Laura H. Raffals

A gene-targeted approach to investigate the intestinal butyrate-producing bacterial community

BackgroundButyrate, which is produced by the human microbiome, is essential for awell-functioning colon. Bacteria that produce butyrate are phylogeneticallydiverse, which hinders their accurate detection based on conventional phylogeneticmarkers. As a result, reliable information on this important bacterial group isoften lacking in microbiome research.ResultsIn this study we describe a gene-targeted approach for 454 pyrotag sequencing andquantitative polymerase chain reaction for the final genes in the two primarybacterial butyrate synthesis pathways, butyryl-CoA:acetate CoA-transferase(but) and butyrate kinase (buk). We monitored theestablishment and early succession of butyrate-producing communities in fourpatients with ulcerative colitis who underwent a colectomy with ileal pouch analanastomosis and compared it with three control samples from healthy colons. Allpatients established an abundant butyrate-producing community (approximately 5% to26% of the total community) in the pouch within the 2-month study, but patternswere distinctive among individuals. Only one patient harbored a community profilesimilar to the healthy controls, in which there was a predominance of butgenes that are similar to reference genes from Acidaminococcus sp.,Eubacterium sp., Faecalibacterium prausnitzii andRoseburia sp., and an almost complete absence of buk genes.Two patients were greatly enriched in buk genes similar to those ofClostridium butyricum and C. perfringens, whereas a fourthpatient displayed abundant communities containing both genes. Most butyrateproducers identified in previous studies were detected and the general patterns oftaxa found were supported by 16S rRNA gene pyrotag analysis, but thegene-targeted approach provided more detail about the potential butyrate-producingmembers of the community.ConclusionsThe presented approach provides quantitative and genotypic insights intobutyrate-producing communities and facilitates a more specific functionalcharacterization of the intestinal microbiome. Furthermore, our analysis refinesbut and buk reference annotations found in centraldatabases.

Effects of Concomitant Immunomodulator Therapy on Efficacy and Safety of Anti–Tumor Necrosis Factor Therapy for Crohn’s Disease: A Meta-analysis of Placebo-controlled Trials

BACKGROUND & AIMS There is debate over whether patients with Crohns disease who start anti-tumor necrosis factor (TNF) therapy after failed immunomodulator therapy should continue to receive concomitant immunomodulators. We conducted a meta-analysis of subgroups from randomized controlled trials (RCTs) of anti-TNF agents to compare the efficacy and safety of concomitant immunomodulator therapy vs anti-TNF monotherapy. METHODS We performed a systematic review of literature published from 1980 through 2008 and identified 11 RCTs of anti-TNF agents in patients with luminal or fistulizing Crohns disease. We excluded RCTs of patients who were naive to anti-TNF and immunomodulator therapy. The primary end points were clinical response at weeks 4-14 and 24-30 and remission at weeks 24-30. Secondary end points included infusion site or injection site reactions and selected adverse events. A priori subgroup analyses were performed to evaluate fistula closure and the efficacy and safety of combination therapy with different anti-TNF agents. RESULTS Overall, combination therapy was no more effective than monotherapy in inducing 6-month remission (odds ratio [OR], 1.02; 95% confidence interval [CI], 0.80-1.31), inducing a response (OR, 1.08; 95% CI, 0.79-1.48), maintaining a response (OR, 1.53; 95% CI, 0.67-3.49), or inducing partial (OR, 1.25; 95% CI, 0.84-1.88) or complete fistula closure (OR, 1.10; 95% CI, 0.68-1.78). In subgroup analyses of individual anti-TNF agents, combination therapy was not more effective than monotherapy in inducing 6-month remission in those treated with infliximab (OR, 1.73; 95% CI, 0.97-3.07), adalimumab (OR, 0.88; 95% CI, 0.58-1.35), or certolizumab (OR, 0.93; 95% CI, 0.65-1.34). Overall, combination therapy was not associated with an increase in adverse events, but inclusion of infliximab was associated with fewer injection site reactions (OR, 0.46; 95% CI, 0.26-0.79.) CONCLUSIONS On the basis of a meta-analysis, continued use of immunomodulator therapy after starting anti-TNF therapy is no more effective than anti-TNF monotherapy in inducing or maintaining response or remission. RCTs are needed to adequately assess the efficacy of continued immunomodulator therapy after anti-TNF therapy is initiated.

