- Email: [email protected]
P.67 THROMBOPHILIA IN INFLAMMATORY BOWEL DISEASE: WHAT IS THE IMPACT OF GENETIC FACTORS?
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Abstracts / Digestive and Liver Disease 42S (2010) S61–S192
sured in control and IBD explants cultured in the presence or absence of inflammatory or anti-inflammatory cytokines respectively. Additionally, IL-25 expression was determined in IBD mucosal explants cultured with anti-TNFα or Smad7 antisense (AS) oligonucleotide. Finally, IL-25 was evaluated in colonic biopsies taken from IBD patients before and after a successful treatment with Infliximab. Results: A diminished production of IL-25 was seen in IBD samples as compared to normal controls. In IBD patients, IL-25 down-regulation was not dependent on the site of lesions or the current therapy. By contrast, a significant increase of IL-25 expression was seen in the gut of patients with active celiac disease. Among inflammatory cytokines, TNF-α reduced IL-25 production in ex vivo organ cultures of normal mucosal explants. Consistently, treatment of IBD biopsies with Infliximab up-regulated IL-25. Similarly, IL-25 expression was enhanced in vivo by Infliximab treatment. Finally, we showed that TGF-β1 positively regulated IL-25 expression in cultures of normal colonic biopsies. Since it is known that, in IBD tissue, there is a disruption of TGF-β1 signaling due to high levels of the inhibitor Smad7, we next determined whether restoring TGF-β1 activity with a specific Smad7 AS oligonucleotide was accompanied by induction of IL-25. Treatment of IBD biopsies with Smad7 AS oligonucleotide enhanced IL-25 expression. Conclusions: Our data indicate that IL-25 production is differently modulated by TNF-α and TGF-β1. To the best of our knowledge this study is the first to show which factors regulate IL-25 expression during IBD. # G. Inflammatory bowel diseases - 1. Basic science
P.65 MESENCHYMAL STEM CELLS INJECTION REDUCES COLITIS SEVERITY AND DOES NOT INCREASE COLITIS-ASSOCIATED COLON CANCER RISK IN MICE F. Scaldaferri ∗ ,1 , M. Puglisi 1 , A. Sgambato 2 , V. Arena 2 , F. Fiore 1 , E. Caredda 2 , G. Andrisani 1 , M. Caristo 3 , A. Piscaglia 1 , A. Papa 1 , A. Gasbarrini 1 1 Catholic University of Rome, Gemelli Hospital, Gastroenterology Division, Rome; 2 Catholic University of Rome, Institute of Pathology, Rome; 3 Catholic University of Rome, Experimental Center, Rome
Background and aim: Mesenchymal stem cells (AMSC) are potent immune regulators, proposed for local and systemic use in human and experimental IBD. Cancer studies found stem cell to be involved in mechanisms of cancer induction and progression, warning the use of stem cell in clinical condition associated to increased cancer risk, such as ulcerative colitis. Despite this, very few information exist on whether and how the use of mesenchymal stem cell influences cancer induction in chronic colitis. Aims: Aim of this study was to evaluate the potential therapeutic effect of AMSC in a murine model of colon cancer associated to chronic colitis. Material and methods: AMSC were isolated from adipose tissue of C57BL/6 mice, analyzed for mesenchymal stem cell markers (CD34, CD90, CD49e, SCA-1) and for adipocyte and osteogenic differentiation, upon exposure to differentiation media in vitro. C57BL/6 mice were injected intraperitoneally with 7 mg/kg of azoxymethane (AOM) and then exposed to 3 cycles of 2.5% Dextran Sodium Sulfate (DSS), given for 7 days in tap water. 400.000 AMSC diluted in 200 μL of buffer solution were injected twice intraperitoneally at day 1 of I and II DSS cycle; control mice received buffer alone. Body weight, occult blood test and stool consistency were measured twice a week and used to calculate the Disease Activity Index (DAI) to assess severity of colitis; survival was expressed as %. Mice were sacrificed at week 10 and colon was analyzed macroscopically and microscopically for number of cancer and degree of inflammation. Results: AMSC retained stem cells properties as they differentiated into adipocytes and osteocytes, and expressed low levels of CD90 together with higher levels of SCA-1 and CD49e. In vivo, AMSC injection significantly reduced DAI in treated mice compared to controls, especially during the first 6 weeks of treatment. AMSC treated mice showed also a lower body weight loss and a better survival rate compared to controls (100% vs 65%). At macroscopic analysis, AMSC treated mice showed a reduced rate of colon cancer development compared to controls (33% vs 67%). However the number
