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Poster presentations
MicroRNA expression in ileal inflammatory bowel disease correlates with disease behavior
S. Ben-Shachar1 *, H. Yanai2, H. Elad2, L. Baram2, H. Sherman Horev2, A. Ofer2, E. Brazowski3, H. Tulchinsky4, M. Pasmanik-Chor5, N. Shomron6, I. Dotan2. 1 Tel Aviv Medical Center affiliated to Sackler Faculty of Medicine, Tel Aviv university, Genetic institute, Tel Aviv, Israel, 2Tel Aviv Medical Center, affiliated to the Sackler Faculty of Medicine, Tel Aviv University, Department of Gastroenterology and Liver Diseases, Tel Aviv, Israel, 3Tel Aviv Sourasky Medical Center, affiliated to Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, Department of Pathology, Tel Aviv, Israel, 4Tel Aviv Medical Center, affiliated to Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, Proctology Unit, Department of Surgery, Tel Aviv, Israel, 5 Tel Aviv University, Tel Aviv, Israel, Bioinformatics unit, G.S.W. Faculty of Life Sciences, Tel Aviv, Israel, 6Tel Aviv University, Sackler Faculty of Medicine, Tel Aviv, Israel
Background: The etiology of inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC) is yet unknown. Inflammation in UC is limited to the large bowel mucosa, thus large bowel resection was expected to be curative. Nonetheless, about 50% of patients undergoing total proctocolectomy with the creation of an ileal pouch (pouch surgery) develop small intestinal inflammation (pouchitis). We have previously shown that gene expression profiles are associated with ileal inflammation and disease behavior in IBD. One mechanism of regulating gene and protein expression is by MicroRNAs (miRNAs). Altered expression of a limited number of miRNAs was previously noted in IBD. We aimed to evaluate global miRNAs expression in ileal IBD, and its correlation with inflammation, and possible mode of action. Methods: Patients with CD ileitis, UC patients (unoperated and after pouch surgery) and normal controls (NC) were recruited. Pouch patients were stratified according to disease behavior into 3 groups: normal pouch (NP), chronic pouchitis (CP), and Crohn's-like disease of the pouch (CLDP). miRNAs expression in Ileal and pouch biopsies was analyzed by parallel massive sequencing (next generation sequencing). Bioinformatics tools were applied for clustering and detection of potential targets. Results: Fifty-Six subjects (10 CD, 12 NP, 12 CP, 4 CLDP and 11 unoperated UC, as well as 7 NC) were recruited. The ileum of unoperated UC patients was comparable to NC. Significant miRNAs alterations (fold change >2, corrected p-value <0.05) were noticed in NP (n = 8), CP (n = 25), and CLDP (n = 124) patients compared to NC. However, only 2 alterations were noticed in CD. Highly overlap of alterations in pouch patients was noticed. The magnitude of alteration change correlated with disease behavior. More than 95% of the altered miRNAs showed increased expression. The magnitude of maximal increase was 47 folds-more pronounced compared with the magnitude of maximal decrease which was only 4 folds. miR-449b, miR-549, and miR-412 had the greatest increase in expression: 47, 44 and 19 fold change in CLDP, respectively. Interestingly, most miRNAs with increased expression were predicted to target mRNA transcripts that were significantly decreased in pouchitis. miRNAs alterations and phenotype clustered in the pouch groups, while no such clustering was noticed among CD patients.
Conclusions: Increased expression of multiple miRNAs occurs in pouchitis and correlates with disease behavior. Thus, miRNAs may have a role in down regulation of mRNA transcripts in IBD. The absence of clustering of miRNAs expression in CD ileitis may reflect the heterogeneity of CD and explain the limited previous findings regarding miRNAs expression alteration in CD.
Macrophage subtypes disrupt the epithelium via deregulated tight junctions and induction of apoptosis
D. Lissner*, M. Schumann, C. May, L.-I. Kredel, A. Batra, A. Kuhl, J.-D. Schulzke, B. Siegmund. Charite, Hepatology and Gastroenterology Campus Benjamin Franklin, Berlin, Germany
Background: We have shown that pro-inflammatory macrophage subtypes, as found in the lamina propria of patients with Crohn's disease (CD), are capable of disrupting the epithelial barrier. The exact mechanism has not been described yet. Methods: Unpolarized macrophages (M0) were isolated from human peripheral blood and subsequently polarized into M1-and M2-macrophages. The effect of these cells types on epithelial resistance and thus integrity was analysed after co-culture with different epithelial cell lines (Caco-2, T-84, HT-29/B6). Tight junction proteins (claudin-1 and -2, JAM-1, ZO-1, E-cadherin) and caspase-3 and -8 were analysed by immunostaining and western blot.
