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Th2 cytokines detected in colonic tissues of inflammatory bowel disease patients but not controls
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Poster Presentations
P314 Mycological stool examination, anti-Candida mannan antibodies and serum concentration of interleukin 10 in ulcerative colitis D. Ksia˛dzyna *, J. Semian´ ow-Wejchert, U. Nawrot, K. Wlodarczyk. Medical University, Wroclaw, Poland Aim: The idea that microorganisms play a role in the aetiology of ulcerative colitis (UC) has gained ground considerably in recent years. Interleukin 10 (IL-10) is an immunomodulatory molecule that inhibits the proinflammatory cytokines, however its colonic activity seems to be insufficient to control inflammation in UC. Some observations suggest that IL-10 might be involved in antifungal immunity. Anti-Candida mannan antibodies (ACMA) and mycological stool examination help to diagnose often latent fungal infections in UC. Although there is evidence for immunomodulation disorders in the response to intestinal flora, published studies dealing with fungi in UC are sparse. Therefore the aim of this study was to assess IL-10 serum concentration, ACMA serum levels and results of mycological stool examination according to the disease activity in UC patients. Materials and Methods: Forty-two consecutive patients (32 females, 10 males; mean age: 43.05±10.22 yrs) with already established diagnosis of UC were subjected to the study. Clinical and endoscopic activity of UC (according to Rachmilewitz’s index) ranged from 0 to 15 (mean 6.51±4.13) points and from 2 to 8 (mean 5.22±2.96) points, respectively. IL-10 serum concentration and serum level of ACMA were assessed with ELISA according to manufacturers’ instructions. IL-10 serum concentration less than 0.5 pg/mL was regarded as negative. The minimal detectable ACMA level was 2.5 U/mL. The quantitive and qualitative mycological stool examinations were performed in all subjects. Significant fungal colonization (SFC) was recognized in case of more than 105 colony forming units (CFU) per 1.0 g of stool. Results: The results of mycological stool culture are shown in the table. All subjects with SFC suffered from active UC with clinical activity index within 9 12 points. Qualitative mycological examination showed marked predominance of Candida albicans that was found in 91.66% patients. Mycological stool culture
UC patients (n = 42)
Negative Positive SFC 1 fungal species 2 fungal species 3 fungal species
6 (14.28%) 36 (85.71%) 3 (8.33%) 21 (58.33%) 11 (30.55%) 4 (11.11%)
UC
ulcerative colitis; SFC
significant fungal colonization.
In 20 patients (7 in remission, 12 with mild and moderate disease activity and 1 with severe course of UC) IL-10 serum concentration was below the test sensitivity. In 11 patients (4 in remission, 2 with mild, 3 with moderate disease activity and 2 with severe course of UC) IL-10 concentrations ranged from 0.78 to 9.43 (mean: 3.38±2.8) pg/mL. IL-10 serum concentration correlated with disease activity only in patients with severe course of UC (r = 0.61 and r = 0.59, respectively). ACMA were observed in 8 (19.04%) patients, all in an active phase of the disease and their levels ranged from 4.4 to 19.5 (mean: 10.72) U/mL. No statistical difference was revealed between serum level of ACMA and diverse disease activity (p < 0.05). No correlation between SFC, ACMA and IL-10 serum concentration was disclosed. Conclusion: According to our study, there is correlation between IL-10 serum concentration and disease activity only in patients with severe course of UC, but not with the level of AMCA and fungal colonization of the gastrointestinal tract.
