- Email: [email protected]
P.61 IL23R, ATG16L1, IRGM, AND OCTNS MRNA EXPRESSION IN INFLAMED AND NON-INFLAMED MUCOSA OF IBD PATIENTS
S124
Abstracts / Digestive and Liver Disease 42S (2010) S61–S192
patients (biopsies and blood). Our findings could suggest a non pathogenetic role of mycobacterium paratuberculosis as a possible infective origin of CD. # G. Inflammatory bowel diseases - 1. Basic science
P.59 HIGH IFN-GAMMA EXPRESSION IS REQUIRED FOR TUMOR PROTECTION IN A MODEL OF COLITIS-ASSOCIATED COLORECTAL CANCER M.C. Fantini ∗ ,1 , A. Rizzo 1 , D. Fina 1 , R. Caruso 1 , M. Sarra 1 , C. Stolfi 1 , C. Becker 2 , T. MacDonald 3 , F. Pallone 1 , M. Neurath 2 , G. Monteleone 1 1 University
of Rome “Tor Vergata”, Rome; 2 University of Erlangen “Friedrich-Alexander”, Erlangen, Germany; 3 Barts and The London School of Medicine and Dentistry, London, UK Background and aim: Patients affected by inflammatory bowel diseases (IBDs) have a higher risk to develop colorectal cancer (CC). In IBDs intestinal inflammation is sustained, at least in part, by the block of the TGF-beta signaling operated by the intracellular inhibitor Smad7 and chronic inflammation of the gut is thought to promote tumor onset by inducing genomic damage in epithelial cells but also by releasing cytokines and growth factors. However IFN-gamma, a proinflammatory cytokine, highly expressed in IBDs, has been shown to activate the immune-surveillance leading to tumor protection in many experimental settings. We have previously observed that in a model of colitis-associated colorectal cancer (CACC) mice over-expressing Smad7 in T cells (Smad7tg) are protected from tumor development even in the presence of a strong Th-1-mediated colonic inflammation. Since IFN-gamma expression results under direct control of the TGF-beta signaling, aim of this study was to investigate the role of IFN-gamma in the protection against CACC. Material and methods: Wt, Smad7tg, IFN-gamma –/– and mice carrying the double mutation (Smad7tg IFN-gamma –/–) underwent the Azoxymethane/ Dextran Sulphate Sodium protocol of CACC. Tumor development was screened by conventional endoscopy and inflammation was graded by histology. Immune cell infiltration in tumoral and peritumoral areas was evaluated by flow cytometry. Expression of cytotoxicity markers was quantified by qPCR. Results: As previously observed, Smad7tg mice resulted protected from tumors even in the presence of strong colitis in comparison to the Wt. In contrast, the loss of IFN-gamma in the Smad7tg restored tumor susceptibility being tumor incidence and tumor score similar to Wt and IFN-gamma –/– groups. Inflammation was stronger in Smad7tg mice in comparison to all the other groups. Analysis of the immune cell infiltrate showed that the loss of IFN-gamma expression in the Smad7tg mice was associated with a lower number of CD8+ T cells infiltrating the tumors and to a reduced number of CD4+ and NKT cells in the tumor surrounding areas. Finally, mice carrying the double mutation had a reduced expression of cytotoxicity markers in both tumoral and peritumoral areas. Conclusions: IFN-gamma mediates, at least in part, the anti-tumoral immune response against CACC generated in the presence of the T cell-specific block of the TGF-beta signaling operated by Smad7 expression. # G. Inflammatory bowel diseases - 1. Basic science
