A426 P0794 EFFECTS OF HUMAN AMNION-DERIVED MESENCHYMAL STEM CELL TRANSPLANTATION AND CONDITIONED MEDIUM ENEMA IN RATS WITH TRINITROBENZENE SULFONIC ACID-INDUCED COLITIS S. Miyamoto1, S. Ohnishi2, I. Tsuchiya3, R. Ohnishi2, H. Hosono2, H. Takeda4, N. Sakamoto2 1 Gastroenterology And Hepatology, Hokkaido University Graduate School of Medicine, Sapporo/Japan 2 Gastroenterology And Hepatology, Hokkaido University Graduate School of Medicine, Sapporo/Japan 3 Laboratory Of Pathophysiology And Therapeutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo/Japan 4 Faculty Of Pharmaceutical Sciences, Hokkaido University, Sapporo/Japan Contact E-mail Address: shuichi0210miyamoto@yahoo.co.jp Introduction: Mesenchymal stem cells (MSCs) have been reported to be a valuable cell source in regenerative medicine, and bone marrow represents a major source of MSCs. Recently, it has been shown that MSC can be easily isolated from human amnion, which is generally discarded after delivery, and a large amount of cells can be obtained. We have previously reported that intravenous administration of human amnion-derived MSCs (hAMSCs) provided significant improvement in rats with colitis induced by dextran sulfate sodium or girradiation. In addition, conditioned medium (CM) obtained from MSCs contains a variety of humoral factors to improve damaged tissues. In this study, we investigated the effects of hAMSCs and CM in rats with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Aims & Methods: hAMSCs were isolated and expanded by digestion with collagenase, followed by culturing in uncoated plastic dishes. CM was collected by culturing subconfluent hAMSCs with serum-free MEMa for 48 hrs. CM gel was prepared by mixing CM with 2% carboxymethyl cellulose. On day 0, 200 l of TNBS (15 mg/rat) in 30% ethanol was intrarectally administered to the ten-week-old male Sprague-Dawley (SD) rats. One-million hAMSCs were intravenously administered 3 hrs after TNBS treatment, and rats were sacrificed on day 7 for histological examination and quantitative PCR. In another experiment, 400 l of CM gel was intrarectally administered 3 hrs after TNBS treatment, and day 1 and day 2. Results: hAMSC transplantation and CM gel enema significantly improved the endoscopical score, and tended to improve the histological score. Quantitative PCR demonstrated that the expression levels of TNF-, CXCL1 and CCL2 tended to be decreased by hAMSC transplantation and CM gel enema. Infiltrations of CD68-positive macrophages and myeloperoxidase-positive neutrophils were significantly decreased by hAMSC transplantation and CM gel enema. Conclusion: Transplantation of hAMSCs and CM gel enema provided significant improvement in rats with colitis induced by TNBS. hAMSCs or CM from hAMSCs may be new therapeutic strategies inflammatory bowel disease. Disclosure of Interest: All authors have declared no conflicts of interest. References 1. Onishi R, et al. Human amnion-derived mesenchymal stem cell transplantation ameliorates dextran sulfate sodium induced severe colitis in rats. Cell Transplantation 2015; 24(12): 2601–14. 2. Ono M, et al. Effects of human amnion-derived mesenchymal stromal cell transplantation in rats with radiation proctitis. Cytotherapy 2015; 17(11): 1545–59. P0795 TERTIARY LYMPHOID ORGANS IN GUT MUCOSA OF NEWLY DIAGNOSED, UNTREATED INFLAMMATORY BOWEL DISEASE PATIENTS C. Smids1, C. S. Horjus Talabur Horje1, J. W.r. Meijer2, M. J. Groenen1, M. K. Rijnders3, E. G. Van Lochem4, P. J. Wahab1 1 Gastroenterology And Hepatology, Rijnstate Hospital Arnhem, Arnhem/ Netherlands 2 Pathology, Rijnstate Hospital, Arnhem/Netherlands 3 Pathology, Rijnstate Hospital Arnhem, Arnhem/Netherlands 4 Microbiology And Immunology, Rijnstate Hospital Arnhem, Arnhem/ Netherlands Contact E-mail Address: csmids@rijnstate.nl Introduction: There is recent evidence of increased naı̈ve and central memory T lymphocytes (TN and TCM) mucosal infiltration of the inflamed gut in Inflammatory Bowel Disease (IBD), while TN and TCM cells were thought to migrate exclusively to secondary lymphoid organs (SLOs). Ectopic formation of tertiary lymphoid organs (TLOs) containing peripheral lymph node adressin (PNAd)þ high endothelial venules (HEVs) might explain the homing of these lymphocytes to the gut, as PNAd is the ligand of L-selectin which is expressed on TN and TCM lymphocytes. Aims & Methods: The aim of this study was to investigate the presence of PNAd expressing HEVs and TLOs in the inflamed intestinal mucosa of newly diagnosed, untreated, IBD patients in relation to the presence of TN and TCM lymphocytes. Thirty-nine newly diagnosed, untreated IBD patients and eight healthy controls were prospectively included. Intestinal biopsy samples were analysed by immunohistochemistry for blood vessels (CD31) and PNAd expression (MECA-79), the density of MECA-79þ vessels was calculated and the presence of lymphoid follicles was assessed. Different lymphocytes subsets in the tissue samples were identified by flowcytometric immunophenotyping, including TN (CD45RAþCD27þ), TCM (CD45RA-CD27þ) and effector memory T cells (CD45RA-CD27-). United European Gastroenterology Journal 3(5S) Results: A statistically significant