Characterization of genomic and functional variation throughout the TNF gene in pateints with IBD

A330 AGA ABSTRACTS

GASTROENTEROLOGY Vol. 118, No, 4

1792

1794

ASSESSMENT OF ANTIBODY STATUS OF MICE INFECTED WITH HELICOBACTER PYLORI PRE· AND POST TREATMENT. Linda M. Best, Donna Hutchison, Sander 1. Veldhuyzen van Zanten,

REAL·TIME FLUOROGENIC RT·PCR ANALYSIS OF 12 CYTO· KINES IN HELICOBACTER PYLORI COLONIZED PATIENTS.

Queen Elizabeth II Health Sci Ctr, Halifax, NS, Canada; Dalhousie Univ, Halifax, NS, Canada. Representative animals are sacrificed to prove infection and eradication in trials using animal models to test the efficacy of antimicrobial agents. Aim: To discover whether the antibody response in mice can indicate infection and eradication without sacrifice. Methods: We assessed the antibody response in uninfected C57IBL6 mice and after infection with H. pylori prior to and following treatment with various regimens. One hundred ILl of blood was drawn from the saphenous vein into a vial, centrifuged, and frozen until tested by a previously validated flow cytometric immunofluorescence method. Polystyrene beads coated with H. pylori whole-cell antigen were added to 5 ILl of serum diluted I :20 in phosphate buffered saline, incubated at 31lC for 20 minutes, washed, added to goat anti-mouse IgG, incubated as above, washed, and analyzed using a flow cytometer. Comparative antibody levels were statistically generated. Results: In H. pylori-infected mice the average increase was 33 units. Four of 200 mice were below the cut-off for infection. The level decreased by an average of 24 units in successfully treated mice at four weeks, and increased by an average of 15 units in untreated and unsuccessfully treated mice. Conclusion: Antibody levels in the mouse readily indicate infection and response to treatment.

Mouse Anti·H. pylori IgGResponse Treatment Failure

Thomas Wigginton, Eric M. Osgard, Jon P. Kushner, Corinne Maydonovitch, William F. Blakely, Andre Dubois, USUHS; Dept of Medicine, Bethesda, MD; Applied Cellular Radiobiology Dept AFRRI, Bethesda, MD.

Helicobacter pylori induces intense inflammation in all colonized subjects and, in addition, causes peptic ulcer disease and/or gastric cancer in 10-15% of these subjects. It has been proposed that excessive inflammation is responsible for disease in those subjects. The goals of the present study were twofold: (1) to validate and use a novel assay to quantify the cytokine profile of colonized and non-colonized patients and (2) to determine whether cytokine mRNA expression is related to gastritis and/or H. pylori colonization. Six H. pylori positive patients with non-ulcer dyspepsia (4 positive by serology, 13C-UBT, CLO test, and culture; 2 by serology alone) were included. Esophagogastroduodenoscopy was performed before and after treatment, and biopsies were harvested and placed either on ice for culture, in 10% formalin for histology (Genta stain using Sydney grading criteria), or flash-frozen in liquid nitrogen within to seconds of sampling. From these frozen biopsies, total RNA was extracted, quantified by UV spectrophotometry, and reverse-transcribed to cDNA using random primers. Samples were analyzed by PeR with fluorogenic probes on human cytokine plates (Perkin Elmer Applied Biosystems) designed to quantify 12 human cytokine targets using the 5-fluorogenic nuclease Taqman TM assay. Cytokine targets, IL-l a, IL-I 13, IL-2, IL-4, IL-5, IL-8, IL-tO, IL-12p35, IL·12p40, IL-15, IFN'Y' and TNFa, were normalized to a control target sequence (18S rRNA). mRNA expression ofIL-lj3, IL-8, IL-tO, and TNFa was detected in 6 of6patients; IL-15 in 5 patients; IL-la and IFN'Y in 4 patients; and IL-2 in 3 patients. A significant direct correlation was observed between gastritis and the increased expression of IL-8, IL-tO, and TNFa (p<.05). A trend towards a decrease in expression of IL-8 and TNFa in the 2 patients positive by serology alone compared to the 4 patients positive by all tests also was observed. These data support the evidence of the direct relationship between histological gastritis and cytokines detected by molecular biology. These observations will need to be reevaluated when additional patients are enrolled, but they indicate that this novel, real-time quantitative PeR assay can automate the detection of multiple cytokines simultaneously. 1795 REFUTING THE DOGMA THAT INFLAMMATORY BOWEL DIS· EASES (IBD) ARE DISEASES OF HIGHER SOCIOECONOMIC STATUS: POPULATION·BASED STUDIES.