Multiphasic analysis of the temporal development of the distal gut microbiota in patients following ileal pouch anal anastomosis.

BackgroundThe indigenous gut microbiota are thought to play a crucial role in the development and maintenance of the abnormal inflammatory responses that are the hallmark of inflammatory bowel disease. Direct tests of the role of the gut microbiome in these disorders are typically limited by the fact that sampling of the microbiota generally occurs once disease has become manifest. This limitation could potentially be circumvented by studying patients who undergo total proctocolectomy with ileal pouch anal anastomosis (IPAA) for the definitive treatment of ulcerative colitis. A subset of patients who undergo IPAA develops an inflammatory condition known as pouchitis, which is thought to mirror the pathogenesis of ulcerative colitis. Following the development of the microbiome of the pouch would allow characterization of the microbial community that predates the development of overt disease.ResultsWe monitored the development of the pouch microbiota in four patients who underwent IPAA. Mucosal and luminal samples were obtained prior to takedown of the diverting ileostomy and compared to samples obtained 2, 4 and 8 weeks after intestinal continuity had been restored. Through the combined analysis of 16S rRNA-encoding gene amplicons, targeted 16S amplification and microbial cultivation, we observed major changes in structure and function of the pouch microbiota following ileostomy. There is a relative increase in anaerobic microorganisms with the capacity for fermentation of complex carbohydrates, which corresponds to the physical stasis of intestinal contents in the ileal pouch. Compared to the microbiome structure encountered in the colonic mucosa of healthy individuals, the pouch microbial community in three of the four individuals was quite distinct. In the fourth patient, a community that was much like that seen in a healthy colon was established, and this patient also had the most benign clinical course of the four patients, without the development of pouchitis 2 years after IPAA.ConclusionsThe microbiota that inhabit the ileal-anal pouch of patients who undergo IPAA for treatment of ulcerative colitis demonstrate significant structural and functional changes related to the restoration of fecal flow. Our preliminary results suggest once the pouch has assumed the physiologic role previously played by the intact colon, the precise structure and function of the pouch microbiome, relative to a normal colonic microbiota, will determine if there is establishment of a stable, healthy mucosal environment or the reinitiation of the pathogenic cascade that results in intestinal inflammation.

Variation in treatment of patients with inflammatory bowel diseases at major referral centers in the United States.

We performed a prospective study of patients with inflammatory bowel diseases to examine variations in treatment among medical centers. In a prospective cohort study of 1659 patients with Crohns disease and 946 patients with ulcerative colitis seen at 7 high-volume referral centers, we collected data on demographics, disease characteristics, and medical and surgical treatments. We used logistic regression to determine differences in treatment among centers, controlling for potential confounders. We found significant variations among centers in the treatment of Crohns disease with immunomodulators (odds ratio [OR], 3.34; 95% confidence interval [CI], 2.09-5.32) but not anti-tumor necrosis factor agents (OR, 1.64; 95% CI, 0.97-2.77). There was less variation in the treatment of ulcerative colitis; we found no difference in use of immunomodulators (OR, 1.83; 95% CI, 1.00-3.36) or anti-tumor necrosis factor therapy (OR, 0.81; 95% CI, 0.40-1.65). The development and implementation of evidence-based standards of care for inflammatory bowel disease may help reduce variation and improve outcomes.

