Background and aims: Ficolin-2 is an acute phase reactant produced by the liver and targeted to recognize N-acetyl-glucosamine which is present in bacterial and fungal cell walls. We recently showed that ficolin-2 serum levels were significantly higher in CD patients compared to healthy controls. We aimed to evaluate serum ficolin-2 concentrations in CD patients regarding their correlation with endoscopic severity and to compare them with clinical activity, fecal calprotectin, and CRP.Methods: Patients provided fecal and blood samples before undergoing ileo-colonoscopy. Disease activity was scored clinically according to the Harvey-Bradshaw Index (HBI) and endoscopically according to the simplified endoscopic score for CD (SES-CD). Ficolin-2 serum levels and fecal calprotectin levels were measured by ELISA.Results: A total of 136 CD patients were prospectively included (mean age at inclusion 41.5 15.4 years, 37.5% females). Median HBI was 3 [2-6] points, median SES-CD was 5 [2-8], median fecal calprotectin was 301 [120-703] mu g/g, and median serum ficolin-2 was 2.69 [2.02-3.83] mu g/mL. SES-CD correlated significantly with calprotectin (R = 0.676, P < 0.001), CRP (R = 0.458, P < 0.001), HBI (R = 0.385, P < 0.001), and serum ficolin-2 levels (R = 0.171, P = 0.047). Ficolin-2 levels were higher in CD patients with mild endoscopic disease compared to patients in endoscopic remission (P = 0.015) but no difference was found between patients with mild, moderate, and severe endoscopic disease.Conclusions: Ficolin-2 serum levels correlate worse with endoscopic CD activity when compared to fecal calprotectin or CRP. (C) 2014 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.

Poster presentationsAccording to our second analysis, CMV colitis is associated with an increased risk of developing VTE (OR = 4.01; 95% CI = 1.4910.78; p = 0.006).Both hospitalization and bowel resection were more common in patients with than without VTE during the follow-up period.None of our VTE patients died.Conclusions: The incidence of VTE seems to be lower in Asian than in Western patients.Higher disease activity is associated with an increased risk of developing VTE and CMV colitis may also increase this risk.IBD patients with CMV colitis and higher disease activity may require vigilant observation to diagnose VTE.We strongly support the use of prophylactic anticoagulation during hospitalization in affected patients.

Results: Significant associations were obtained between IBD overall and SNP markers in the genetic regions of MAGI3 (P = 0.002), MAGI2 (P = 0.010), F11R (P = 0.019), and MAGI1 (P = 0.024).Markers in the genetic regions of MAGI2 (P = 0.011) and MAGI1 (P = 0.037) were significantly associated with CD.Additionally, markers in the genetic regions of MAGI3 (P = 0.003), MAGI2 (P = 0.010), and F11R (P = 0.026) showed significant associations to UC. Conclusions: Four out of six investigated TJ-related genes showed association to IBD overall as well as CD and UC.Our findings add further support for a genetically impaired intestinal epithelial barrier as one predisposing factor in the etiology of IBD.In-depth exploration of these TJ-components, may lead to a better understanding of the pathogenic mechanisms underlying IBD.

Background and aims: Mannan-binding lectin (MBL) and ficolins are microbial pattern recognition molecules that activate the lectin pathway of complement. We previously reported the association of MBL deficiency with anti-Saccharomyces cerevisiae antibodies (ASCA) in patients with Crohn's disease (CD). However, ASCA are also frequently found in MBL-proficient CD patients. Here we addressed expression/function of ficolins and MBL-associated serine protease-2 (MASP-2) regarding potential association with ASCA. Methods: ASCA titers and MBL, ficolin and MASP-2 concentrations were determined by ELISA in the serum of patients with CD, ulcerative colitis (UC), and in healthy controls. MASP-2 activity was determined by measuring complement C4b-fixation. Anti-MBL autoantibodies were detected by ELISA. Results: In CD and UC patients, L-ficolin concentrations were significantly higher compared to healthy controls (p < 0.001 and p = 0.029). In contrast, H-ficolin concentrations were slightly reduced in CD and UC compared to healthy controls (p = 0.037 for UC vs. hc). CD patients with high ASCA titers had significantly lower H-ficolin concentrations compared to ASCA-low/negative CD patients (p = 0.009). However, MASP-2 activity was not different in ASCA-negative and ASCA-positive CD patients upon both, ficolin- or MBL-mediated MASP-2 activation. Finally, anti-MBL autoantibodies were not over-represented in MBL-proficient ASCA-positive CD patients. Conclusions: Our results suggest that low expression of H-ficolin may promote elevated ASCA titers in the ASCA-positive subgroup of CD patients. However, unlike MBL deficiency, we found no evidence for low expression of serum ficolins or reduced MASP-2 activity that may predispose to ASCA development.

