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Early infection with measles virus is associated with an increased risk of ulcerative colitis
GASTROENTEROLOGYVol. 114, No. 4
A1058 AGA ABSTRACTS
• G4330 EARLY INFECTION WITH MEASLES VIRUS IS ASSOCIATED WITH AN INCREASED RISK OF ULCERATIVE COLITIS. DS Pardi, WJ Tremaine, WJ Sandboru, EV Loftus Jr, GA Poland, AR Zinsmeister, and LJ Melton. Mayo Clinic and Foundation, Rochester, MN. Background: An association between wild measles virus infection and inflammatory bowel disease (IBD) has been reported (Wakefield, Gastroenterology 1995; 108:911-916). Aim: To determine if early infection with measles is associated with an increased risk of IBD. Methods: The Mayo Clinic diagnostic index was used to identify all patients less than five years old diagnosed with measles between 1950 and 1966. This period was chosen to allow at least 30 years follow up for the diagnoses of ulcerative colitis (UC) or Crohn's disease (CD), which were determined by a questionnaire that was mailed to each subject. Cases of IBD were confirmed using records from the patients' local physicians. The identified number of cases of IBD in this cohort was compared to that expected using age and gender-specific population-based rates from Olmsted County, MN. Results: 1501 patients with measles infection prior to age five were identified, and 840 (56%) completed the survey. There were 32,978 person-years of follow up. IBD type CD UC Total
Observed Cases 5 7 12
Expected Cases 2.4 2.5 4.8
Relative Risk (95% CI) 2.1 (0.7-4.9) 2.8 (1.1-5.8) 2.5 (1.3-4.3)
Conclusion: Early infection with measles appears to be associated with an increased risk of IBD, and of ulcerative colitis in particular. There was a similar trend towards an increased risk of Crohn's disease, but this was not statistically significant.
• G4331 FOCAL GASTRITIS IN CROHN'S DISEASE (CD): PREVALENCE, IMMUNOHISTOCHEMICAL CHARACTERISTICS, AND CLINICO /ANATOMICAL DETERMINANTS. F.Parente, S. Bollani, C. Cucino, S.Ardizzone, L.Vago, P.Duca, G.Bianchi Porto. Gastrointestinal and Pathology Unit, L.Sacco University Hospital, Milan, Italy. Background. To date, very few studies have evaluated gastric histology in patients with CD. Aim of this prospective study was to establish the frequency of focal gastritis in a large series of CD patients as well as to evaluate its immunohistochemical characteristics and clinico/anatomical determinants. Methods. 124 consecutive patients with known CD of the large and/or small bowel underwent upper GI endoscopy and serum anti-H.pylori lgG assay. Biopsy specimens taken from the antrum, angulus and body were evaluated by histology and immunohistochemistry. A series of variables, including CD activity index, disease length and extent, number of recurrences, previous and current medical therapy as well as the presence of dyspeptic symptoms, evaluated by means of a validated symptom questionnaire, and mucosal lesions at endoscopy were determined in all CD patients. The relationship between focal gastritis and the aforesaid variables was assessed by univariate and multivariate analysis. Results. H.pylori-associated gastritis was found in 40 patients with CD (32%). In H.pylori-negative (histology and serology) CD patients granulomas and/or focal gastritis were found in 39% of cases (33•84). The latter was characterized by a focal perifoveolar or periglandular lympho-monocytic infiltrate, with CD8 + cells predominance. There were no correlations between the occurrence of focal gastritis and clinico/anatomical CD features, although it was more frequently associated with dyspeptic symptoms and macroscopic mucosal lesions at endoscopy. Conclusions. Focal gastritis is common in CD patients. Its recognition should be part of the diagnostic work-up of any patient with suspected or indeterminate inflammatory bowel disease since it makes a