of tumors per mouse did not differ significantly among mice which actually developed cancer (1.7 vs 1.5 tumors/mouse). # G. Inflammatory bowel diseases - 1. Basic science
P.66 MUCOSAL ASSESSMENT OF TUMOR NECROSIS FACTOR (TNF)-ALPHA LEVELS: A COMPARISON BETWEEN IMMUNOHISTOCHEMISTRY (IHC) AND REAL TIME POLYMERASE CHAIN REACTION (PCR) ON PARAFFINED SAMPLES F. Giorgio, M. Zotti, R. Rosania, S. Prencipe, N. Della Valle, M.C. Nacchiero, N. Muscatiello, C. Panella, E. Ierardi ∗ A.O.U. Ospedali Riuniti, Foggia Background and aim: TNF-alpha levels characterize intestinal mucosal damage in inflammatory bowel diseases (IBD).Therefore its evaluation has been performed by both IHC and real time PCR to evaluate inflammatory response and biologic drug efficacy in fresh tissue samples. Aim of this study was to correlate the simultaneous expression of TNF-alpha in patients affected by IBD with either IHC and real time PCR on archived paraffin-embedded material. Material and methods: Samples from 60 subjects were retrieved and divided into 3 groups: ileo-colic Crohn’s disease, severe ulcerative colitis and irritable bowel syndrome (control group). Real time PCR allowed obtaining the quantification of mRNA codifying for the synthesis of TNF-alpha expressed by a numerical value (i.e. the fold change compared to control). RNA was extracted from five 10 micron sections, using QIAgen RNA mini kit (QIAGEN GmbH, Germany). Two-step reverse transcription PCR was performed using first-strand cDNA with a final concentration of 1 × TaqMan gene expression assay, i.e. TNF-alpha and GAPDH (Applied Biosystems, CA). The final reaction volume was 25 μl and analyzed in triplicate (all experiments were repeated twice). A non-template control (Rnase-free water) was included on every plate. Specific thermal cycler conditions were employed by real time PCR System (Applied Biosystems, CA). A standard curve plus validation experiment were performed for each primer/probe set. A series of 6 serial dilutions (20 to 0.1 ng/μl) of tissue cDNA were used as a template for each primer/probe set. IHC was performed with monoclonal mouse antibody (Santa Cruz, CA) plus avitin-biotin based development system (Vector Laboratories, A) and expressed by the labelling index (% of positive stromal cells). Results: We observed a strong correlation between the two methods in each group: CD (p=0.02; r=0.9; Pearson’s test), severe UC (p=0.02; r=0.9) and controls (p=0.02; r=0.8). Moreover, TNF-alpha molecular analysis was significantly raised in both CD and severe UC in comparison to the control group (5.5±2.1 > 3.1±1.2 > 1.1±0.7 respectively, p=0.02, ANOVA plus Student-Neumann-Keuls). Conclusions: Our experience demonstrates that TNF-alpha mucosal assay in IBD may be feasible using real time PCR in archived material from paraffin embedded biopsy samples. Its appliance offers perspectives for retrospective studies. # G. Inflammatory bowel diseases - 1. Basic science
P.67 THROMBOPHILIA IN INFLAMMATORY BOWEL DISEASE: WHAT IS THE IMPACT OF GENETIC FACTORS? M. Cappello ∗ , I. Bravatà, C. Randazzo, S. Grimaudo, R. Pipitone, A. Di Cristina, A. Licata, C. Cammà, A. Craxì Cattedra di Gastroenterologia, Di.Bi. M.I.S., Università di Palermo, Palermo Background and aim: Inflammatory bowel disease (IBD) is linked to a definite risk in thromboembolic events (TE), but data on the most frequent prothrombotic genetic mutations are conflicting. We aimed to investigate a comprehensive panel of 14 genetic factors involved in TE pathogenesis in a homogeneous cohort of IBD Sicilian patients with and without TE and in healthy controls. Material and methods: Forty consecutive IBD patients (14/26 M/F, mean age
