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P039 Mucosal interleukin-33 levels in patients with moderate to severe ulcerative colitis before and after induction of endoscopic remission by infliximab
Basic Science after the beginning of the study. As control, group Control was administrated with isotype of anti-CTLA-4 in the same way. Results: Compared with group Control, mice in group Mab showed amelioration of colitis with lower disease activity index, MPO activity and histological scores of colonic tissue as well as less shortened length of colon (p < 0.05, respectively) (Figure 1); Th17 cells decreased (p = 0.029) and Treg cells increased in mesenteric lymph node (p = 0.006) (Figure 2). Protein expression of JAK-2, RORgt and IL-17A was reduced but level of STAT-3, phosphorylated STAT-3 and Foxp3 was upregulated (p < 0.05, respectively).
Figure 1. After induction of colitis, disease activity index (DAI) was evaluated (b), H&E stained sections of colon was pictured (a), histological analysis was made (d) and MPO activity was detected (c). Colon length was examined (e and f).
S79 P038 Neuropeptide S: effects on motility, contractility and inflammation in the rat and human gastrointestinal tract M.A. Halim1 *, L. Gillberg1 , U. Karlbom2 , M. Sundbom2 , E. Rosenqvist3 , S. Saudi3 , M. Sj¨ oblom3 , A. Sommansson3 , E. N¨ aslund4 , T. Feldreich1 , D.-L. Webb1 , P.M. Hellstr¨ om1 . 1 Uppsala University, Medical sciences, gastroenterology & hepatology, Uppsala, Sweden, 2 Uppsala University, Surgical sciences, surgery, Uppsala, Sweden, 3 Uppsala University, Neuroscience, Physiology, Uppsala, Sweden, 4 Karolinska Institutet, Clinical sciences, Danderyd hospital, Stockholm, Sweden Background: Neuropeptide S (NPS) is expressed by gastrointestinal (GI) enteroendocrine cells and macrophages in rat and man. Polymorphisms of the NPS receptor are linked to increased risk of inflammatory bowel disease as well as motor and sensory disturbances of the gut, suggesting a role for NPS in GI disorders. Further knowledge of NPS effects on motility and inflammation is needed. Methods: Studies of motility were carried out in rats with electrodes implanted in the small bowel. NPS was infused IV for 60 min and effects on myoelectrical activity were recorded. Motility effects of NPS were further studied as luminal pressure changes in anaesthetized rats where the proximal small intestine with intact blood supply was perfused with saline for motility recordings. Tissue samples were obtained from rats for evaluation of gene expression and protein elaboration of inflammatory biomarkers. Muscle strips of the human stomach, small intestine and colon were used for pharmacological analysis in organ baths of motility responses to NPS. Localization of the NPS receptor was done with fluorescent-protein tagging using Cy3-NPS. Plasma levels of NPS were measured using ELISA in 5 healthy and 14 IBD patients before and after meal. Results: In conscious rats, NPS 1 nmol kg-1min-1 increased irregular spiking, 4 nmol kg-1min-1 reduced spiking and increased the MMC cycle length (P = 0.005). In anesthetized rats, NPS 0.01 1 nmol kg-1min-1 dose-dependently reduced small bowel motility (P < 0.001). In rats, NPS also increased the mRNA expression of iNOS, and CXCL1 and IL-1beta at the protein level. In human, fluorescent protein-tagging showed the NPS receptor primarily to be present in enolase-positive nerve fibers, but some scattered also in muscle tissue. In organ baths, NPS 1 100 nM caused TTX-dependent relaxations of the smooth muscle. We found no detectable levels of NPS in plasma of neither healthy subjects nor IBD patients. Conclusions: NPS has a smooth muscle-relaxing effect in the GI tract and dampens myoelectrical and contractile motor activity of the small bowel. This is achieved primarily through an inhibition of nerve-mediated relaxation of smooth muscle cells. Circulating NPS levels in humans are undetectable in healthy subjects and in active IBD. This implies that NPS exerts its inhibitory function by a paracrine or neurocrine mechanism in the gut. NPS seems also capable of intervening with inflammation by increasing gut cytokines and iNOS expression. P039 Mucosal interleukin-33 levels in patients with moderate to severe ulcerative colitis before and after induction of endoscopic remission by infliximab
Figure 2. The results of CD3+CD8 IL-17A+ and CD4+Foxp3+ cells in mesenteric lymph node examined by flow cytometry was shown (a), and the percentage of them was compared in colitis with or without anti-CTLA-4 treating (b).