P315 Fecal biomarkers as assessment of inflammatory bowel disease activity A. Vieira Sr.1 *, C.B. Fang Sr.1 , E.G. Rolim Sr.1 , W.A. Klug Sr.1 , F. Steinwurz Sr.2 , L. Rossini1 , P.A. Candel´ aria Sr.1 . 1 Santa Casa de S˜ ao Paulo, S˜ ao Paulo, Brazil, 2 Albert Einstein Hospital, S˜ ao Paulo, Brazil Research has shown that fecal biomarkers are useful to assess the activity of inflammatory bowel disease (IBD). The aim of the this study is: (1) to evaluate the efficacy of the fecal lactoferrin and calprotectin as indicators of inflammatory activity; (2) to verify whether the combination of these two tests improves the performance of inflammatory activity assessment; and (3) to observe whether there are different patterns in the results of patients with Crohn’s disease (CD) or Ulcerative Colitis (UC).A total of 78 patients presenting inflammatory bowel disease were evaluated. The Crohn’s disease Activity Index (CDAI), Modified Mayo Disease Activity Index (MMDAI), and Crohn’s disease Endoscopic Index of Severity (CDEIS) were used for the clinical and endoscopic evaluation. Two tests were performed on the fecal samples, to check the levels of calprotectin and lactoferrin. Histological evaluation was considered to be the gold standard for the detection of inflammation. Correlations were made among the following variables: fecal biomarkers, histological evaluation, CDAI, MMDAI and CDEIS.A total of 52 patient’s samples whose histological evaluations showed inflammation, 49 were lactoferrinpositive, and 40 were calprotectin-positive (p = 0.000). Fecal calprotectin levels correlated with the degree of inflammation observed (p = 0.000). Fecal lactoferrin and calprotectin levels correlated with CDAI values (p = 0.043; 0.010) and CDEIS values in DC cases (p = 0.000; 0.000), and correlated with MMDAI values in UC cases (p = 0.000). Calprotectin presence correlated with lactoferrin presence at a highly significant level (p < 0.001). The results were similar for the two groups of patients. Fecal lactoferrin and calprotectin are highly sensitive and specific markers for detecting intestinal inflammation. Levels of fecal calprotectin have a proportional correlation to the degree of inflammation of the intestinal mucosa. However, combining the two fecal markers does not enhance the detection rate of inflammatory activity, indicating that either alone can be considered adequate. Markers are similarly useful for both CD and UC. P316 TNF-a production and differential release of Th1/Th2 cytokines detected in colonic tissues of inflammatory bowel disease patients but not controls S.C. Ng1 *, N.E. McCarthy1 , S. Plamondon1 , M.A. Kamm1 , A.J. Stagg2 , S.C. Knight1 . 1 Antigen Presentation Research Group, St Mark’s Hospital, London, United Kingdom, 2 Bart’s and the Royal London Hospital, London, United Kingdom Background: Inflammatory bowel disease (IBD) comprises Crohn’s disease (CD) and ulcerative colitis (UC). CD predominantly exhibits Th1 profile while UC is weakly associated with Th2 immunity. Better understanding of inflammatory profiles accompanying distinct forms of IBD will aid development of patient-specific therapies. Aims and Methods: Colonic biopsies were obtained from patients with CD, UC, irritable bowel syndrome (IBS) and healthy controls. Tissue was incubated overnight in tissue culture medium and cytokine content was analysed by multiplex bead array. Trends in cytokine production and associations between mediators were determined for each patient subtype (UC, CD, IBS or control) and in a subgroup of patients treated with probiotics. Results: Increased levels of IL-6 were seen in IBD patients compared with IBS patients or controls. Supernatants from all biopsies showed production of cytokines associated with
Abstracts of the 4th Congress of ECCO
the European Crohn’s and Colitis Organisation
tissue trauma; IL-1b, IL-10 and IL-6, which together exhibited a significant positive correlation. Production of IL-1b, a key mediator of epithelial inflammation, correlated significantly with TNF-a production, but only in samples from CD or UC. TNF-a release was further associated with IL-12p70(Th1) production in UC, and IL-5(Th2) production in CD. UC patients who had probiotics showed significant decreases in IL-2 levels, suggesting general suppression of T-cell activity. Conclusions: Cytokines associated with tissue trauma were produced from all gut biopsies, but accompanied by TNFa only in tissue from IBD patients. TNF-a in IBD was further associated with production of Th1/Th2 cytokines of opposing bias to the inflammatory profile thought to predominate in these patients probably indicating the counter-balancing of pathologic immune responses in the gut. P317 Mucosa-associated Escherichia coli and Faecalibacterium prausnitzii quantification by real-time PCR and its potential use as complementary diagnostic tool for inflammatory bowel diseases M. Lopez-Siles1 *, M. Martinez-Medina1 , X. Aldeguer2 , L. Garcia-Gil1 . 