979 Impact of Concomitant Immunomodulator Treatment on Efficacy and Safety of Anti-TNF Therapy in Crohn's Disease: A Meta-Analysis of Placebo Controlled Trials With Individual Patient-Level Data

Background: Objective markers of Crohns Disease (CD) activity have been sought as diagnostic, phenotypic, prognostic and disease activity markers. Complications such as stricture and fistula and characteristics such as TNF-antagonist responsiveness have been suggested as discreet mechanistic CD subtypes. This study explored the ability of genome wide expression profiling in whole blood to differentiate CD sub-populations. Methods: In the previously reported Phase 2b ustekinumab CERTIFI study of patients with moderate to severely active CDwho had failed or were intolerant to TNF-antagonists, whole blood samples were collected from a subset for mRNA expression profiling using Affymetrix HG-U133+ PM arrays. Baseline expression profiles were compared between patient sub-groups characterized by defined baseline disease attributes; and compared with those from samples obtained independently from healthy subjects. Expression modulations of .+/-1.5x and false discovery rate (FDR) p-value , 0.05 were considered significant. Results: Patients (n=204) with moderate to severe CD had significant expression modulations in 1725 transcripts in the whole blood compared with healthy subjects (n=49), including genes involved in inflammatory response and connective tissue disorders. A panel of 20 transcripts (including GAB2 and IL18R1) discriminated patients with only colonic (n=49) vs. strictly ileal (n=60) disease involvement. Significantly different expression modulations of 169, 321, and 151 transcripts, respectively, were identified in patients with high baseline CRP ( ≥10 mg/dL, n=97), fecal calprotectin (≥850 mg/g, n=80) or lactoferrin (. 100 mg/g, n=89) compared with patients with low baseline CRP ( ≤3 mg/dL, n=45), fecal calprotectin (≤250 mg/g, n=58), or lactoferrin (≤100 mg/g, n=107). As expected, patients with high baseline CRP, fecal calprotectin, or lactoferrin had elevated gene expressions in inflammatory pathways such as IL-6 and acute phase response signaling. In contrast, gene expression profiles did not differentiate between patients with different durations of disease (long [≥15yrs] vs. short [≤5yrs]); prior treatment response (Primary responder vs. non-responder) and treatment history (number of TNFs failed); and the presence or absence of complications (stricture/stenosis, fistula). Conclusion: Genomewide expression profiling of peripheral blood samples provides the understanding of CD at the molecular level in circulation. This is a new, non-invasive method that can be used to identify systemic markers of local pathological alterations in CD and to discriminate clinically between different CD sub-types. Table: Number of significant expression modulations between CD sub-populations

Role of Intestinal Bacteria in the Pathogenesis of Pouchitis

Abstract:Pouchitis is a common complication seen in patients with ulcerative colitis who undergo total proctocolectomy with ileal pouch anal anastomosis. Bacteria seem to play an important role in the development of pouchitis, although this role is not well defined. Because technology has advanced, we are able to apply molecular techniques to describe the structure and function of the pouch microbial community. In recent years, several studies have been performed comparing the pouch microbiota in patients with ulcerative colitis with healthy pouches and pouchitis. Many of these studies have suggested that pouchitis is characterized by dysbiosis and/or decreased microbial diversity. There has not been a clear pattern identifying a pathogenic organism or a group of organisms responsible for pouchitis. This review summarizes recent studies exploring the pouch microbiota in health and disease, the relationship of bacterial metabolites and pouchitis, and the role of antibiotics and probiotics for the treatment and prevention of pouchitis.

Early Transcriptomic Changes in the Ileal Pouch Provide Insight into the Molecular Pathogenesis of Pouchitis and Ulcerative Colitis