P.60 HOMING SIGNALS OF ENDOTHELIAL PROGENITORS CELLS IN IBD PATIENTS D. Checchin ∗ ,1 , A. Garolla 2 , M. Scarpa 3 , L. De Toni 2 , A. Michielan 1 , G. Sturniolo 1 , C. Foresta 2 , R. D’Incà 1 1 Department of Surgical Gastroenterological Science, Azienda Ospedaliera University of Padua, Padova; 2 Department of Histology, Microbiology and Medical Biotechnologies, Center for Male Gamete Cryopreservation, University of Padua, Padova; 3 Department of Surgery, Veneto Oncological Institute, Padova
Background and aim: A reduced number of circulating endothelial progenitor cells (EPC) is an important independent risk factor for cardiovascular
disease. IBD patients in remission have a marked reduction in the number of circulating EPC and with high rate of early apoptotic markers. EPC mobilization is driven by enhanced expression of VEGF and SDF1a/CXCR4 homing signals. Aim of this study is to inquire EPC homing signals in IBD patients. Material and methods: In 40 IBD patients (18 ulcerative colitis (UC) and 22 Crohn’s disease (CD)) and7 healthy controls, EPC number, CXC motif receptor 4 (CXCR4) expression, the percentage of apoptotic circulating EPCs, and the number of colony-forming units were evaluated. Endothelial dysfunction was assessed by luteinizing hormone (LH), follicle stimulating hormone (FSH), and testosterone levels, and in a subgroup of patients, brachial artery flow-mediated dilation (FMD) was measured. VEGF plasma levels and CXCL12/SDF-1a were quantified with ELISA. Kruskal–Wallis ANOVA (analysis of variance), Mann–Whitney U two-tailed, and Kendall t rank correlation tests were used. Results: EGF plasma levels were higher in UC (13.9 pg/ml (IQR 8.7-28.5)) and CD (28.7 (IQR9.6-100.7)) patients compared to healthy controls (0.0 (IQR 0.0-0.0)); (p<0.0001). VEGF was inversely correlated with apoptosis (t=-0.47; p=0.032) and history of extra-intestinal manifestations (t=-0.22; p=0.041), and, directly, with FSH levels (t=0.26; p=0.028). SDF1a plasma levels were similar in UC (2077pg/ml (IQR 1798-2348)), CD (2185 (IQR1878-2410)) patients and healthy controls (2136 (IQR 1981-2162)); (p=0.84). SDF1a was inversely correlated with disease duration (t=-0.29; p=0.008) and stricturing phenotype for CD (t=-0.22; p=0.041). It was directly correlated with active smoking (t=0.22; p=0.044, use of Azathioprine (t=0.22; p=0.041) and FSH levels (t=-0.23; p=0.049). Conclusions: Our results showed that VEGF is significantly more expressed in IBD patients and it is inversely correlated with EPC apoptosis. Probably this phenomenon is related to a chronic active EPC homing toward the inflamed bowel or with inadequate EPC mobilization. Although SFD1a plasma levels were similar in IBD patients and healthy controls its expression seems related to peculiar phenotypes. # G. Inflammatory bowel diseases - 1. Basic science
P.61 IL23R, ATG16L1, IRGM, AND OCTNS MRNA EXPRESSION IN INFLAMED AND NON-INFLAMED MUCOSA OF IBD PATIENTS O. Palmieri ∗ ,1 , A. Latiano 1 , F. Bossa 1 , E. Colombo 1 , D. Scimeca 1 , G. Biscaglia 1 , M. Valvano 1 , G. Corritore 1 , T. Latiano 1 , G. Martino 1 , A. Andriulli 1 , V. Annese 2 1
IRCCS, Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG); Careggi Hospital, Firenze
