Mary L. Harris

MicroRNAs Are Differentially Expressed in Ulcerative Colitis and Alter Expression of Macrophage Inflammatory Peptide-2α

BACKGROUND & AIMSnChronic inflammatory bowel diseases such as ulcerative colitis (UC) are associated with differential expression of genes involved in inflammation and tissue remodeling. MicroRNAs (miRNAs), which direct mRNA degradation and translational inhibition, influence a number of disease processes. We examined whether miRNAs are differentially expressed in UC tissues and are associated with expression of genes that regulate inflammation.nnnMETHODSnmiRNA expression was assessed in patients with active UC, inactive UC, Crohns disease, irritable bowel syndrome, infectious colitis, and microscopic colitis, as well as in healthy subjects by microarray, quantitative reverse transcription-polymerase chain reaction and in situ hybridization analyses. Colonic epithelial cell (HT29) expression of miRNAs was assessed. Regulation of gene expression by miRNAs was assessed by luciferase reporter construct assays and transfection of specific miRNA mimics.nnnRESULTSnActive UC was associated with the differential expression of 11 miRNAs; 3 were significantly decreased and 8 were significantly increased in UC tissues. In situ hybridization analysis indicated that miR-192, an miRNA with decreased expression in active UC, was predominantly localized to colonic epithelial cells. Macrophage inflammatory peptide (MIP)-2 alpha, a chemokine expressed by epithelial cells, was identified as a target of miR-192. In colon epithelial cells, induction of MIP-2 alpha expression by tumor necrosis factor-alpha was accompanied by a concomitant reduction in miR-192 expression and miR-192 was observed to regulate the expression of MIP-2 alpha.nnnCONCLUSIONSnThese findings expand the known roles of miRNAs, indicating that tissues from patients with UC, and possibly other chronic inflammatory diseases, have altered miRNA expression patterns. These findings also demonstrate that miRNAs regulate colonic epithelial cell-derived chemokine expression.

A national survey of the prevalence and impact of Clostridium difficile infection among hospitalized inflammatory bowel disease patients.

BACKGROUND:We sought to determine nationwide, population-based trends in rates of Clostridium difficile (C. difficile) infection among hospitalized inflammatory bowel disease (IBD) patients in the United States, and to determine its mortality and economic impact.METHODS:We analyzed discharge records from the Nationwide Inpatient Sample, and used the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) codes to identify Crohns disease (CD) and ulcerative colitis (UC) cases, and cases of C. difficile infection between 1998 and 2004. Temporal patterns of C. difficile incidence in IBD patients were compared to non-IBD gastroenterology patients and all-hospitalized patients. The impact of C. difficile on in-hospital mortality and resource utilization was quantified using multiple regression analysis.RESULTS:The prevalence of C. difficile among UC patients (37.3 per 1,000, 95% confidence interval [CI] 34.0–40.7 per 1,000) was higher than that among CD patients (10.9 per 1,000, 95% CI 9.9–12.0 per 1,000), non-IBD gastrointestinal (GI) patients (4.8 per 1,000, 95% CI 4.6–5.0 per 1,000), and general medical patients (4.5 per 1,000, 95% CI 4.2–4.7 per 1,000). C. difficile incidence nearly doubled among UC patients (26.6 per 1,000 to 51.2 per 1,000) over 7 yr. After adjustment for confounders, C. difficile infection was associated with greater mortality among patients with UC (odds ratio [OR] 3.79, 95% CI 2.84–5.06), but not CD (OR 1.66, 95% CI 0.75–3.66). C. difficile was also associated with 65% and 46% longer lengths of stay, which correlated with 63% and 46% higher average hospital charges, for CD and UC patients, respectively.CONCLUSIONS:C. difficile infection is a growing public health issue among hospitalized IBD patients, especially those with UC, and is associated with higher mortality and resource utilization, prompting the need for better preventative measures and early detection.

Identification of microRNAs associated with ileal and colonic Crohn's disease.

