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P046 Mucosal immune activation in diarrhea-predominant irritable bowel syndrome: comparison with health controls and ulcerative colitis
S28 were variables between normal colon mucosa and inflammatory mucosa of UC patients. Conclusions: We can obtain the distribution image throughout entire depths in the intact live colon tissue of ulcerative colitis patients. This method using multiphoton microscope maybe a new research tool to understand the role of H2S in disease progression and healing process of inflammation in ulcerative colitis patients. P045 Mycobacterium avium subsp. paratuberculosis survival in macrophages from inflammatory bowel disease patients N. Nazareth1 , F. Magro2 *, R. Appelberg3 , F. Vilas-Boas2 , G. Macedo2 , A. Sarmento1 . 1 University Fernando Pessoa, Faculty of Health Sciences, Porto, Portugal, 2 Hospital de S˜ ao Jo˜ ao, Gastroenterology Department, Porto, Portugal, 3 Institute for Molecular and Cell Biology (IBMC), Microbiology and Immunology of Infection, Porto, Portugal Background: Mycobacterium avium subsp. paratuberculosis (MAP) has long been implicated in Crohn’s disease (CD). Increased MAP prevalence was detected in CD patients, suggesting defective handling of MAP. We studied MAP survival in macrophage cultures from healthy controls (HC) and inflammatory bowel disease (IBD) patients. Co-localization of MAP with LAMP-1 was used to evaluate the nature of intracellular MAP-containing vacuoles. Methods: 21 HC, 29 CD patients, 11 UC patients and also 22 CD patients and 22 UC patients undergoing infliximab treatment (anti-TNF-a; CD-IFX and UC-IFX), were enrolled in this study. Peripheral blood mononuclear cells were isolated using Histopaque density gradient (SIGMA). The monocyte fraction was enriched by adherence and cells were cultured for 7 days to allow differentiation into macrophages. Cultures were infected with MAP and bacterial growth was evaluated by CFU counts at infection day 7 (T7d) and day 0 (3 hours postinfection; T3h). Pre-16Sr RNA/DNA ratio was also determined at T3h and T7d, in order to confirm CFU results. For LAMP-1 co-localization studies, macrophages were infected for 24h with Syto BC-labelled MAP, fixed with 4% paraformaldehyde, permeabilized and stained with anti-human LAMP-1 antibody. Results were obtained by confocal microscopy followed by image analysis using Image J co-localization plug-in. Results: In macrophages MAP showed constant CFU counts over 7 days. However, evaluation of the pre-16Sr RNA/DNA ratio indicated that macrophages from HC and UC patients, but not macrophages from CD patients, were able to eliminate MAP organisms shortly after infection (T3h). IFX treatment resulted in further decreased MAP killing by CD or UC macrophages. Macrophages from CD patients exhibited higher co-localization of MAP with LAMP-1, as compared to macrophages from HC. Treatment of CD or UC patients with IFX resulted in decreased MAP co-localization with LAMP-1. Conclusions: Although MAP showed nearly constant CFU counts in macrophage cultures, results from pre-16Sr RNA/DNA ratio suggested that CD macrophages had decreased ability to eliminate MAP. The discrepancy observed between CFU and RNA/DNA ratio results might be due to the effect of clumps on MAP culturability. IFX treatment decreased MAP elimination by macrophages, suggesting a deleterious effect of IFX in MAPcolonized patients. Co-localization of MAP with LAMP-1-bearing vesicles did not correlate with MAP growth or elimination by macrophages.
Poster presentations P046 Mucosal immune activation in diarrhea-predominant irritable bowel syndrome: comparison with health controls and ulcerative colitis C.H. Choi1 *, J.Y. Ahn1 , K.H. Lee1 , J.W. Kim1 , S.K. Chang1 . 1 Chung-Ang University College of Medicine, Internal Medicine, Seoul, South Korea Background: Mucosal immune activation is suggested to have a role in the pathogenesis of irritable bowel syndrome (IBS), especially in diarrheal type. We tried to evaluate mast cell and T cell activation in colonic mucosa of diarrhea-predominant IBS (D-IBS) patients in comparison with healthy controls (HCs) and ulcerative colitis (UC). Methods: Between November 2007 and May 2010, 83 patients with D-IBS satisfying the Rome III criteria, 49 with UC in remission (UCR, n = 28) or mild activity (UCMA, n = 21), and 25 HCs were recruited. In all cases, biopsies were taken from each of ascending colon, transverse colon, descending colon, sigmoid colon and rectum by colonoscopy, and questionnaires, including symptoms and health-related quality of life (HRQOL) were checked. The numbers of mast cells per one high power field and intraepithelial lymphocytes (IELs) and lamina proprial lymphocytes (LPLs) per 300 cells were counted and analyzed by immunohistochemistry with anti-human mast cell tryptase and anti-CD3 antibodies. The relationship between the immune cell counts and symptoms was analyzed in D-IBS group. Results: Mean values of mucosal mast cells, IELs and LPLs of all colonic segments were significantly increased in the D-IBS (32.4±15.2, 20.0±9.3 and 31.4±16.4, respectively), UCR (28.2±15.2, 23.0±10.9 and 29.9±17.3, respectively) and UCMA (38.2±16.6, 31.2±10.9 and 51.3±27.4, respectively) patients compared to HCs (14.8±6.7, 10.2±8.1 and 19.9±8.7, respectively) (all P values <0.001). The mucosal mast cell and T cell counts were similar between D-IBS and UCR except a few segments of colon. The IELs and LPLs counts were significantly higher in UCMA than in all other patient groups throughout all colonic segments, especially in lamina propria of rectum. Most scores of abdominal symptoms and HRQOL in D-IBS were significantly higher than those in HCs, and similar with UC. However, the symptom severities were not correlated with the immune cell counts in D-IBS patients. Conclusions: Colonic mucosal immune cells in D-IBS were increased significantly compared to HCs, and similar with those in UCR. The symptoms in D-IBS and UCR might be originated from low grade inflammation in colonic mucosa. P047 Metformin inhibits NF-úB signaling in intestinal epithelial cells, and ameliorates experimental colitis and colitis-associated colon cancer in mice S.