BackgroundAvocados (Persea Americana) are a high fat, high fiber albeit low energy density food. Our goal was to examine the effect of incorporating avocados in meals on post‐meal peptide hormone concentrations.MethodsA randomized 3×3 crossover design was used to measure peptide hormones in 26 healthy overweight (BMI 28.1±2.4 kg/m2) adults. Subjects consumed in random order a standardized breakfast meal providing 25% of their energy need (EN) on each of 3 days, followed by one of three test lunch meal: control (C) avocado‐free (35% of EN); avocado‐inclusive (AI) a meal containing avocado and matched with C for energy; and avocado‐added (AA) a meal in which a portion of avocados was added to C to provide 41% of EN. Blood samples obtained before and at 0.5 h, 1 h, 2 h and 3 h following test meals were assayed for ghrelin, leptin, gastric inhibitory peptide, glucagon‐like peptide‐1, and peptide YY by ELISA. A mixed model statistical approach was used to compare differences among test meals adjusting for time and treating participants as random effects.ResultsThere was a significant difference between the 3 test meals in plasma leptin responses at 0.5 h (P<0.05) and at 3 h (P<0.0001), and an overall test meal x time interaction (P=0.05).ConclusionThe inclusion or addition of avocado to a meal influences post meal leptin responses over a 3 hour period.Funded by the Hass Avocado Board.

of local inflammatory signals to develop gut antigen-specific Th17 and Treg cells that may be found systemically after the resolution of inflammation.These insights will ultimately help elucidate how the gut environment and pathogen-associated molecular patterns steer the development of adaptive immune responses during colitis and how pathogen-recognition receptors can be used to manipulate the development or resolution of inflammation to treat gastrointestinal disease.

Background In Crohn's disease (CD) the deficiency of mannan-binding lectin (MBL) is associated with an increased prevalence of anti-Saccharomyces cerevisiae antibodies (ASCA) and with complicated phenotypes of the disease. However, the role of MBL in intestinal inflammation is currently unclear. A study was undertaken to analyse local MBL expression in human intestine and the consequences of MBL deficiency in experimental colitis and yeast infection. Methods ASCA were measured by ELISA. MBL was assessed by ELISA and quantitative PCR. Wild type and MBL-deficient mice were administered dextran sulfate sodium (DSS) in the presence or absence of viable Candida albicans or adhesive invasive Escherichia coli (AIEC). Mice were infected with C albicans to assess generation of anti-yeast mannan antibodies. Results MBL expression was virtually undetectable in the intestinal mucosa of both healthy controls and patients with CD, irrespective of macroscopic inflammation, indicating that systemic MBL must be responsible for the reduced risk of complicated disease in MBL-competent patients with CD. MBL-deficient mice showed enhanced DSS colitis upon oral challenge with C albicans or AIEC. C albicans could be recovered from the kidneys of colitic/C albicans-fed MBL-deficient, but not wild type mice. Infection with C albicans induced high titres of anti-C albicans mannan IgM and IgG in MBL-deficient mice but only a modest and transient IgM response with no class switch to IgG in wild type mice. Cross-reactive ASCA IgM continuously increased in MBL-deficient mice but rapidly declined after transient induction in wild type mice. In MBL-deficient mice, increased C albicans dissemination correlated with reduced early retention in the circulation. Conclusions These results suggest that systemic MBL helps to prevent excessive inflammation upon access of normally mild pathogens across the damaged intestinal epithelium. Lack of this innate defence promotes antibody responses with cross-reactive potential against common mannan epitopes. These interpretations are compatible with the increased prevalence of ASCA and complicated disease phenotypes in MBL-deficient patients with CD.

