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PA.144 ENTERIC GLIAL PROTEIN S100B UP-REGULATION STIMULATES NITRIC OXIDE PRODUCTION IN ULCERATIVE COLITIS
Abstracts / Digestive and Liver Disease 40S (2008), S1–S195 due to a disruption of Tight Junction (TJ) has been proposed as an etiological factor of IBD. Previous studies suggested that a disruption of intestinal barrier allows traslocation of toxic luminal antigen promoting intestinal inflammation. TNF-a is a proinflammatory cytokine that plays a central role in the intestinal inflammation of IBD. In vitro studies have demonstrated that TNF-a increases intestinal permeability through an enhanced myosin light chain kinase (MLCK) protein expression. The resulting alteration in intestinal permeability has been proposed to be an important proinflammatory mechanism contributing to intestinal inflammation in Crohn’s disease and other inflammatory conditions. The aim of this study was to analyze MLCK expression on protein extracts of endoscopic biopsies in patients with IBD. Material and methods: Specimens were obtained from 18 patients (10 M, 8 F), mean age 46 years (range 26-69), who underwent colonoscopy, at the University “Magna Graecia” Catanzaro. Ten patients with UC, 8 patients with CD and 2 controls were included. Mucosal biopsies were obtained from inflamed and non inflamed areas. Biopsy specimens were snap frozen in liquid nitrogen within one minute of endoscopic resection and stored at -80°C until utilization. A total tissue protein extract was obtained by homogenization and protein concentration
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Fig. 4. Western blot analysis to evaluate MLCK expression in patients with UC. There is the control with Tubulin. P = biopsy of tissue affected by disease. N = biopsy of normal tissue. CT = control (patient with irritable BS).
was measured by Bradford assay. Western immunoblot analysis was performed to evaluate MLCK expression. Results: MLCK protein expression is increased in involved areas compared with uninvolved areas. The enhanced expression of MLCK was associated with severe disease activity. MLCK expression was not found in non inflammed tissue of patients with IBD and in controls. Conclusions: Data demonstrate that MLCK protein expression is up-regulated in inflammed tissue of patients with IBD and that the enhanced expression correlates with disease activity. Moreover, it can be speculated that MLCK up regulation may be locally controlled and that MLCK expression is specific of inflammatory condition. # L. Inflammatory bowel diseases 1. Basic science
PA.144 ENTERIC GLIAL PROTEIN S100B UP-REGULATION STIMULATES NITRIC OXIDE PRODUCTION IN ULCERATIVE COLITIS Fig. 1. Western blot analysis to evaluate MLCK expression in patients with CD. There is the control with Tubulin. P = biopsy of tissue affected by disease. N = biopsy of normal tissue. CT = control (patient with irritable BS).
C. Cirillo ∗ ,1 , G. Sarnelli 1 , G. Esposito 2 , M. Grosso 1 , G. Mandarini 1 , R. Petruzzelli 1 , T. Iuvone 1 , R. Cuomo 1 1 University
Fig. 2. Western blot analysis to evaluate MLCK expression in patients with CD. There is the control with Tubulin. P = biopsy of tissue affected by disease. N = biopsy of normal tissue. CT = control (patient with irritable BS).
Fig. 3. Western blot analysis to evaluate MLCK expression in patients with UC. There is the control with Tubulin. P = biopsy of tissue affected by disease. N = biopsy of normal tissue. CT = control (patient with irritable BS).