2 A.O.U.
Background and aim: Inflammatory Bowel disease (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory disorders caused by a disruptive interaction between the immune system and gut luminal factors. Recently published GWAS report over 50 IBD loci been identified, however functional studies, such as different mucosal expression of candidate genes are scarce. Aim: This study was designed to investigate the expression of IL23R, ATG16L1, IRGM, OCTN1, and OCTN2 mRNAs in the colonic mucosa of patients with IBD during active phase of disease. Material and methods: In 62 consecutive IBD patients (31 CD and 31 UC) biopsies were taken at inflamed area of the gut, and compared to non-inflamed area by RT-PCR. The GAPDH was the housekeeping gene. Gene expression levels were determined by using the comparative 2ˆ-(delta delta CT), and 2ˆ-(DCt) method. Data were expressed as medians and mean. The Mann Whitney U-test or Wilcoxon test were used when appropriated. Results: The whole mucosal mRNAs expression of IL23R, ATG16L1, IRGM, and OCTN1 were not significantly different between inflamed and noninflamed mucosa, both in CD and UC patients, also after stratifying carriers of the risk genotypes for variation rs7517847 and rs11209026 (IL23R), rs1000113 and rs4958847 (IRGM), rs2241880 (ATG16L1), and rs1050152 (OCTN1). However, the normalized quantities expression levels of IL23R mRNA were significantly reduced between inflamed (CD: Mean = 0.50; DS= 0.46; UC: Mean = 0.59; DS = 0.37) and non-inflamed groups (CD: Mean = 0.68; DS= 0.46; UC: Mean = 0.70; DS = 0.31) (CD: p=0.01; UC: p=0.04). In addition, the whole expression level of OCTN2 mRNA at the inflamed mucosa
Abstracts / Digestive and Liver Disease 42S (2010) S61–S192 was significantly reduced compared to non-inflamed areas, both in CD and UC patients, (CD: Median = 0.34, Q1-Q3 = 0.17-0.74, p=0.001; UC: Median = 0.47, Q1-Q3 = 0.24-0.77, p=0.0004), irrespective of the risk genotypes for the rs2631367 variant. The differences remained still significant when comparing the normalized quantities (CD: p=0.0001; UC: p=0.000056). Conclusions: The OCTN2 and IL23R mRNA levels are significantly reduced in inflamed colonic mucosa, in both CD and UC patients, irrespective of patients’ genotypes. # G. Inflammatory bowel diseases - 1. Basic science
P.62 INFLIXIMAB MODULATES MUCOSAL CYTOKINE PROFILE IN PATIENTS WITH INFLAMMATORY BOWEL DISEASES F. Caprioli ∗ ,1 , F. Bosè 2 , L. Raeli 2 , C. Viganò 3 , G. Basilisco 1 , D. Conte 3 , S. Abrignani 2 , E. Reali 2 1 Gastroenterology
Unit 2. Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano; 2 INGM-National Institute of Molecular Genetics, Milano; 3 Department of Medical Sciences, Università degli Studi di Milano, Milano Background and aim: Monoclonal antibodies against tumor necrosis factoralpha (TNFα) are highly effective in the treatment of patients with inflammatory bowel diseases (IBD), even if their mechanism of action has yet to be fully elucidated. In patients with psoriasis, TNFα neutralization reduces dermal expression of proinflammatory cytokines produced by Th1 and Th17 lymphocytes, T cell lineages critically involved also in the pathogenesis of IBD. In this study, we aimed to evaluate the effect of TNFα neutralization by infliximab in modulating mucosal cytokine profile in patients with IBD. Material and methods: Twelve patients with active left-sided colonic IBD (6 patients with Crohn’s disease (CD) and 6 with ulcerative colitis, 7M, median age 34 yrs, range 25-50), and naïve to anti-TNF therapy were enrolled in the study. All patients underwent induction therapy with infliximab (5 mg/kg administered at week 0, 2 and 6). At enrolment and at week 6 a sigmoidoscopy was performed, and samples were obtained from the sigmoid colon. RNA was extracted from mucosal samples and quantitative expression of 96 inflammation-related genes was evaluated by qPCR, by means of TaqMan Low Density Array Human Immune Panel (Applied Biosystems). Mucosal gene expression was evaluated before and after therapy, and compared with results obtained from 16 controls with normal-appearing mucosa (6M, median age 38 yrs, range 20-44). Results: