuation of inflammation at distant sites. Diversion of the faecal stream away from this region abrogates the distant inflammation without affecting the colitis. To assess the role of the ileecaecal region in arthropathy in Crohn's disease we compared the incidence of arthropathy in patients pre- and postileocaecal resection (ICR) with patients who had never undergone surgery. Methods - Data on length of follow up, dates and nature of surgery and presence of Type 1 and Type 2 peripheral arthropathy and ankylosing spondylitis were obtained by case note review and patient interview of all patients attending the Oxford inflammatory bowel disease (IBD) clinic. The incidence of arthropathy pre-and post-surgery was compared for patients who had undergone ICR with patients who had never undergone surgery. Results were expressed in number of years of patient follow up per joint complication. Periods of remission induced by surgery were not counted as years of follow up. Results - 217 patients who had never had surgery and 194 who had undergone ICR were identified. Median length of follow-up was 10.7 years and 10.2 years respectively. In the non-surgery group there was 1 articular complication per 76 years of patient follow up compared to 80 years of patient follow up in the surgical patients pre-surgery. Post- ICR the incidence of articular complications was 1 per 656 years of patient follow up. Conclusion - The incidence of articular complications is significantly lower after ICR even after correction for surgically induced remission. One explanation for these findings may be that the change in intestinal flora after ICR reduces the risk of inflammation at extraintestinal sites. G4323
AN ANIMAL MODEL OF H. PYLORI-POSITIVE CHRONIC ACTIVE GASTRITIS USING NEONATAL THYMECTOMIZED MICE. C. Oshima K.Okazaki, K. Uchida, Y.Matsushima, T.Chiba. Department of Gastroenterology, Hepatology, and Endoscopy, Kyoto University, Kyoto, Japan. Aim: The immunological interaction of the host with H.pylori seems to play an important role in the pathophysiology of H.pylori-infected gastric diseases, However, it is still unknown whether autoimmune mechanism may be involved in H.pylori-infected chronic gastritis or not. To clarify it, we have established an animal model using neonatal thymectomized mice having autoimmune gastritis, similar to type A human gastritis. Methods and Materials: Ten Balb/c mice in each group were thymectomized on the 3rd day after birth (3dTx). To confirm whether they have autoimmune gastritis or not, serum levels of autoantibody against parietal cells were measured by ELISA system. At the period of 6-8 weeks old, mice were orally
GASTROENTEROLOGY Vol. 114, No. 4
infected with l0 s of H.pylori and sacrificed after 2, 4, and 6 month. Three groups (normal mice with or without H.pylori, and 3dTx-mice without H.pylori) were served as controls. Antibodies against parietal cells and H.pylori were serially measured. We studied histological findings of the stomach, and the subsets and intracellular cytokines (interferon-'/and IL-4) of infiltrating lymphocytes by immuno-histochemistry and flow cytometic analysis. Gene expressions of interferon-'/and IL-4 were also analyzed by RT-PCR. Results: In each group of H.pylori-infected 3dTx-mice, histological findings showed chronic active gastritis with lymph follicles in the gastric mucosa. Four months after H.pylori infection, formation of germinal center occurred in one of ten mice. Flow cytometric analysis of infiltrating lymphocytes showed 30, 3, and 6% as B cells, CD4, and CD8 positive cells, respectively. Paneth or goblet-like cells, suggesting intestinal metaplasia of the gastric mucosa, were found in some cases. Expression of interferon-'/ was identified in all of H.pylori-infected 3dTx-mice, but IL-4 was equivocal. The population of lymphocytas by flow cytometric analysis, staining intracellular cytokines, were consistent with the data of RT-PCR. On the other hand, infiltrating cells were few in the control mice. Serum levels of autoantibody were not correlated with the severity of gastritis. Conclusion: Autoimmune mechanism and predominant Thl type response may be involved in chronic active gasttitis in H.pylori-infected 3dTx Balb/c mice. G4324