1 Laboratori de Microbiologia Molecular, Departament de Biologia, Universitat de Girona, Girona, Spain, 2 Departament de Gastroenterologia, Hospital Dr. Josep Trueta, Girona, Spain Background: It has been demonstrated that CD patients are distinguishable from C subjects according to their intestinal microbial composition, as revealed by qualitative molecular methods [1]. Escherichia coli and Faecalibacterium prausnitzii have been reported to be the main representatives of CD dysbiosis. Aim: The aim of this study was to determine if the quantification of E.coli and F.prausnitzii by real-time PCR could be used as complementary tool for diagnostic purposes in inflammatory bowel diseases. Materials and Methods: E.coli and F.prausnitzii were quantified using a previously reported real-time PCR assay [2] and a new real-time PCR assay designed in our laboratory, respectively. The abundance of E.coli was determined in 26 CD patients, 17 C subjects and 8 patients suffering from UC, whereas F.prausnitzii was quantified in 11 C subjects and 10 CD patients. Both studies were focused on the quantification of those bacteria intimately adhered to the mucosa. Thus, transient and loosely attached bacteria were discarded by mild sonication. Moreover, a quantification of human cells was performed in order to correct variability due to sample size and efficiency of DNA extraction (data are expressed as log bacterial cells/106 human cells). Results: Irrespectively of the zone sampled, CD patients carried higher quantity of E.coli (6.54±1.97 log E.coli /106 human cells) than C (4.97±2.29) and UC patients (4.49±0.71) (p = 0.010), which is in agreement with previous studies [3]. Among CD patients, those with I-CD showed highest E.coli abundance in comparison with those patients with other affectations (I-CD: 8.1±1.55, IC-CD: 4.96±0.72, C-CD: 5.97±1.32; p = 0.001). Concerning F.prausnitzii, CD patients harboured a lower quantity of F.prausnitzii (3.10±1.40 log F.prausnitzii /106 human cells) than C subjects (3.88±0.76) (p = 0.036). For this bacterial indicator, differences among distinct CD affectations were also observed (I-CD: 2.58±1.28, IC-CD: 2.80±1.33, C-CD: 4.64±0.49; p = 0.035). Conclusions: Our methodological approach revealed that in CD patients, E.coli numbers were higher than in C subjects, whereas F.prausnitzii numbers were lower, which makes this a potential diagnostic tool. Differences were especially clear for CD patients with ileal involvement, which is in agreement with previous reports [3]. Since current trends distinguish between I-CD and C-CD as two different pathological entities [4,5],
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additional bacterial indicators must be searched to find effective tools for C-CD and UC differential diagnose. Abbreviations: CD: Crohn’s disease, UC: ulcerative colitis, C: control, I-CD: Crohn’s ileitis, C-CD: Crohn’s colitis, IC-CD: Crohn’s disease with ileo-colonic affectation. Reference(s) [1] Martinez-Medina, M., et al. Inflamm Bowel Dis 2006; 12: 1136 45. [2] Huijsdens, X.W., et al. J Clin Microbiol 2002; 40: 4423 7. [3] Baumgart, M., et al., The ISME Journal. 2007; 1: 403 18. [4] Hanauer, S.B., et al, Inflamm Bowel Dis 2006; 12(Suppl 1): S3 9. [5] Sartor R.B., et al, Nat Clin Pract Gastroenterol Hepatol 2006; 3: 390 407. P318 Pouchitis and bacterial flora M. Scarpa1,2 *, A. Grillo2 , I. Castagliuolo2 , D. Faggian3 , E. Bonello4 , C. Ruffolo5 , R. D’Inc` a5 , I. Angriman5 . 1 Veneto Oncological Institute, Dept of Surgery, Padova, Italy, 2 Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Padova, Italy, 3 Dept. of Diagnostic Sciences and Special Therapy, University of Padova, Italy, Padova, Italy, 4 4Dept. of Surgical and Gastroenterological Sciences, University of Padova, Padova, Italy, 5 Dept. of Surgical and Gastroenterological Sciences, University of Padova, Padova, Italy Background: Pouchitis is an idiopathic chronic inflammatory disease. Cumulative risk of suffering of one or more episodes of pouchitis is almost 50% within the first five years after restorative proctocolectomy for ulcerative colitis. Aims of our study were to assess the relations between pouch