Results: Compared to regulatory M2-macrophages, pro-inflammatory M0- and M1 macrophages lead to a massive decrease in epithelial resistance. Mainly, this is due to deregulation of tight junction proteins: The pore-forming protein claudin-2 is significantly up-regulated in M0- and M1-exposed epithelial cells, whereas E-cadherin tends to be decreased in these conditions and increased in M2-exposed cells. Additionally, increased caspase-3 in MO-treated epithelial cells reveals induction of apoptosis as a further mechanism. Here, elevated amounts of active caspase-8 suggest death receptor mediated apoptosis, rather than via mitochondrial regulation.
Conclusions: The disrupting effect of pro-inflammatory macrophages, as found in the lamina propria of patients with CD, on the epithelial barrier is due to deregulated tight junction proteins and induction of death receptor mediated apoptosis.
Lymphocytic and collagenous colitis: two clinically similar entities but with a distinct immunological pattern
A. Carrasco1 *, M. Esteve1, E. Pedrosa1, M. Rosinach1, M. Aceituno1, X. Andujar1, C. Loras1, Y. Zabana1, M. Forne1, A. Salas2, F. Fernandez-Banares1. 1Hospital Universitari Mutua Terrassa. UB, Gastroenterology, Terrassa, Spain, 2Hospital Universitari MUtua Terrassa, UB, Pathology, Terrassa, Spain
Background: The immunology of microscopic colitis (MC) is poorly understood and it is unknown whether the two forms of presentation, collagenous colitis (CC) and lymphocytic colitis (LC) share common mechanisms.
Methods: 15 patients with CC and 7 with LC with untreated active disease were included. As a control group 10 patients with normal colonoscopy and colonic histology were also included. Left colon biopsies were taken for analysis of lymphocyte subpopulations by flow cytometry and cytokine production by qPCR.
Results: The absolute number of cells was significantly higher and the number of apoptotic cells (Caspase3+) lower in both CC and LC compared to controls. There were significant differences in lymphocyte subpopulations between CC and LC: the percentage of CD3+, CD3+CD8+, CD3+CD4+TCRgS+, CD3+CD4-CD8- was increased in LC as compared to CC and controls, whereas the percentage of CD3+CD4+ and CD3+CD4+CD8+ was decreased in LC compared to CC and controls. In CC the percentages of these cell types did not differ from the control group. Moreover, Treg cells (CD4+CD25+FoxP3+) were significantly increased in both the Cc and LC. In addition, an increase in CD4+CD25-FoxP3+ was observed in CL. Gene expression (measured as 'fold Increase' compared to controls) of IFNg and IL-17Awas increased in both types of MC, being higher in LC. In addition, IL15 mRNA was
Basic Science
lower in both CC and LC, whereas IL10 was found increased in CL but not in CC.
Conclusions: We have detected remarkable differences in cellular immune response between CC and LC, suggesting that they do not share the same pathophysiological mechanisms. The decrease of apoptosis may play a role in the increased cellularity observed in both CC and Lc. Sponsored by a Basic Research Grant from Academia de Ciencies Médiques, Barcelona.
Involvement of PSGL1 and its ligands P-, E- and L-selectins in inflammatory bowel disease (IBD)
A. Perez-Frías1 *, M.E. Fernandez-Contreras2, R. González-Tajuelo1, P.M. Linares2, M. Guijarro-Rojas3, A. Algaba4, M. Chaparro2, J.P. Gisbert2, A. Urzainqui1. 1Hospital Universitario de La Princesa and IP, Immunology Unit, Madrid, Spain,2Hospital Universitario de La Princesa, lP and CIBERehd, Gastroenterology Unit, Madrid, Spain, 3Hospital Universitario de La Princesa and IP, Pathology Unit, Madrid, Spain, 4Hospital Universitario de Fuenlabrada, Gastroenterology Unit, Fuenlabrada, Spain
Background: PSGL1 and L-Selectin on leukocytes and P- and E-Selectins on endothelial cells are responsible for the initial steps of leukocyte extravasation to the inflamed tissue. Our group has shown that PSGL1/P-Selectin interaction triggers tolerance signals in human monocyte-derived dendritic cells and that PSGL1 acts as a tolerogenic receptor, essential to maintain the colonic lamina propria homeostasis in mice. Our aims were: (1) To quantify soluble PSGL1 and its ligands P-, E- and L-selectins in patients with ulcerative colitis (UC) and Crohn's disease (CD), in order to find a characteristic profile of these molecules in inactive and active IBD. (2) To study the expression of PSGL1 in the colonic mucosa of patients with inactive and active UC and CD.
Methods: PSGL1 and P-, E- and L-Selectins levels were measured by ELISA in serum samples from patients with IBD and healthy volunteers. PSGL1 tissue expression was studied by immunohistochemistry in colon biopsies from patients with IBD and patients without immune-mediated diseases as controls. Clinical IBD activity was assessed by the Mayo score for ulcerative colitis (UC), and by the Harvey-Bradshaw index for Crohn's disease (CD). Tissue IBD activity was determined by histological criteria.
