Timothy S. Sadiq

MicroRNAs Classify Different Disease Behavior Phenotypes of Crohn's Disease and May Have Prognostic Utility.

Background:There is a dire need for reliable prognostic markers that can guide effective therapeutic intervention in Crohns disease (CD). We examined whether different phenotypes in CD can be classified based on colonic microRNA (miRNA) expression and whether miRNAs have prognostic utility for CD. Methods:High-throughput sequencing of small and total RNA isolated from colon tissue from patients with CD and controls without Inflammatory Bowel Disease (non-IBD) was performed. To identify miRNAs associated with specific phenotypes of CD, patients were stratified according to disease behavior (nonstricturing, nonpenetrating; stricturing; penetrating), and miRNA profiles in each subset were compared with those of the non-IBD group. Validation assays were performed using quantitative reverse transcription polymerase chain reaction. These miRNAs were further evaluated by quantitative reverse transcriptase polymerase chain reaction on formalin-fixed, paraffin-embedded tissue (index biopsies) of patients with nonpenetrating CD at the time of diagnosis that either retained the nonpenetrating phenotype or progressed to penetrating/fistulizing CD. Results:We found a suite of miRNAs, including miR-31-5p, miR-215, miR-223-3p, miR-196b-5p, and miR-203 that stratify patients with CD according to disease behavior independent of the effect of inflammation. Furthermore, we also demonstrated that expression levels of miR-215 in index biopsies of patients with CD might predict the likelihood of progression to penetrating/fistulizing CD. Finally, using a novel statistical simulation approach applied to colonic RNA-sequencing data for patients with CD and non-IBD controls, we identified miR-31-5p and miR-203 as candidate master regulators of gene expression profiles associated with CD. Conclusions:miRNAs may serve as clinically useful prognostic markers guiding initial therapy and identifying patients who would benefit most from effective intervention.

Molecular classification of Crohn's disease reveals two clinically relevant subtypes

Objective The clinical presentation and course of Crohns disease (CD) is highly variable. We sought to better understand the cellular and molecular mechanisms that guide this heterogeneity, and characterise the cellular processes associated with disease phenotypes. Design We examined both gene expression and gene regulation (chromatin accessibility) in non-inflamed colon tissue from a cohort of adult patients with CD and control patients. To support the generality of our findings, we analysed previously published expression data from a large cohort of treatment-naïve paediatric CD and control ileum. Results We found that adult patients with CD clearly segregated into two classes based on colon tissue gene expression—one that largely resembled the normal colon and one where certain genes showed expression patterns normally specific to the ileum. These classes were supported by changes in gene regulatory profiles observed at the level of chromatin accessibility, reflective of a fundamental shift in underlying molecular phenotypes. Furthermore, gene expression from the ilea of a treatment-naïve cohort of paediatric patients with CD could be similarly subdivided into colon-like and ileum-like classes. Finally, expression patterns within these CD subclasses highlight large-scale differences in the immune response and aspects of cellular metabolism, and were associated with multiple clinical phenotypes describing disease behaviour, including rectal disease and need for colectomy. Conclusions Our results strongly suggest that these molecular signatures define two clinically relevant forms of CD irrespective of tissue sampling location, patient age or treatment status.

Alterations to chromatin in intestinal macrophages link IL‐10 deficiency to inappropriate inflammatory responses

Intestinal macrophages (IMs) are uniquely programmed to tolerate exposure to bacteria without mounting potent inflammatory responses. The cytokine IL‐10 maintains the macrophage anti‐inflammatory response such that loss of IL‐10 results in chronic intestinal inflammation. To investigate how IL‐10‐deficiency alters IM programming and bacterial tolerance, we studied changes in chromatin accessibility in response to bacteria in macrophages from two distinct niches, the intestine and bone‐marrow, from both wild‐type and IL‐10‐deficient (Il10−/−) mice. We identified chromatin accessibility changes associated with bacterial exposure and IL‐10 deficiency in both bone marrow derived macrophages and IMs. Surprisingly, Il10−/− IMs adopted chromatin and gene expression patterns characteristic of an inflammatory response, even in the absence of bacteria. Further, when recombinant IL‐10 was added to Il10−/− cells, it could not revert the chromatin landscape to a normal state. Our results demonstrate that IL‐10 deficiency results in stable chromatin alterations in macrophages, even in the absence of bacteria. This supports a model in which IL‐10‐deficiency leads to chromatin alterations that contribute to a loss of IM tolerance to bacteria, which is a primary initiating event in chronic intestinal inflammation.

