Pathophysiology Of Ulcerative Colitis Research Articles

P019. Informing ulcerative colitis pathophysiology and outcomes through metabolic pofiling

P019. Informing ulcerative colitis pathophysiology and outcomes through metabolic pofiling

Altered microRNA Expression and Immunosuppressive Cytokine Production by Regulatory T Cells of Ulcerative Colitis Patients

ABSTRACTRegulatory T (Treg) cells are essential for maintenance of peripheral tolerance and prevention of autoimmune diseases in part by producing immunosuppressive cytokines. Recently, microRNAs (miRNAs) have also been involved in autoimmune disorders, not least for their crucial role in the regulation of Treg biology and function. We simultaneously investigated the concentration of IL-35, IL-10, TGF-β, and sCD25 in supernatant of cell culture and the expression patterns of several miRNAs in CD4+CD25+ CD127−/low FoxP3+ Tregs of ulcerative colitis (UC) patients. Significantly lower levels of IL-10 and IL-35 were observed in Treg cultures of UC patients. miR-21, miR-146a, and miR-155 levels were downregulated and miR-31 level was upregulated in Tregs of patients. Our results suggest that microRNAs may serve as a novel regulator in function and homoeostasis of UC Treg cells, providing a key role for them in pathophysiology of UC.

Successful Use of Tocilizumab in a Patient with Coexisting Rheumatoid Arthritis and Ulcerative Colitis.

Tocilizumab is an interleukin-6 receptor inhibitor licensed for moderate to severe rheumatoid arthritis (RA). We report a case of Tocilizumab monotherapy for severe active RA in a patient with coexisting ulcerative colitis (UC). The patient was intolerant to multiple disease-modifying drugs, so Tocilizumab monotherapy was commenced. We found clinical improvement in both RA and UC. There was no major adverse event after 2 years. Manufacturer advised caution in using Tocilizumab in patient with gastrointestinal ulceration due to an increased risk of bowel perforation. However, alternative treatments such as glucocorticoid and nonsteroidal anti-inflammatory drugs may carry a higher bowel perforation risk. The presence of gastrointestinal ulceration therefore should not constitute an absolute contraindication for Tocilizumab therapy. Future studies of registry data will inform clinician of the Tocilizumab-related risk of gastrointestinal toxicity in “real-life” settings. Contrary to previous case report, we found Tocilizumab therapy to have a positive effect on UC. Laboratory studies supported a role for interleukin-6 in the pathophysiology of UC. Further clinical trial to evaluate the therapeutic role of Tocilizumab in UC would be warranted.

Key Mucosal Helper T Cell-Related Gene Signature Paralleling Disease Activity in Ulcerative Colitis Is Underlain by Different Interactions Between IL-17A and IL-17F With Other Helper T Cell-Related Genes

Introduction: T helper (Th) and T regulatory (Treg) cell-related cytokines are involved in the pathogenesis of inflammatory bowel diseases, such as ulcerative colitis (UC). However, in inflamed mucosae these molecules seem to be upregulated almost unselectively, and thus predominant molecules contributing to the aberrant immune response and disease activity in UC is undetermined. Methods: Forty-seven UC patients undergoing colonoscopy were assessed with Rachmilewitz endoscopic index (REI, 0-12), which scores mucosal damage. Biopsies obtained from inflamed (REI=3-12) and noninflamed (REI=0-2) area were analysed for mRNA expression of cytokines and transcription factors related to Th1 (TNF-α, IFN-γ, IL-12p35, IL- 12p40, and T-bet), Th2 (IL-4, IL-13, IL-33, and GATA3), Th17 (IL-17A, IL-17F, IL-23p19, IL-21, IL-22, IL-6, and RORC) and Treg (TGF-β and Foxp3) by quantitative PCR. Their expression pattern paralleling higher REI was sought by univariate and multivariate analyses. Results: No target expression univariately correlated with REI in inflamed samples despite their general upregulation. Multiple regression analysis, however, identified a significant REI-predictive model (P=0.0011, RSq=0.472), consisting of IL-17A, IL-17F, IL-21, IL-22, RORC, and Foxp3 levels, with major individual contribution of IL-17A (P=0.0001) and IL-17F (P < 0.0001), which shifted REI upward and downward, respectively. Partial correlation analysis validated this result and further indicated some differeces in correlations between IL-17A and IL-17F with other targets when influences of al the other targets levels were taken into account. Ultimately, simply IL-17A/IL-17F ratio, correlated with REI substantially(r=0.5120, P=0.0007), whereas levels of IL-13, IFN-γ, TNF-α and etc. did not essentially serve as significant parameters in REI-predictive models. Conclusion: Mucosal IL-17A/IL-17F ratio significantly correlated with endoscopic score in UC, indicating different roles of IL-17A and IL-17F in the immune response. Their disparate interactions with other Th/Treg-related genes underlying this finding were elucidated through untangling the complex correlations among simultaneously upregulated genes. Predominant roles of Th17 cytokines over IL-13 or Th1 cytokines in the pathophysiology of UC were also suggested from the present study.