higher number of extra-follicular PNAdþ vessels were found in the inflamed colon of patients with ulcerative colitis (median density of 3.05 PNAdþ vessels/mm2 (IQR 0–6.39)) and ileum of patients with Crohn’s disease (median density of 1.40 PNAdþ vessels/mm2 (IQR 0–4.34)) compared with healthy controls (median density of colon: 0 PNADþ vessels/mm2 (IQR 0–0, p ¼ 0.033) and ileum: 0 PNAdþ vessels/mm2 (IQR 0–0.50, p ¼ 0.033)). The heterogeneity of extra-follicular PNAdþ vessels in IBD patients allowed classification in two different groups: HEVhigh and HEVlow. A high density of PNAdþ HEV-like vessels was associated with increased numbers of TN and TCM in the inflamed gut mucosa (median 87% (IQR 82–93%) of total T cell population), compared with the inflamed mucosa of patients from the HEVlow group (58% (IQR 38–81%) p ¼ 0.003). The number of colonic follicles was higher in HEVhigh patients (median 0.54/mm2 (IQR 0.28–0.84)) when compared with HEVlow patients (median 0.25/mm2 (IQR 0.08–0.45) p ¼ 0.031) and controls (0.31/mm2 (IQR 0.23–0.45) p ¼ 0.043). Conclusion: For the first time, evidence has been delivered of extra-follicular HEVlike vessels and TLOs, strongly associated with TN and TCM cell mucosal infiltration, in a subgroup of newly diagnosed IBD patients. Different T cell migration phenotypes based on TLO formation in the early phase of IBD might allow riskstratification of patients and enable more effective, individualized treatment. Disclosure of Interest: All authors have declared no conflicts of interest. P0796 TOLL-LIKE RECEPTOR SEROTONERGIC SYSTEM 9 MODIFIES INTESTINAL E. Layunta1, E. Latorre2, R. Forcén1, L. Grasa1, J. Pardo3, M. Castro4, M.A. Plaza1, M.P. Arruebo1, J.E. Mesonero1 1 Pharmacology And Physiology Department, University of Zaragoza, Zaragoza/ Spain 2 Institute Of Biomedical & Clinical Science, University of Exeter Medical School, Exeter/United Kingdom 3 Biochemistry And Molecular Biology, University of Zaragoza, Zaragoza/Spain 4 Instituto Agroalimentario De Aragón IaUniversity of Zaragoza, Zaragoza/ Spain Contact E-mail Address: elena.layunta@unizar.es Introduction: Toll-like receptor 9 (TLR9) is expressed in intestinal epithelial cells, which recognize microbiota developing different responses 1. Several studies have shown that TLR9 seems to be involved in Inflammatory Bowel Diseases (IBD) due to an inappropriate defensive response against microorganisms 2. Moreover, intestinal serotonergic system is also altered in IBD, where extracellular serotonin (5–HT) levels are increased 3. 5-HT bioavailability is mainly regulated by the serotonin transporter (SERT), expressed in enterocytes 4. Aims & Methods: The aim of the present study was to analyse whether TLR9 activation affects SERT expression and activity, and expression of other elements from the serotonergic system (TPH1, TPH2 and 5-HT receptors). Human enterocyte-like Caco-2 cells, and ileum and colon from TLR9-/- mice and Dextran Sulphate Sodium (DSS) mouse colitis model were used as experimental models. mRNA expression was determined by RT-qPCR, and protein expression by western blot. Results: TLR9 activation in Caco-2 cells decreased SERT mRNA and protein expression. TLR9 activation also reduced SERT activity by different intracellular pathways, depending on activation period. Indeed, TLR9 long-time activation altered 5-HT uptake through ERK pathway, whereas short-time activation modified SERT by p38/MAPK pathway. Moreover, 5-HT addition to culture media increased TLR9 protein expression in the brush-border membrane of Caco-2 cells. In TLR9-/- mice were observed different expression patterns. SERT was not modified in ileum, but increased in colon. TPH1 was increased in ileum, and TPH2, in colon. Regarding 5-HT receptors, 5HT2A, 5-HT2B, and 5-HT3 were increased in ileum; however, 5-HT1A, 5-HT2A and 5-HT4 were increased in colon. In both ileum and colon of DSS mice, TLR9 and 5-HT7 mRNA expression were increased, whereas SERT expression was diminished. Conclusion: Our results suggest that TLR9 modulates intestinal serotonergic system. Indeed, TLR9 activation decreases SERT function and expression by different pathways. In part, this is corroborated by the decreased expression of SERT in colon of TLR9 knockout mice and altered 5-HT receptors expression. Finally, SERT and TLR9 are inversely disturbed in DSS mice colitis model. Disclosure of Interest: All authors have declared no conflicts of interest. References 1. McDermott AJ and Huffnagle GB. The microbiome and regulation of mucosal immunity. Immunology 2014 May; 142(1): 24–31. doi: 10.1111/ imm.12231. 2. Sánchez-Muñoz F, Fonseca-Camarillo G, Villeda-Ramı́rez, MAMirandaPérez E, Mendivil EJ, Barreto-Zúñiga R, Uribe M, Bojalil R, Domı́nguezLópez A and Yamamoto-Furusho JK. Transcript levels of Toll-Like Receptors 5, 8 and 9 correlate with inflammatory activity in Ulcerative Colitis. BMC Gastroenterol 2011; 11: 138. 3. Shajib and MSKhan WI. The role of serotonin and its receptors in activation of immune responses and inflammation. Acta Physiol (Oxf) 2015; 213(3): 561–74. 4. Gershon. 5-Hydroxytryptamine (serotonin) in the gastrointestinal tract. Curr Opin Endocrinol Diabetes Obes 2013; 20(1): 14–21MD.