Abstracts / Digestive and Liver Disease 42S (2010) S61–S192 47.1±17.2 years) seen for a follow-up visit between December 2007 and May 2008 were studied. Nineteen had ulcerative colitis (UC) and 21 Crohn’s disease (CD). 20 healthy individuals with a personal and family history negative for TE and IBD were used as healthy controls. Data regarding age, sex, IBD type, disease localization, duration and activity, extraintestinal manifestations, therapy, previous TE, familial predisposition for IBD and/or TE and risk factors for TE (smoking, hypertension, diabetes mellitus, dyslipidemia, oral contraceptives and hormone-replacement therapy) were registered. Genomic DNA was extracted from peripheral blood samples and tested using Cardio and Thrombo Test (Nuclear Laser Medicine) which analyzes 14 polymorphisms involved in thrombophilia and cholesterol metabolism. The rate of genetic polymorphisms and mutations was compared between IBD patients with and without previous history of TE and healthy controls. Results: 9 IBD patients (22.5%) had history of previous TE. No significant difference in allelic frequency was found between IBD patients and controls except ACE D/D and AGT T/T. 8 out of 9 patients with previous history of TE had more than one polymorphism. In patients with IBD the mutation AGT T/T was related with male sex (p<0.0259) and, marginally, with arterial hypertension (p<0.06) and diabetes (p<0.09). Conclusions: Our data confirm a definite risk of TE in IBD (22.5% of our series). An increased frequency of the genotypes ACE D/D and AGT T/T, never reported so far, was present in our series. In IBD patients with TE the expression of multiple polymorphisms and mutations suggests a multifactorial genesis of TE with the involvement of several genes as well as of acquired factors. Genetic screening for a panel of prothrombotic factors could help segregate IBD patients at higher risk of TE. # G. Inflammatory bowel diseases - 1. Basic science
P.68 TRANSGLUTAMINASE TYPE 2 INHIBITION REDUCES THE SEVERITY OF INFLAMMATION IN A RAT MODEL OF INFLAMMATORY BOWEL DISEASE L. Elli ∗ ,1 , M. Ciulla 2 , L. Roncoroni 3 , G. Busca 2 , C. Maioli 4 , S. Ferrero 4 , M. Bardella 1 , A. Bonura 1 , C. Terrani 5 , R. Paliotti 2 , P. Braidotti 4 1 Centro per la Prevenzione e Diagnosi della Malattia Celiaca, Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano; 2 Dipartimento Toracopolmonare e Cardiocircolatorio, Università degli Studi di Milano, Milano; 3 Dipartimento di Scienze Mediche, Università degli Studi di Milano, Milano; 4 Dipartimento di Medicina, Chirurgia e Odontoiatria, Ospedale San Paolo e Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Università degli Studi di Milano, Milano; 5 U.O. Gastroenterologia, Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano
Background and aim: Transglutaminase type 2 (TG2) is an enzyme with pleiotropic functions, involved in different pathways of inflammation and central in the pathogenesis of some human disorders as the inflammatory bowel disease (IBD). Aim of the study was to evaluate the effect of cystamine (Cys)-mediated inhibition of TG2 in an IBD model. Material and methods: Rats were divided into 4 groups: controls with no treatment (CTR; n = 7); receiving the 2, 4, 6-trinitrobenzene sulfonic acid enema (TNBS group; n = 8); treated with TNBS enema plus oral Cys (TNBSCys group; n = 8); treated with Cys (Cys group; n = 7). At sacrifice, bowel inflammation was evaluated applying specific scores. TG2 and tumor necrosis factor alpha (TNFα) were measured on tissue colonic homogenates (ELISA and immunohistochemistry). TG2 was also evaluated on the luminal side of the colon by immunoautoradiography. Colonic samples from IBD patients were compared to animal results. Results: The TNBS-Cys group developed a less severe colitis compared to the TNBS group (macroscopic score 0.43±0.78 vs 3.28±0.95, microscopic score 6.62±12.01 vs 19.25±6.04, p<0.05 respectively) associated with a statistically significant decrease of TG2 activity (-50%) and TNFα expression (-80%). No statistically significant differences were found between Cys and CTR groups. The colonic immunolocalization of TG2 was comparable in humans affected by IBD and TNBS administered animals. Conclusions: This is the first demonstration that treatment with a TG2 inhibitor has an antinflammatory effect reducing severity of colitis in a rat