bacterial populations and disease activity, systemic and local inflammation, local cytokines network and intracellular inflammatory cascade. Patients and Methods: In this cross-sectional study 32 consecutive patients who underwent restorative proctocolectomy, coming for follow up endoscopy in our out-patient department were recruited. Clinical disease activity was classified using Pouchitis Disease Activity Index. Systemic inflammatory status was graded according to: blood cell count, ESR, CRP, and albuminemia. Local inflammatory status was assessed with faecal lactoferrin levels analyzed by quantitative ELISA. During pouch endoscopy biopsies from the ileal pouch were obtained: two samples to culture bacteria adherent to the mucosa, two for conventional histology, two for cytokine pattern assessment, two for molecular biology. Serum and mucosal levels of IL-1b, IL-6 and TNF-a were measured with immunometric assays. Finally, mRNA level for Toll like receptors for bacterial lipopolysaccharide (TLR4) and peptidoglycan (TLR2) were measured by quantitative Real Time RT-PCR. Results: Histological diagnosis of pouchitis was made in 20 patients while clinical diagnosis (PDAI > 7) was made in 10 patients. In patients with histological diagnosis of pouchitis mucosal IL-1b was more expressed than in those with healthy mucosa (p = 0.018). Mucosal IL-1b correlated directly with the number of CFU of Thermosinus Carboxydivorans (r = 0.52, p = 0.018) adherent to the mucosa. Granulocytes and monocytes mucosal infiltrate were more severe in patients with histological diagnosis of pouchitis (p < 0.001 for both). Monocytes and granulocytes infiltrate correlated directly with the number of CFU of Bacteriodiaceae adherent to the mucosa (r = 0.52, p = 0.019, r = 0.53, p = 0.009). Moreover, granulocytes infiltrate inversely correlated with the number of CFU of Enterobacteriaceae adherent to the mucosa (r = 0.45, p = 0.044). In patients with clinical diagnosis of pouchitis the total number of CFU of Enterococcaceae and of Enterobacteriaceae in the mucus was lower than in healthy patients (p = 0.023 and p = 0.018).
Poster Presentations
P314 Mycological stool examination, anti-Candida mannan antibodies and serum concentration of interleukin 10 in ulcerative colitis D. Ksia˛dzyna *, J. Semian´ ow-Wejchert, U. Nawrot, K. Wlodarczyk. Medical University, Wroclaw, Poland Aim: The idea that microorganisms play a role in the aetiology of ulcerative colitis (UC) has gained ground considerably in recent years. Interleukin 10 (IL-10) is an immunomodulatory molecule that inhibits the proinflammatory cytokines, however its colonic activity seems to be insufficient to control inflammation in UC. Some observations suggest that IL-10 might be involved in antifungal immunity. Anti-Candida mannan antibodies (ACMA) and mycological stool examination help to diagnose often latent fungal infections in UC. Although there is evidence for immunomodulation disorders in the response to intestinal flora, published studies dealing with fungi in UC are sparse. Therefore the aim of this study was to assess IL-10 serum concentration, ACMA serum levels and results of mycological stool examination according to the disease activity in UC patients. Materials and Methods: Forty-two consecutive patients (32 females, 10 males; mean age: 43.05±10.22 yrs) with already established diagnosis of UC were subjected to the study. Clinical and endoscopic activity of UC (according to Rachmilewitz’s index) ranged from 0 to 15 (mean 6.51±4.13) points and from 2 to 8 (mean 5.22±2.96) points, respectively. IL-10 serum concentration and serum level of ACMA were assessed with ELISA according to manufacturers’ instructions. IL-10 serum concentration less than 0.5 pg/mL was regarded as negative. The minimal detectable ACMA level was 2.5 U/mL. The quantitive and qualitative mycological stool examinations were performed in all subjects. Significant fungal colonization (SFC) was recognized in case of more than 105 colony forming units (CFU) per 1.0 g of stool. Results: The results of mycological stool culture are shown in the table. All subjects with SFC suffered from active UC with clinical activity index within 9 12 points. Qualitative mycological examination showed marked predominance of Candida albicans that was found in 91.66% patients. Mycological stool culture
UC patients (n = 42)
Negative Positive SFC 1 fungal species 2 fungal species 3 fungal species
6 (14.28%) 36 (85.71%) 3 (8.33%) 21 (58.33%) 11 (30.55%) 4 (11.11%)
UC
ulcerative colitis; SFC
significant fungal colonization.