“Federico II”, Naples; 2 University “La Sapienza”, Roma
Background and aim: In the central nervous system S100B protein has been associated with inflammation via nitric oxide (NO) production. Enteroglial cells (EGC) activation has been described in celiac disease, but its role in inflammatory bowel disease has been poorly investigated in humans. We evaluated the role of S100B and its association with NO production in ulcerative colitis (UC). Material and methods: Basal S100B mRNA and protein expression, iNOS expression and NO production were evaluated in rectal biopsies from 40 controls and 35 UC patients. To verify the correlation between EGC activation and NO production, biopsies were stimulated with exogenous S100B (0.005-5µM), either alone or in the presence of budesonide (BUD, 30µM) and receptor for advanced glycation endproducts antibody (RAGE, 1:1000). To test the role of EGC in experimental inflammation, S100B and iNOS expression were evaluated after incubation with lipopolysaccharides (LPS, 10µg/ml)+interferongamma (IFN-γ 300U/mL) either in the presence or absence of BUD (30µM); iNOS expression was evaluated in the presence of anti-RAGE (1:1000) or anti-S100B (1:1000) antibodies. Results: In biopsies from UC patients, S100B immunoreactivity, mRNA (10720%), protein expression (1476%) and release (537%) were significantly higher than in controls, parallel to iNOS expression and NO levels (1030 and 268%). Exogenous S100B induced a significant and concentration-dependent increase of both iNOS expression (174; 672; 914 and 1438%) and NO production (90; 228; 412 and 766%), which were inhibited by anti-RAGE antibody (-65 and -61%, respectively). In biopsies from controls and UC patients, LPS/IFN-γ induced a significant increase of S100B expression (1.4 and 10 fold
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Abstracts / Digestive and Liver Disease 40S (2008), S1–S195
increase vs basal) and release (0.05 and 0.31 ng/ml), which were not affected by BUD. LPS/IFN-γ stimulated iNOS expression and NO production (2.9 and 4.1 and 1.5 and 2.5 fold increase vs basal respectively), which were reduced by BUD, both in control and UC patients (-64 and -46% respectively); an inhibition was also obtained by anti-RAGE and anti-S100B antibodies (-61 and -68, -47 and -63% in controls and UC, respectively). Conclusions: We showed that EGC activation stimulates NO production in UC via S100B up-regulation and via activation of RAGE in a steroid insensitive pathway. Our data highlight a pivotal role of S100B in the mechanism of NO production underlying the involvement of EGC in gut inflammation. # L. Inflammatory bowel diseases 1. Basic science
PA.145 INCREASED THROMBIN GENERATION IN INFLAMMATORY BOWEL DISEASES V. Saladino ∗ ,1 , F. Villa 1 , S. Saibeni 2 , B. Savino 2 , M. Vecchi 3 , C. Sei 1 , R. de Franchis 1 , P.M. Mannucci 1 , V. Chantarangkul 1 , A. Tripodi 1 1 IRCCS
Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano; 2 Azienda Ospedaliera Fatebenefratelli e Oftalmico, Milano; 3 IRCCS Policlinico San Donato and University of Milan, San Donato Milanese
Background and aim: Inflammatory Bowel Diseases (IBD) are characterized by an increased thrombotic risk. Endogenous Thrombin Potential (ETP), a parameter of the thrombin generation curve, represents a new tool in the evaluation of thrombotic and bleeding disorders. Aim: to study ETP in IBD patients and to correlate the results with clinical and biochemical features. Material and methods: 74 IBD patients (37 ulcerative colitis and 37 Crohn’s disease) and 57 healthy controls enrolled. ETP values, measured with or without thrombomodulin, are expressed as nM thrombin times minutes. Results: In the presence of thrombomodulin, IBD patients with increased C-reactive protein (CRP) had significantly higher mean (± SD) ETP values (1,721.3±458.0 nM·min) than either patients with normal CRP (1,356.6±394.5 nM·min) and controls (1,277.1 ± 402.7 nM·min) (p<0.001). A significant correlation was observed between ETP and CRP (r=0.28, 95% C.I., 0.06-0.48, p=0.015) and erythrocyte sedimentation rate (ESR) (r=0.26, 95% C.I. 0.04-0.47, p=0.022). ETP values higher than the 95th percentile of control values were significantly more frequent in IBD patients than in controls (RR 1.71, 95% C.I. 1.33-2.20; p<0.002). Conclusions: ETP is increased in IBD patients, markedly in those with increased acute-phase reactants. ETP may help to identify IBD patients at increased thrombotic risk, regardless the underlying causes. # L. Inflammatory bowel diseases 1. Basic science
PA.146 UNDERSTANDING OF DISEASE IN PATIENTS WITH A RECENT DIAGNOSIS OF INFLAMMATORY BOWEL DISEASE M. Martinato ∗ , M. Ferrante, B. Burattin, V. Di Leo, L. Oliva, P. Bertomoro, M. Piovanello, T. Slongo, G.C. Sturniolo, R. D’Incà Azienda Ospedaliera - Università di Padova, Padova Background and aim: Inflammatory bowel diseases (IBD) have a negative impact on patients’ daily activities and health-related quality of life (HRQOL). Information about the disease may improve their HRQOL, reducing anxiety and depression, facilitating adherence to treatment, containing health-related costs and improving outcomes.