S127
model. Cys could be tested as possible treatment or co-treatment in IBD therapeutic trials. # G. Inflammatory bowel diseases - 1. Basic science
P.69 A “REAL LIVE” EXPERIENCE OF INFLIXIMAB USE FOR CROHN’S DISEASE IN SOUTHERN ITALY M. Lo Presti ∗ ,1 , A. Privitera 2 , M. Cappello 3 , A. Lauria 4 , M. Principi 5 , N. Della Valle 6 , L. Grossi 7 , W. Fries 1 1 Dipartimento
di Medicina Interna, Policlinico, Messina; 2 Dipartimento di Scienze Chirurgiche, Trapianti d’Organo e Tecnologie Avanzate, U.O.C. di Chirurgia Generale e Serv. Endoscopia, Az. Osp. per l’Emergenza “Cannizzaro”, Università di Catania;, Catania; 3 Gastroenterologia ed Epatologia, Policlinico Palermo, Università di Palermo, Palermo; 4 Gastroenterologia e Endoscopia Digestiva A. O. Bianchi, Melacrino, Morelli, Reggio Calabria; 5 Sezione di Gastroenterologia ed Endoscopia Digestiva, Policlinico, Università di Bari, Bari; 6 Gastroenterologia, Policlinico, Università di Foggia, Foggia; 7 Unità di Fisiopatologia Digestiva all’Ospedale Spirito Santo di Pescara, Università di Chieti-Pescara, Pescara Background and aim: The biologic agent Infliximab is used now widely in Italy for the therapy of Crohn’s disease (CD). Available data are mainly from referral centres or from centres which participated at clinical trials. The open field use of this anti-TNF agent includes frequently patients with concomitant pathologies. The aim of this study was to evaluate the clinical and endoscopic response to infliximab (Ifx) in non-referral centres. Material and methods: Data from 7 centres located mainly in Southern Italy were collected between 2003–2008 and analysed. Demographic data, Harvey-Bradshaw index (HBI), C-reactive protein (CRP) levels, indications for treatment, concomitant therapies, and endoscopy, before Ifx therapy and at 3 months, 1 and 2 years; side effects and withdrawal of therapy were recorded. Data are mean values ± SE. Statistics were performed with ANOVA, paired t-test and chi-square test. Results: A total of 179 CD patients (90 F/89 M; mean age 41±2 yrs) were included. Indications were steroid-dependency, intolerance or non-response to immunomodulators (IM) and perianal fistulas. A total of 2106 infusions were administered with a median of 9 infusions/patient. 64% of patients were on steroids before therapy; at 3 months 71% of them was off steroids, and 15% of patients reduced steroid dose. At 1 year, 80% of patients were off steroids. The mean HBI declined from a pre-Ifx value of 10.4 to 1.92 at 3 months, to 1.6 at 1 year and remained stable with 1.75 at 2 years (all p<0.01 or less vs baseline). CRP was significantly reduced at 3 and at 12 months after Ifx (pre-therapy 11.4±4.2 mg/l; 6 months: 6.9±3.1 mg/l, p<0.0 vs baseline; at 12 months: 5.04±2.9 mg/dl, p<0.01). 37 patients with perianal fistulas were evaluated at 6 and at 12 months achieving fistula closure in 32 and 54%, respectively, and a reduction of drainage in 62% and 32%, respectively. An endoscopic evaluation after 1 year was available from 86 patients revealing mucosal healing in 44 patients (51%). A total of 71 adverse reactions were observed leading to interruption of therapy in 23 patients (13%); no drug-related death occurred in this cohort. Conclusions: In this real live experience the benefits achieved with Ifx were comparable with those achieved in referral centres and in clinical studies with respect to steroid withdrawal/reduction, fistula healing and endoscopic healing. Adverse events do not occur more frequently than in referral centres. # G. Inflammatory bowel diseases - 2. Ulcerative colitis and Crohn’s disease diagnosis/therapy
