Colitis Samples Research Articles

Identification of Candidate Genes Related to Inflammatory Bowel Disease Using Minimum Redundancy Maximum Relevance, Incremental Feature Selection, and the Shortest-Path Approach.

Identification of disease genes is a hot topic in biomedicine and genomics. However, it is a challenging problem because of the complexity of diseases. Inflammatory bowel disease (IBD) is an idiopathic disease caused by a dysregulated immune response to host intestinal microflora. It has been proven to be associated with the development of intestinal malignancies. Although the specific pathological characteristics and genetic background of IBD have been partially revealed, it is still an overdetermined disease and the blueprint of all genetic variants still needs to be improved. In this study, a novel computational method was built to identify genes related to IBD. Samples from two subtypes of IBD (ulcerative colitis and Crohn's disease) and normal samples were employed. By analyzing the gene expression profiles of these samples using minimum redundancy maximum relevance and incremental feature selection, 21 genes were obtained that could effectively distinguish samples from the two subtypes of IBD and the normal samples. Then, the shortest-path approach was used to search for an additional 20 genes in a large network constructed using protein-protein interactions based on the above-mentioned 21 genes. Analyses of the 41 genes obtained indicate that they are closely associated with this disease.

East Meets West: The Increasing Incidence of Inflammatory Bowel Disease in Asia as a Paradigm for Environmental Effects on the Pathogenesis of Immune-Mediated Disease

East Meets West: The Increasing Incidence of Inflammatory Bowel Disease in Asia as a Paradigm for Environmental Effects on the Pathogenesis of Immune-Mediated Disease

A new role for reticulon-4B/NOGO-B in the intestinal epithelial barrier function and inflammatory bowel disease.

Inflammatory bowel disease (IBD) is characterized by an impaired intestinal barrier function. We aimed to investigate the role of reticulon-4B (RTN-4B/NOGO-B), a structural protein of the endoplasmic reticulum, in intestinal barrier function and IBD. We used immunohistochemistry, confocal microscopy, real-time PCR, and Western blotting to study tissue distribution and expression levels of RTN-4B/NOGO-B in control and IBD samples from mouse and humans. We also targeted RTN-4B/NOGO-B using siRNAs in cultured human intestinal epithelial cell (IECs). Epithelial barrier permeability was assessed by transepithelial electrical resistance (TEER) measurement. RTN-4B/NOGO-B is expressed in the intestine mainly by IECs. Confocal microscopy revealed a colocalization of RTN-4B, E-cadherin, and polymerized actin fibers in tissue and cultured IECs. RTN-4B mRNA and protein expression were lower in the colon of IL-10(-/-) compared with wild-type mice. Colocalization of RTN-4B/E-cadherin/actin was reduced in the colon of IL-10(-/-) mice. Analysis of endoscopic biopsies from IBD patients showed a significant reduction of RTN-4B/NOGO-B expression in inflamed mucosa compared with control. Treatment of IECs with H2O2 reduced TEER values and triggered phosphorylation of RTN-4B in serine 107 residues as well as downregulation of RTN-4B expression. Acute RTN-4B/NOGO-B knockdown by siRNAs resulted in a decreased TEER values and reduction of E-cadherin and α-catenin expression and in the amount of F-actin-rich filaments in IECs. Epithelial RTN-4B/NOGO-B was downregulated in human and experimental IBD. RTN-4B participates in the intestinal epithelial barrier function, most likely via its involvement in E-cadherin, α-catenin expression, and actin cytoskeleton organization at sites of cell-to-cell contacts.

Reduced expression of aquaporins in human intestinal mucosa in early stage inflammatory bowel disease