In 20 patients (7 in remission, 12 with mild and moderate disease activity and 1 with severe course of UC) IL-10 serum concentration was below the test sensitivity. In 11 patients (4 in remission, 2 with mild, 3 with moderate disease activity and 2 with severe course of UC) IL-10 concentrations ranged from 0.78 to 9.43 (mean: 3.38±2.8) pg/mL. IL-10 serum concentration correlated with disease activity only in patients with severe course of UC (r = 0.61 and r = 0.59, respectively). ACMA were observed in 8 (19.04%) patients, all in an active phase of the disease and their levels ranged from 4.4 to 19.5 (mean: 10.72) U/mL. No statistical difference was revealed between serum level of ACMA and diverse disease activity (p < 0.05). No correlation between SFC, ACMA and IL-10 serum concentration was disclosed. Conclusion: According to our study, there is correlation between IL-10 serum concentration and disease activity only in patients with severe course of UC, but not with the level of AMCA and fungal colonization of the gastrointestinal tract.
P315 Fecal biomarkers as assessment of inflammatory bowel disease activity A. Vieira Sr.1 *, C.B. Fang Sr.1 , E.G. Rolim Sr.1 , W.A. Klug Sr.1 , F. Steinwurz Sr.2 , L. Rossini1 , P.A. Candel´ aria Sr.1 . 1 Santa Casa de S˜ ao Paulo, S˜ ao Paulo, Brazil, 2 Albert Einstein Hospital, S˜ ao Paulo, Brazil Research has shown that fecal biomarkers are useful to assess the activity of inflammatory bowel disease (IBD). The aim of the this study is: (1) to evaluate the efficacy of the fecal lactoferrin and calprotectin as indicators of inflammatory activity; (2) to verify whether the combination of these two tests improves the performance of inflammatory activity assessment; and (3) to observe whether there are different patterns in the results of patients with Crohn’s disease (CD) or Ulcerative Colitis (UC).A total of 78 patients presenting inflammatory bowel disease were evaluated. The Crohn’s disease Activity Index (CDAI), Modified Mayo Disease Activity Index (MMDAI), and Crohn’s disease Endoscopic Index of Severity (CDEIS) were used for the clinical and endoscopic evaluation. Two tests were performed on the fecal samples, to check the levels of calprotectin and lactoferrin. Histological evaluation was considered to be the gold standard for the detection of inflammation. Correlations were made among the following variables: fecal biomarkers, histological evaluation, CDAI, MMDAI and CDEIS.A total of 52 patient’s samples whose histological evaluations showed inflammation, 49 were lactoferrinpositive, and 40 were calprotectin-positive (p = 0.000). Fecal calprotectin levels correlated with the degree of inflammation observed (p = 0.000). Fecal lactoferrin and calprotectin levels correlated with CDAI values (p = 0.043; 0.010) and CDEIS values in DC cases (p = 0.000; 0.000), and correlated with MMDAI values in UC cases (p = 0.000). Calprotectin presence correlated with lactoferrin presence at a highly significant level (p < 0.001). The results were similar for the two groups of patients. Fecal lactoferrin and calprotectin are highly sensitive and specific markers for detecting intestinal inflammation. Levels of fecal calprotectin have a proportional correlation to the degree of inflammation of the intestinal mucosa. However, combining the two fecal markers does not enhance the detection rate of inflammatory activity, indicating that either alone can be considered adequate. Markers are similarly useful for both CD and UC. P316 TNF-a production and differential release of Th1/Th2 cytokines detected in colonic tissues of inflammatory bowel disease patients but not controls S.C. Ng1 *, N.E. McCarthy1 , S. Plamondon1 , M.A. Kamm1 , A.J. Stagg2 , S.C. Knight1 . 