This study aims to identify the features of IBD patients exposed to the risk of knowing little about their disease and to establish which patients would benefit from educational programs. Material and methods: All IBD patients followed up at a tertiary referral centre, diagnosed less than 18 months previously and with no prior major surgery (ileostomy, ileo-anal pouch) or other concurrent chronic diseases, were tested with the Crohn’s and Colitis Knowledge (CCKNOW) Score, a valid index with a high internal consistency and a good level of reliability for assessing IBD patients’ understanding of their disease. Quality of life was investigated with the Short Inflammatory Bowel Disease Quality of Life (S-IBDQ) questionnaire. Disease activity was assessed using the MTWSI (Modified Truelove and Witts Severity Index) for ulcerative colitis (UC) and the Harvey Bradshaw Index for Crohn’s disease (CD). Results: Forty-nine patients gave their written informed consent and completed both questionnaires, and had their disease activity index assessed. More than half the patients had an appropriate understanding of IBD diagnostic tests, symptoms and general issues. Questions on medical treatment, disease aetiology, bowel anatomy and extra-intestinal manifestations were answered correctly by 25-30% of patients, while 15% knew about the side effects of medical therapy and 4% had some knowledge of the areas relating to fertility, pregnancy and surgery. The patients’ level of understanding of IBD correlated with their schooling (Spearman’s rho=0.40;p=0.01) and age (Spearman’s rho=0.30; p=0.05). Conclusions: Educational measures should target elderly patients, and those without higher education, focus particularly on the type of medical therapy and its side effects, and on the complications of surgery. Information provided during specialist consultations or hospital stays appears to have little effect on the patients’ understanding of IBD, so ad hoc patient education schemes should be implemented soon after a new case of IBD has been diagnosed. # L. Inflammatory bowel diseases 1. Basic science
PA.147 PROLONGED SCHEDULED THERAPY WITH INFLIXIMAB IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE: EVALUATION OF CLINICAL REMISSION AND MUCOSAL HEALING L. Grossi ∗ , M. Spezzaferro, L.F. Sacco, M.E. Serio, M. Amitrano, B. Cerasa, L. Marzio G d’Annunzio University School of Gastroenterology c/o Digestive Physiopathology, Ospedale Spirito Santo, Pescara Background and aim: The use of infliximab for one year is known to guarantee remission in a good percentage of patients with Crohn’s Disease(CD) and Ulcerative Colitis(UC) refractory or intolerant to traditional medical therapies. There is still a paucity of data regarding similar therapies prolonged after the conventional 12 months regimen in IBD patients. Aim of our study was to analyse the clinical, serological and endoscopic patterns in IBD patients receiving scheduled infliximab infusions for more than 1 year. Material and methods: Data are related to IBD patients of our Unit receiving scheduled infliximab for periods longer than 12 months, evaluating the clinical remission, mucosal healing, CRP levels and steroid sparing. Each of these patients received 8-weekly scheduled 5 mg/kg infliximab treatment, with endoscopic examination performed every 12 months. Laboratory exams were also performed every 3 months. In case of reduced effect the time interval between the infusions was reduced to 6 weeks, with the dose subsequently increased to 10 mg/kg every 8 weeks if further needed. The treatment was then stopped in case of lack of response. Results: A total of 39 patients, 23 CD and 16 UC were treated for a mean period of 34 months (range CD: 20-80 months, UC:16-26 months), obtaining the following results: Pts. in Clinical Remission: CD 21/23 (91%), UC 12/16 (75%); Pts with Mucosal healing (%):
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Fig. 4. Western blot analysis to evaluate MLCK expression in patients with UC. There is the control with Tubulin. P = biopsy of tissue affected by disease. N = biopsy of normal tissue. CT = control (patient with irritable BS).
was measured by Bradford assay. Western immunoblot analysis was performed to evaluate MLCK expression. Results: MLCK protein expression is increased in involved areas compared with uninvolved areas. The enhanced expression of MLCK was associated with severe disease activity. MLCK expression was not found in non inflammed tissue of patients with IBD and in controls. Conclusions: Data demonstrate that MLCK protein expression is up-regulated in inflammed tissue of patients with IBD and that the enhanced expression correlates with disease activity. Moreover, it can be speculated that MLCK up regulation may be locally controlled and that MLCK expression is specific of inflammatory condition. # L. Inflammatory bowel diseases 1. Basic science
PA.144 ENTERIC GLIAL PROTEIN S100B UP-REGULATION STIMULATES NITRIC OXIDE PRODUCTION IN ULCERATIVE COLITIS Fig. 1. Western blot analysis to evaluate MLCK expression in patients with CD. There is the control with Tubulin. P = biopsy of tissue affected by disease. N = biopsy of normal tissue. CT = control (patient with irritable BS).
C. Cirillo ∗ ,1 , G. Sarnelli 1 , G. Esposito 2 , M. Grosso 1 , G. Mandarini 1 , R. Petruzzelli 1 , T. Iuvone 1 , R. Cuomo 1 1 University
Fig. 2. Western blot analysis to evaluate MLCK expression in patients with CD. There is the control with Tubulin. P = biopsy of tissue affected by disease. N = biopsy of normal tissue. CT = control (patient with irritable BS).