-J. Koh1 *, J.M. Kim2 , J.P. Im3 , C. Lee3 , H.C. Jung3 , J.S. Kim3 . 1 Seoul National University Boramae Hospital, Seoul Korea, Internal Medicine, Seoul, South Korea, 2 Hanyang University College of Medicine, Seoul, South Korea, 3 Seoul National University College of Medicine, Internal Medicine, Seoul, South Korea Background: Metformin, a hypoglycemic agent, is known to demonstrate anti-inflammatory and anti-cancer activity. However, little information is available in the effect of metformin on intestinal inflammation and colitis-associated colon cancer. Methods: COLO 205 intestinal epithelial cells (IECs) were pretreated with metformin and then stimulated with TNF-a. IL-8 transcriptional activity and expression was determined by luciferase reporter assay and real-time RT-PCR/ELISA. IúBa phosphorylation/degradation and DNA binding activity of NFúB were evaluated by Western blot analysis and electrophretic mobility shift assay (EMSA), respectively. In the acute colitis model, mice were given 4% dextran sulfate sodium (DSS)
Poster presentations P046 Mucosal immune activation in diarrhea-predominant irritable bowel syndrome: comparison with health controls and ulcerative colitis C.H. Choi1 *, J.Y. Ahn1 , K.H. Lee1 , J.W. Kim1 , S.K. Chang1 . 1 Chung-Ang University College of Medicine, Internal Medicine, Seoul, South Korea Background: Mucosal immune activation is suggested to have a role in the pathogenesis of irritable bowel syndrome (IBS), especially in diarrheal type. We tried to evaluate mast cell and T cell activation in colonic mucosa of diarrhea-predominant IBS (D-IBS) patients in comparison with healthy controls (HCs) and ulcerative colitis (UC). Methods: Between November 2007 and May 2010, 83 patients with D-IBS satisfying the Rome III criteria, 49 with UC in remission (UCR, n = 28) or mild activity (UCMA, n = 21), and 25 HCs were recruited. In all cases, biopsies were taken from each of ascending colon, transverse colon, descending colon, sigmoid colon and rectum by colonoscopy, and questionnaires, including symptoms and health-related quality of life (HRQOL) were checked. The numbers of mast cells per one high power field and intraepithelial lymphocytes (IELs) and lamina proprial lymphocytes (LPLs) per 300 cells were counted and analyzed by immunohistochemistry with anti-human mast cell tryptase and anti-CD3 antibodies. The relationship between the immune cell counts and symptoms was analyzed in D-IBS group. Results: Mean values of mucosal mast cells, IELs and LPLs of all colonic segments were significantly increased in the D-IBS (32.4±15.2, 20.0±9.3 and 31.4±16.4, respectively), UCR (28.2±15.2, 23.0±10.9 and 29.9±17.3, respectively) and UCMA (38.2±16.6, 31.2±10.9 and 51.3±27.4, respectively) patients compared to HCs (14.8±6.7, 10.2±8.1 and 19.9±8.7, respectively) (all P values <0.001). The mucosal mast cell and T cell counts were similar between D-IBS and UCR except a few segments of colon. The IELs and LPLs counts were significantly higher in UCMA than in all other patient groups throughout all colonic segments, especially in lamina propria of rectum. Most scores of abdominal symptoms and HRQOL in D-IBS were significantly higher than those in HCs, and similar with UC. However, the symptom severities were not correlated with the immune cell counts in D-IBS patients. Conclusions: Colonic mucosal immune cells in D-IBS were increased significantly compared to HCs, and similar with those in UCR. The symptoms in D-IBS and UCR might be originated from low grade inflammation in colonic mucosa. P047 Metformin inhibits NF-úB signaling in intestinal epithelial cells, and ameliorates experimental colitis and colitis-associated colon cancer in mice S.-J. Koh1 *, J.M. Kim2 , J.P. Im3 , C. Lee3 , H.C. Jung3 , J.S. Kim3 . 1 Seoul National University Boramae Hospital, Seoul Korea, Internal Medicine, Seoul, South Korea, 2 Hanyang University College of Medicine, Seoul, South Korea, 3 Seoul National University College of Medicine, Internal Medicine, Seoul, South Korea Background: Metformin, a hypoglycemic agent, is known to demonstrate anti-inflammatory and anti-cancer activity. However, little information is available in the effect of metformin on intestinal inflammation and colitis-associated colon cancer. Methods: COLO 205 intestinal epithelial cells (IECs) were pretreated with metformin and then stimulated with TNF-a. IL-8 transcriptional activity and expression was determined by luciferase reporter assay and real-time RT-PCR/ELISA. IúBa phosphorylation/degradation and DNA binding activity of NFúB were evaluated by Western blot analysis and electrophretic mobility shift assay (EMSA), respectively. In the acute colitis model, mice were given 4% dextran sulfate sodium (DSS)