1 Antigen Presentation Research Group, St Mark’s Hospital, London, United Kingdom, 2 Bart’s and the Royal London Hospital, London, United Kingdom Background: Inflammatory bowel disease (IBD) comprises Crohn’s disease (CD) and ulcerative colitis (UC). CD predominantly exhibits Th1 profile while UC is weakly associated with Th2 immunity. Better understanding of inflammatory profiles accompanying distinct forms of IBD will aid development of patient-specific therapies. Aims and Methods: Colonic biopsies were obtained from patients with CD, UC, irritable bowel syndrome (IBS) and healthy controls. Tissue was incubated overnight in tissue culture medium and cytokine content was analysed by multiplex bead array. Trends in cytokine production and associations between mediators were determined for each patient subtype (UC, CD, IBS or control) and in a subgroup of patients treated with probiotics. Results: Increased levels of IL-6 were seen in IBD patients compared with IBS patients or controls. Supernatants from all biopsies showed production of cytokines associated with
Abstracts of the 4th Congress of ECCO
the European Crohn’s and Colitis Organisation
tissue trauma; IL-1b, IL-10 and IL-6, which together exhibited a significant positive correlation. Production of IL-1b, a key mediator of epithelial inflammation, correlated significantly with TNF-a production, but only in samples from CD or UC. TNF-a release was further associated with IL-12p70(Th1) production in UC, and IL-5(Th2) production in CD. UC patients who had probiotics showed significant decreases in IL-2 levels, suggesting general suppression of T-cell activity. Conclusions: Cytokines associated with tissue trauma were produced from all gut biopsies, but accompanied by TNFa only in tissue from IBD patients. TNF-a in IBD was further associated with production of Th1/Th2 cytokines of opposing bias to the inflammatory profile thought to predominate in these patients probably indicating the counter-balancing of pathologic immune responses in the gut. P317 Mucosa-associated Escherichia coli and Faecalibacterium prausnitzii quantification by real-time PCR and its potential use as complementary diagnostic tool for inflammatory bowel diseases M. Lopez-Siles1 *, M. Martinez-Medina1 , X. Aldeguer2 , L. Garcia-Gil1 . 1 Laboratori de Microbiologia Molecular, Departament de Biologia, Universitat de Girona, Girona, Spain, 2 Departament de Gastroenterologia, Hospital Dr. Josep Trueta, Girona, Spain Background: It has been demonstrated that CD patients are distinguishable from C subjects according to their intestinal microbial composition, as revealed by qualitative molecular methods [1]. Escherichia coli and Faecalibacterium prausnitzii have been reported to be the main representatives of CD dysbiosis. Aim: The aim of this study was to determine if the quantification of E.coli and F.prausnitzii by real-time PCR could be used as complementary tool for diagnostic purposes in inflammatory bowel diseases. Materials and Methods: E.coli and F.prausnitzii were quantified using a previously reported real-time PCR assay [2] and a new real-time PCR assay designed in our laboratory, respectively. The abundance of E.coli was determined in 26 CD patients, 17 C subjects and 8 patients suffering from UC, whereas F.prausnitzii was quantified in 11 C subjects and 10 CD patients. Both studies were focused on the quantification of those bacteria intimately adhered to the mucosa. Thus, transient and loosely attached bacteria were discarded by mild sonication. Moreover, a quantification of human cells was performed in order to correct variability due to sample size and efficiency of DNA extraction (data are expressed as log bacterial cells/106 human cells). Results: Irrespectively of the zone sampled, CD patients carried higher quantity of E.coli (6.54±1.97 log E.coli /106 human cells) than C (4.97±2.29) and UC patients (4.49±0.71) (p = 0.010), which is in agreement with previous studies [3]. Among CD patients, those with I-CD showed highest E.coli abundance in comparison with those patients with other affectations (I-CD: 8.1±1.55, IC-CD: 4.96±0.72, C-CD: 5.97±1.32; p = 0.001). Concerning F.prausnitzii, CD patients harboured a lower quantity of F.prausnitzii (3.10±1.40 log F.prausnitzii /106 human cells) than C subjects (3.88±0.76) (p = 0.036). For this bacterial indicator, differences among distinct CD affectations were also observed (I-CD: 2.58±1.28, IC-CD: 2.80±1.33, C-CD: 4.64±0.49; p = 0.035). Conclusions: Our methodological approach revealed that in CD patients, E.coli numbers were higher than in C subjects, whereas F.prausnitzii numbers were lower, which makes this a potential diagnostic tool. Differences were especially clear for CD patients with ileal involvement, which is in agreement with previous reports [3]. Since current trends distinguish between I-CD and C-CD as two different pathological entities [4,5],