Conclusions: Blocking CTLA-4 function with anti-CTLA-4 alleviated DSS-induced colitis in mice through changing the immune responses of Th17-Treg profile, which suggests antiCTLA-4 as a potential therapeutic method for the therapy of ulcerative colitis.
M.D. Gundersen1 *, R. Goll1,2 , G. Haraldsen3 , J.R. Florholmen1,2 . 1 University of Tromsø, The Arctic University of Norway, Research group of Gastroenterology and Nutrition, Institute of Clinical Medicine, Tromsø, Norway, 2 University Hospital of North Norway, Department of Medical Gastroenterology, Tromsø, Norway, 3 Oslo University Hospital, Department of Pathology, Rikshospitalet, Oslo, Norway Background: Interleukin-33 (IL-33) is a known mediator in inflammatory disease and has been shown to be upregulated
S80 in acute lesions of inflammation including ulcerative colitis. Its role is not fully understood, especially its importance in wound and mucosal healing in inflammatory bowel disease [1,2]. Methods: We included 57 patients with moderate to severe ulcerative colitis from the University Hospital of North Norway. Patients were treated with infliximab to clinical and endoscopic remission defined by the ulcerative disease activity index as a score of 2 or less and with an endoscopic sub score of 0 or 1. Mucosal gene expression levels of IL-33 and tumour necrosis factor alpha (TNF-a) levels where determined in colon mucosal biopsies prior to infliximab therapy and at endoscopic remission using real-time PCR. Immunohistochemistry analysis was performed for IL-33 in 10 patients and 10 healthy controls. Results: No differences in IL-33 mucosal levels were found for the whole study population when comparing active disease with endoscopic remission, however a significant reduction of IL-33 was found in a subgroup of 17 patients (30%) who achieved normalisation of TNF-a expression (fold change 0.44, p value = 0.039). Infliximab therapy was stopped in 55 patients following endoscopic remission, at 1 year follow up 26 patients (47%) where still in disease remission. Immunohistochemistry showed IL-33 positive cells in the acute disease phase localised in the lamina propria, endothelial cells and also in basalcryptcells, the latter was not seen in endoscopic remission or in the healthy controls. Conclusions: Induction of endoscopic remission by infliximab in patients with ulcerative colitis significantly reduced the mucosal IL-33 levels only when TNF-a was normalised. Our data support the hypothesis of IL-33 as a pivotal mediator of inflammation in ulcerative colitis and is tightly associated to the mucosal expression of TNF-a. Reference(s) [1] Sponheim J, Pollheimer J, Oslen T et al, (2010), Inflammatory bowel disease-associated interleukin-33 is preferentially expressed in ulceration-associated myofibroblasts, Am J Pathol, 2010; 177(6): 2804 2815. [2] Haraldsen G, Balogh J, Pollhemier J et al, (2009), Interleukin-33 cytokine of dual function or novel alarmin? Trends in Immunology, 2009; 30(5): 227 233. P040 Mucosal T cell profiles in newly diagnosed, untreated inflammatory bowel disease patients C. Horjus1 , M.J. Groenen2 , P.J. Wahab3 *, E. van Lochem4 . Radboud University, Gastroenterology and hepatology, Nijmegen, Netherlands, 2 Rijnstate Hospital, Gastroenterology and hepatology, Arnhem, Netherlands, 3 Rijnstate Hospital, Gastroenterology and hepatology, Nijmegen, Netherlands, 4 Rijnstate Hospital, Medical Microbiology and Immunology, Arnhem, Netherlands 1
Background: Inflammatory bowel disease (IBD) represents a heterogeneous group of disorders associated with imbalances in the intestinal adaptive immune response. In an attempt to explain its diversity in clinical presentation and disease course, we set out to investigate the phenotype of the T cell subpopulations and the related cytokine network in the gastrointestinal mucosa of naïve, untreated IBD patients. Methods: Mucosal biopsies from duodenum, ileum and colon mucosa of newly diagnosed, untreated patients with Crohn’s disease (CD, n = 51), Ulcerative Colitis (UC, n = 14) and controls (n = 14) were obtained. Flow cytometry was used to analyse the expression of maturation and activation markers on gutinfiltrating T cells. The remaining mucosal lymphocytes were stimulated overnight with PMA and ionomycin where after the supernatants were analyzed for proinflammatory cytokines by cytometric bead array. The endoscopic disease activity was scored by using the Simple Endoscopy Score in CD and the endoscopic Mayo score in UC.