Among the panel of 96 inflammation-related genes, 36 were found to be significantly upregulated in the mucosa of patients with active IBD in comparison to controls. As compared to baseline, 6 weeks therapy with infliximab induced a significant downregulation of the expression of interleukin (IL)17A, IL-1β, IL-1α, IL-6, IL-8 and NOS2A (all P<0.05). In the CD patients subgroup, TNFα neutralization led to a reduced expression of interferongamma (IFNγ) and lymphotoxin-α, and to an upregulation of the expression of IL-4. With the exception of IL-8 and NOS2A, no significant differences were observed between the post-therapy levels of the above-mentioned genes and those measured in controls. Conclusions: Our data demonstrate that TNFα neutralization rapidly normalizes mucosal expression of several inflammation-related genes, including the signature Th1 and Th17-cytokines IFNγ and IL-17A. These results provide further insights on the mechanisms of anti-TNF agents in controlling mucosal inflammation. # G. Inflammatory bowel diseases - 1. Basic science
S125
P.63 INTERLEUKIN (IL)-17A BUT NOT IL-17E IS PROFIBROTIC IN CROHN’S DISEASE L. Rovedatti ∗ ,1 , A. Di Sabatino 1 , F. Vidali 1 , D. Fina 2 , P. Biancheri 1 , C. Ubezio 1 , A. Pasini 1 , G. Sampietro 3 , C. Alvisi 4 , M. Perego 4 , D. Foschi 3 , T. MacDonald 5 , G. Monteleone 2 , G. Corazza 1 1 First Department of Medicine, Fondazione IRCCS Policlinico S. Matteo, Centro per lo Studio e la Cura delle Malattie Infiammatorie Croniche Intestinali, University of Pavia, Pavia; 2 Department of Internal Medicine, University Tor Vergata, Roma; 3 Surgery Division, Department of Clinical Sciences, Luigi Sacco University Hospital, Milano; 4 Endoscopy Unit, Fondazione IRCCS Policlinico S. Matteo, Centro per lo Studio e la Cura delle Malattie Infiammatorie Croniche Intestinali, University of Pavia, Pavia; 5 Centre for Immunology and Infectious Disease, Bicms, Barts and The London School of Medicine and Dentistry, London, UK
Background and aim: While interleukin (IL)-17A is pro-inflammatory, IL17E (also known as IL-25) is anti-inflammatory in Crohn’s disease (CD). There are no studies investigating the role of IL-17E in modulating extracellular matrix in the gut, while it is known that IL-17A increases matrix metalloproteinase (MMP) production by intestinal fibroblasts. Here we have studied the role of both IL-17A and IL-17E in CD fibrogenesis. Material and methods: Fibroblasts were isolated from colonic surgical specimens collected from strictured and non-strictured areas of 10 patients with fibrostenotic CD, and normal areas of 10 control subjects. The expression of IL-17R, which is common to both IL-17A and IL-17E, was determined on fibroblast lysates by immunoblotting. Fibroblasts were cultured for 24 hours with recombinant human IL-17A and IL-17E. Then supernatants were used for detection of soluble collagen by Sircoll Collagen Assay and tissue inhibitor of MMPs (TIMP-1) by immunoblotting. Fibroblast migration was assessed using the in vitro wound-healing scratch assay. Results: IL-17R was expressed on CD strictured, CD non-strictured and control fibroblasts, with no significant differences between the three cell populations. IL-17A but not IL-17E significantly increased collagen production and TIMP-1 expression, mainly in the supernatants of CD strictured fibroblasts. Migration of CD strictured, CD non-strictured and control fibroblasts was significantly inhibited by IL-17A but not IL-17E, with no significant differences between the three cell populations. Conclusions: Our results suggest that IL-17A but not IL-17E is profibrotic in CD. Further studies are needed to clarify the molecular mechanisms underlying this profibrotic action. # G. Inflammatory bowel diseases - 1. Basic science
P.64 INTERLEUKIN-25 PRODUCTION IS DIFFERENTLY REGULATED BY TNF-α AND TGF-β1 IN THE HUMAN GUT D. Fina ∗ ,1 , E. Franzè 1 , L. Rovedatti 2 , G. Corazza 2 , P. Sileri 3 , T. MacDonald 4 , F. Pallone 1 , A. Di Sabatino 2 , G. Monteleone 1 1 Dipartimento
di Medicina Interna, Università “Tor Vergata”, Roma; Medica I, Fondazione IRCCS Policlinico, San Matteo, Università di Pavia, Pavia; 3 Dipartimento di Chirurgia, Università “Tor Vergata”, Roma; 4 Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, London 2 Clinica