ObjectivesThe aim of this study was to investigate the relationship between aquaporin (AQP) water channel expression and the pathological features of early untreated inflammatory bowel disease (IBD) in humans.MethodsPatients suspected to have IBD on the basis of predefined symptoms, including abdominal pain, diarrhea, and/or blood in stool for more than 10 days, were examined at the local hospital. Colonoscopy with biopsies was performed and blood samples were taken. Patients who did not meet the diagnostic criteria for IBD and who displayed no evidence of infection or other pathology in the gut were included as symptomatic non-IBD controls. AQP1, 3, 4, 5, 7, 8, and 9 messenger RNA (mRNA) levels were quantified in biopsies from the distal ileum and colon by quantitative real-time polymerase chain reaction. Protein expression of selected AQPs was assessed by confocal microscopy. Through multiple alignments of the deduced amino acid sequences, the putative three-dimensional structures of AQP1, 3, 7, and 8 were modeled.ResultsAQP1, 3, 7, and 8 mRNAs were detected in all parts of the intestinal mucosa. Notably, AQP1 and AQP3 mRNA levels were reduced in the ileum of patients with Crohn’s disease, and AQP7 and AQP8 mRNA levels were reduced in the ileum and the colon of patients with ulcerative colitis. Immunofluorescence confocal microscopy showed localization of AQP3, 7, and 8 at the mucosal epithelium, whereas the expression of AQP1 was mainly confined to the endothelial cells and erythrocytes. The reduction in the level of AQP3, 7, and 8 mRNA was confirmed by immunofluorescence, which also indicated a reduction of apical immunolabeling for AQP8 in the colonic surface epithelium and crypts of the IBD samples. This could indicate loss of epithelial polarity in IBD, leading to disrupted barrier function.ConclusionAQPs 1 and 8 and the aquaglyceroporins AQPs 3 and 7 are the AQPs predominantly expressed in the lower intestinal tract of humans. Their expression is significantly reduced in patients with IBD, and they are differentially expressed in specific bowel segments in patients with Crohn’s disease and ulcerative colitis. The data present a link between gut inflammation and water/solute homeostasis, suggesting that AQPs may play a significant role in IBD pathophysiology.

Antigen Up-take by Intestinal Epithelial Cells in IBD

Table 1. Percentage of the cells stained with CD45 (leukocytes) and EpCAM (epithelial cells) antibodies for flow cytometry analysis of the mucosal fractions obtained from surgical specimens from UC inflamed. UC non-inflamed. CD and NL patients. Statistic comparisons performed between UC I, CD and NL (***p<0.001. **p<0.01 and *p<0.05). Previous work of our group showed that inflammatory bowel disease (IBD) patients could not be orally tolerized against keyhole limpet hemocyanin (KLH) [1]. Our aim is to test in vitro the antigen up-take ability of the same antigen in isolated IECs from IBD patients and non-inflammatory controls. 1. Kraus TA et al. Failure to induce oral tolerance to a soluble protein in patients with inflammatory bowel disease. Gastroenterology (2004) 126: 1771-1778. Surgical specimens were obtained from intestinal resections performed in Mount Sinai Medical Center. Five ulcerative colitis (with inflamed tissue (UC I) and four out of them also with non-inflamed tissue (UC NI)), and five Crohn's disease (CD) patients and 8 normal mucosal samples (NL) were included. Briefly, the protocol of IECs isolation consisted in intense cleaning of surgical pieces with PBS; the mucosa was stripped from the submucosa in small pieces (3-5 mm). The pieces were incubated in DTT (1 mM in RPMI medium) for 15 min followed by 2 incubations of 30 min each in 25 mL of Dispase II (1.5 mg/mL in RPMI) at 37 °C. After washing and centrifugation, IECs were recovered from the supernatant and were counted and adjusted to a final concentration of 106 cells/mL in RPMI (plus antibiotics and Fungizone). 105 cells/well were plated in 96 wells plates in a total volume of 200 μl/well of RPMI with or without 100 μg of KLH. At different time points (0.5, 1, 2, 4 and 18 h) IECs were surface stained for flow cytometry analysis with CD45 (as a marker of leukocytes) and Ep-CAM (as a marker of IECs) antibodies, fixed and permeabilized and intra-cellular stained with an anti-KLH antibody. In 4 UC inflamed, 4 CD and 7 NL samples from same patients MHCII expression was analyzed by flow cytometry. In other set of experiments, after overnight (ON) incubation with or without KLH, MHCII expression was analyzed by flow cytometry in Ep-CAM + cells from 3 NL and 3 UC inflamed samples. Comparisons between groups were statistically analyzed by Mann-Whitney test for unpaired samples. As showed in Table 1, the proportion of leukocytes and IECs in mucosa reflected the inflammatory state of the tissue with increased proportion of leukocytes and decreased levels of IECs in IBD samples. IECs from UC inflamed showed an increased earlier up-take of KLH compared to CD, however CD IECs had larger overnight uptake of KLH than UC inflamed (Fig. 1). The percentage of IECs that are MHCII + is significantly higher in inflamed UC patients compared to NL, although KLH incubation does not have any effect on% of IECs expressing MHCII+ (Fig. 2). The different kinetics of KLH up-take by IECs from CD and UC patients can affect the way through which KLH can be presented to immune cells. Although KLH presence does not seem to be important to IECs expression of MHCII molecules, differences in MHCII expression suggests a difference in the ability of IECs from both types of IBD to stimulate different immune cells subsets.