diagnosis of CD more likely. • G4332 PRECISION MAPPING OF CHROMOSOME 12 LINKAGE IN IBD: EVIDENCE FOR A HAPLOTYPE ASSOCIATION. M ['arkes, J Satsangi, A Merriman, DP Jewell. Gastroenterology Unit and Wellcome Trust Centre For Human Genetics, University of Oxford, UK. Introduction and Aims: The 40 centiMorgan (cM) region of linkage which we originally identified on Chr 12 has been found in at least two additional Caucasian IBD datasets, but pinpointing the IBD susceptibility locus among the 1000 or more genes in this region remains a major challenge. Our aim has been to precision map this region using linkage and association studies, including haplotype analysis. Methods: in stage 1 DNA from 198 pairs of siblings with IBD (CD=89, UC=69, mixed--40) was genotyped at 24 microsatellite markers using standard ABI protocols, the data being analysed for linkage by multipoint analysis (ASPEX 1.9) and for association using the transmission disequilbrium test (TDT). In stage 2, a 1.5 cM region of interest was saturation-mapped using an additional 13 markers: only alleles in linkage
disequilibrium with those showing a positive TDT result in stage 1 (at d12s83 & d12s90) were considered in the analysis. Results Stage 1: multipoint analysis of the 24 markers using the whole IBD dataset narrows the linkage to an 11 cM segment between d12s1677 and d12s1686; and analysing the disease subgroups separately suggests tighter linkage to UC (max. lod score of 2.08 at d12s1724) than CD (max lod score of 0.91 at d12s90), although tests for heterogeneity fail to reach significance. Results of the TDT were positive at two loci, lying 1.5 cM apart near the peak of the linkage curve: allele 4 of d12s83 (p=0.008) and allele 5 of d12s90 (p=0.015) showed distortion of transmission, but no linkage disequilibrium despite the proximity of their loci. Stage 2: among the 13 additional markers studied linkage disequilibrium was now seen, between allele 4 of d12s83 and both allele 1 of d12s1662 (p-9xl0 -7) and allele 3 of d12s1655 (p=7.2xl0-S). These markers lie in the same linkage group on the Genethon map. Although neither allele alone appeared more tightly associated than allele 4 of d12s83, transmission of the 4-1-3 haplotype (representing 7.4% of possible parental haplotypes) to affected offspring showed striking distortion - again particularly for UC. 4-1-3 haplotype Transmitted Not transmitted g2 (p value)
i
UC 23 6 5.5 (0.02)
CD 13 6 1.3 (0.24)
IBD 39 15 5.6 (0.018)
No haplotype around d 12s90 showed significant distortion. Conclusion: this is the first reported study in IBD to successfully detect allelic and haplotype association within a region of linkage, and precision maps the Chr 12 susceptibility locus to within 0.5 cM of d12s83. • G4333 INCREASED NEUROTENSIN RECEPTOR (NTR) mRNA IN THE COLONIC MUCOSA DURING CLOSTRIUDIUM DIFFICILE PSEUDOMEMBRANOUS COLITIS (PMC) IN HUMANS. Pasha A.. C Wang, S Nikulasson*, C Pothoulakis, I Castagliuolo. Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School;* Boston University School of Medicine, Boston MA,. We recently reported that a NTR antagonist inhibits C. difficile toxin A (TxA)-induced colonic secretion and inflammation in rats. Since neurotensin (NT) and NTR levels are increased during experimental TxA colitis and because both TxA and B (TxB) cause damage in human colon we 1) investigated whether C. difficile TxA or TxB or the proinflammatory cytokine IL-113 up-regulate NTR expression in human colonic epithelial (HT-29) cells, and 2) examined the level of expression and the cellular localization of NTR mRNA in colonic biopsies from patients with C. difficile colitis. Methods: HT-29 cells were exposed to TxA or TxB (10 nM) or IL-113 ( I 0 ng/ml). After 1-6 hr total RNA was extracted and NTR mRNA was determined by semiquantitative RT-PCR. In situ hybridization for NTR-mRNA was performed on colonic biopsies