Background and aim: IL-25 has been recently shown to suppress the production of inflammatory cytokines by gut antigen presenting cells, and to inhibit Th1 cell-driven inflammation in experimental colitis. Interestingly, a diminished production of IL-25 occurs in the inflamed gut of IBD patients. These findings raise the possibility that IL-25 down-regulation can contribute to amplify and/or maintain the ongoing tissue-damaging immune response in IBD. Factors/mechanisms underlying the defective production of IL-25 in IBD remain however unknown. We investigated whether the decreased production of IL-25 is a hallmark of IBD, and which factors/mechanisms control IL-25 production. Material and methods: IL-25 was examined in mucosal samples taken from IBD, celiac disease, and normal controls by ELISA. IL-25 was also mea-
Abstracts / Digestive and Liver Disease 42S (2010) S61–S192
patients (biopsies and blood). Our findings could suggest a non pathogenetic role of mycobacterium paratuberculosis as a possible infective origin of CD. # G. Inflammatory bowel diseases - 1. Basic science
P.59 HIGH IFN-GAMMA EXPRESSION IS REQUIRED FOR TUMOR PROTECTION IN A MODEL OF COLITIS-ASSOCIATED COLORECTAL CANCER M.C. Fantini ∗ ,1 , A. Rizzo 1 , D. Fina 1 , R. Caruso 1 , M. Sarra 1 , C. Stolfi 1 , C. Becker 2 , T. MacDonald 3 , F. Pallone 1 , M. Neurath 2 , G. Monteleone 1 1 University
of Rome “Tor Vergata”, Rome; 2 University of Erlangen “Friedrich-Alexander”, Erlangen, Germany; 3 Barts and The London School of Medicine and Dentistry, London, UK Background and aim: Patients affected by inflammatory bowel diseases (IBDs) have a higher risk to develop colorectal cancer (CC). In IBDs intestinal inflammation is sustained, at least in part, by the block of the TGF-beta signaling operated by the intracellular inhibitor Smad7 and chronic inflammation of the gut is thought to promote tumor onset by inducing genomic damage in epithelial cells but also by releasing cytokines and growth factors. However IFN-gamma, a proinflammatory cytokine, highly expressed in IBDs, has been shown to activate the immune-surveillance leading to tumor protection in many experimental settings. We have previously observed that in a model of colitis-associated colorectal cancer (CACC) mice over-expressing Smad7 in T cells (Smad7tg) are protected from tumor development even in the presence of a strong Th-1-mediated colonic inflammation. Since IFN-gamma expression results under direct control of the TGF-beta signaling, aim of this study was to investigate the role of IFN-gamma in the protection against CACC. Material and methods: Wt, Smad7tg, IFN-gamma –/– and mice carrying the double mutation (Smad7tg IFN-gamma –/–) underwent the Azoxymethane/ Dextran Sulphate Sodium protocol of CACC. Tumor development was screened by conventional endoscopy and inflammation was graded by histology. Immune cell infiltration in tumoral and peritumoral areas was evaluated by flow cytometry. Expression of cytotoxicity markers was quantified by qPCR. Results: As previously observed, Smad7tg mice resulted protected from tumors even in the presence of strong colitis in comparison to the Wt. In contrast, the loss of IFN-gamma in the Smad7tg restored tumor susceptibility being tumor incidence and tumor score similar to Wt and IFN-gamma –/– groups. Inflammation was stronger in Smad7tg mice in comparison to all the other groups. Analysis of the immune cell infiltrate showed that the loss of IFN-gamma expression in the Smad7tg mice was associated with a lower number of CD8+ T cells infiltrating the tumors and to a reduced number of CD4+ and NKT cells in the tumor surrounding areas. Finally, mice carrying the double mutation had a reduced expression of cytotoxicity markers in both tumoral and peritumoral areas. Conclusions: IFN-gamma mediates, at least in part, the anti-tumoral immune response against CACC generated in the presence of the T cell-specific block of the TGF-beta signaling operated by Smad7 expression. # G. Inflammatory bowel diseases - 1. Basic science