Prevalence of Enterotoxigenic Bacteroides fragilis Detected in Stool Samples from Pediatric Inflammatory Bowel Disease Patients

Purpose: Enterotoxigenic Bacteroides fragilis (ETBF) are anaerobic enteric bacteria known to cause an acute, inflammatory diarrhea in both children and adults via its primary virulence factor the B. fragilis toxin (BFT). In murine models, ETBF causes asymptomatic, chronic colitis in C57BL/6 mice and increases colon tumorigenesis in multiple intestinal neoplasia (Min Apc+/-) mice. Prior human studies suggest an association between ETBF infection and active inflammatory bowel disease (IBD), though these studies are small in sample size. Our study aims to characterize the prevalence of ETBF colonization among pediatric control and IBD patients using combined anaerobic culture and fecal PCR diagnostic approaches. Methods: We obtained stool samples (n=259) and correlating clinical data from 105 prospectively enrolled pediatric patients with ulcerative colitis or Crohn's disease from two academic IBD centers. A single fecal sample was similarly collected from each of 105 confirmed non-IBD patients presenting to the same pediatric GI clinics. Frozen fecal samples were anaerobically cultured on Bacteroides Bile Esculin (BBE) agar to help promote Bacteroides spp. growth. Samples that did not grow on BBE or that grew poorly were plated on less selective Brucella anaerobic blood plates and/or LKV (Brucella Laked Blood Agar with Kanamycin and Vancomycin) plates in attempt to coax growth of Bacteroides spp. that may have been “stunned” by storage conditions. Any growth on Brucella blood agar or LKV plates was then transferred onto BBE. For all samples that grew on BBE, 16 single colonies were isolated and tested by touchdown PCR for the bft gene. Results: To date, 22 IBD samples have been tested. 17 grew on BBE, of which 10 samples contained isolates positive for bft by touchdown PCR (range of positive isolates per sample 1-8, average number of positive isolates per sample 3.6). In comparison, of the 14 control samples that were tested, 12 grew on BBE and 3 of these contained bft+ isolates (range 1-6, average 2.67). Conclusion: Our current data suggests that fecal carriage of ETBF is approximately twice as common in IBD patients compared to controls (45% vs. 21%, p=0.175). Analysis of our fecal bank is ongoing; updated microbiology results will be presented. We further plan to analyze the associated clinical data to determine if ETBF carriage correlates with clinical manifestations of IBD, such as disease location, severity, or timing of flares.

Comparative Expression of Vitamin D Receptor and Cathelicidin in Human Intestinal Mucosa in Persons With and Without Inflammatory Bowel Disease