Background: Ulcerative colitis (UC) only involves the colonic mucosa. Yet, nearly 50% of patients with UC who undergo total proctocolectomy with ileal pouch anal anastomosis develop UC-like inflammation of the ileal pouch (pouchitis). By contrast, patients with familial adenomatous polyposis (FAP) with ileal pouch anal anastomosis develop pouchitis far less frequently. We hypothesized that pathogenic events associated with the development of UC are recapitulated by colonic-metaplastic transcriptomic reprogramming of the UC pouch. Methods: We prospectively sampled pouch and prepouch ileum mucosal biopsies in patients with UC with ileal pouch anal anastomosis 4, 8, and 12 months after their pouch was in continuity. Mucosal samples were also obtained from patients with FAP. Transcriptional profiles of the UC and FAP pouch and prepouch ileum were investigated via RNA sequencing and compared with data from a previously published microarray study. Results: Unlike patients with FAP, subjects with UC exhibited a large set of differentially expressed genes between the pouch and prepouch ileum as early as 4 months after pouch functionalization. Functional pathway analysis of differentially expressed genes in the UC pouch revealed an enhanced state of immune/inflammatory response and extracellular matrix remodeling. Moreover, >70% of differentially expressed genes mapped to published inflammatory bowel diseases microarray data sets displayed directional changes consistent with active UC but not with Crohns disease. Conclusions: The UC pouch, well before histologic inflammation, already displays a systems-level gain of colon-associated genes and loss of ileum-associated genes. Patients with UC exhibit a unique transcriptomic response to ileal pouch creation that can be observed well before disease and may in part explain their susceptibility to the development of pouchitis.BACKGROUND Ulcerative colitis (UC) only involves the colonic mucosa. Yet, nearly 50% of patients with UC who undergo total proctocolectomy with ileal pouch anal anastomosis develop UC-like inflammation of the ileal pouch (pouchitis). By contrast, patients with familial adenomatous polyposis (FAP) with ileal pouch anal anastomosis develop pouchitis far less frequently. We hypothesized that pathogenic events associated with the development of UC are recapitulated by colonic-metaplastic transcriptomic reprogramming of the UC pouch. METHODS We prospectively sampled pouch and prepouch ileum mucosal biopsies in patients with UC with ileal pouch anal anastomosis 4, 8, and 12 months after their pouch was in continuity. Mucosal samples were also obtained from patients with FAP. Transcriptional profiles of the UC and FAP pouch and prepouch ileum were investigated via RNA sequencing and compared with data from a previously published microarray study. RESULTS Unlike patients with FAP, subjects with UC exhibited a large set of differentially expressed genes between the pouch and prepouch ileum as early as 4 months after pouch functionalization. Functional pathway analysis of differentially expressed genes in the UC pouch revealed an enhanced state of immune/inflammatory response and extracellular matrix remodeling. Moreover, >70% of differentially expressed genes mapped to published inflammatory bowel diseases microarray data sets displayed directional changes consistent with active UC but not with Crohns disease. CONCLUSIONS The UC pouch, well before histologic inflammation, already displays a systems-level gain of colon-associated genes and loss of ileum-associated genes. Patients with UC exhibit a unique transcriptomic response to ileal pouch creation that can be observed well before disease and may in part explain their susceptibility to the development of pouchitis.

561 Inflammatory Bowel Disease and Non-Relaxing Pelvic Floor Dysfunction

Symptoms of non-relaxing pelvic floor dysfunction may overlap with those of inflammatory bowel disease (IBD) and include abdominal and pelvic pain, bloating, and straining with bowel movements. Aim: To assess the prevalence of non-relaxing pelvic floor dysfunction (N-RPFD), clinical features, and response to pelvic floor biofeedback therapy in patients with ulcerative colitis (UC), Crohns Disease (CD) and ileal J pouch dysfunction (IJPD) after Ileal j-pouch anal anastomosis(IPAA) with symptoms suggestive of disordered stool evacuation.. Methods: We reviewed the medical records of patients with IBD at our institution who underwent high resolution anorectal manometry (ARM) for clinical indications from January 2007-April 2012. We recorded and summarized data from their medical records. N-RPFD was characterized by high anal sphincter pressures (ASP) at rest (maximum .90mmHg), or inability to expel a 50 mL balloon from the rectum despite addition of .200g weight. Results: ARM was performed in 177 patients with IBD including 66 with CD, 62 with UC, and 49 with IJPD. There were 125 females and 52 males. The median age was 48 years, range 13-83. ARM was abnormal in 82/177 patients (46%): 80 had NRPFD and 2 patients had low anal pressures and fecal incontinence. In patients with IJPD, 35/49 (71%) had pouchitis and 20/35 pouchitis patients (57%) had N-RPFD. Among the14/ 49 (29%) who did not have pouchitis, 4/14 (29%) had N-RPFD. Among 22 patients with CD and UC who did not have diarrhea or rectal bleeding, 8 (36%) had N-RPFD. Twenty of 80 patients with N-RPFD underwent multiple pelvic floor retraining sessions with rectal sensor and EMG monitoring for anorectal biofeedback: 12/20 (60%) had improvement in symptoms, including 8/13 (62%) with IJPD. Conclusions: Non-relaxing pelvic floor dysfunction was confirmed in 45% of patients with IBD referred for a suspected disorder of stool evacuation. The prevalence of N-RPFD is highest in patients with pouchitis after IPAA. Importantly, 60% of patients with IBD and non-relaxing pelvic floor dysfunction improved with pelvic floor retraining. These data support the hypothesis that PFDmay be a contributing factor and in some cases a major cause of symptoms in patients with IBD.