S135
additional bacterial indicators must be searched to find effective tools for C-CD and UC differential diagnose. Abbreviations: CD: Crohn’s disease, UC: ulcerative colitis, C: control, I-CD: Crohn’s ileitis, C-CD: Crohn’s colitis, IC-CD: Crohn’s disease with ileo-colonic affectation. Reference(s) [1] Martinez-Medina, M., et al. Inflamm Bowel Dis 2006; 12: 1136 45. [2] Huijsdens, X.W., et al. J Clin Microbiol 2002; 40: 4423 7. [3] Baumgart, M., et al., The ISME Journal. 2007; 1: 403 18. [4] Hanauer, S.B., et al, Inflamm Bowel Dis 2006; 12(Suppl 1): S3 9. [5] Sartor R.B., et al, Nat Clin Pract Gastroenterol Hepatol 2006; 3: 390 407. P318 Pouchitis and bacterial flora M. Scarpa1,2 *, A. Grillo2 , I. Castagliuolo2 , D. Faggian3 , E. Bonello4 , C. Ruffolo5 , R. D’Inc` a5 , I. Angriman5 . 1 Veneto Oncological Institute, Dept of Surgery, Padova, Italy, 2 Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Padova, Italy, 3 Dept. of Diagnostic Sciences and Special Therapy, University of Padova, Italy, Padova, Italy, 4 4Dept. of Surgical and Gastroenterological Sciences, University of Padova, Padova, Italy, 5 Dept. of Surgical and Gastroenterological Sciences, University of Padova, Padova, Italy Background: Pouchitis is an idiopathic chronic inflammatory disease. Cumulative risk of suffering of one or more episodes of pouchitis is almost 50% within the first five years after restorative proctocolectomy for ulcerative colitis. Aims of our study were to assess the relations between pouch bacterial populations and disease activity, systemic and local inflammation, local cytokines network and intracellular inflammatory cascade. Patients and Methods: In this cross-sectional study 32 consecutive patients who underwent restorative proctocolectomy, coming for follow up endoscopy in our out-patient department were recruited. Clinical disease activity was classified using Pouchitis Disease Activity Index. Systemic inflammatory status was graded according to: blood cell count, ESR, CRP, and albuminemia. Local inflammatory status was assessed with faecal lactoferrin levels analyzed by quantitative ELISA. During pouch endoscopy biopsies from the ileal pouch were obtained: two samples to culture bacteria adherent to the mucosa, two for conventional histology, two for cytokine pattern assessment, two for molecular biology. Serum and mucosal levels of IL-1b, IL-6 and TNF-a were measured with immunometric assays. Finally, mRNA level for Toll like receptors for bacterial lipopolysaccharide (TLR4) and peptidoglycan (TLR2) were measured by quantitative Real Time RT-PCR. Results: Histological diagnosis of pouchitis was made in 20 patients while clinical diagnosis (PDAI > 7) was made in 10 patients. In patients with histological diagnosis of pouchitis mucosal IL-1b was more expressed than in those with healthy mucosa (p = 0.018). Mucosal IL-1b correlated directly with the number of CFU of Thermosinus Carboxydivorans (r = 0.52, p = 0.018) adherent to the mucosa. Granulocytes and monocytes mucosal infiltrate were more severe in patients with histological diagnosis of pouchitis (p < 0.001 for both). Monocytes and granulocytes infiltrate correlated directly with the number of CFU of Bacteriodiaceae adherent to the mucosa (r = 0.52, p = 0.019, r = 0.53, p = 0.009). Moreover, granulocytes infiltrate inversely correlated with the number of CFU of Enterobacteriaceae adherent to the mucosa (r = 0.45, p = 0.044). In patients with clinical diagnosis of pouchitis the total number of CFU of Enterococcaceae and of Enterobacteriaceae in the mucus was lower than in healthy patients (p = 0.023 and p = 0.018).