IL-37b gene transfer enhances the therapeutic efficacy of mesenchumal stromal cells in DSS-induced colitis mice.

Aim:To investigate whether the transfer of the IL-37b gene, a newly identified inhibitor of both innate and adaptive immunity, could improve the therapeutic efficacy of mesenchumal stromal cells (MSCs) in inflammatory bowel disease (IBD).Methods:The expression of IL-37 in biopsied specimens of the patients with active ulcerative colitis (UC) was detected using RT-PCR and immunohistochemistry. Mice were treated with 3% dextran sulfate sodium (DSS) for 8 days to induce colitis. Before DSS treatment, the mice were injected with MSCs, MSC-eGFP or MSC-IL37b. Their body weight was measured each day, and the colons and spleens were harvested on d 10 for pathological and biochemical analyses.Results:In biopsied specimens of the patients with active UC, the expression of IL-37 was dramatically elevated in inflamed mucosa, mainly in epithelial cells and infiltrating immune cells. Compared to MSC-eGFP or MSCs, MSC-IL37b administration significantly attenuated the body weight and colon length reduction, and decreased the histological score in DSS-induced colitis mice. Furthermore, MSC-IL37b administration increased the percentage of myeloid-derived suppressor cells (MDSCs) among total splenic mononuclear cells as well as the percentage of regulatory T cells (Tregs) among splenic CD4+ T cells in the mice. Moreover, MSC-IL37b administration increased the IL-2+ cells and decreased the IFN-γ+ cells among splenic CD4+ T cells.Conclusion:IL-37 is involved in the pathophysiology of UC. IL-37b gene transfer enhances the therapeutic efficacy of MSCs in DSS-induced colitis mice by inducing Tregs and MDSCs and regulating cytokine production.

PTU-069 The fate of epithelial keratins in active ulcerative colitis

<h3>Introduction</h3> Intermediate filaments (IF), which mainly consist of keratins (K), are one of the main components of the human cell cytoskeleton. K8, K18 and K19 constitute the main keratins in the intestinal epithelial cells. Keratin alterations may play a role in the pathophysiology of ulcerative colitis (UC). We have previously shown reduced expression of Keratins in IF fraction from distal active UC relative to proximal inactive mucosa as well as controls using proteomic iTRAQ-based analysis.¹Whether this reduced expression is due to change in solubilisation, reduced total expression and/or degradation, is unknown. We aimed to clarify the fate of epithelial keratins during UC disease activity. <h3>Method</h3> Soluble proteome fractions were extracted from rectal biopsies in patients (n = 10) with active distal colitis (ACT) as well as endoscopically and histologically uninflamed proximal colonic mucosa (INACT) in the same patients. Median histological activity index in ACT and INACT were 2 (range 1–3) and 0 (range 0). Control colonic biopsies (n = 7) from normal individuals were fractionated similarly. An iTRAQ-compatible extraction protocol for soluble proteins was developed and applied. Labelled peptides from pooled patients in each group were analysed by SCX-LC-MS/MS and data reconstituted in GeneBio Phenyx. Inter-group comparisons were made using in-house algorithms based on t-testing with multiple test correction. Validation of iTRAQ results was carried out with immunoblotting on pooled and individual samples using monoclonal antibody against K8. These outcomes were then compared with our previous insoluble proteome analyses.¹ <h3>Results</h3> Comparative iTRAQ analysis of the soluble fraction showed significantly lower log fold changes in K8, K18 and K19 levels in ACT relative to INACT (0.42, 0.53, 0.42) and normal controls (0.61, 0.76, 0.41), respectively. The results were similar to our recent iTRAQ analysis of the IF fraction.¹Immunoblotting further confirmed these findings: median relative K8 concentration in IF/soluble proteome fractions in individual ACT and INACT samples was 0.18/1 (p = 0.02) and 1.21/1.02 (p = 0.02), respectively. <h3>Conclusion</h3> This study suggests relative total reduction in expression of epithelial keratins in actively inflamed colonic mucosa of UC patients compared to un-inflamed proximal mucosa and normal controls. Keratins may have utility as a tissue biomarker of UC disease activity. <h3>Disclosure of interest</h3> None Declared. <h3>Reference</h3> Corfe BM, Majumdar D, Assadsangabi A, Marsh AMR, Cross SS, Connolly JB, Evans CA, Lobo AJ. Inflammation decreases keratin level in ulcerative colitis; inadequate restoration associates with increased risk of colitis-associated-cancer. <i>BMJ Open Gastroenterol.</i>, In press