Background: Crohns disease (CD) and ulcerative colitis (UC) are associated with expression differences in genes involved in immune function, wound healing, and tissue remodeling. MicroRNAs (miRNAs) are small, noncoding RNAs that act as potent negative regulators of gene expression and are differentially expressed in chronic inflammatory diseases, including UC. We examined the expression of miRNAs in tissues from different intestinal regions and in patients with active ileal and colonic CD. Methods: Colonoscopic pinch biopsies were obtained from the terminal ileum, cecum, transverse colon, sigmoid colon, and rectum of normal, healthy adults and from the ileum and sigmoid colon of patients with active ileal and colonic CD. miRNA expression was assessed using miRNA microarray and validated by mature miRNA quantitative reverse‐transcription polymerase chain reaction (RT‐PCR). Results: Ten intestine region‐specific miRNAs were identified. Three miRNAs were increased and one miRNA was decreased in the terminal ileum as compared to the colon. Six other miRNAs expressed varying levels of expression among the colon regions. Five miRNAs were found to be differentially expressed in tissues of patients with active colonic CD, with three increased and two decreased as compared to normal, healthy controls. Similarly, four miRNAs were found to be significantly increased in tissues of patients with active ileal CD. Conclusions: The expression differences between ileal CD, colonic CD, and previously identified UC‐associated miRNAs support the likelihood that miRNAs influence differing inflammation‐related gene expression in each inflammatory bowel disease (IBD) subtype and may form the basis for future diagnostic tests and therapeutic targets for IBD. Inflamm Bowel Dis 2010

Inflammatory bowel disease characteristics among African Americans, Hispanics, and non-Hispanic Whites: characterization of a large North American cohort.

OBJECTIVES:Inflammatory bowel disease (IBD), comprising primarily of Crohns disease (CD) and ulcerative colitis (UC), is increasingly prevalent in racial and ethnic minorities. This study was undertaken to characterize racial differences in disease phenotype in a predominantly adult population.METHODS:Phenotype data on 830 non-Hispanic white, 127 non-Hispanic African American, and 169 Hispanic IBD patients, recruited from six academic centers, were abstracted from medical records and compiled in the NIDDK-IBD Genetics Consortium repository. We characterized racial differences in family history, disease location and behavior, surgical history, and extraintestinal manifestations (EIMs) using standardized definitions.RESULTS:African American CD patients were more likely than whites to develop esophagogastroduodenal CD (OR = 2.8; 95% CI: 1.4–5.5), colorectal disease (OR = 1.9; 95% CI: 1.1–3.4), perianal disease (OR = 1.7; 95% CI: 1.03–2.8), but less likely to have ileal involvement (OR = 0.55; 95% CI: 0.32–0.96). They were also at higher risk for uveitis (OR = 5.5; 95% CI: 2.3–13.0) and sacroiliitis (OR = 4.0; 95% CI: 1.55–10.1). Hispanics had higher prevalence of perianal CD (OR = 2.9; 95% CI: 1.8–4.6) and erythema nodosum (3.3; 95% CI: 1.7–6.4). Among UC patients, Hispanics had more proximal disease extent. Both African American and Hispanic CD patients, but not UC patients, had lower prevalences of family history of IBD than their white counterparts.CONCLUSIONS:There are racial differences in IBD family history, disease location, and EIMs that may reflect underlying genetic variations and have important implications for diagnosis and management of disease. These findings underscore the need for further studies in minority populations.

Genome‐wide gene expression differences in Crohn's disease and ulcerative colitis from endoscopic pinch biopsies: Insights into distinctive pathogenesis

Background: Ulcerative colitis (UC) and Crohns disease (CD) are inflammatory bowel diseases (IBD) with variable, overlapping clinical features and complex pathophysiologies. Methods: To identify pathogenic processes underlying these disease subtypes, we used single endoscopic pinch biopsies to elucidate patterns of gene expression in active and inactive areas of UC and CD and compared these to infectious colitis and healthy control samples. Results: Unsupervised classification of a total of 36 samples yielded promising separation between the affected IBD, unaffected IBD, non‐IBD colitis, and normal control samples, suggesting each sample type had a distinctive gene expression pattern. Genes differentially expressed in the CD samples compared to in the controls were related to IFN&ggr;‐inducible TH1 processes (IFITM1, IFITM3, STAT1, and STAT3) and antigen presentation (TAP1, PSME2, PSMB8). The most noticeable change in the UC samples was reduced expression of genes regulating biosynthesis, metabolism, and electrolyte transport (HNF4G, KLF5, AQP8, ATP2B1, and SLC16A). Twenty‐five percent of genes down‐regulated in the UC samples were also down‐regulated in the infectious colitis samples. Unaffected biopsy samples of IBD patients also registered differences expression of genes compared to in the normal controls. Of these differentially expressed genes, only 2 were up‐regulated, PSKH1, a regulator of mRNA processing, and PPID, a suppressor of apoptosis. Conclusions: The study shows that the gene expression patterns of IBD, CD in particular, are quite different from those of infectious colitis, highlighting distinctive expression of genes and pathways in UC and CD. (Inflamm Bowel Dis 2007)

NOD2 Mutations and Anti-Saccharomyces cerevisiae Antibodies Are Risk Factors for Crohn's Disease in African Americans