Poster presentations Results: Irrespective of the IBD phenotype, the frequency of B cells, CD4+CD103 and FoxP3+/CD25high T cells was much higher in the colon/ileum than in duodenum. We identified four mucosal T cell profiles according to the expression of maturation and activation markers CD45RA+and CD27+: profile A: high percentages (>40%) CD45RA+CD27+ T cells; profile B: high percentages (>40%) CD45RA CD27+ T cells; profile C: high percentages (>40%) CD45RA CD27 T cells, and profile D: equal relative numbers of these before mentioned subpopulations. Within the ileum/colon mucosa, profile D was only seen in controls while profile A was only seen in patients, both CD and UC. However, profile A was associated with upper gastrointestinal location and perianal disease in CD. Surprisingly, profile A expressed a significantly higher amount of TNFalfa (30% vs. 17%, p = 0.004 in CD; 32% vs. 19%, p 0.118 in UC) and a lower amount of IFN-gamma (47% vs. 66%, p = 0.003 in CD; 47% vs. 63%, p = 0.340 in UC) than profile C. There was no correlation between these profiles and the endoscopic disease activity. Conclusions: Our results suggest that there are different mucosal T cell maturation profiles with distinct cytokine responses, in the very early phase of untreated IBD. This could in part explain the heterogeneity of the clinical presentation and response to immunomodulating therapies in IBD. P041 Modulation of inflammatory processes in mouse colitis models and gastrointestinal acute radiation syndrome with AVX-470m, an oral polyclonal anti-TNF antibody D. Hartman *, K. Bhol, B. Lemos, E. Erlich, D. Keane, D. Tracey, B. Fox. Avaxia Biologics, Inc, Research, Lexington, United States Background: Improved therapies for inflammatory bowel disease (IBD) require animal models that elucidate underlying mechanisms of disease. DSS-induced colitis is a well-established mouse model of the dysregulated inflammatory response in IBD. Acute radiation syndrome (ARS) also has devastating consequences in the gastrointestinal (GI) tract driven in part by cytokine-mediated inflammation, which closely resembles the pathophysiology of IBD. Tumor Necrosis Factor (TNF) is strongly implicated in both IBD and GI ARS. In this study AVX-470m, a bovine polyclonal neutralizing antibody for murine TNF, is used to characterize the pathogenesis of GI damage in models of GI ARS and IBD. Methods: In C57BL/6 mice, colitis was induced by including 3% DSS in drinking water for five days. GI ARS was induced in C57BL/6 mice by irradiation with 15.9 Gy (~LD80/30) from a 60-Cobalt gamma radiation source, with partial bone marrow shielding. AVX-470m (10 mg/day) or saline was administered BID by oral gavage for up to 14 days. Protein expression of inflammatory and immune cell markers was analyzed by immunohistochemistry in paraffin embedded, formalin fixed tissue sections, and relative mRNA expression measured by RTPCR. Results: In both the colitis and GI ARS models, mRNA and protein levels of TNF were significantly increased, in colon and jejunum tissue, respectively. IL-1-beta, IL-6, MMP9, ICAM-1, and TNFR2 mRNA levels were increased in both models; increased IL12p40 was detectable only in the colitis model. MPO protein expression was also increased in both models. CD68 and CD3 protein were increased in the colitis model, but were not detected in GI ARS, perhaps due to cell destruction by ionizing radiation. Treatment with AVX-470m in both colitis and GI ARS models resulted in a significant decrease in TNF mRNA and protein expression in colon and jejunum tissues, respectively. AVX-470m decreased MPO protein expression, and reduced MMP9 mRNA expression, in both colitis and GI ARS models.