Background: Vitamin D receptor (VDR) expression has been demonstrated to be lower in explanted colons of patients with Ulcerative Colitis (UC) compared to patients without Inflammatory Bowel Disease (IBD). Similar studies are not available with Crohn's disease (CD). VitaminD has been shown to induce expression of the antimicrobial protein cathelicidin in multiple cell lines, whereas expression of cathelicidin has been shown to be blunted in patients with CD. Objectives: To quantify the expression of the VDR, CYP24A and CYP27B, and cathelicidin in colonic and ileal mucosa of patients with IBD when compared to healthy controls, and to correlate this expression with endoscopically visualized mucosal inflammation in patients with IBD. Methods: Patient undergoing colonoscopy for colorectal cancer screening were invited to participate in this study, as were patients undergoing colonoscopy for either diagnosis or follow-up of IBD. Endoscopic findings were noted. Biopsies were obtained from the rectum and terminal ileum in all patients. Biopsies were obtained from areas of active inflammation in patients with IBD. Pathology reports were reviewed when biopsies were obtained for diagnostic or surveillance purposes. Real Time PCR was performed on samples for VDR, CYP24A, CYP27B and cathelicidin. Immunofluorescent staining was also performed for VDR expression. Results: Forty-two patients were recruited: 27 without IBD, 10 with UC, and 5 with CD. Five patients had endoscopic evidence of active disease with erythema, nodularity, or ulceration. PCR revealed measurable expression of VDR, CYP24A, and cathelicidin in controls and subjects. Minimal expression of CYP27B was present in either controls or subjects. Intestinal mucosa from patients with IBD had decreased mRNA transcripts of VDR compared to those without IBD (p <0.05). There was a trend of increased mRNA transcripts of cathelicidin in the intestinal mucosa from subjects with IBD compared to controls. Results were not statistically significant. Immunostaining revealed prominent VDR expression in normal intestinal epithelium. Intestinal epithelial VDR expression by immunostaining is decreased in endoscopically apparent areas of active IBD when compared with controls. Epithelial VDR expression by immunostaining in IBD samples without active disease was similar to controls. Conclusions: VDR is expressed in healthy intestinal epithelial cells. Intestinal epithelial VDR expression is decreased in patients with active UC and CD when compared to healthy controls, and when compared to IBD patients with inactive disease. Since vitamin D administration enhances VDR expression, a clinical study with vitamin D administration in patients with UC and CD is warranted to confirm the role of VDR in the pathophysiology of IBD.

Immunohistochemical expression of CDX2, β-catenin, and TP53 in inflammatory bowel disease-associated colorectal cancer

Inflammatory bowel disease (IBD) exposes patients to an increased risk of colorectal cancer (i-CRC) and differences between i-CRC and sporadic colorectal cancer (s-CRC) pathogenesis were reported. In s-CRC, studies indicate abnormalities in the tumor-suppressor gene Cdx2. This study compared CDX2, β-catenin, and TP53 expression in i-CRC, s-CRC, noncancer IBD, and normal control colonic mucosa. Expression was investigated by immunohistochemistry in 10 normal, 20 s-CRC, 11 noncancer colonic IBD and 30 i-CRC samples, and in four samples of Crohn's disease (CD)-associated small bowel adenocarcinoma (i-SBA). In normal and noncancer IBD samples, CDX2 was confined to the colonocytes nuclei. CDX2 expression was normal in 90% of i-CRC, regardless of tumor differentiation or inflammation intensity. By contrast, CDX2 expression was altered in 45% s-CRC, particularly at the front of invasion in undifferentiated tumors. β-Catenin was restricted to cell membrane in all controls, in 91% noncancer IBD, and in 84% i-CRC samples, whereas 35% s-CRC showed cytoplasmic redistribution and exclusive nuclear staining at the front of invasion. TP53 was strongly and homogeneously expressed in i-CRC nuclei compared to normal control or s-CRC, and increases with inflammation intensity. Nested or diffuse TP53 was found in 81.8% of noncancer IBD samples with a higher proportion of TP53-expressing cells in the most inflamed samples. CDX2, β-catenin, and TP53 expression in CD-associated SBA appears similar to that of i-CRC. Neither Cdx2 nor β-catenin alterations are prominent features of i-CRC. In i-CRC and CD-associated SBA, carcinogenesis is associated early with p53 mutations and to inflammation intensity.