Mo1688 Interobserver and Intraobserver Agreement of the Mayo Score of Endoscopic Activity in Ulcerative Colitis

After ileocecal resection, there is a high risk of recurrence on the ileocolic anastomosis and / or in the neoterminal ileum. The severity of endoscopic recurrence in the first year, assessed by the Rutgeerts score, can predict the risk of clinical recurrence and surgery. Treatments can be started immediately after surgery or in the presence of moderate or severe endoscopic lesions. Recent studies show that anti-TNF antibodies are very effective in preventing recurrence. However, there are only few data on the efficacy of anti-TNF antibodies initiated for a severe endoscopic recurrence. The main objective of this study was to evaluate the efficacy of anti-TNF on mucosal healing in patients with severe endoscopic recurrence (i3-i4). Methods: It is a retrospective, multicenter study from the GETAID. All GETAID centers were contacted to identify eligible cases (patients treated with anti-TNF for postoperative endoscopic recurrence). Inclusion criteria were: diagnosis of CD ileocecal resection with no residual macroscopic lesions Rutgeerts score (i3-i4) at ileocolonoscopy performed after surgery treatment with infliximab or adalimumab assessment of mucosal healing (Rutgeerts score). Results: Twenty-eight patients were included, 14 (50%) treated with adalimumab and 14 (50%) with infliximab. Partial mucosal healing (score i0 or i1) was achieved in 14 patients (50%). Complete mucosal healing (score i0) was obtained in 8 patients (29%) and improvement of endoscopic score (decrease of at least 1 point) was observed in 16 patients (57%). Fifty % of patients in the adalimumab group had received anti-TNF therapy before surgery versus 21% in the infliximab group (p=0.11). In multivariate analysis, there was a significantly better response on the partial mucosal healing in the group of patients treated with infliximab (p=0.03). Conclusion: Anti-TNFmonoclonal antibodies initiated after surgery because of a severe endoscopic recurrence (i3, i4) induce a partial mucosal healing in 50% of patients.

AlphaE Integrin Expression Is Increased in the Ileum Relative to the Colon and Unaffected by Inflammation

Abstract Background Recent findings suggest that αE expression is enriched on effector T cells and that intestinal αE+ T cells have increased expression of inflammatory cytokines. αE integrin expression is a potential predictive biomarker for response to etrolizumab, a monoclonal antibody against β7 integrin that targets both α4β7 and αEβ7. We evaluated the prevalence and localization of αE+ cells as well as total αE gene expression in healthy and inflammatory bowel disease patients. Methods αE+ cells were identified in ileal and colonic biopsies by immunohistochemistry and counted using an automated algorithm. Gene expression was assessed by quantitative reverse-transcriptase polymerase chain reaction. Results In both healthy and inflammatory bowel disease patients, significantly more αE+ cells were present in the epithelium and lamina propria of ileal compared with colonic biopsies. αE gene expression levels were also significantly higher in ileal compared with colonic biopsies. Paired biopsies from the same patient showed moderate correlation of αE expression between the ileum and colon. Inflammation did not affect αE expression, and neither endoscopy nor histology scores correlated with αE gene expression. αE expression was not different between patients based on concomitant medication use except 5-aminosalicylic acid. Conclusion αE+ cells, which have been shown to have inflammatory potential, are increased in the ileum in comparison with the colon in both Crohn’s disease and ulcerative colitis, as well as in healthy subjects. In inflammatory bowel disease patients, αE levels are stable, regardless of inflammatory status or most concomitant medications, which could support its use as a biomarker for etrolizumab.