Figure 1. After induction of colitis, disease activity index (DAI) was evaluated (b), H&E stained sections of colon was pictured (a), histological analysis was made (d) and MPO activity was detected (c). Colon length was examined (e and f).
S79 P038 Neuropeptide S: effects on motility, contractility and inflammation in the rat and human gastrointestinal tract M.A. Halim1 *, L. Gillberg1 , U. Karlbom2 , M. Sundbom2 , E. Rosenqvist3 , S. Saudi3 , M. Sj¨ oblom3 , A. Sommansson3 , E. N¨ aslund4 , T. Feldreich1 , D.-L. Webb1 , P.M. Hellstr¨ om1 . 1 Uppsala University, Medical sciences, gastroenterology & hepatology, Uppsala, Sweden, 2 Uppsala University, Surgical sciences, surgery, Uppsala, Sweden, 3 Uppsala University, Neuroscience, Physiology, Uppsala, Sweden, 4 Karolinska Institutet, Clinical sciences, Danderyd hospital, Stockholm, Sweden Background: Neuropeptide S (NPS) is expressed by gastrointestinal (GI) enteroendocrine cells and macrophages in rat and man. Polymorphisms of the NPS receptor are linked to increased risk of inflammatory bowel disease as well as motor and sensory disturbances of the gut, suggesting a role for NPS in GI disorders. Further knowledge of NPS effects on motility and inflammation is needed. Methods: Studies of motility were carried out in rats with electrodes implanted in the small bowel. NPS was infused IV for 60 min and effects on myoelectrical activity were recorded. Motility effects of NPS were further studied as luminal pressure changes in anaesthetized rats where the proximal small intestine with intact blood supply was perfused with saline for motility recordings. Tissue samples were obtained from rats for evaluation of gene expression and protein elaboration of inflammatory biomarkers. Muscle strips of the human stomach, small intestine and colon were used for pharmacological analysis in organ baths of motility responses to NPS. Localization of the NPS receptor was done with fluorescent-protein tagging using Cy3-NPS. Plasma levels of NPS were measured using ELISA in 5 healthy and 14 IBD patients before and after meal. Results: In conscious rats, NPS 1 nmol kg-1min-1 increased irregular spiking, 4 nmol kg-1min-1 reduced spiking and increased the MMC cycle length (P = 0.005). In anesthetized rats, NPS 0.01 1 nmol kg-1min-1 dose-dependently reduced small bowel motility (P < 0.001). In rats, NPS also increased the mRNA expression of iNOS, and CXCL1 and IL-1beta at the protein level. In human, fluorescent protein-tagging showed the NPS receptor primarily to be present in enolase-positive nerve fibers, but some scattered also in muscle tissue. In organ baths, NPS 1 100 nM caused TTX-dependent relaxations of the smooth muscle. We found no detectable levels of NPS in plasma of neither healthy subjects nor IBD patients. Conclusions: NPS has a smooth muscle-relaxing effect in the GI tract and dampens myoelectrical and contractile motor activity of the small bowel. This is achieved primarily through an inhibition of nerve-mediated relaxation of smooth muscle cells. Circulating NPS levels in humans are undetectable in healthy subjects and in active IBD. This implies that NPS exerts its inhibitory function by a paracrine or neurocrine mechanism in the gut. NPS seems also capable of intervening with inflammation by increasing gut cytokines and iNOS expression. P039 Mucosal interleukin-33 levels in patients with moderate to severe ulcerative colitis before and after induction of endoscopic remission by infliximab
Figure 2. The results of CD3+CD8 IL-17A+ and CD4+Foxp3+ cells in mesenteric lymph node examined by flow cytometry was shown (a), and the percentage of them was compared in colitis with or without anti-CTLA-4 treating (b).