Presentationsbeen proposed for this: induction of IL-13Ra2 expression on the surface membrane by signalling of IL-13 through the IL-13Ra1 receptor in the presence of TNF-a, followed by IL-13 signalling through IL-13Ra2.Signalling via this route in macrophages induced production of TGF-b, potentially leading to collagen synthesis and fibrosis.Therefore, the aim of this study is to determine whether this two-step process occurs in human intestine and particularly if it occurs in collagen-producing intestinal fibroblasts.Using intestinal tissue taken from patients with CD, ulcerative colitis (UC) or cancer, the preliminary immunohistology data suggests that both IL-13 receptors are expressed in the intestinal muscle and mucosa of all patients, and appear to be co-expressed on the same cells which, phenotypically, are stromal or epithelial cells.There seems to be little difference in either the number of IL-13R + cells present, or the appearance of the cells, between cancer and UC tissue.However, in the muscle layer of fibrotic areas of CD tissue, IL-13Ra1/Ra2 double positive cells appear to be enlarged.Fibroblast lines generated from all tissue samples retain the expression of IL-13Ra1 but Ra2 expression was variable, as determined by immunofluorescence.These receptors are able to signal since induction of phosphorylated stat 6 was detected by Western blotting in cell lysates generated from cell lines treated with IL-13, with maximum stimulation at 60 minutes, while there was no change in the level of total stat 6 over the same period.Initial studies in cell lines treated with IL-13 indicate increased collagen synthesis and decreased TIMP-1 and TIMP-2, as detected by ELISA on cell culture supernatants.Upregulation of IL-13Ra2 was observed in cell lines treated with IL-13 and TNF-a, in comparison with untreated cells, quantified by Western blotting.In summary, our data are consistent with the hypothesis that signalling through IL-13 receptors in mesenchymal cells contributes to the fibrotic process in CD.We propose, therefore, that IL-13 and its receptors may be considered as targets for future therapy.

OBJECTIVES:Mannan-binding lectin (MBL) acts as a pattern-recognition molecule directed against oligomannan, which is part of the cell wall of yeasts and various bacteria. We have previously shown an association between MBL deficiency and anti-Saccharomyces cerevisiae mannan antibody (ASCA) positivity. This study aims at evaluating whether MBL deficiency is associated with distinct Crohn's disease (CD) phenotypes.METHODS:Serum concentrations of MBL and ASCA were measured using ELISA (enzyme-linked immunosorbent assay) in 427 patients with CD, 70 with ulcerative colitis, and 76 healthy controls. CD phenotypes were grouped according to the Montreal Classification as follows: non-stricturing, non-penetrating (B1, n=182), stricturing (B2, n=113), penetrating (B3, n=67), and perianal disease (p, n=65). MBL was classified as deficient (<100 ng/ml), low (100-500 ng/ml), and normal (500 ng/ml).RESULTS:Mean MBL was lower in B2 and B3 CD patients (1,503+/-1,358 ng/ml) compared with that in B1 phenotypes (1,909+/-1,392 ng/ml, P=0.013). B2 and B3 patients more frequently had low or deficient MBL and ASCA positivity compared with B1 patients (P=0.004 and P<0.001). Mean MBL was lower in ASCA-positive CD patients (1,562+/-1,319 ng/ml) compared with that in ASCA-negative CD patients (1,871+/-1,320 ng/ml, P=0.038). In multivariate logistic regression modeling, low or deficient MBL was associated significantly with B1 (negative association), complicated disease (B2+B3), and ASCA. MBL levels did not correlate with disease duration.CONCLUSIONS:Low or deficient MBL serum levels are significantly associated with complicated (stricturing and penetrating) CD phenotypes but are negatively associated with the non-stricturing, non-penetrating group. Furthermore, CD patients with low or deficient MBL are significantly more often ASCA positive, possibly reflecting delayed clearance of oligomannan-containing microorganisms by the innate immune system in the absence of MBL.

Background: Distinct Crohn's disease (CD) phenotypes correlate with antibody reactivity to microbial antigens. We examined the association between antibody response to 2 new flagellins called A4-Fla2 and Fla-X, anti-Saccharomyces cerevisiae antibodies (ASCA). anti-neutrophil cytoplasmic antibodies (p-ANCA), anti-pancreas antibodies (PAB), NOD2 mutations (R702W, G908R, and L1007fsinsC), and clinical CD phenotypes (according to Vienna criteria).Methods: All the above-mentioned antibodies as well as NOD2 Mutations were determined in 252 CD patients. 53 With ulcerative colitis (UC), and 43 healthy controls (HC) and correlated with clinical data.Results: A seroreactivity for A4-Fla2/Fla-X/ASCA/p-ANCA/PAB (in percent) was found in 59/57/62/12/22 of CD patients, 6/6/4/51/0 of UC patients. and 0/2/5/0/0 of healthy controls. CD behavior: 37% B1, 36% B2, and 27% B3. In multivariate logistic regression, antibodies to A4-Fla2, Fla-X, and ASCA were significantly associated with stricturing phenotype (P = 0.027, P = 0.041, P < 0.001). negative associations were found with inflammatory phenotype (P 0.001. P = 0.005, P < 0.001). Antibodies to A4-Fla2, Fla-X. ASCA, and NOD2 mutations were significantly associated with small bowel disease (P = 0.013, P = 0.01. P < 0.001, P = 0.04). whereas ASCA was correlated with fistulizing, disease (P = 0.007) and small bowel surgery (P = 0.009). Multiple antibody responses against microbial antigens were associated with stricturing (P < 0.001), fistulizing disease (P = 0.002), and small bowel Surgery (P = 0.002).Conclusions: Anti-flagellin antibodies and ASCA are strongly associated with complicated CD phenotypes. CD patients with serum reactivity against multiple microbes have the greatest frequency of strictures, perforations, and small bowel surgery. Further prospective longitudinal studies are needed to show that antibody-based risk stratification improves the clinical outcome of CD patients.