from normal subjects (n=4) and patients with C. difficile PMC (n=10). Results: Exposure of HT-29 cells to either TxA or TxB induced a time-dependant increase in NTR mRNA which peaked after 2 hr (3.1-and 8.5-fold increase, respectively, p<0.01). IL-113also caused a 2.8-fold increase in NTR mRNA after 2 hr incubation (p<0.05). In situ hybridization using a digoxigenin-labelled NTR mRNA riboprobe and confocal microscopy showed low, but detectable signal present in epithelial cells and cells of the colonic lamina propria in normal colon. In colon from patients with PMC there was a marked increase in NTR mRNA with increased expression in both colonic epithelial cells and lamina propria ceils. Conclusions: Our results indicate that C. difficile toxins and IL-113 upregulate NTR in human colonocytes. We speculate that NT released from the colon during C. difficile colitis stimulates colonic secretion and inflammation by interacting with its receptor on colonocytes. Supported by the Crohn's and Colitis Foundation of America, Inc. G4334 ULCERATIVE COLITIS AND COLONIC LAVAGE: EFFECT ON MUCOSALLY-ASSOCIATED FLORA. S.Pathmakanthan, J.Thornley, D.Jenkins, C.J Hawkey. Division of Gastroenterology, University Hospital, Nottingham NG7 2UH, UK. Background: There is growing evidence for colonic bacteria implicated in the
aetiology of ulcerative colitis (UC) either as a single pathogen or the focus of an abnormal immune response. Mucosally associated flora (MAF) is closely associated with the colonic epithelial cell and may play a major role in the initiation or perpetuation of UC. Aims: To quantify and qualitate MAF aerobically, anaerobically and microaerophilically on selective media from normal and UC mucosa and determine the effects of 3 colonic lavage solutions on MAF. Methods: Biopsies (10 normal, 10 UC) were obtained during colonoscopy for culture of MAF and scanning electron microscopy (SEM). Biopsies were snap frozen in glycerol after washing and subsequently homogenised and cultured on selective media under anaerobic, aerobic and microaerophilic conditions. Quantitative estimations with gram stain and morphology were performed on all growths. Three colonic lavage solutions (Fleet®, Klean Prep® and
A1058 AGA ABSTRACTS
• G4330 EARLY INFECTION WITH MEASLES VIRUS IS ASSOCIATED WITH AN INCREASED RISK OF ULCERATIVE COLITIS. DS Pardi, WJ Tremaine, WJ Sandboru, EV Loftus Jr, GA Poland, AR Zinsmeister, and LJ Melton. Mayo Clinic and Foundation, Rochester, MN. Background: An association between wild measles virus infection and inflammatory bowel disease (IBD) has been reported (Wakefield, Gastroenterology 1995; 108:911-916). Aim: To determine if early infection with measles is associated with an increased risk of IBD. Methods: The Mayo Clinic diagnostic index was used to identify all patients less than five years old diagnosed with measles between 1950 and 1966. This period was chosen to allow at least 30 years follow up for the diagnoses of ulcerative colitis (UC) or Crohn's disease (CD), which were determined by a questionnaire that was mailed to each subject. Cases of IBD were confirmed using records from the patients' local physicians. The identified number of cases of IBD in this cohort was compared to that expected using age and gender-specific population-based rates from Olmsted County, MN. Results: 1501 patients with measles infection prior to age five were identified, and 840 (56%) completed the survey. There were 32,978 person-years of follow up. IBD type CD UC Total
Observed Cases 5 7 12
Expected Cases 2.4 2.5 4.8
Relative Risk (95% CI) 2.1 (0.7-4.9) 2.8 (1.1-5.8) 2.5 (1.3-4.3)
Conclusion: Early infection with measles appears to be associated with an increased risk of IBD, and of ulcerative colitis in particular. There was a similar trend towards an increased risk of Crohn's disease, but this was not statistically significant.