Conclusions: Blocking CTLA-4 function with anti-CTLA-4 alleviated DSS-induced colitis in mice through changing the immune responses of Th17-Treg profile, which suggests antiCTLA-4 as a potential therapeutic method for the therapy of ulcerative colitis.
M.D. Gundersen1 *, R. Goll1,2 , G. Haraldsen3 , J.R. Florholmen1,2 . 1 University of Tromsø, The Arctic University of Norway, Research group of Gastroenterology and Nutrition, Institute of Clinical Medicine, Tromsø, Norway, 2 University Hospital of North Norway, Department of Medical Gastroenterology, Tromsø, Norway, 3 Oslo University Hospital, Department of Pathology, Rikshospitalet, Oslo, Norway Background: Interleukin-33 (IL-33) is a known mediator in inflammatory disease and has been shown to be upregulated
S80 in acute lesions of inflammation including ulcerative colitis. Its role is not fully understood, especially its importance in wound and mucosal healing in inflammatory bowel disease [1,2]. Methods: We included 57 patients with moderate to severe ulcerative colitis from the University Hospital of North Norway. Patients were treated with infliximab to clinical and endoscopic remission defined by the ulcerative disease activity index as a score of 2 or less and with an endoscopic sub score of 0 or 1. Mucosal gene expression levels of IL-33 and tumour necrosis factor alpha (TNF-a) levels where determined in colon mucosal biopsies prior to infliximab therapy and at endoscopic remission using real-time PCR. Immunohistochemistry analysis was performed for IL-33 in 10 patients and 10 healthy controls. Results: No differences in IL-33 mucosal levels were found for the whole study population when comparing active disease with endoscopic remission, however a significant reduction of IL-33 was found in a subgroup of 17 patients (30%) who achieved normalisation of TNF-a expression (fold change 0.44, p value = 0.039). Infliximab therapy was stopped in 55 patients following endoscopic remission, at 1 year follow up 26 patients (47%) where still in disease remission. Immunohistochemistry showed IL-33 positive cells in the acute disease phase localised in the lamina propria, endothelial cells and also in basalcryptcells, the latter was not seen in endoscopic remission or in the healthy controls. Conclusions: Induction of endoscopic remission by infliximab in patients with ulcerative colitis significantly reduced the mucosal IL-33 levels only when TNF-a was normalised. Our data support the hypothesis of IL-33 as a pivotal mediator of inflammation in ulcerative colitis and is tightly associated to the mucosal expression of TNF-a. Reference(s) [1] Sponheim J, Pollheimer J, Oslen T et al, (2010), Inflammatory bowel disease-associated interleukin-33 is preferentially expressed in ulceration-associated myofibroblasts, Am J Pathol, 2010; 177(6): 2804 2815. [2] Haraldsen G, Balogh J, Pollhemier J et al, (2009), Interleukin-33 cytokine of dual function or novel alarmin? Trends in Immunology, 2009; 30(5): 227 233. P040 Mucosal T cell profiles in newly diagnosed, untreated inflammatory bowel disease patients C. Horjus1 , M.J. Groenen2 , P.J. Wahab3 *, E. van Lochem4 . Radboud University, Gastroenterology and hepatology, Nijmegen, Netherlands, 2 Rijnstate Hospital, Gastroenterology and hepatology, Arnhem, Netherlands, 3 Rijnstate Hospital, Gastroenterology and hepatology, Nijmegen, Netherlands, 4 Rijnstate Hospital, Medical Microbiology and Immunology, Arnhem, Netherlands 1
Background: Inflammatory bowel disease (IBD) represents a heterogeneous group of disorders associated with imbalances in the intestinal adaptive immune response. In an attempt to explain its diversity in clinical presentation and disease course, we set out to investigate the phenotype of the T cell subpopulations and the related cytokine network in the gastrointestinal mucosa of naïve, untreated IBD patients. Methods: Mucosal biopsies from duodenum, ileum and colon mucosa of newly diagnosed, untreated patients with Crohn’s disease (CD, n = 51), Ulcerative Colitis (UC, n = 14) and controls (n = 14) were obtained. Flow cytometry was used to analyse the expression of maturation and activation markers on gutinfiltrating T cells. The remaining mucosal lymphocytes were stimulated overnight with PMA and ionomycin where after the supernatants were analyzed for proinflammatory cytokines by cytometric bead array. The endoscopic disease activity was scored by using the Simple Endoscopy Score in CD and the endoscopic Mayo score in UC.