PTH-080 Informing ulcerative colitis pathophysiology and outcomes through metabolic profiling

Introduction Ulcerative colitis (UC) is a chronic gastrointestinal disorder involving a complex interplay between host genetics, mucosal immunology, gut microbiome and the environment. A complete understanding of aetiology and biomarkers predicting outcome are lacking. We used High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance (HR MAS NMR) spectroscopy to generate metabolic profiles from intact colonic biopsies from UC patients and controls, to inform us about disease pathophysiology and severity. Method Colonic tissue biopsies were acquired from 20 individuals (10 UC patients, and 10 controls who had normal mucosa on colonoscopy) at the time of routine colonoscopy. Metabolic signatures were obtained by HR-MAS NMR followed by multivariate pattern-recognition analysis to investigate differences between cohorts. 2 dimensional correlation spectroscopy (COSY) was performed to identify metabolites. Results Principal component analysis (PCA) identified one outlier which was excluded from further analysis. Orthogonal projection to latent structures analysis (OPLS) was applied to remaining cases and distinguished UC and control cohorts with significant predictive accuracy. The OPLS model supported good fit (R2Y 0.8362) and predictability (Q2Y 0.5026) parameters. The data analysis showed that colonic biopsies from UC patients contained relatively higher levels of antioxidants (ascorbate) and ethanolamine compared to those obtained from controls. A novel finding was that the model was capable of stratifying the samples based on severity (quiescent, mild, moderate or severe), which has not been demonstrated in previous metabonomic studies on colonic samples of ulcerative colitis. ![Abstract PTH-080 Figure 1][1] Abstract PTH-080 Figure 1 Conclusion HR-MAS NMR based metabolic profiling has identified distinct differences in UC patients and controls, in particular increased levels of antioxidants and amino acid which may be required in greater quantities during catabolic conditions such as UC. This study shows the utility of HR-MAS NMR in exploring diagnostic possibilities, disease severity stratification and generating a better understanding of UC pathophysiology. Disclosure of interest None Declared. Reference 1. Bjerrum JT, Nielsen OH, Hao F et al . Metabonomics in ulcerative colitis: diagnostics, biomarker identification, and insight into the pathophysiology. Journal of Proteome Research 2010;9(2):954–962 [1]: pending:yes

From colon-sparing techniques to pelvic ileal pouch: history and evolution of surgery for ulcerative colitis

Since the first decription of ulcerative colitis(UC) by Sir Wilkins from the London Guy’s Hospital in 1859, medical and surgical management have gradually evolved. Aims of this review are to describe the milestones and pioneers of surgery for UC, taking the readers on a journey through the steps that led to the development of restorative proctocolectomy with ileal pelvic pouch. This is an historical, narrative review. We reviewed scientific literature and historical archives, seeking for documentation of surgery of UC. Information were integrated with our personal experience, taking into accout patients’ perspective. Different surgical approaches have been attempted over time, grounding on different concepts. As long as pathophysiology of UC has become clearer, surgeons have profoundly modified their attitudes toward this disease. Early approaches aimed at preserving organs, with “damage-control” surgery. Advances in knowledge and surgical skills led surgeons to demolitive treatments, which proved to be very effective, but with poor quality of life in most patients, especially in young, due to permanent stoma—even if continent ileostomy ameliorated patient satisfaction. On the other hand, straight ileoanal anastomosis, despite preserving trans-anal defaecation, resulted in inacceptably high bowel frequency. Restorative proctocolectomy was the turning point. By combining the concepts of disease removal, continence, and physiological evacuation, it has become the surgical treatment of choice in most UC patients. Choice and timing of treatment of UC need to be individualized. Besides surgical knowledge and techniques, the patient’s perspective must be examined closely. Restorative proctocolectomy is the mainstay.