Abstracts / Digestive and Liver Disease 42S (2010) S61–S192
sured in control and IBD explants cultured in the presence or absence of inflammatory or anti-inflammatory cytokines respectively. Additionally, IL-25 expression was determined in IBD mucosal explants cultured with anti-TNFα or Smad7 antisense (AS) oligonucleotide. Finally, IL-25 was evaluated in colonic biopsies taken from IBD patients before and after a successful treatment with Infliximab. Results: A diminished production of IL-25 was seen in IBD samples as compared to normal controls. In IBD patients, IL-25 down-regulation was not dependent on the site of lesions or the current therapy. By contrast, a significant increase of IL-25 expression was seen in the gut of patients with active celiac disease. Among inflammatory cytokines, TNF-α reduced IL-25 production in ex vivo organ cultures of normal mucosal explants. Consistently, treatment of IBD biopsies with Infliximab up-regulated IL-25. Similarly, IL-25 expression was enhanced in vivo by Infliximab treatment. Finally, we showed that TGF-β1 positively regulated IL-25 expression in cultures of normal colonic biopsies. Since it is known that, in IBD tissue, there is a disruption of TGF-β1 signaling due to high levels of the inhibitor Smad7, we next determined whether restoring TGF-β1 activity with a specific Smad7 AS oligonucleotide was accompanied by induction of IL-25. Treatment of IBD biopsies with Smad7 AS oligonucleotide enhanced IL-25 expression. Conclusions: Our data indicate that IL-25 production is differently modulated by TNF-α and TGF-β1. To the best of our knowledge this study is the first to show which factors regulate IL-25 expression during IBD. # G. Inflammatory bowel diseases - 1. Basic science
P.65 MESENCHYMAL STEM CELLS INJECTION REDUCES COLITIS SEVERITY AND DOES NOT INCREASE COLITIS-ASSOCIATED COLON CANCER RISK IN MICE F. Scaldaferri ∗ ,1 , M. Puglisi 1 , A. Sgambato 2 , V. Arena 2 , F. Fiore 1 , E. Caredda 2 , G. Andrisani 1 , M. Caristo 3 , A. Piscaglia 1 , A. Papa 1 , A. Gasbarrini 1 1 Catholic University of Rome, Gemelli Hospital, Gastroenterology Division, Rome; 2 Catholic University of Rome, Institute of Pathology, Rome; 3 Catholic University of Rome, Experimental Center, Rome
Background and aim: Mesenchymal stem cells (AMSC) are potent immune regulators, proposed for local and systemic use in human and experimental IBD. Cancer studies found stem cell to be involved in mechanisms of cancer induction and progression, warning the use of stem cell in clinical condition associated to increased cancer risk, such as ulcerative colitis. Despite this, very few information exist on whether and how the use of mesenchymal stem cell influences cancer induction in chronic colitis. Aims: Aim of this study was to evaluate the potential therapeutic effect of AMSC in a murine model of colon cancer associated to chronic colitis. Material and methods: AMSC were isolated from adipose tissue of C57BL/6 mice, analyzed for mesenchymal stem cell markers (CD34, CD90, CD49e, SCA-1) and for adipocyte and osteogenic differentiation, upon exposure to differentiation media in vitro. C57BL/6 mice were injected intraperitoneally with 7 mg/kg of azoxymethane (AOM) and then exposed to 3 cycles of 2.5% Dextran Sodium Sulfate (DSS), given for 7 days in tap water. 400.000 AMSC diluted in 200 μL of buffer solution were injected twice intraperitoneally at day 1 of I and II DSS cycle; control mice received buffer alone. Body weight, occult blood test and stool consistency were measured twice a week and used to calculate the Disease Activity Index (DAI) to assess severity of colitis; survival was expressed as %. Mice were sacrificed at week 10 and colon was analyzed macroscopically and microscopically for number of cancer and degree of inflammation. Results: AMSC retained stem cells properties as they differentiated into adipocytes and osteocytes, and expressed low levels of CD90 together with higher levels of SCA-1 and CD49e. In vivo, AMSC injection significantly reduced DAI in treated mice compared to controls, especially during the first 6 weeks of treatment. AMSC treated mice showed also a lower body weight loss and a better survival rate compared to controls (100% vs 65%). At macroscopic analysis, AMSC treated mice showed a reduced rate of colon cancer development compared to controls (33% vs 67%). However the number