AUTOIMMUNITY INDUCES A SEVERE RESPONSE TO HELICOBACTER FELIS IN NEONATALLY THYMECTOMIZED MICE. C. Oshima. K. Okazaki, Y. Matsushima, T. Chiba Department of Gastroenterology, Hepatology, and Endoscopy, Kyoto University, Kyoto, Japan. Aim: In humans, atrophic gastritis is classified as two types, type A and B. We have reported that neonatally thymectomized (nTx) mice have autoimmune gastritis (AIG), similar to human type A gastritis. On the other hand, it has been suggested that Helicobacter pylori is involved in type B chronic gastritis. Helicobacter fells (H.f) colonizes in the normal Balb/c mouse. In this study, we investigated influence of autoimmunity on pathophysiology, and host immune response to H.felis, using nTx mice. Material and methods: Ten Balb/c mice in each group were thymectomized on the 3rd day after birth. To confirm whether they have AIG or not, serum levels of autoantibody against parietal cells were measured by ELISA system. Group 1 (AIG (+), H.f (+)) and Group 2 (AIG (-), H.f (+)) mice were orally infected with 10s of H. fells at 8 weeks old. They were sacrificed at one and three months, and histopathological findings were compared with those of the control groups, Group3 (AIG (+), H.f (-)) and Group4 (AIG (-), H.f (-)). Antibodies to parietal cells and H.f were serially measured. Results: In Group1, both the corpus and antrai gastric mucosa showed more severe inflamation than that in other three groups. Serum levels of antibodies to parietal cells and H.fin Groupl were higher than those in other three groups. Conclusion: These data suggested that autoimmune mechanism may be involved in more severe gastritis with H.f positive nTx Baib/c mice. G4325
ALTERNATION OF DECAY-ACCELERATING FACTOR EXPRESSION ON GUINEA PIG GASTRIC MUCOSA AFTER ISCHEMIA REPERFUSION. T.Oshima, T.Joh, K.Seno, M.Ikai, M.Sasaki, H.Katanka, Y.Yokoyama, M.Nonaka*, N.Okada*, H.Okada*, T. Tada**, M.Itoh. 1st Dept. Int. Med., *Dept. of Molecular Biology and **Pathology. Nagoya City University Medical School, Nagoya, Japan. The complement regulatory protein, decay-accelerating factor (DAF), protects host tissues from damage due to autologous complement activation, We have previously shown that expression of human gastric mucosai DAF was markedly enhanced relative to the degree of inflammation (Gastroenterol 112: A 1082, 1997). However, expression of DAF in gastric mucosa has not been well investigated using experimental models. We recently identified 6 isoforms of guinea pig (GP) DAF (transmembrane types; TM-a, TM-ab, TM-abc, and glycosylphosphatidylinositol anchored types; GPI-a, GPI-ab, GPI-abc). In this study, we investigated the changes in DAF gene and protein expression following GP gastric injury induced by ischemia reperfusion (I/R). Methods: Male Hartley guinea pigs (250-300g) were subjected to an overnight fast and anesthetized. Gastric ischemia was induced by clamping the left gastric and epiploic arteries for 30 min. Prior to sacrifice, stomachs were removed 0, 6, 12, 24 hr, 3 and 7 days after I/R. Total RNA was extracted from half of each sample collected. Polyadenylated RNA was isolated as mRNA, and the amount of DAF mRNA was evaluated by Northern blot analysis. The other half of each sample was fixed in acetone, and immunohistochemical staining was performed with a specific monoclonal antibody to GP DAF (J. Immunol. 154:6103). Expression of DAF in gastric mucosa was semiquantitatively evaluated by scoring using 3 grades and data was then statistically analyzed, Expression of DAF isoform mRNA was assessed by RT-PCR with isoform specific primers. Results: Mucosai erosion was observed from 6 hr to 3 days after I/R (p<0,05), but was not detected at 7 days. Expression of DAF mRNA was constitutively demonstrated in normal GP gastric mucosa and was most intense 6 hr after I/R, however, it was detected at normal levels at 24 hr, 3 and 7 days. Expression of DAF protein was not significant from 0 to 12 hr after I/R, but it