PTU-064 Increased Mucosal Expression Of Insoluble Keratins 8, 18 And 19 In Long-standing Ulcerative Colitis In Comparison To Recent-onset Ulcerative Colitis: Validation Of Mass Spectrometry Data

IntroductionIntermediate filaments (IF) are one of the main components of the human cell cytoskeleton with keratins (K) being the largest component. K8, K18 and K19 constitute the main keratins in...

Sa1783 Nicotine Ameliorates Colonic Inflammation via Down-Regulation of Vascular Endothelial Cell Adhesion Molecules

Background Ulcerative colitis (UC) is an intractable disease. Lymphocytes migration to colonic mucosa through endotherial venule like vessel is considered to be largely involved in pathophysiology of UC, and anti-adhesion molecule therapy is one of the established therapy. Although smoking has been reported to have a beneficial effect on UC, pathophysiology of nicotine from the point of lymphocytes trafficking is not studied. We investigated the involvement of nicotine in the colonic inflammation in the following three studies: 1) in vitro effect of nicotine on vascular endothelial cells, 2) in vivo effect of nicotine on inflamed mucosa of murine colitis model, and 3) intravital observation of lymphocyte migration to microvessels. Method 1) Microvascular endothelial cell line, bEnd3, was used. The bEnd3 cells (2x105) were cultured for 3days, and were treated with 5ng/ml TNFα and/or 200μg/ ml nicotine for 5 hours. Degree of mRNA expression of ICAM-1, VCAM-1 and MAdCAM1 was determined by quantitative RT-PCR. 2) C57BL/6J mice were used. For induction of colitis, mice were treated with 3% dextran sodium sulfate (DSS, 40kDa) in drinking water for 7 days. In some group, mice were treated with 0.1mg/ml nicotine in drinking water for 7 days. Disease activity of colitis was assessed by DAI score. We measured mRNA expressions of VCAM-1, ICAM-1 and MAdCAM-1 in the colonic tissue. 3) C57BL/6J mice were treated with 0.1mg/ml nicotine in drinking water 1 day after 3% DSS treatment for 7 days. Mice were injected FITC-labeld lymphocytes (3x107 dissoleved in 0.3ml) obtained from spleen of another mouse, via cervical vein. Microcirculation in the colonic mucosa was observed with a fluorescence microscope. Lymphocytes that moved outside the vessel were counted as transmigrated cells. Result In in vitro study, expression of ICAM-1, VCAM-1 andMAdCAM1 was increased by TNFα. Increase in expression of ICAM-1 by TNFα was not changed by nicotine. However, increase in expression of VCAM-1 and MAdCAM-1 was significantly attenuated by nicotine. In in vivo study, nicotine improved DAI score. Nicotine alone treatment did not affect expression of ICAM-1, VCAM-1 and MAdCAM-1, and DSS treatment significantly increased them. Addition of nicotine treatment to DSS significantly attenuated the increased expression of VCAM-1 and MAdCAM-1 induced by DSS, and tended to inhibit ICAM-1, although not statistically significant. By intravital observation, DSS increased the number of lymphocytes adhering to vascular endothelium and migrating out of vessel. Increase in lymphocytes migration was attenuated by nicotine. Conclusion Microvascular endothelial expression of adhesion molecules, especially VCAM-1 and MAdCAM-1 was significantly inihibited by exposure to nicotine. Down-regulation of vascular adhesion molecules by nicotine may play one of attenuative roles in colonic inflammation.