OBJECTIVES:NOD2 mutations and anti-Saccharomyces cerevisiae antibodies (ASCAs) are established risk factors of Crohns disease (CD) in whites but have not been assessed in African-American (AA) adults with CD.METHODS:AAs with CD and controls were recruited by the Mid-Atlantic African-American IBD Study as part of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) IBD Genetics Consortium. Genotyping for the three common CD NOD2 mutations (Leu1007fsinsC, G908R/2722g>c, and R702W/2104c>t) and ASCA enzyme-linked immunosorbent assays were performed in 183 AA CD patients and in 143 controls. Logistic regression was used to calculate adjusted odds ratios (ORs) for the association between ASCA and disease phenotype.RESULTS:ASCA sensitivity and specificity values were 70.5 and 70.4%, respectively. On univariate analysis, ASCA was significantly associated with younger age at diagnosis, ileal involvement, and complicated (stricturing/penetrating) behavior. On multivariate analysis, ASCA titer (per 25u2009Units) was associated with ileal involvement (OR 1.18, 95% confidence interval (CI): 1.04–1.34), complicated behavior (OR 1.13, 95% CI: 1.01–1.28), and surgery (hazard ratio: 1.11, 95% CI: 1.02–1.21). Cigarette smoking and CD family history were also significantly associated with surgery. NOD2 carriers (all heterozygotes) were more common among CD cases than controls (8.2 vs. 2.1%; OR 4.17%, 95% CI: 1.18–14.69). The NOD2 mutation population attributable risk was 6.2%.CONCLUSIONS:In comparison with whites, ASCA in AAs has a similar sensitivity but a lower specificity for CD. ASCA is associated with ileal involvement, complicated behavior, and surgery in AAs with CD. NOD2 is a risk gene for AA CD, although mutation frequency and population attributable risk are much lower than in whites.

Contribution of higher risk genes and European admixture to Crohn's disease in African Americans

Background: African Americans (AAs) are an admixed population of West African (WA) and European ancestry (EA). Crohns disease (CD) susceptibility genes have not been established. We therefore evaluated the contribution of European admixture and major established risk genes to AA CD. Methods: Ninety‐seven admixture informative markers were genotyped for ancestry estimates using STRUCTURE. Overall, 354 AA CD cases and 354 ethnicity‐matched controls were genotyped for total 21 single nucleotide polymorphisms (SNPs) in ATG16L1, NOD2, IBD5, IL23R and IRGM by TaqMan or direct sequencing. Association was evaluated by logistic regression, adjusted for ancestry. Results: Mean EA was similar among the CD cases and controls (20.9% and 20.4%, respectively, P = 0.58). No significant admixture differences were observed among 211 to 227 cases stratified by phenotypic subclassifications including onset, surgery, site, and behavior. CD was associated with NOD2 carrier (6.93% CD, 2.15% Controls, P = 0.007), ATG16L1 Thr300Ala (36.1% CD, 29.3% Controls, P = 0.003), SLC22A4 and SLC22A5 (IBD5 locus) functional SNPs (Leu503Phe [10.5% CD, 7.6% Controls, P = 0.05] and g‐207c [41.3% CD, 35.7% Controls, P = 0.03], respectively), and IL23R rs2201841 (18.2% CD, 13.8% Controls, P = 0.03), but not IRGM variants, nor three African ancestral NOD2 nonsynonymous variants. IBD5 risk was recessive. An all‐minor allele IBD5 haplotype from EA was associated (P = 0.05), whereas a more common haplotype isolating g‐207c was not. Conclusions: Specific functional gene variations contribute significantly to AA CD risk. Established NOD2, SLC22A4‐A5, and ATG16L1 variants show increased CD risk, with IBD5 recessive. Although CD is more common in whites, European admixture is similar among AA cases and controls. (Inflamm Bowel Dis 2012;)

In vitro cell aggregation and cell adhesion molecules in Crohn's disease

BACKGROUNDnLack of a suitable model has hindered efforts to understand inflammation and granuloma formation in Crohns disease.nnnMETHODSnGranulomalike aggregates of circulating mononuclear cells are produced in vitro by cultures of cells with polyacrylamide beads. To identify features of in vitro aggregates, which are similar to tissue granulomas of Crohns disease, the gross morphology and immunohistological appearance of the aggregates produced with peripheral blood mononuclear cells from patients with Crohns disease were analyzed, and the size of in vitro aggregates was correlated with clinical activity of the disease. Blocking antibodies were used to evaluate the role of cell-adhesion molecules in the formation of in vitro aggregates.nnnRESULTSnThe size of in vitro aggregates correlates very significantly with clinical activity (P < 0.001). In active Crohns disease, in vitro aggregates show immunohistological features of hypersensitivity type granulomas. Blocking antibodies against leukocyte function associated antigen LFA-2 (CD2), LFA-3 (CD58), and Mac-1 (CD11b/CD18) inhibit in vitro aggregate formation.nnnCONCLUSIONnIn vitro aggregates model in vivo granulomas in size and organization. Cell adhesion molecules like CD2, CD58, and CD11b/CD18 may be involved in granuloma-formation of Crohns disease.