• G4331 FOCAL GASTRITIS IN CROHN'S DISEASE (CD): PREVALENCE, IMMUNOHISTOCHEMICAL CHARACTERISTICS, AND CLINICO /ANATOMICAL DETERMINANTS. F.Parente, S. Bollani, C. Cucino, S.Ardizzone, L.Vago, P.Duca, G.Bianchi Porto. Gastrointestinal and Pathology Unit, L.Sacco University Hospital, Milan, Italy. Background. To date, very few studies have evaluated gastric histology in patients with CD. Aim of this prospective study was to establish the frequency of focal gastritis in a large series of CD patients as well as to evaluate its immunohistochemical characteristics and clinico/anatomical determinants. Methods. 124 consecutive patients with known CD of the large and/or small bowel underwent upper GI endoscopy and serum anti-H.pylori lgG assay. Biopsy specimens taken from the antrum, angulus and body were evaluated by histology and immunohistochemistry. A series of variables, including CD activity index, disease length and extent, number of recurrences, previous and current medical therapy as well as the presence of dyspeptic symptoms, evaluated by means of a validated symptom questionnaire, and mucosal lesions at endoscopy were determined in all CD patients. The relationship between focal gastritis and the aforesaid variables was assessed by univariate and multivariate analysis. Results. H.pylori-associated gastritis was found in 40 patients with CD (32%). In H.pylori-negative (histology and serology) CD patients granulomas and/or focal gastritis were found in 39% of cases (33•84). The latter was characterized by a focal perifoveolar or periglandular lympho-monocytic infiltrate, with CD8 + cells predominance. There were no correlations between the occurrence of focal gastritis and clinico/anatomical CD features, although it was more frequently associated with dyspeptic symptoms and macroscopic mucosal lesions at endoscopy. Conclusions. Focal gastritis is common in CD patients. Its recognition should be part of the diagnostic work-up of any patient with suspected or indeterminate inflammatory bowel disease since it makes a diagnosis of CD more likely. • G4332 PRECISION MAPPING OF CHROMOSOME 12 LINKAGE IN IBD: EVIDENCE FOR A HAPLOTYPE ASSOCIATION. M ['arkes, J Satsangi, A Merriman, DP Jewell. Gastroenterology Unit and Wellcome Trust Centre For Human Genetics, University of Oxford, UK. Introduction and Aims: The 40 centiMorgan (cM) region of linkage which we originally identified on Chr 12 has been found in at least two additional Caucasian IBD datasets, but pinpointing the IBD susceptibility locus among the 1000 or more genes in this region remains a major challenge. Our aim has been to precision map this region using linkage and association studies, including haplotype analysis. Methods: in stage 1 DNA from 198 pairs of siblings with IBD (CD=89, UC=69, mixed--40) was genotyped at 24 microsatellite markers using standard ABI protocols, the data being analysed for linkage by multipoint analysis (ASPEX 1.9) and for association using the transmission disequilbrium test (TDT). In stage 2, a 1.5 cM region of interest was saturation-mapped using an additional 13 markers: only alleles in linkage
disequilibrium with those showing a positive TDT result in stage 1 (at d12s83 & d12s90) were considered in the analysis. Results Stage 1: multipoint analysis of the 24 markers using the whole IBD dataset narrows the linkage to an 11 cM segment between d12s1677 and d12s1686; and analysing the disease subgroups separately suggests tighter linkage to UC (max. lod score of 2.08 at d12s1724) than CD (max lod score of 0.91 at d12s90), although tests for heterogeneity fail to reach significance. Results of the TDT were positive at two loci, lying 1.5 cM apart near the peak of the linkage curve: allele 4 of d12s83 (p=0.008) and allele 5 of d12s90 (p=0.015) showed distortion of transmission, but no linkage disequilibrium despite the proximity of their loci. Stage 2: among the 13 additional markers studied linkage disequilibrium was now seen, between allele 4 of d12s83 and both allele 1 of d12s1662 (p-9xl0 -7) and allele 3 of d12s1655 (p=7.2xl0-S). These markers lie in the same linkage group on the Genethon map. Although neither allele alone appeared more tightly associated than allele 4 of d12s83, transmission of the 4-1-3 haplotype (representing 7.4% of possible parental haplotypes) to affected offspring showed striking distortion - again particularly for UC. 4-1-3 haplotype Transmitted Not transmitted g2 (p value)