Aberrant Expression of MUC1 Mucin in Pediatric Inflammatory Bowel Disease

Mucin 1 (MUC1) glycoprotein is normally expressed at low levels on the luminal side of healthy colonic epithelial cells. In colon cancer and other epithelial tumors, MUC1 is overexpressed and hypoglycosylated. Antibodies specific for this "tumor form" of MUC1 are found in cancer patients. We hypothesized that MUC1 expression might be altered in chronic inflammation, such as in inflammatory bowel disease (IBD). Furthermore, we hypothesized that these alterations might induce antibody responses. The aims of this study were to characterize MUC1 expression in IBD and to examine whether pediatric patients with IBD have an MUC1-specific antibody. Colon biopsies were examined for MUC1 expression by immunochemistry using anti-MUC1 antibodies that detect normal or abnormal forms of MUC1. Sera were analyzed by Enzyme Linked Immunosorbent Assay (ELISA) for evidence of anti-MUC1 antibody. We found marked overexpression of MUC1 in IBD, most of which was hypoglycosylated. On colon specimens from healthy age-matched controls, we found low levels of luminal MUC1 and no alteration in its glycosylation. We detected antibody to MUC1 in sera of IBD patients as well as controls, and in a limited number of IBD samples examined longitudinally, we could correlate the rise and fall of antibody levels with clinical disease severity. MUC1 is overexpressed and hypoglycosylated in pediatric IBD and may play an important role in the pathogenesis of IBD, and thus warrants further study as a potential therapeutic target. Similarly, antibodies to MUC1 may influence IBD and should be explored as potential diagnostic or prognostic markers.

Epigenetic Regulation of WNT Signaling Pathway Genes in Inflammatory Bowel Disease (IBD) Associated Neoplasia

IntroductionWNT signaling pathway dysregulation is an important event in the pathogenesis of colorectal cancer (CRC) with APC mutations seen in more than 80% of sporadic CRC. However, such mutations in the WNT signaling pathway genes are rare in inflammatory bowel disease (IBD) associated neoplasia (dysplasia and cancer). This study examined the role of epigenetic silencing of WNT signaling pathway genes in the pathogenesis of IBD-associated neoplasia. MethodsParaffin-embedded tissue samples were obtained and methylation of ten WNT signaling pathway genes, including APC1A, APC2, SFRP1, SFRP2, SFRP4, SFRP5, DKK1, DKK3, WIF1 and LKB1, was analyzed. Methylation analysis was performed on 41 IBD samples, 27 normal colon samples (NCs), and 24 sporadic CRC samples. ResultsMethylation of WNT signaling pathway genes is a frequent and early event in IBD and IBD-associated neoplasia. A progressive increase in the percentage of methylated genes in the WNT signaling pathway from NCs (4.2%) to IBD colitis (39.7%) to IBD-associated neoplasia (63.4%) was seen (NCs vs. IBD colitis, p < 0.01; IBD colitis vs. IBD-associated neoplasia, p = 0.01). In the univariate logistic regression model, methylation of APC2 (OR 4.7, 95% CI: 1.1–20.63, p = 0.04), SFRP1 (OR 5.1, 95% CI: 1.1–31.9, p = 0.04), and SFRP2 (OR 5.1, 95% CI: 1.1–32.3, p = 0.04) was associated with progression from IBD colitis to IBD-associated neoplasia, while APC1A methylation was borderline significant (OR 4.1, 95% CI: 0.95–17.5, p = 0.06). In the multivariate logistic regression model, methylation of APC1A and APC2 was more likely to be associated with IBD-associated neoplasia than IBD colitis. (OR APC1A: 6.4, 95% CI: 1.1–37.7 p = 0.04; OR APC2 9.1, 95% CI: 1.3–61.7, p = 0.02). SummaryMethylation of the WNT signaling genes is an early event seen in patients with IBD colitis and there is a progressive increase in methylation of the WNT signaling genes during development of IBD-associated neoplasia. Moreover, methylation of APC1A, APC2, SFRP1, and SFRP2 appears to mark progression from IBD colitis to IBD-associated neoplasia, and these genes may serve as biomarkers for IBD-associated neoplasia.