P.60 HOMING SIGNALS OF ENDOTHELIAL PROGENITORS CELLS IN IBD PATIENTS D. Checchin ∗ ,1 , A. Garolla 2 , M. Scarpa 3 , L. De Toni 2 , A. Michielan 1 , G. Sturniolo 1 , C. Foresta 2 , R. D’Incà 1 1 Department of Surgical Gastroenterological Science, Azienda Ospedaliera University of Padua, Padova; 2 Department of Histology, Microbiology and Medical Biotechnologies, Center for Male Gamete Cryopreservation, University of Padua, Padova; 3 Department of Surgery, Veneto Oncological Institute, Padova
Background and aim: A reduced number of circulating endothelial progenitor cells (EPC) is an important independent risk factor for cardiovascular
disease. IBD patients in remission have a marked reduction in the number of circulating EPC and with high rate of early apoptotic markers. EPC mobilization is driven by enhanced expression of VEGF and SDF1a/CXCR4 homing signals. Aim of this study is to inquire EPC homing signals in IBD patients. Material and methods: In 40 IBD patients (18 ulcerative colitis (UC) and 22 Crohn’s disease (CD)) and7 healthy controls, EPC number, CXC motif receptor 4 (CXCR4) expression, the percentage of apoptotic circulating EPCs, and the number of colony-forming units were evaluated. Endothelial dysfunction was assessed by luteinizing hormone (LH), follicle stimulating hormone (FSH), and testosterone levels, and in a subgroup of patients, brachial artery flow-mediated dilation (FMD) was measured. VEGF plasma levels and CXCL12/SDF-1a were quantified with ELISA. Kruskal–Wallis ANOVA (analysis of variance), Mann–Whitney U two-tailed, and Kendall t rank correlation tests were used. Results: EGF plasma levels were higher in UC (13.9 pg/ml (IQR 8.7-28.5)) and CD (28.7 (IQR9.6-100.7)) patients compared to healthy controls (0.0 (IQR 0.0-0.0)); (p<0.0001). VEGF was inversely correlated with apoptosis (t=-0.47; p=0.032) and history of extra-intestinal manifestations (t=-0.22; p=0.041), and, directly, with FSH levels (t=0.26; p=0.028). SDF1a plasma levels were similar in UC (2077pg/ml (IQR 1798-2348)), CD (2185 (IQR1878-2410)) patients and healthy controls (2136 (IQR 1981-2162)); (p=0.84). SDF1a was inversely correlated with disease duration (t=-0.29; p=0.008) and stricturing phenotype for CD (t=-0.22; p=0.041). It was directly correlated with active smoking (t=0.22; p=0.044, use of Azathioprine (t=0.22; p=0.041) and FSH levels (t=-0.23; p=0.049). Conclusions: Our results showed that VEGF is significantly more expressed in IBD patients and it is inversely correlated with EPC apoptosis. Probably this phenomenon is related to a chronic active EPC homing toward the inflamed bowel or with inadequate EPC mobilization. Although SFD1a plasma levels were similar in IBD patients and healthy controls its expression seems related to peculiar phenotypes. # G. Inflammatory bowel diseases - 1. Basic science
P.61 IL23R, ATG16L1, IRGM, AND OCTNS MRNA EXPRESSION IN INFLAMED AND NON-INFLAMED MUCOSA OF IBD PATIENTS O. Palmieri ∗ ,1 , A. Latiano 1 , F. Bossa 1 , E. Colombo 1 , D. Scimeca 1 , G. Biscaglia 1 , M. Valvano 1 , G. Corritore 1 , T. Latiano 1 , G. Martino 1 , A. Andriulli 1 , V. Annese 2 1
IRCCS, Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG); Careggi Hospital, Firenze
2 A.O.U.
Background and aim: Inflammatory Bowel disease (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory disorders caused by a disruptive interaction between the immune system and gut luminal factors. Recently published GWAS report over 50 IBD loci been identified, however functional studies, such as different mucosal expression of candidate genes are scarce. Aim: This study was designed to investigate the expression of IL23R, ATG16L1, IRGM, OCTN1, and OCTN2 mRNAs in the colonic mucosa of patients with IBD during active phase of disease. Material and methods: In 62 consecutive IBD patients (31 CD and 31 UC) biopsies were taken at inflamed area of the gut, and compared to non-inflamed area by RT-PCR. The GAPDH was the housekeeping gene. Gene expression levels were determined by using the comparative 2ˆ-(delta delta CT), and 2ˆ-(DCt) method. Data were expressed as medians and mean. The Mann Whitney U-test or Wilcoxon test were used