i
UC 23 6 5.5 (0.02)
CD 13 6 1.3 (0.24)
IBD 39 15 5.6 (0.018)
No haplotype around d 12s90 showed significant distortion. Conclusion: this is the first reported study in IBD to successfully detect allelic and haplotype association within a region of linkage, and precision maps the Chr 12 susceptibility locus to within 0.5 cM of d12s83. • G4333 INCREASED NEUROTENSIN RECEPTOR (NTR) mRNA IN THE COLONIC MUCOSA DURING CLOSTRIUDIUM DIFFICILE PSEUDOMEMBRANOUS COLITIS (PMC) IN HUMANS. Pasha A.. C Wang, S Nikulasson*, C Pothoulakis, I Castagliuolo. Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School;* Boston University School of Medicine, Boston MA,. We recently reported that a NTR antagonist inhibits C. difficile toxin A (TxA)-induced colonic secretion and inflammation in rats. Since neurotensin (NT) and NTR levels are increased during experimental TxA colitis and because both TxA and B (TxB) cause damage in human colon we 1) investigated whether C. difficile TxA or TxB or the proinflammatory cytokine IL-113 up-regulate NTR expression in human colonic epithelial (HT-29) cells, and 2) examined the level of expression and the cellular localization of NTR mRNA in colonic biopsies from patients with C. difficile colitis. Methods: HT-29 cells were exposed to TxA or TxB (10 nM) or IL-113 ( I 0 ng/ml). After 1-6 hr total RNA was extracted and NTR mRNA was determined by semiquantitative RT-PCR. In situ hybridization for NTR-mRNA was performed on colonic biopsies from normal subjects (n=4) and patients with C. difficile PMC (n=10). Results: Exposure of HT-29 cells to either TxA or TxB induced a time-dependant increase in NTR mRNA which peaked after 2 hr (3.1-and 8.5-fold increase, respectively, p<0.01). IL-113also caused a 2.8-fold increase in NTR mRNA after 2 hr incubation (p<0.05). In situ hybridization using a digoxigenin-labelled NTR mRNA riboprobe and confocal microscopy showed low, but detectable signal present in epithelial cells and cells of the colonic lamina propria in normal colon. In colon from patients with PMC there was a marked increase in NTR mRNA with increased expression in both colonic epithelial cells and lamina propria ceils. Conclusions: Our results indicate that C. difficile toxins and IL-113 upregulate NTR in human colonocytes. We speculate that NT released from the colon during C. difficile colitis stimulates colonic secretion and inflammation by interacting with its receptor on colonocytes. Supported by the Crohn's and Colitis Foundation of America, Inc. G4334 ULCERATIVE COLITIS AND COLONIC LAVAGE: EFFECT ON MUCOSALLY-ASSOCIATED FLORA. S.Pathmakanthan, J.Thornley, D.Jenkins, C.J Hawkey. Division of Gastroenterology, University Hospital, Nottingham NG7 2UH, UK. Background: There is growing evidence for colonic bacteria implicated in the
aetiology of ulcerative colitis (UC) either as a single pathogen or the focus of an abnormal immune response. Mucosally associated flora (MAF) is closely associated with the colonic epithelial cell and may play a major role in the initiation or perpetuation of UC. Aims: To quantify and qualitate MAF aerobically, anaerobically and microaerophilically on selective media from normal and UC mucosa and determine the effects of 3 colonic lavage solutions on MAF. Methods: Biopsies (10 normal, 10 UC) were obtained during colonoscopy for culture of MAF and scanning electron microscopy (SEM). Biopsies were snap frozen in glycerol after washing and subsequently homogenised and cultured on selective media under anaerobic, aerobic and microaerophilic conditions. Quantitative estimations with gram stain and morphology were performed on all growths. Three colonic lavage solutions (Fleet®, Klean Prep® and