Fig. 3. Western blot analysis to evaluate MLCK expression in patients with UC. There is the control with Tubulin. P = biopsy of tissue affected by disease. N = biopsy of normal tissue. CT = control (patient with irritable BS).
“Federico II”, Naples; 2 University “La Sapienza”, Roma
Background and aim: In the central nervous system S100B protein has been associated with inflammation via nitric oxide (NO) production. Enteroglial cells (EGC) activation has been described in celiac disease, but its role in inflammatory bowel disease has been poorly investigated in humans. We evaluated the role of S100B and its association with NO production in ulcerative colitis (UC). Material and methods: Basal S100B mRNA and protein expression, iNOS expression and NO production were evaluated in rectal biopsies from 40 controls and 35 UC patients. To verify the correlation between EGC activation and NO production, biopsies were stimulated with exogenous S100B (0.005-5µM), either alone or in the presence of budesonide (BUD, 30µM) and receptor for advanced glycation endproducts antibody (RAGE, 1:1000). To test the role of EGC in experimental inflammation, S100B and iNOS expression were evaluated after incubation with lipopolysaccharides (LPS, 10µg/ml)+interferongamma (IFN-γ 300U/mL) either in the presence or absence of BUD (30µM); iNOS expression was evaluated in the presence of anti-RAGE (1:1000) or anti-S100B (1:1000) antibodies. Results: In biopsies from UC patients, S100B immunoreactivity, mRNA (10720%), protein expression (1476%) and release (537%) were significantly higher than in controls, parallel to iNOS expression and NO levels (1030 and 268%). Exogenous S100B induced a significant and concentration-dependent increase of both iNOS expression (174; 672; 914 and 1438%) and NO production (90; 228; 412 and 766%), which were inhibited by anti-RAGE antibody (-65 and -61%, respectively). In biopsies from controls and UC patients, LPS/IFN-γ induced a significant increase of S100B expression (1.4 and 10 fold
S128
Abstracts / Digestive and Liver Disease 40S (2008), S1–S195
increase vs basal) and release (0.05 and 0.31 ng/ml), which were not affected by BUD. LPS/IFN-γ stimulated iNOS expression and NO production (2.9 and 4.1 and 1.5 and 2.5 fold increase vs basal respectively), which were reduced by BUD, both in control and UC patients (-64 and -46% respectively); an inhibition was also obtained by anti-RAGE and anti-S100B antibodies (-61 and -68, -47 and -63% in controls and UC, respectively). Conclusions: We showed that EGC activation stimulates NO production in UC via S100B up-regulation and via activation of RAGE in a steroid insensitive pathway. Our data highlight a pivotal role of S100B in the mechanism of NO production underlying the involvement of EGC in gut inflammation. # L. Inflammatory bowel diseases 1. Basic science
PA.145 INCREASED THROMBIN GENERATION IN INFLAMMATORY BOWEL DISEASES V. Saladino ∗ ,1 , F. Villa 1 , S. Saibeni 2 , B. Savino 2 , M. Vecchi 3 , C. Sei 1 , R. de Franchis 1 , P.M. Mannucci 1 , V. Chantarangkul 1 , A. Tripodi 1 1 IRCCS
Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano; 2 Azienda Ospedaliera Fatebenefratelli e Oftalmico, Milano; 3 IRCCS Policlinico San Donato and University of Milan, San Donato Milanese
Background and aim: Inflammatory Bowel Diseases (IBD) are characterized by an increased thrombotic risk. Endogenous Thrombin Potential (ETP), a parameter of the thrombin generation curve, represents a new tool in the evaluation of thrombotic and bleeding disorders. Aim: to study ETP in IBD patients and to correlate the results with clinical and biochemical features. Material and methods: 74 IBD patients (37 ulcerative colitis and 37 Crohn’s disease) and 57 healthy controls enrolled. ETP values, measured with or without thrombomodulin, are expressed as nM thrombin times minutes. Results: In the presence of thrombomodulin, IBD patients with increased C-reactive protein (CRP) had significantly higher mean (± SD) ETP values (1,721.3±458.0 nM·min) than either patients with normal CRP (1,356.6±394.5 nM·min) and controls (1,277.1 ± 402.7 nM·min) (p<0.001). A significant correlation was observed between ETP and CRP (r=0.28, 95% C.I., 0.06-0.48, p=0.015) and erythrocyte sedimentation rate (ESR) (r=0.26, 95% C.I. 0.04-0.47, p=0.022). ETP values higher than the 95th percentile of control values were significantly more frequent in IBD patients than in controls (RR 1.71, 95% C.I. 1.33-2.20; p<0.002). Conclusions: ETP is increased in IBD patients, markedly in those with increased acute-phase reactants. ETP may help to identify IBD patients at increased thrombotic risk, regardless the underlying causes. # L. Inflammatory bowel diseases 1. Basic science
PA.146 UNDERSTANDING OF DISEASE IN PATIENTS WITH A RECENT DIAGNOSIS OF INFLAMMATORY BOWEL DISEASE M. Martinato ∗ , M. Ferrante, B. Burattin, V. Di Leo, L. Oliva, P. Bertomoro, M. Piovanello, T. Slongo, G.C. Sturniolo, R. D’Incà Azienda Ospedaliera - Università di Padova, Padova Background and aim: Inflammatory bowel diseases (IBD) have a negative impact on patients’ daily activities and health-related quality of life (HRQOL). Information about the disease may improve their HRQOL, reducing anxiety and depression, facilitating adherence to treatment, containing health-related costs and improving outcomes.