OBJECTIVES Reactivation of latent tuberculosis (TB) in inflammatory bowel disease (IBD) patients treated with antitumor necrosis factor-alpha medication is a serious problem. Currently, TB screening includes chest x-rays and a tuberculin skin test (TST). The interferon-gamma release assay (IGRA) QuantiFERON-TB Gold In-Tube (QFT-G-IT) shows better specificity for diagnosing TB than the skin test. This study evaluates the two test methods among IBD patients. METHODS Both TST and IGRA were performed on 212 subjects (114 Crohn's disease, 44 ulcerative colitis, 10 indeterminate colitis, 44 controls). RESULTS Eighty-one percent of IBD patients were under immunosuppressive therapy; 71% of all subjects were vaccinated with Bacille Calmette Guérin; 18% of IBD patients and 43% of controls tested positive with the skin test (P < 0.0001). Vaccinated controls tested positive more often with the skin test (52%) than did vaccinated IBD patients (23%) (P= 0.011). Significantly fewer immunosuppressed patients tested positive with the skin test than did patients not receiving therapy (P= 0.007); 8% of patients tested positive with the QFT-G-IT test (14/168) compared to 9% (4/44) of controls. Test agreement was significantly higher in the controls (P= 0.044) compared to the IBD group. CONCLUSIONS Agreement between the two test methods is poor in IBD patients. In contrast to the QFT-G-IT test, the TST is negatively influenced by immunosuppressive medication and vaccination status, and should thus be replaced by the IGRA for TB screening in immunosuppressed patients having IBD.

AIM:To test whether humoral immune reaction against mycobacteria may play a role in anti-Saccharomyces cerevisiae antibodies (ASCA) generation in Crohn's disease (CD) and/or whether it correlates with clinical subtypes.METHODS:The dominant ASCA epitope was detected by Galanthus nivalis lectin (GNL)-binding assay. ASCA and IgG against mycobacterial lysates (M avium, M smegmatis, M chelonae, M bovis BCG, M avium ssp. paratuberculosis (MAP)] or purified lipoarabinomannans (LAM) were detected by ELISA. ASCA and anti-mycobacterial antibodies were affinity purified to assess cross-reactivities. Anti-mycobacterial IgG were induced by BCG-infection of mice.RESULTS:GNL bound to different extents to mycobacterial lysates, abundantly to purified mannose-capped (Man) LAM from M tuberculosis, but not to uncapped LAM from M smegmatis. Fifteen to 45% of CD patients but only 0%-6% of controls were seropositive against different mycobacterial antigens. Anti-mycobacterial IgG correlated with ASCA (r = 0.37-0.64; P = 0.003-P < 0.001). ASCA-positivity and deficiency for mannan-binding lectin synergistically associated with anti-mycobacterial IgG. In some patients, anti-mycobacterial antibodies represent cross-reactive ASCA. Vice-versa, the predominant fraction of ASCA did not cross-react with mycobacteria. Finally, fistulizing disease associated with antibodies against M avium, M smegmatis and MAP (P = 0.024, 0.004 and 0.045, respectively).CONCLUSION:Similar to ASCA, seroreactivity against mycobacteria may define CD patients with complicated disease and a predisposition for immune responses against ubiquitous antigens. While in some patients anti-mycobacterial antibodies strongly cross-react with yeast mannan; these cross-reactive antibodies only represent a minor fraction of total ASCA. Thus, mycobacterial infection unlikely plays a role in ASCA induction.