Regulation of eotaxin-3/CC chemokine ligand 26 expression by T helper type 2 cytokines in human colonic myofibroblasts

Eotaxins induce the trafficking of eosinophils to the sites of inflammation via CC chemokine receptor 3 (CCR3). In this study, we investigated eotaxin-3/CC chemokine ligand 26 (CCL26) expression in the inflamed mucosa of patients with inflammatory bowel disease (IBD), and characterized the molecular mechanisms responsible for eotaxin-3 expression in human colonic myofibroblasts. Eotaxin-3 mRNA and protein expression was evaluated by real time-polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Eotaxin-3 mRNA expression was elevated significantly in the active lesions of ulcerative colitis (UC) patients. Significant elevations were also observed in the active lesions of Crohn's disease (CD) patients, but this was significantly lower than that detected in the active UC lesions. There were no significant increases in the inactive lesions of UC or CD patients. Colonic myofibroblasts were identified as a major source of eotaxin-3 in the colonic mucosa, and interleukin (IL)-4 and IL-13 enhanced eotaxin-3 mRNA and protein expression significantly in these cells. There was a significant positive correlation between mucosal eotaxin-3 and IL-4 mRNA expression in the active lesions of IBD patients. The IL-4- and IL-13-induced eotaxin-3 mRNA expression was regulated by the signal transducer and activator of transcription-6 (STAT-6) and suppressor of cytokine signalling (SOCS)1-mediated pathways. Interferon (IFN)-γ acts as a negative regulator on the IL-4- and IL-13-induced eotaxin-3 expression via STAT-1 activation. Eotaxin-3 expression was elevated specifically in the active lesions of IBD, in particular UC. Eotaxin-3 derived from colonic myofibroblasts may play an important role in the pathophysiology of UC.

The Role of Bacteria in the Pathogenesis of Ulcerative Colitis

Factors implicated in the pathophysiology of ulcerative colitis (UC) are an abnormal immune response, defect in intestinal epithelial barrier function, and gut microbiota. Currently, it is unclear whether specific bacterial strains are responsible for the induction of intestinal inflammation, but increased bacterial tissue invasion has been described in affected UC patients. Further, a quantitative and qualitative microbial imbalance in UC, defined as dysbiosis, has been characterized by an increase in Rhodococcus spp., Shigella spp., and Escherichia spp., but a decrease in certain Bacteroides spp.. More specifically, Campylobacter spp., Enterobacteriae, and enterohepatic Helicobacter were more prevalent in tissue sample from UC patients subjected to molecular detection methods, but not controls. In addition, serologic testing identified Fusobacterim varium as a potential contributor to the intestinal inflammation in UC. Interestingly, in-situ hybridization studies have shown anti-inflammatory Lactobacillus spp. and Pediococcus spp. were absent in samples from subjects affected by UC. Therefore, dysbiosis is a factor in the pathogenesis of UC.

Peripheral lymphocyte DNA damage and oxidative stress in patients with ulcerative colitis

Ulcerative colitis (UC) is a fairly common chronic inflammatory disorder. Chronic inflammation may contribute to the risk of colorectal cancer through the accumulation of specific products resulting from DNA damage. Previous studies reported that DNA damage and oxidative stress play a significant role in the pathophysiology of UC, but the results are inconsistent. In the present study, we investigated peripheral DNA damage and oxidative stress in patients with UC. The study included 20 patients with UC and 20 controls. Peripheral lymphocyte DNA damage was measured using the alkaline comet assay. Plasma total antioxidant capacity (TAC), total oxidant status (TOS), and oxidative stress index (OSI) were determined. DNA damage levels, TOS, and OSI were significantly higher in patients with UC than in controls (P <0.001 for all parameters), while TAC was significantly lower (P <0.001). DNA damage was significantly correlated with TOS, TAC, and OSI (r = 0.604, P <0.001; r = -0.593, P <0.001; and r = 0.716, P <0.001, respectively). Moreover, TAC levels were significantly correlated with TOS and OSI (r = 0.604, P <0.001; r = -0.399, P <0.05; and r = -0.513, P <0.05, respectively). Our results show that increased peripheral DNA damage and oxidative stress seem to be associated with decreased antioxidant levels and thus may in part contribute to the development of colorectal cancer associated with UC.