This study aims to identify the features of IBD patients exposed to the risk of knowing little about their disease and to establish which patients would benefit from educational programs. Material and methods: All IBD patients followed up at a tertiary referral centre, diagnosed less than 18 months previously and with no prior major surgery (ileostomy, ileo-anal pouch) or other concurrent chronic diseases, were tested with the Crohn’s and Colitis Knowledge (CCKNOW) Score, a valid index with a high internal consistency and a good level of reliability for assessing IBD patients’ understanding of their disease. Quality of life was investigated with the Short Inflammatory Bowel Disease Quality of Life (S-IBDQ) questionnaire. Disease activity was assessed using the MTWSI (Modified Truelove and Witts Severity Index) for ulcerative colitis (UC) and the Harvey Bradshaw Index for Crohn’s disease (CD). Results: Forty-nine patients gave their written informed consent and completed both questionnaires, and had their disease activity index assessed. More than half the patients had an appropriate understanding of IBD diagnostic tests, symptoms and general issues. Questions on medical treatment, disease aetiology, bowel anatomy and extra-intestinal manifestations were answered correctly by 25-30% of patients, while 15% knew about the side effects of medical therapy and 4% had some knowledge of the areas relating to fertility, pregnancy and surgery. The patients’ level of understanding of IBD correlated with their schooling (Spearman’s rho=0.40;p=0.01) and age (Spearman’s rho=0.30; p=0.05). Conclusions: Educational measures should target elderly patients, and those without higher education, focus particularly on the type of medical therapy and its side effects, and on the complications of surgery. Information provided during specialist consultations or hospital stays appears to have little effect on the patients’ understanding of IBD, so ad hoc patient education schemes should be implemented soon after a new case of IBD has been diagnosed. # L. Inflammatory bowel diseases 1. Basic science
PA.147 PROLONGED SCHEDULED THERAPY WITH INFLIXIMAB IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE: EVALUATION OF CLINICAL REMISSION AND MUCOSAL HEALING L. Grossi ∗ , M. Spezzaferro, L.F. Sacco, M.E. Serio, M. Amitrano, B. Cerasa, L. Marzio G d’Annunzio University School of Gastroenterology c/o Digestive Physiopathology, Ospedale Spirito Santo, Pescara Background and aim: The use of infliximab for one year is known to guarantee remission in a good percentage of patients with Crohn’s Disease(CD) and Ulcerative Colitis(UC) refractory or intolerant to traditional medical therapies. There is still a paucity of data regarding similar therapies prolonged after the conventional 12 months regimen in IBD patients. Aim of our study was to analyse the clinical, serological and endoscopic patterns in IBD patients receiving scheduled infliximab infusions for more than 1 year. Material and methods: Data are related to IBD patients of our Unit receiving scheduled infliximab for periods longer than 12 months, evaluating the clinical remission, mucosal healing, CRP levels and steroid sparing. Each of these patients received 8-weekly scheduled 5 mg/kg infliximab treatment, with endoscopic examination performed every 12 months. Laboratory exams were also performed every 3 months. In case of reduced effect the time interval between the infusions was reduced to 6 weeks, with the dose subsequently increased to 10 mg/kg every 8 weeks if further needed. The treatment was then stopped in case of lack of response. Results: A total of 39 patients, 23 CD and 16 UC were treated for a mean period of 34 months (range CD: 20-80 months, UC:16-26 months), obtaining the following results: Pts. in Clinical Remission: CD 21/23 (91%), UC 12/16 (75%); Pts with Mucosal healing (%):