when appropriated. Results: The whole mucosal mRNAs expression of IL23R, ATG16L1, IRGM, and OCTN1 were not significantly different between inflamed and noninflamed mucosa, both in CD and UC patients, also after stratifying carriers of the risk genotypes for variation rs7517847 and rs11209026 (IL23R), rs1000113 and rs4958847 (IRGM), rs2241880 (ATG16L1), and rs1050152 (OCTN1). However, the normalized quantities expression levels of IL23R mRNA were significantly reduced between inflamed (CD: Mean = 0.50; DS= 0.46; UC: Mean = 0.59; DS = 0.37) and non-inflamed groups (CD: Mean = 0.68; DS= 0.46; UC: Mean = 0.70; DS = 0.31) (CD: p=0.01; UC: p=0.04). In addition, the whole expression level of OCTN2 mRNA at the inflamed mucosa
Abstracts / Digestive and Liver Disease 42S (2010) S61–S192 was significantly reduced compared to non-inflamed areas, both in CD and UC patients, (CD: Median = 0.34, Q1-Q3 = 0.17-0.74, p=0.001; UC: Median = 0.47, Q1-Q3 = 0.24-0.77, p=0.0004), irrespective of the risk genotypes for the rs2631367 variant. The differences remained still significant when comparing the normalized quantities (CD: p=0.0001; UC: p=0.000056). Conclusions: The OCTN2 and IL23R mRNA levels are significantly reduced in inflamed colonic mucosa, in both CD and UC patients, irrespective of patients’ genotypes. # G. Inflammatory bowel diseases - 1. Basic science
P.62 INFLIXIMAB MODULATES MUCOSAL CYTOKINE PROFILE IN PATIENTS WITH INFLAMMATORY BOWEL DISEASES F. Caprioli ∗ ,1 , F. Bosè 2 , L. Raeli 2 , C. Viganò 3 , G. Basilisco 1 , D. Conte 3 , S. Abrignani 2 , E. Reali 2 1 Gastroenterology
Unit 2. Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano; 2 INGM-National Institute of Molecular Genetics, Milano; 3 Department of Medical Sciences, Università degli Studi di Milano, Milano Background and aim: Monoclonal antibodies against tumor necrosis factoralpha (TNFα) are highly effective in the treatment of patients with inflammatory bowel diseases (IBD), even if their mechanism of action has yet to be fully elucidated. In patients with psoriasis, TNFα neutralization reduces dermal expression of proinflammatory cytokines produced by Th1 and Th17 lymphocytes, T cell lineages critically involved also in the pathogenesis of IBD. In this study, we aimed to evaluate the effect of TNFα neutralization by infliximab in modulating mucosal cytokine profile in patients with IBD. Material and methods: Twelve patients with active left-sided colonic IBD (6 patients with Crohn’s disease (CD) and 6 with ulcerative colitis, 7M, median age 34 yrs, range 25-50), and naïve to anti-TNF therapy were enrolled in the study. All patients underwent induction therapy with infliximab (5 mg/kg administered at week 0, 2 and 6). At enrolment and at week 6 a sigmoidoscopy was performed, and samples were obtained from the sigmoid colon. RNA was extracted from mucosal samples and quantitative expression of 96 inflammation-related genes was evaluated by qPCR, by means of TaqMan Low Density Array Human Immune Panel (Applied Biosystems). Mucosal gene expression was evaluated before and after therapy, and compared with results obtained from 16 controls with normal-appearing mucosa (6M, median age 38 yrs, range 20-44). Results: Among the panel of 96 inflammation-related genes, 36 were found to be significantly upregulated in the mucosa of patients with active IBD in comparison to controls. As compared to baseline, 6 weeks therapy with infliximab induced a significant downregulation of the expression of interleukin (IL)17A, IL-1β, IL-1α, IL-6, IL-8 and NOS2A (all P<0.05). In the CD patients subgroup, TNFα neutralization led to a reduced expression of interferongamma (IFNγ) and lymphotoxin-α, and to an upregulation of the expression of IL-4. With the exception of IL-8 and NOS2A, no significant differences were observed between the post-therapy levels of the above-mentioned genes and those measured in controls. Conclusions: Our data demonstrate that TNFα neutralization rapidly normalizes mucosal expression of several inflammation-related genes, including the signature Th1 and Th17-cytokines IFNγ and IL-17A. These results provide further insights on the mechanisms of anti-TNF agents in controlling mucosal inflammation. # G. Inflammatory bowel diseases - 1. Basic science