Results: 48 serum samples were obtained from 16 controls, 22 patients with CD (11 active and 11 inactive) and 10 patients with UC (5 active and 5 inactive). Biopsies were taken from 5 controls, 22 patients with UC (8 inactive and 14 with histological activity) and 7 CD (3 inactive and 4 active). Our preliminary results show that patients with UC have lower serum concentration of PSGL1 and higher levels of P-Selectin than controls, regardless of IBD activity, while L-Selectin is increased in the active disease. Patients with CD have lower concentration of PSGL1 but levels of P-, E- and L-Selectins are not different from controls. Regarding tissue expression, PSGL1 is present in the membrane of colonic mucosa leukocytes from controls and patients with inactive IBD. Membrane pattern disappears as IBD histological activity increases. Conclusions: Decreased PSGL1 and increased P-Selectin serum levels are associated with UC, regardless of the disease activity, while high levels of L-Selectin are indicative of active UC. CD is associated with lower concentration of PSGL1, without changes in Selectins levels. Membrane PSGL1 expression in colonic mucosa leukocytes is lost as IBD histological activity increases.
In vitro pancreas toxicity by azathioprine but not 6-mercaptopurine
M. Broekman*, H.M. Roelofs, F. Hoentjen, T. Demir, W.H. Peters, G.J. Wanten, D.J. de Jong. Radboud UMC, Gastroenterology & Hepatology, Nijmegen, Netherlands
Background: Among drugs often used in the treatment of inflammatory bowel disease (IBD), thiopurines and
5-aminosalicylic acid (5-ASA) can cause pancreatitis. The underlying mechanism remains largely unclear, but may include an immune mediated drug reaction. Knowing that azathioprine (AZA) can stimulate pancreas secretion, we postulate that, like in the cerulein model of acute pancreatitis, this might cause acinar cell damage and consequently to autodigestion of the pancreas by its proteases. To bolster this hypothesis, we evaluated in vitro cytotoxic effects of thiopurines and 5-ASA, tested as single drugs and as combination treatment, on three pancreatic cell lines.
Methods: The human pancreatic cell lines PANC-1 (epithelioid carcinoma), AsPC-1 and Capan-1 (both adenocarcinoma) were cultured in Dulbecco's modified Eagle's medium (DMEM) (PANC-1) or RPMI medium (AsPC-1 and Capan-1). After seeding in 96-wells plates (1.48x105 cells/cm2), cells were incubated with the single drugs AZA, 6-mercaptopurine (6-MP), tioguanine (TG) or 5-ASA in the concentration range of 3.9^000mM. Every 24 hours culture medium and drugs were refreshed. After 24, 48 or 72 hours cytotoxicity was established by performing the water-soluble tetrazolium salt-1 (WST-1) assay. Cell survival curves were obtained and half maximal inhibitory concentrations (IC50) were calculated. Next, combination experiments were conducted, with various concentrations AZA,
6-MP or TG (3.9^000 mM) in combination with a fixed non toxic concentration of 5-ASA (200mM). Three independent experiments were conducted in triplicate.
Results: IC50 values of AZA were between 300 and 530mM after 72 hours of incubation in the three cell lines. Incubation with TG resulted in IC50 values, ranging from 360 to 2508 mM, depending on the cell line used. Incubations with 6-MP or
5-ASA did not result in decreased cell survival. Combinations of thiopurines with 5-ASA resulted in increased toxicity of AZA and TG in AsPC-1 cells, and decreased toxicity of AZA in Capan-1 cells. TMPT genotyping did not reveal polymorphisms associated with decreased TPMT activity in the three cell lines.
Conclusions: AZA, and to a much lesser extent TG, but not 6-MP and 5-ASA, exerted a cytotoxic effect on the tested pancreatic cell lines in the present study. The difference in toxicity between AZA and 6-MP could be explained by differences in their metabolism. A large comparative study between AZA and
6-MP would be necessary to verify if there is also an in vivo difference.
Investigation into the binding affinity of certolizumab pegol to FcRn and functional consequences for FcRn-mediated transcytosis: comparison to infliximab, adalimumab and etanercept
T. Baker, L. Kevorkian, A. Nesbitt*. UCB Pharma, Slough, United Kingdom
Background: Certolizumab pegol (CZP) is an anti-TNF that lacks the monoclonal antibody Fc portion. In contrast, anti-TNF antibodies infliximab (IFX) and adalimumab (ADA) and receptor fusion protein etanercept (ETA) all possess an IgG1 Fc. It has been reported that lower levels of CZP, compared to ADA/IFX, are transferred from treated mothers to the neonate. This transfer differential may be due to the one-way active transport of antibodies across the placenta thought to be mediated by the neonatal Fc receptor (FcRn). The objective