What Are the Indications for Resection After an Episode of Sigmoid Diverticulitis

Diverticulitis is a common and costly health care problem that has received renewed attention over the past decade, particularly with regard to surgical management. The historical indication for sigmoid resection after a recurrent episode of uncomplicated diverticulitis has been replaced by an individualized approach. The role of the Hartmann procedure in the management of acute diverticulitis complicated by perforation is diminishing in favor of antibiotics with or without percutaneous drainage followed by elective single stage resection in Hinchey I/ IIA disease and in favor of resection with primary anastomosis/protecting loop ileostomy in Hinchey III/IV cases. Although much has been learned over the past 10 years, it has largely been through retrospective analysis.

Laparoscopic Ladd Procedure for Correction of Pediatric Malrotation: Initial Experience

Introduction: Malrotation without midgut volvulus can cause chronic, intermittent symptoms and produce potentially disastrous consequences if left untreated. Upper gastrointestinal x-ray series (UGI) in such patients often suggest a rotational anomaly but may not be completely diagnostic. Methods: Thirteen children, aged 3 months to 14 years, suspected of having malrotation without volvulus based on preoperative symptoms and UGI, underwent laparoscopic exploration by a single surgeon over a 21 month period. Results: Ten of 13 (77%) were confirmed to have malrotation and underwent laparoscopic correction. Operative time ranged from 80 to 209 minutes (mean, 134 minutes) with minimal blood loss. Feedings were started within 24 hours in 6 (60%) and within 72 hours in the remaining 4 (40%). Full feedings were tolerated by postoperative day (POD) 4 in all patients. Hospital stay ranged from 1 to 4 days (mean, 2.8 days). Two of the three children with negative laparoscopies were discharged by POD 1. Followup has...

O-004 Analysis of Chromatin and Transcriptional Profiles in Crohn's Disease Reveals Molecular Subclasses and Highlights Functional Regulatory Regions Implicated in Disease.

Background:Crohns disease (CD) is a chronic inflammatory disease of the gastrointestinal tract, characterized by an inappropriate immune response to the enteric microbiota. Chromatin based assays have successfully identified cell type and disease-specific regions of DNA accessibility, and have been linked to differential transcription of target genes, but this approach has yet to be explored in CD. In this study we performed formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) to identify nucleosome-depleted regions associated with regulatory elements, and RNA-seq to quantify expression, to characterize disease-relevant changes in chromatin and establish links to putative target genes. Methods:Mucosal Biopsies for 21 CD and 12 non-IBD individuals were taken from macroscopically uninflamed portions of the ascending colon at time of surgery. We performed FAIRE-seq and RNA-seq on each tissue sample. Reads were aligned to personalized genomes constructed with genotypes assayed on the Illumina Immunochip platform. A genome-wide scan for differential regions and genes was performed using 2 sided t-tests on normalized read counts in 300bp regions (FAIRE-seq) and RPKM (RNA-seq). Results:RNA-seq data analysis across all samples identified 2 distinct classes of patients associated with epithelial cell lipid transport (class I, n = 10, CD; n = 1, non-IBD) and maintenance of cellular and luminal pH (class II, n = 11, CD, n = 11, non-IBD). To unravel potential gene regulatory mechanisms we analyzed chromatin profiles using FAIRE-seq and histone annotations using tissue specific ChIP-seq data sets. Class I patients showed increased accessibility at distal sites marked for enhancer activity in small intestine, and reduced accessibility at sites marked as enhancers in colon, suggesting 2 distinct regulatory programs driving the expression of differential genes. This pattern was reversed in class II samples. In an analysis restricting to class II, which was characterized by normal colon enhancer activity, we found regulatory regions specific to CD and non-IBD, respectively, as well as 51 and 507 genes upregulated in the respective cohorts. Top ontologies of genes upregulated in CD included host response to bacteria and immune response. Differential regulatory genes were enriched near TSSs of differentially expressed genes, implying putative causal effect. In an integrative analysis, we identified 72 regulatory regions that associate with both disease status and expression of nearby genes. A retrospective analysis of patients in class I and II revealed that the majority of patients (10/11) in class I required post-operative anti-TNF therapy for disease recurrence compared to only 1/11 CD patients in the class II group. Conclusions:We used chromatin status as a novel marker of disease in Crohns. We identified a subset of CD patients that are characterized at the chromatin level in the colon by enrichment of enhancer marks specific to small intestine, and loss of enhancer activity specific to colon. Among individuals with enhancer activity indicative of normal colon, we integrated data from well-described transcriptional markers with novel chromatin variables to identify molecular associations and interactions that affect disease predisposition. Despite small patient numbers we were able to use chromatin profiles to identify patients that required post-operative management with anti-TNF agents.