Poster presentations Results: Irrespective of the IBD phenotype, the frequency of B cells, CD4+CD103 and FoxP3+/CD25high T cells was much higher in the colon/ileum than in duodenum. We identified four mucosal T cell profiles according to the expression of maturation and activation markers CD45RA+and CD27+: profile A: high percentages (>40%) CD45RA+CD27+ T cells; profile B: high percentages (>40%) CD45RA CD27+ T cells; profile C: high percentages (>40%) CD45RA CD27 T cells, and profile D: equal relative numbers of these before mentioned subpopulations. Within the ileum/colon mucosa, profile D was only seen in controls while profile A was only seen in patients, both CD and UC. However, profile A was associated with upper gastrointestinal location and perianal disease in CD. Surprisingly, profile A expressed a significantly higher amount of TNFalfa (30% vs. 17%, p = 0.004 in CD; 32% vs. 19%, p 0.118 in UC) and a lower amount of IFN-gamma (47% vs. 66%, p = 0.003 in CD; 47% vs. 63%, p = 0.340 in UC) than profile C. There was no correlation between these profiles and the endoscopic disease activity. Conclusions: Our results suggest that there are different mucosal T cell maturation profiles with distinct cytokine responses, in the very early phase of untreated IBD. This could in part explain the heterogeneity of the clinical presentation and response to immunomodulating therapies in IBD. P041 Modulation of inflammatory processes in mouse colitis models and gastrointestinal acute radiation syndrome with AVX-470m, an oral polyclonal anti-TNF antibody D. Hartman *, K. Bhol, B. Lemos, E. Erlich, D. Keane, D. Tracey, B. Fox. Avaxia Biologics, Inc, Research, Lexington, United States Background: Improved therapies for inflammatory bowel disease (IBD) require animal models that elucidate underlying mechanisms of disease. DSS-induced colitis is a well-established mouse model of the dysregulated inflammatory response in IBD. Acute radiation syndrome (ARS) also has devastating consequences in the gastrointestinal (GI) tract driven in part by cytokine-mediated inflammation, which closely resembles the pathophysiology of IBD. Tumor Necrosis Factor (TNF) is strongly implicated in both IBD and GI ARS. In this study AVX-470m, a bovine polyclonal neutralizing antibody for murine TNF, is used to characterize the pathogenesis of GI damage in models of GI ARS and IBD. Methods: In C57BL/6 mice, colitis was induced by including 3% DSS in drinking water for five days. GI ARS was induced in C57BL/6 mice by irradiation with 15.9 Gy (~LD80/30) from a 60-Cobalt gamma radiation source, with partial bone marrow shielding. AVX-470m (10 mg/day) or saline was administered BID by oral gavage for up to 14 days. Protein expression of inflammatory and immune cell markers was analyzed by immunohistochemistry in paraffin embedded, formalin fixed tissue sections, and relative mRNA expression measured by RTPCR. Results: In both the colitis and GI ARS models, mRNA and protein levels of TNF were significantly increased, in colon and jejunum tissue, respectively. IL-1-beta, IL-6, MMP9, ICAM-1, and TNFR2 mRNA levels were increased in both models; increased IL12p40 was detectable only in the colitis model. MPO protein expression was also increased in both models. CD68 and CD3 protein were increased in the colitis model, but were not detected in GI ARS, perhaps due to cell destruction by ionizing radiation. Treatment with AVX-470m in both colitis and GI ARS models resulted in a significant decrease in TNF mRNA and protein expression in colon and jejunum tissues, respectively. AVX-470m decreased MPO protein expression, and reduced MMP9 mRNA expression, in both colitis and GI ARS models.