Peri-Appendiceal Red Patch and Pathogenesis of the Appendix in Ulcerative Colitis

To the Editor,We read with interest the article by Rubin and Roth [1]on the peri-appendiceal red patch (PARP) in ulcerativecolitis (UC). In 367 UC patients with distal colitis, 29(7.9%) patients had PARP. In the 29 patients, 23 (79%)were male, none had prior appendectomy, 20 of 30 (67%)biopsy findings showed that the parallel histologic activityin the PARP and the distal colitis, and 11 of 21 patients(52%) with endoscopic follow-up progressed to moreextensive disease. We believe that the pathogenesis of theappendix in UC patients should be highlighted.Although UC is characterized by continuous and diffuseinflammation extending proximally from the rectum, PARPhas been increasingly recognized in 48–86% patients withdistal UC [2–4], as in the study of Rubin and Rothe [1].Patients with PARP experience a more aggressive andrelapsing disease courses compared with patients withoutPARP [3]. Many case–control studies suggest that previousappendectomy is rare in UC patients [2, 3]. Patients withprevious appendectomy have a delayed onset of UC, areduced need for immunomodulators and proctocolectomy,and a reduced relapse rate and extent of UC [5]. Moreover,we and several investigators have reported the improve-ment of UC after appendectomy, especially in youngpatients with PARP [2, 6].The pathogenesis of UC has not been determined, but anabnormal mucosal immune response plays a major role inthe occurrence and pathophysiology of UC [2, 4]. Extensiveinfiltration of lymphocytes, especially CD4? T cells, hasbeen observed in the inflamed mucosa of UC patients.Activated CD4? T cells exhibit increased cytotoxic activityand secrete cytokines that enhance the inflammatory state,resulting in tissue injury. We have disclosed that the pro-portion of CD4? early-but-not-mature-activated T cells issignificantly increased in the appendix of UC patients [4, 7],and suspect that the appendix may be a priming site in theoccurrence of UC. We therefore believe that the appendixshould no longer be considered an evolutionary redundancy,especially in UC patients with PARP.References

Eosinophils Express Muscarinic Receptors and Corticotropin-Releasing Factor to Disrupt the Mucosal Barrier in Ulcerative Colitis

Altered intestinal barrier function has been implicated in the pathophysiology of ulcerative colitis (UC) in genetic, functional, and epidemiological studies. Mast cells and corticotropin-releasing factor (CRF) regulate the mucosal barrier in human colon. Because eosinophils are often increased in colon tissues of patients with UC, we assessed interactions among mast cells, CRF, and eosinophils in the mucosal barrier of these patients. Transmucosal fluxes of protein antigens (horseradish peroxidase) and paracellular markers ((51)Cr-EDTA, fluorescein isothiocyanate-dextran 4000) were studied in noninflamed, colonic mucosal biopsy samples collected from 26 patients with UC and 53 healthy volunteers (controls); samples were mounted in Ussing chambers. We also performed fluorescence and electron microscopy of human tissue samples, assessed isolated eosinophils, and performed mechanistic studies using in vitro cocultured eosinophils (15HL-60), mast cells (HMC-1), and a colonic epithelial cell line (T84). Colon tissues from patients with UC had significant increases in permeability to protein antigens compared with controls. Permeability was blocked by atropine (a muscarinic receptor antagonist), α-helical CRF(9-41) (a CRF receptor antagonist), and lodoxamide (a mast-cell stabilizer). Eosinophils were increased in number in UC tissues (compared with controls), expressed the most M2 and M3 muscarinic receptors of any mucosal cell type, and had immunoreactivity to CRF. In coculture studies, carbachol activation of eosinophils caused production of CRF and activation of mast cells, which increased permeability of T84 epithelial cells to macromolecules. We identified a neuroimmune intercellular circuit (from cholinergic nerves, via eosinophils to mast cells) that mediates colonic mucosal barrier dysfunction in patients with UC. This circuit might exacerbate mucosal inflammation.