High acceptance rate of anal pap screening despite limited knowledge about anal dysplasia among HIV+ MSM

Background Anal cancer in the general population is more prevalent in women, but in most HIV populations, MSM have the highest risk. Data suggest that screening can prevent invasive carcinoma. Use of routine cervical pap smears resulted in an 80% reduction in cervical cancer rates. The current study examines the effectiveness of a clinical intervention designed to increase anal dysplasia education, screening, and treatment for HIV+ MSM.

Association Between Colonic Level of microRNA 31 and Subtypes of Adult and Pediatric Crohn's Disease

Crohn9s disease (CD) is highly heterogeneous, due in large part to variability in cellular processes that underlie the natural history of CD, thereby confounding effective therapy. There is a critical need to advance understanding of the cellular mechanisms that drive CD heterogeneity. In this study, small RNA-sequencing and microRNA profiling in the colon revealed two distinct molecular subtypes, each with different clinical associations, in both adult and treatment-naive pediatric CD patients. Notably, we found that microRNA-31 (miR-31) expression by itself can stratify patients into these two subtypes. Through detailed analysis of several colonic mucosa cell types from adult patients, we found that differential levels of miR-31 are particularly pronounced in epithelial cells. We generated patient crypt-derived epithelial colonoids and showed that miR-31 expression differences preserved in this ex-vivo system. In adult patients, low colonic miR-31 expression levels at the time of surgery are associated with post-operative recurrence of ileal disease. In pediatric patients, lower miR-31 expression at the time of diagnosis is associated with the future development of fibrostenotic ileal CD requiring surgery. These findings represent an important step forward in designing more effective clinical trials and developing personalized therapies for CD.Background & Aims The course of Crohns disease (CD) is heterogeneous, confounding effective therapy. An analysis of differences in colonic gene expression between patients with vs without CD revealed 2 subsets of patients—a group characterized by genes more highly expressed in the colon (colonlike CD) and a group with increased expression of ileum marker genes (ileum-like CD). We compared differences in microRNAs between these groups. Methods We performed genome-wide microRNA profile analyses of colon tissues from 18 adults with CD and 12 adults without CD (controls). We performed principal component analyses to associate levels of microRNAs with CD subtypes. Colonic epithelial cells and lamina propria immune cells were isolated from intestinal tissues and levels of microRNA 31 (MIR31 or miR-31) were measured by real-time quantitative PCR. We validated the differential expression of miR-31 between the subtypes by measuring miR-31 levels in an independent cohort of 32 adult patients with CD and 23 controls. We generated epithelial colonoid cultures from controls and patients with CD, and measured levels of miR-31 in crypts. We performed genome-wide microRNA profile analyses of formalin-fixed paraffin-embedded colon and ileum biopsies from 76 treatment-naïve pediatric patients with CD and 51 controls and collected data on disease features and outcomes. Results In comparing miRNA expression profiles between 9 patients with colon-like CD and 9 patients with ileum-like CD, we identified 19 miRNAs with significant differences in levels. We observed a 13.5-fold difference in level of miR-31-5p between tissues from patients with colon-like vs ileum-like CD (Padj = 1.43 x 10-18). Principal component analysis found miR-31 to be the top contributor to the variance observed. Levels of miR-31 were increased 60-fold in tissues from patients with ileum-like CD compared with controls (Padj = 2.59 × 10-51). We validated the differential expression of miR-31 between the subtypes in the independent set of tissues. Colonoids derived from patients with CD had significantly higher levels of miR-31 than colonoids derived from control tissues (day 2 P=.041 and day 6 P=.0095). Levels of miR-31 were significantly increased in colon tissues from pediatric patients with CD compared with controls (~7.8-fold, P=4.64 ×10-7) and in ileum tissues from patients with CD vs controls (~1.5-fold, P=9.97 × 10-7). A low level of miR-31 in index biopsies from pediatric patients with only inflammation and no other complications at time of diagnosis associated with development of fibrostenotic ileal CD. Conclusions We identified differences in miR-31 levels in colon tissues from adult and pediatric patients with CD compared with controls, and in patients with ileum-like CD compared with colon-like CD. Further studies are needed to determine the mechanisms by which miR-31 might contribute to pathogenesis of this subtype of CD, or affect response to therapy.