S125
P.63 INTERLEUKIN (IL)-17A BUT NOT IL-17E IS PROFIBROTIC IN CROHN’S DISEASE L. Rovedatti ∗ ,1 , A. Di Sabatino 1 , F. Vidali 1 , D. Fina 2 , P. Biancheri 1 , C. Ubezio 1 , A. Pasini 1 , G. Sampietro 3 , C. Alvisi 4 , M. Perego 4 , D. Foschi 3 , T. MacDonald 5 , G. Monteleone 2 , G. Corazza 1 1 First Department of Medicine, Fondazione IRCCS Policlinico S. Matteo, Centro per lo Studio e la Cura delle Malattie Infiammatorie Croniche Intestinali, University of Pavia, Pavia; 2 Department of Internal Medicine, University Tor Vergata, Roma; 3 Surgery Division, Department of Clinical Sciences, Luigi Sacco University Hospital, Milano; 4 Endoscopy Unit, Fondazione IRCCS Policlinico S. Matteo, Centro per lo Studio e la Cura delle Malattie Infiammatorie Croniche Intestinali, University of Pavia, Pavia; 5 Centre for Immunology and Infectious Disease, Bicms, Barts and The London School of Medicine and Dentistry, London, UK
Background and aim: While interleukin (IL)-17A is pro-inflammatory, IL17E (also known as IL-25) is anti-inflammatory in Crohn’s disease (CD). There are no studies investigating the role of IL-17E in modulating extracellular matrix in the gut, while it is known that IL-17A increases matrix metalloproteinase (MMP) production by intestinal fibroblasts. Here we have studied the role of both IL-17A and IL-17E in CD fibrogenesis. Material and methods: Fibroblasts were isolated from colonic surgical specimens collected from strictured and non-strictured areas of 10 patients with fibrostenotic CD, and normal areas of 10 control subjects. The expression of IL-17R, which is common to both IL-17A and IL-17E, was determined on fibroblast lysates by immunoblotting. Fibroblasts were cultured for 24 hours with recombinant human IL-17A and IL-17E. Then supernatants were used for detection of soluble collagen by Sircoll Collagen Assay and tissue inhibitor of MMPs (TIMP-1) by immunoblotting. Fibroblast migration was assessed using the in vitro wound-healing scratch assay. Results: IL-17R was expressed on CD strictured, CD non-strictured and control fibroblasts, with no significant differences between the three cell populations. IL-17A but not IL-17E significantly increased collagen production and TIMP-1 expression, mainly in the supernatants of CD strictured fibroblasts. Migration of CD strictured, CD non-strictured and control fibroblasts was significantly inhibited by IL-17A but not IL-17E, with no significant differences between the three cell populations. Conclusions: Our results suggest that IL-17A but not IL-17E is profibrotic in CD. Further studies are needed to clarify the molecular mechanisms underlying this profibrotic action. # G. Inflammatory bowel diseases - 1. Basic science
P.64 INTERLEUKIN-25 PRODUCTION IS DIFFERENTLY REGULATED BY TNF-α AND TGF-β1 IN THE HUMAN GUT D. Fina ∗ ,1 , E. Franzè 1 , L. Rovedatti 2 , G. Corazza 2 , P. Sileri 3 , T. MacDonald 4 , F. Pallone 1 , A. Di Sabatino 2 , G. Monteleone 1 1 Dipartimento
di Medicina Interna, Università “Tor Vergata”, Roma; Medica I, Fondazione IRCCS Policlinico, San Matteo, Università di Pavia, Pavia; 3 Dipartimento di Chirurgia, Università “Tor Vergata”, Roma; 4 Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, London 2 Clinica
Background and aim: IL-25 has been recently shown to suppress the production of inflammatory cytokines by gut antigen presenting cells, and to inhibit Th1 cell-driven inflammation in experimental colitis. Interestingly, a diminished production of IL-25 occurs in the inflamed gut of IBD patients. These findings raise the possibility that IL-25 down-regulation can contribute to amplify and/or maintain the ongoing tissue-damaging immune response in IBD. Factors/mechanisms underlying the defective production of IL-25 in IBD remain however unknown. We investigated whether the decreased production of IL-25 is a hallmark of IBD, and which factors/mechanisms control IL-25 production. Material and methods: IL-25 was examined in mucosal samples taken from IBD, celiac disease, and normal controls by ELISA. IL-25 was also mea-