Musculoskeletal Complications of Crohn's Disease: The Role of Computed Tomography in Diagnosis and Patient Management

The delayed diagnosis of musculoskeletal complications of Crohns disease may produce major morbidity in patients. This study compared abdominal and pelvic computed tomography (CT) with conventional radiography in the diagnosis of musculoskeletal complications in 23 of 552 patients with Crohns disease examined by CT over a 7-year period. Surgical confirmation was available in 15 of 21 patients. The clinical features of psoas/gluteal abscesses, abdominal wall fistulae, and sacral osteomyelitis are described. Because the clinical manifestations of these musculoskeletal complications are often nonspecific, CT is often useful in diagnosing and directing therapeutic interventions.

Dietary antigens as aggravating factors in Crohn's disease

In this issue Drs. Allen L. Ginsberg and Michael B. Albert enter the emotional mine field of suggesting a cause for Crohns disease, at least in one patient. They feel milk was an etiologic factor in a 35-year-old man with recurrent ileitis after an ileoright colon resection. This hypersensitivity to milk decreased with achievement of a remission. Since the dictionary defines etiology as a noun meaning an assigning of a cause, perhaps Ginsberg and Albert would not object if we compromised on milk protein as an aggravating factor in this man when his Crohns disease was somewhat active but not when he was in remission. Presumably cows milk acted as an antigen when it could permeate the small intestine rendered more permeable or more reactive by ulceration and tissue inflammation. Enhanced intestinal permeability to various sized sugars as well as to protein probes has been demonstrated in Crohns disease (1). A relatively recent (but as yet still unconfirmed) report by Hollander and colleagues suggested that not only was the mucosa of Crohns disease patients more permeable to polyethylene glycol but that seemingly healthy relatives also shared this permeability defect. They also ventured into the etiology area when suggesting that an inherited intestinal permeability defect was the underlying cause of Crohns disease (2). There is evidence that the damaged intestinal mucosa is permeable to dietary and bacterial antigens. The increased permeability for dietary and bacterial antigens through the damaged intestine can be documented by the finding of elevated serum levels of antibodies to cows milk proteins in patients with inflammatory bowel disease. Interestingly, there was a good correlation of disease activity and antibody titers against casein in Crohns disease patients (3). Even though milk allergy had been postulated as an important factor in the pathogenesis of ulcerative colitis by Andersen (4), and later by Wright and Truelove (5), the instances in which antigen-antibody complexes or dietary antibodies are important in disease pathogenesis are not numerous. The best known examples of cows milk allergy are in infants and in some children, especially those with other signs of atopy. Some infants also seem to become sensitized to cows milk protein after an infectious enteritis. Loss of mucosal peptidases and other enzymes that might be expected to destroy luminal antigens may also contribute to the process of bowel damage. There may also be a heightened immune recognition and presentation system in the small bowel of patients with Crohns disease. Class II antigen expression on lymphocytes and on the monocyte/ macrophage is essential for such cells to function as antigen presenting cells (6). Normal intestinal epithelial cells express class II antigens (7, 8) and function as accessory cells in processing and presenting soluble antigens to immunocompetent T cells. Intestinal epithelial cells selectively enhance proliferation of antigen nonspecific suppressor T cells in contrast to conventional antigen presenting cells, which preferentially stimulate helper/inducer T cells. Selby et al (9), using tissue from patients with IBD, demonstrated a marked increase in DR expression. However, epithelium overlying active diverticulitis or ischemic colitis also demonstrated enhanced DR expression. Thus enhanced expression of class II antigens occurs in the presence of active inflammation and the enhancement is not IBD specific. Nevertheless, in the Crohns disease tissue, DR expression was significantly greater than other inflammatory diseases (9). Taking another perspective: Can dietary and luminal factors be important factors in keeping Crohns disease active? More evidence indicates a positive answer to this question. Uncomplicated small bowel Crohns disease will go into remission in over three fourths of patients if the bowel is put at rest with total parenteral nutrition or with an elemental diet (10). In other types of studies, two thirds of patients with Crohns colitis will go into remission if the fecal stream is diverted with a double-barreled ileostomy (11). As a negative note, a recent editorial pompously trumpeted Total parenteral nutrition as primary treatment in Crohns disease--RIP? (12). In the article accompanying this recent editorial over 80% of the patients went into remission on bowel rest, but, as should have been expected, some relapsed when fed a regular diet without antiinflammatory medications to help sustain the remission