Colonic epithelial miR-31 associates with the development of Crohn’s phenotypes

BACKGROUND Crohns disease (CD) is highly heterogeneous, due in large part to variability in cellular processes that underlie the natural history of CD, thereby confounding effective therapy. There is a critical need to advance understanding of the cellular mechanisms that drive CD heterogeneity. METHODS We performed small RNA sequencing of adult colon tissue from CD and NIBD controls. Colonic epithelial cells and immune cells were isolated from colonic tissues, and microRNA-31 (miR-31) expression was measured. miR-31 expression was measured in colonoid cultures generated from controls and patients with CD. We performed small RNA-sequencing of formalin-fixed paraffin-embedded colon and ileum biopsies from treatment-naive pediatric patients with CD and controls and collected data on disease features and outcomes. RESULTS Small RNA-sequencing and microRNA profiling in the colon revealed 2 distinct molecular subtypes, each with different clinical associations. Notably, we found that miR-31 expression was a driver of these 2 subtypes and, further, that miR-31 expression was particularly pronounced in epithelial cells. Colonoids revealed that miR-31 expression differences are preserved in this ex vivo system. In adult patients, low colonic miR-31 expression levels at the time of surgery were associated with worse disease outcome as measured by need for an end ileostomy and recurrence of disease in the neoterminal ileum. In pediatric patients, lower miR-31 expression at the time of diagnosis was associated with future development of fibrostenotic ileal CD requiring surgeryCONCLUSIONS. These findings represent an important step forward in designing more effective clinical trials and developing personalized CD therapies. FUNDING This work was supported by CCF Career Development Award (SZS), R01-ES024983 from NIEHS (SZS and TSF), 1R01DK104828-01A1 from NIDDK (SZS and TSF), P01-DK094779-01A1 from NIDDK (SZS), P30-DK034987 from NIDDK (SZS), 1-16-ACE-47 ADA Pathway Award (PS), UNC Nutrition Obesity Research Center Pilot & Feasibility Grant P30DK056350 (PS), CCF PRO-KIIDS NETWORK (SZS and PS), UNC CGIBD T32 Training Grant from NIDDK (JBB), T32 Training Grant (5T32GM007092-42) from NIGMS (MH), and SHARE from the Helmsley Trust (SZS). The UNC Translational Pathology Laboratory is supported, in part, by grants from the National Cancer Institute (3P30CA016086) and the UNC University Cancer Research Fund (UCRF) (PS).