of tumors per mouse did not differ significantly among mice which actually developed cancer (1.7 vs 1.5 tumors/mouse). # G. Inflammatory bowel diseases - 1. Basic science
P.66 MUCOSAL ASSESSMENT OF TUMOR NECROSIS FACTOR (TNF)-ALPHA LEVELS: A COMPARISON BETWEEN IMMUNOHISTOCHEMISTRY (IHC) AND REAL TIME POLYMERASE CHAIN REACTION (PCR) ON PARAFFINED SAMPLES F. Giorgio, M. Zotti, R. Rosania, S. Prencipe, N. Della Valle, M.C. Nacchiero, N. Muscatiello, C. Panella, E. Ierardi ∗ A.O.U. Ospedali Riuniti, Foggia Background and aim: TNF-alpha levels characterize intestinal mucosal damage in inflammatory bowel diseases (IBD).Therefore its evaluation has been performed by both IHC and real time PCR to evaluate inflammatory response and biologic drug efficacy in fresh tissue samples. Aim of this study was to correlate the simultaneous expression of TNF-alpha in patients affected by IBD with either IHC and real time PCR on archived paraffin-embedded material. Material and methods: Samples from 60 subjects were retrieved and divided into 3 groups: ileo-colic Crohn’s disease, severe ulcerative colitis and irritable bowel syndrome (control group). Real time PCR allowed obtaining the quantification of mRNA codifying for the synthesis of TNF-alpha expressed by a numerical value (i.e. the fold change compared to control). RNA was extracted from five 10 micron sections, using QIAgen RNA mini kit (QIAGEN GmbH, Germany). Two-step reverse transcription PCR was performed using first-strand cDNA with a final concentration of 1 × TaqMan gene expression assay, i.e. TNF-alpha and GAPDH (Applied Biosystems, CA). The final reaction volume was 25 μl and analyzed in triplicate (all experiments were repeated twice). A non-template control (Rnase-free water) was included on every plate. Specific thermal cycler conditions were employed by real time PCR System (Applied Biosystems, CA). A standard curve plus validation experiment were performed for each primer/probe set. A series of 6 serial dilutions (20 to 0.1 ng/μl) of tissue cDNA were used as a template for each primer/probe set. IHC was performed with monoclonal mouse antibody (Santa Cruz, CA) plus avitin-biotin based development system (Vector Laboratories, A) and expressed by the labelling index (% of positive stromal cells). Results: We observed a strong correlation between the two methods in each group: CD (p=0.02; r=0.9; Pearson’s test), severe UC (p=0.02; r=0.9) and controls (p=0.02; r=0.8). Moreover, TNF-alpha molecular analysis was significantly raised in both CD and severe UC in comparison to the control group (5.5±2.1 > 3.1±1.2 > 1.1±0.7 respectively, p=0.02, ANOVA plus Student-Neumann-Keuls). Conclusions: Our experience demonstrates that TNF-alpha mucosal assay in IBD may be feasible using real time PCR in archived material from paraffin embedded biopsy samples. Its appliance offers perspectives for retrospective studies. # G. Inflammatory bowel diseases - 1. Basic science
P.67 THROMBOPHILIA IN INFLAMMATORY BOWEL DISEASE: WHAT IS THE IMPACT OF GENETIC FACTORS? M. Cappello ∗ , I. Bravatà, C. Randazzo, S. Grimaudo, R. Pipitone, A. Di Cristina, A. Licata, C. Cammà, A. Craxì Cattedra di Gastroenterologia, Di.Bi. M.I.S., Università di Palermo, Palermo Background and aim: Inflammatory bowel disease (IBD) is linked to a definite risk in thromboembolic events (TE), but data on the most frequent prothrombotic genetic mutations are conflicting. We aimed to investigate a comprehensive panel of 14 genetic factors involved in TE pathogenesis in a homogeneous cohort of IBD Sicilian patients with and without TE and in healthy controls. Material and methods: Forty consecutive IBD patients (14/26 M/F, mean age