Comparison of the fecal microbiota profiles between ulcerative colitis and Crohn’s disease using terminal restriction fragment length polymorphism analysis

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a powerful tool to assess the diversity of a microbial community. In this study, we performed T-RFLP analysis of the fecal microbiota from patients with ulcerative colitis (UC) and those with Crohn's disease (CD). Thirty-one patients with UC, 31 patients with CD, and 30 healthy individuals were enrolled. The polymerase chain reaction (PCR) products obtained from the 16S rRNA genes of fecal samples were digested with BslI, and T-RF lengths were determined. The fecal microbial communities were classified into 5 clusters. Twenty-eight of the 30 healthy individuals and 17 of the 18 patients with inactive UC were classified into clusters I, II, and III, but these clusters included a small number of patients with active UC and inactive/active CD. In contrast, 8 of the 13 patients with active UC and the majority of CD patients (12 of the 16 patients with inactive CD, and 11 of the 15 patients with active CD) were included in clusters IV and V. Based on the BslI-digested T-RFLP database, the bacteria showed a significant decrease in the Clostridium family in patients with active UC and inactive/active CD. In contrast, Bacteroides were significantly increased in CD patients. No significant differences were observed between patients with active UC and those with active CD. The fecal microbial communities of IBD patients were different from those of healthy individuals. The gut microbiota of patients with inactive UC tended to be closer to that of healthy individuals, suggesting different roles for the fecal microbiota in the pathophysiology of UC and CD.

DMBT1 is a novel gene induced by IL-22 in ulcerative colitis

Interleukin (IL)-22 is a recently identified cytokine that is suggested to play pivotal roles in various inflammatory diseases. Although the IL-22 receptor 1 (IL-22R1) is restrictively expressed in epithelial cells in the colon, the role of IL-22 in colonic diseases still remains unclear. In this study microarray analyses revealed that deleted in malignant brain tumors 1 (DMBT1) is a novel upregulated gene in IL-22-stimulated colon cancer cells. Therefore, we investigated the involvement of DMBT1 and IL-22 in ulcerative colitis (UC) tissues and examined the mechanism regulating the expression of DMBT1 in response to IL-22 stimulation. Changes of gene expression in IL-22-stimulated SW403 cells were investigated by microarray analyses. The effects of IL-22 on DMBT1 expression were examined in SW403 cells using a small interfering RNA (si)RNA for STAT3 or inhibitors for MEK, PI3K, and nuclear factor kappa B (NF-κB). The element responsible for IL-22-induced DMBT1 promoter activation was determined by a promoter deletion and electrophoretic mobility shift assay (EMSA). Expression of IL-22, IL-22R1, and DMBT1 in UC tissues was analyzed by real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. IL-22 treatment enhanced the expression of DMBT1 through STAT3 tyrosine phosphorylation and NF-κB activation in colon cancer cells. The IL-22-responsive element was located between -187 and -179 in the DMBT1 promoter region. In the UC mucosa the levels of DMBT1 and IL-22 mRNA expression were significantly enhanced and positively correlated, the numbers of IL-22-positive lymphocytes were increased, and the expression of IL-22R1 and DMBT1 was enhanced in the inflamed epithelium. The IL-22/DMBT1 axis may play a pivotal role in the pathophysiology of UC.

W1321 Long-Term Clinical Outcomes After the First Course of Corticosteroid Therapy in Patients With Moderate to Severe Intestinal Behget's Disease

[Background & Aims]The granulocytapheresis (GCAP) for ulcerative colitis (UC) has been developed in Japan. GCAP is a non-pharmacological therapy thereby does not have severe adverse effect. The mechanism of GCAP is expected to reduce inflammatory cytokines and the production of L-selectin by absorbing selected active granulocytes. Weakness of GCAP is the difficulty of the anticipation of treatment effect in early treatment stage. In this study, we focused on adhesion molecules which are thought to be associated with the pathophysiology of UC. We examined 5 SNPs in 3 adhesion molecule genes. [Methods] Twenty eight UC patients (M12, F16, averaged age 37.9y.o.) who received GCAP in past 5 years were examined. These patients were 21 pancolitis, 5 left sided colitis and 2 proctitis. DNA was extracted from all patients by colonic biopsy or blood sample and examined about the polymorphisms by PCR-RFLP. Genes and polymorphisms were L-selectin -642C>T and 725C>T, PECAM-1 codon125 V>L, E-selectin 98G>T in exon2 and S128R in exon4. The differences of the clinical activity index (CAI) scores of preand post-treatment of each 1 course (5 times of GCAP performed) or 2 course (10times of GCAP performed) were used for the evaluation of UC activity. Statistical analysis was done by Mann-Whitney U test. [Results] L-selectin-642C>T C carrier tended to be contributed to better treatment results after 2 course. L-selectin 725C>T polymorphism was not associated with treatment effect. PECAM-1 codon125 V>L, L/L or L/V was significantly associated with better treatment effect within 1 course treatment than V/V (p T and S128R polymorphisms showed no effect about GCAP treatment. [Conclusions] In this study, a significant effect of polymorphisms in PECAM-1 was found. Future study will be needed to confirm this result in next step.