Genetic Analysis of Innate Immunity in Crohn's Disease and Ulcerative Colitis Identifies Two Susceptibility Loci Harboring CARD9 and IL18RAP

The two main phenotypes of inflammatory bowel disease (IBD)--Crohn's disease (CD) and ulcerative colitis (UC)--are chronic intestinal inflammatory disorders with a complex genetic background. Using a three-stage design, we performed a functional candidate-gene analysis of innate immune pathway in IBD. In phase I, we typed 354 SNPs from 85 innate immunity genes in 520 Dutch IBD patients (284 CD, 236 UC) and 808 controls. In phase II, ten autosomal SNPs showing association at p < 0.006 in phase I were replicated in a second cohort of 545 IBD patients (326 CD, 219 UC) and 360 controls. In phase III, four SNPs with p < 0.01 in the combined phase I and phase II analysis were genotyped in an additional 786 IBD samples (452 CD, 334 UC) and 768 independent controls. Joint analysis of 1851 IBD patients (1062 CD, 789 UC) and 1936 controls demonstrated strong association to the IL18RAP rs917997 SNP for both CD and UC (p(IBD) 1.9 x 10(-8); OR 1.35). Association in CD is independently supported by the Crohn's disease dataset of the Wellcome Trust Case Control Consortium (imputed SNP rs917997, p = 9.19 x 10(-4)). In addition, an association of the CARD9 rs10870077 SNP to CD and UC was observed (p(IBD) = 3.25 x 10(-5); OR 1.21). Both genes are located in extended haplotype blocks on 2q11-2q12 and 9q34.3, respectively. Our results indicate two IBD loci and further support the importance of the innate immune system in the predisposition to both CD and UC.

This Month in Gastroenterology

This Month in Gastroenterology

Cytokine/chemokine messenger-RNA expression profiles in ulcerative colitis and Crohn's disease.

To define mediator profiles in inflamed and noninflamed areas in inflammatory bowel disease (IBD) we analyzed the expression of 35 messenger-RNAs (mRNAs) encoding cytokines, chemokines, and some related molecules in transmural gut tissues (n=138) from patients with ulcerative colitis (UC), Crohn's disease (CD), and inflammatory and normal controls by real-time quantitative reverse transcription polymerase chain reaction. Using sample collectives with a comparable degree of inflammation, most parameters investigated showed similarly increased mRNA expression levels in both active UC and CD. This included proinflammatory cytokines, but also interferon (IFN) gamma and several IFN-gamma inducible chemokines. Only macrophage inflammatory protein (MIP)-2alpha mRNA was expressed at higher levels in inflamed UC vs. CD. IH revealed that MIP-2alpha protein was produced mainly by intestinal epithelial cells. Importantly, in histologically noninflamed/inactive IBD samples mRNAs for several mediators were significantly enhanced, accompanied by elevated levels of migration-inhibition factor related protein (MRP) 14 transcripts. CD14 positive macrophages were found especially in noninflamed/inactive UC, many of which coexpressed the RFD-7 antigen. Our results indicate a substantial overlap in cytokine/chemokine mRNA expression in UC and CD. Elevated mediator expression is evident in noninflamed/inactive areas in both diseases. Local recruitment of MRP-14 positive leukocytes might contribute to this phenomenon. In inactive UC a phenotypically altered population of macrophages expressing CD14 might play an additional role.

Colonic explant production of IL-1and its receptor antagonist is imbalanced in inflammatory bowel disease (IBD).

IBD is associated with an increased activation of intestinal immune cells, which causes overproduction of proinflammatory cytokines such as IL-1beta. IL-1beta is implicated in mediating the sustained inflammatory response. IL-1 receptor antagonist (IL-1Ra), the naturally occurring inhibitor of IL-1, has been shown to have beneficial effects in experimental models of colitis. In this study we investigated the hypothesis that an imbalance between IL-1 and IL-1Ra exists in IBD by measuring their secretion by explant cultures of colonic biopsies. Freshly homogenized biopsies from involved tissue in IBD patients exhibited significantly lower IL-1Ra/IL-1beta ratios than control and uninvolved IBD mucosal tissue. Using explant cultures, in vitro production of IL-1beta and IL-1Ra increased progressively during the 4-18-h culture periods. IL-1beta secretion was higher in supernatants from involved Crohn's disease (CD) and ulcerative colitis tissue compared with control tissue, and IL-1beta levels increased with severity of inflammation. IL-1Ra secretion was not elevated in involved IBD samples, but significantly higher levels were released when moderate to severely involved tissue samples were compared with noninflammatory controls. Similar to freshly homogenized tissue, explant studies showed that the IL-1Ra/IL-1beta ratios were significantly decreased in involved IBD tissue, but not in uninvolved CD or inflammatory control specimens. These data support the hypothesis of an imbalance between IL-1beta and IL-1Ra in IBD.