Abstracts / Digestive and Liver Disease 42S (2010) S61–S192 47.1±17.2 years) seen for a follow-up visit between December 2007 and May 2008 were studied. Nineteen had ulcerative colitis (UC) and 21 Crohn’s disease (CD). 20 healthy individuals with a personal and family history negative for TE and IBD were used as healthy controls. Data regarding age, sex, IBD type, disease localization, duration and activity, extraintestinal manifestations, therapy, previous TE, familial predisposition for IBD and/or TE and risk factors for TE (smoking, hypertension, diabetes mellitus, dyslipidemia, oral contraceptives and hormone-replacement therapy) were registered. Genomic DNA was extracted from peripheral blood samples and tested using Cardio and Thrombo Test (Nuclear Laser Medicine) which analyzes 14 polymorphisms involved in thrombophilia and cholesterol metabolism. The rate of genetic polymorphisms and mutations was compared between IBD patients with and without previous history of TE and healthy controls. Results: 9 IBD patients (22.5%) had history of previous TE. No significant difference in allelic frequency was found between IBD patients and controls except ACE D/D and AGT T/T. 8 out of 9 patients with previous history of TE had more than one polymorphism. In patients with IBD the mutation AGT T/T was related with male sex (p<0.0259) and, marginally, with arterial hypertension (p<0.06) and diabetes (p<0.09). Conclusions: Our data confirm a definite risk of TE in IBD (22.5% of our series). An increased frequency of the genotypes ACE D/D and AGT T/T, never reported so far, was present in our series. In IBD patients with TE the expression of multiple polymorphisms and mutations suggests a multifactorial genesis of TE with the involvement of several genes as well as of acquired factors. Genetic screening for a panel of prothrombotic factors could help segregate IBD patients at higher risk of TE. # G. Inflammatory bowel diseases - 1. Basic science
P.68 TRANSGLUTAMINASE TYPE 2 INHIBITION REDUCES THE SEVERITY OF INFLAMMATION IN A RAT MODEL OF INFLAMMATORY BOWEL DISEASE L. Elli ∗ ,1 , M. Ciulla 2 , L. Roncoroni 3 , G. Busca 2 , C. Maioli 4 , S. Ferrero 4 , M. Bardella 1 , A. Bonura 1 , C. Terrani 5 , R. Paliotti 2 , P. Braidotti 4 1 Centro per la Prevenzione e Diagnosi della Malattia Celiaca, Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano; 2 Dipartimento Toracopolmonare e Cardiocircolatorio, Università degli Studi di Milano, Milano; 3 Dipartimento di Scienze Mediche, Università degli Studi di Milano, Milano; 4 Dipartimento di Medicina, Chirurgia e Odontoiatria, Ospedale San Paolo e Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Università degli Studi di Milano, Milano; 5 U.O. Gastroenterologia, Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano
Background and aim: Transglutaminase type 2 (TG2) is an enzyme with pleiotropic functions, involved in different pathways of inflammation and central in the pathogenesis of some human disorders as the inflammatory bowel disease (IBD). Aim of the study was to evaluate the effect of cystamine (Cys)-mediated inhibition of TG2 in an IBD model. Material and methods: Rats were divided into 4 groups: controls with no treatment (CTR; n = 7); receiving the 2, 4, 6-trinitrobenzene sulfonic acid enema (TNBS group; n = 8); treated with TNBS enema plus oral Cys (TNBSCys group; n = 8); treated with Cys (Cys group; n = 7). At sacrifice, bowel inflammation was evaluated applying specific scores. TG2 and tumor necrosis factor alpha (TNFα) were measured on tissue colonic homogenates (ELISA and immunohistochemistry). TG2 was also evaluated on the luminal side of the colon by immunoautoradiography. Colonic samples from IBD patients were compared to animal results. Results: The TNBS-Cys group developed a less severe colitis compared to the TNBS group (macroscopic score 0.43±0.78 vs 3.28±0.95, microscopic score 6.62±12.01 vs 19.25±6.04, p<0.05 respectively) associated with a statistically significant decrease of TG2 activity (-50%) and TNFα expression (-80%). No statistically significant differences were found between Cys and CTR groups. The colonic immunolocalization of TG2 was comparable in humans affected by IBD and TNBS administered animals. Conclusions: This is the first demonstration that treatment with a TG2 inhibitor has an antinflammatory effect reducing severity of colitis in a rat