W1320 Natalizumab is Effective in the Treatment of Moderate to Severe Crohn's Disease Unresponsive to Anti-TNFα Agents

[Background & Aims]The granulocytapheresis (GCAP) for ulcerative colitis (UC) has been developed in Japan. GCAP is a non-pharmacological therapy thereby does not have severe adverse effect. The mechanism of GCAP is expected to reduce inflammatory cytokines and the production of L-selectin by absorbing selected active granulocytes. Weakness of GCAP is the difficulty of the anticipation of treatment effect in early treatment stage. In this study, we focused on adhesion molecules which are thought to be associated with the pathophysiology of UC. We examined 5 SNPs in 3 adhesion molecule genes. [Methods] Twenty eight UC patients (M12, F16, averaged age 37.9y.o.) who received GCAP in past 5 years were examined. These patients were 21 pancolitis, 5 left sided colitis and 2 proctitis. DNA was extracted from all patients by colonic biopsy or blood sample and examined about the polymorphisms by PCR-RFLP. Genes and polymorphisms were L-selectin -642C>T and 725C>T, PECAM-1 codon125 V>L, E-selectin 98G>T in exon2 and S128R in exon4. The differences of the clinical activity index (CAI) scores of preand post-treatment of each 1 course (5 times of GCAP performed) or 2 course (10times of GCAP performed) were used for the evaluation of UC activity. Statistical analysis was done by Mann-Whitney U test. [Results] L-selectin-642C>T C carrier tended to be contributed to better treatment results after 2 course. L-selectin 725C>T polymorphism was not associated with treatment effect. PECAM-1 codon125 V>L, L/L or L/V was significantly associated with better treatment effect within 1 course treatment than V/V (p T and S128R polymorphisms showed no effect about GCAP treatment. [Conclusions] In this study, a significant effect of polymorphisms in PECAM-1 was found. Future study will be needed to confirm this result in next step.

W1316 Genetic Polymorphisms of Adhesion Molecules and the Effect of Granulocytapheresis Treatment for Ulcerative Colitis

[Background & Aims]The granulocytapheresis (GCAP) for ulcerative colitis (UC) has been developed in Japan. GCAP is a non-pharmacological therapy thereby does not have severe adverse effect. The mechanism of GCAP is expected to reduce inflammatory cytokines and the production of L-selectin by absorbing selected active granulocytes. Weakness of GCAP is the difficulty of the anticipation of treatment effect in early treatment stage. In this study, we focused on adhesion molecules which are thought to be associated with the pathophysiology of UC. We examined 5 SNPs in 3 adhesion molecule genes. [Methods] Twenty eight UC patients (M12, F16, averaged age 37.9y.o.) who received GCAP in past 5 years were examined. These patients were 21 pancolitis, 5 left sided colitis and 2 proctitis. DNA was extracted from all patients by colonic biopsy or blood sample and examined about the polymorphisms by PCR-RFLP. Genes and polymorphisms were L-selectin -642C>T and 725C>T, PECAM-1 codon125 V>L, E-selectin 98G>T in exon2 and S128R in exon4. The differences of the clinical activity index (CAI) scores of preand post-treatment of each 1 course (5 times of GCAP performed) or 2 course (10times of GCAP performed) were used for the evaluation of UC activity. Statistical analysis was done by Mann-Whitney U test. [Results] L-selectin-642C>T C carrier tended to be contributed to better treatment results after 2 course. L-selectin 725C>T polymorphism was not associated with treatment effect. PECAM-1 codon125 V>L, L/L or L/V was significantly associated with better treatment effect within 1 course treatment than V/V (p T and S128R polymorphisms showed no effect about GCAP treatment. [Conclusions] In this study, a significant effect of polymorphisms in PECAM-1 was found. Future study will be needed to confirm this result in next step.