A Genomewide Analysis Provides Evidence for Novel Linkages in Inflammatory Bowel Disease in a Large European Cohort

Inflammatory bowel disease (IBD) is characterized by a chronic relapsing intestinal inflammation, typically starting in early adulthood. IBD is subdivided into two subtypes, on the basis of clinical and histologic features: Crohn disease and ulcerative colitis (UC). Previous genomewide searches identified regions harboring susceptibility loci on chromosomes 1, 3, 4, 7, 12, and 16. To expand our understanding of the genetic risk profile, we performed a 9-cM genomewide search for susceptibility loci in 268 families containing 353 affected sibling pairs. Previous linkages on chromosomes 12 and 16 were replicated, and the chromosome 4 linkage was extended in this sample. New suggestive evidence for autosomal linkages was observed on chromosomes 1, 6, 10, and 22, with LOD scores of 2.08, 2.07, 2.30, and 1.52, respectively. A maximum LOD score of 1.76 was observed on the X chromosome, for UC, which is consistent with the clinical association of IBD with Ullrich-Turner syndrome. The linkage finding on chromosome 6p is of interest, given the possible contribution of human leukocyte antigen and tumor necrosis-factor genes in IBD. This genomewide linkage scan, done with a large family cohort, has confirmed three previous IBD linkages and has provided evidence for five additional regions that may harbor IBD predisposition genes.

Measles virus RNA is not detected in inflammatory bowel disease using hybrid capture and reverse transcription followed by the polymerase chain reaction

Recent epidemiological and immunohistochemical studies have indicated a possible link between measles virus and inflammatory bowel disease (IBD). The aim of this study was to use a sensitive and robust method for the detection of measles virus RNA in IBD and control clinical samples. Peripheral blood mononuclear cells and intestinal resection tissue from IBD and control patients were studied. Two methods were used to determine the presence of measles virus RNA: hybrid capture, using measles virus-specific oligonucleotides linked to paramagnetic solid-phase supports, was carried out on total cellular RNA to enrich for measles virus RNA sequences. Reverse transcription followed by the polymerase chain reaction (RT-PCR) using rTth DNA polymerase was employed for amplification of measles virus N-gene sequences amongst the enriched species. Total RNA was also used for RT-PCR of a housekeeping mRNA species to assess RNA quality. RT-PCR for another region of the measles genome (the haemagglutinin (H) gene) was also undertaken in order to confirm the results obtained using N-gene primers for analysis of these samples. None of the samples were positive for measles N- or H-gene RNA using RT-PCR. Positive control samples confirmed the sensitivity of the methods employed. These results show that either measles virus RNA was not present in the samples, or was present below the sensitivity limits known to have been achieved.

Measles virus RNA is not detected in inflammatory bowel disease using hybrid capture and reverse transcription followed by the polymerase chain reaction

Recent epidemiological and immunohistochemical studies have indicated a possible link between measles virus and inflammatory bowel disease (IBD). The aim of this study was to use a sensitive and robust method for the detection of measles virus RNA in IBD and control clinical samples. Peripheral blood mononuclear cells and intestinal resection tissue from IBD and control patients were studied. Two methods were used to determine the presence of measles virus RNA: hybrid capture, using measles virus-specific oligonucleotides linked to paramagnetic solid-phase supports, was carried out on total cellular RNA to enrich for measles virus RNA sequences. Reverse transcription followed by the polymerase chain reaction (RT-PCR) using rTth DNA polymerase was employed for amplification of measles virus N-gene sequences amongst the enriched species. Total RNA was also used for RT-PCR of a housekeeping mRNA species to assess RNA quality. RT-PCR for another region of the measles genome (the haemagglutinin (H) gene) was also undertaken in order to confirm the results obtained using N-gene primers for analysis of these samples. None of the samples were positive for measles N- or H-gene RNA using RT-PCR. Positive control samples confirmed the sensitivity of the methods employed. These results show that either measles virus RNA was not present in the samples, or was present below the sensitivity limits known to have been achieved. J. Med. Virol. 55:305–311, 1998. © 1998 Wiley-Liss, Inc.