S127
model. Cys could be tested as possible treatment or co-treatment in IBD therapeutic trials. # G. Inflammatory bowel diseases - 1. Basic science
P.69 A “REAL LIVE” EXPERIENCE OF INFLIXIMAB USE FOR CROHN’S DISEASE IN SOUTHERN ITALY M. Lo Presti ∗ ,1 , A. Privitera 2 , M. Cappello 3 , A. Lauria 4 , M. Principi 5 , N. Della Valle 6 , L. Grossi 7 , W. Fries 1 1 Dipartimento
di Medicina Interna, Policlinico, Messina; 2 Dipartimento di Scienze Chirurgiche, Trapianti d’Organo e Tecnologie Avanzate, U.O.C. di Chirurgia Generale e Serv. Endoscopia, Az. Osp. per l’Emergenza “Cannizzaro”, Università di Catania;, Catania; 3 Gastroenterologia ed Epatologia, Policlinico Palermo, Università di Palermo, Palermo; 4 Gastroenterologia e Endoscopia Digestiva A. O. Bianchi, Melacrino, Morelli, Reggio Calabria; 5 Sezione di Gastroenterologia ed Endoscopia Digestiva, Policlinico, Università di Bari, Bari; 6 Gastroenterologia, Policlinico, Università di Foggia, Foggia; 7 Unità di Fisiopatologia Digestiva all’Ospedale Spirito Santo di Pescara, Università di Chieti-Pescara, Pescara Background and aim: The biologic agent Infliximab is used now widely in Italy for the therapy of Crohn’s disease (CD). Available data are mainly from referral centres or from centres which participated at clinical trials. The open field use of this anti-TNF agent includes frequently patients with concomitant pathologies. The aim of this study was to evaluate the clinical and endoscopic response to infliximab (Ifx) in non-referral centres. Material and methods: Data from 7 centres located mainly in Southern Italy were collected between 2003–2008 and analysed. Demographic data, Harvey-Bradshaw index (HBI), C-reactive protein (CRP) levels, indications for treatment, concomitant therapies, and endoscopy, before Ifx therapy and at 3 months, 1 and 2 years; side effects and withdrawal of therapy were recorded. Data are mean values ± SE. Statistics were performed with ANOVA, paired t-test and chi-square test. Results: A total of 179 CD patients (90 F/89 M; mean age 41±2 yrs) were included. Indications were steroid-dependency, intolerance or non-response to immunomodulators (IM) and perianal fistulas. A total of 2106 infusions were administered with a median of 9 infusions/patient. 64% of patients were on steroids before therapy; at 3 months 71% of them was off steroids, and 15% of patients reduced steroid dose. At 1 year, 80% of patients were off steroids. The mean HBI declined from a pre-Ifx value of 10.4 to 1.92 at 3 months, to 1.6 at 1 year and remained stable with 1.75 at 2 years (all p<0.01 or less vs baseline). CRP was significantly reduced at 3 and at 12 months after Ifx (pre-therapy 11.4±4.2 mg/l; 6 months: 6.9±3.1 mg/l, p<0.0 vs baseline; at 12 months: 5.04±2.9 mg/dl, p<0.01). 37 patients with perianal fistulas were evaluated at 6 and at 12 months achieving fistula closure in 32 and 54%, respectively, and a reduction of drainage in 62% and 32%, respectively. An endoscopic evaluation after 1 year was available from 86 patients revealing mucosal healing in 44 patients (51%). A total of 71 adverse reactions were observed leading to interruption of therapy in 23 patients (13%); no drug-related death occurred in this cohort. Conclusions: In this real live experience the benefits achieved with Ifx were comparable with those achieved in referral centres and in clinical studies with respect to steroid withdrawal/reduction, fistula healing and endoscopic healing. Adverse events do not occur more frequently than in referral centres. # G. Inflammatory bowel diseases - 2. Ulcerative colitis and Crohn’s disease diagnosis/therapy