10'

10'

10

1793 COMPARISON BETWEEN ORAL AND NASAL IMMUNIZATION AGAINST H. PYLORI WITH CO·ADMINISTRATION OF RECOM· BINANT CHOLERATOXIN B SUBUNIT. Katsushi Watanabe, Takashi Joh, Katsuyuki Miyashita, Isami Todoroki, Nobuo Takahashi, Hideo Suzuki, Hiromi Kataoka, Makoto Sasaki, Kyouji Seno, Yoshifumi Yokoyama, Kunio Tochikubo, Makoto Itoh, Nagoya City Univ Med Sch, Nagoya, Aichi, Japan. Background and Aims: Choleratoxin (CT) is often used as an experimental mucosal immuno-adjuvant, while cr possesses the strong toxicity to human intestinal mucosa. We have previously reported that recombinant choleratoxin B subunit (rCTB), which was obtained from Bacillus brevis carrying CTB gene-inserted plasmid pNU212, can be used as a non-toxic adjuvant against H. pylori (Hp). However, it has not been clarified which route is best for the mucosal immunization against Hp with rCTB. In this study, we investigated the mucosal and systemic immuno-adjuvant activities of rCTB by oral and nasal immunization. Methods: Female C57IBL6 mice aged 7 weeks were used for this study. Mice were administered I mg of Hp sonicate plus 50 JJ.g of rCTB for oral immunization, or 40 JJ.g of Hp sonicate plus 10 ILg of rCTB for nasal immunization on days 0, 7, 14 and 21. Mice were starved and sacrificed on day 42. Blood and lavage fluid of stomach and small intestine were collected. Hp-specific IgG or IgA antibody titers in serum and lavage fluids were estimated using ELISA. Hp-specific IgE activity in serum was estimated by passive cutaneous anaphylaxis test. Results: When Hp was administered with rCTB by oral or nasal route, significant increases in IgG and IgA were detected (see the table). However, no Hp-specific IgE activity was detected, although such an IgE activity was detected in the mice in which Hp + CT were administered. Conclusion: I) In nasal as well as oral immunization, co-administration of CTB demonstrated significant adjuvant properties for stimulating IgG and IgA, but not IgE immune responses to Hp antigens. 2) Nasal immunization induced predominant IgG response in the stomach. 3) These results indicated that rCTB can be used as non-toxic and non-allergic mucosal adjuvant for vaccine against Hp. Immunogloblin responses after oral ornasal immunization with Hpplus rCTS serum IgG

IgA

stomach IgA IgG

2/6

0/6 4/6 0/4 4/4

0/6 4/6 0/4 4/4

positive I totalmice

oralimmunization oralimmunization nasal immunization nasal immunization

Hp only Hp +rCTS Hp only Hp +rCTS

6/6 0/4 4/4

0/6 4/6 0/4 0/4

small intestine IgA IgG

0/6 4/6 0/4

0/6 5/6

0/4

Charles N. Bernstein, Allen Kraut, James F. Blanchard, Patricia Rawsthorne, Nancy Yu, Randy Walld, Cameron Mustard, Univ of Manitoba, Winnipeg, MB, Canada. Background ffiD may impact on employment, education & marital status. It has long been held that ffiD patients are of a higher socioeconomic strata & more educated than the general population. We aimed to determine the influence of ffiD on work status, income, education level and marital status compared to population controls. Methods: 2 studies are reported herein. Study A: Questionnaire responses (n=2759) from the population-based Uof M ffiD Database (1996) were compared to employment, education, & marital status data from the 1996 National Population Health Survey (n=14,177 for Manitoba). Study B: we used the linked Manitoba Health Statistics Canada database (utilizes the 1986 Canada Census) & applied our administrative definition of ffiD. We extracted all working age (18-64 yrs) IBD patients (n=80) to be compared with the non-ffiD cohort (n=25,554). Labor force participants were either employed or actively seeking work. Results: Study A: Combining rates of unemployment & disability ffiD males and females, respectively, were more likely to be out of work in 1996 (12.5%,11.8%) than at diagnosis (5.8%, 6.5%) or than controls (8.4%, 7.2%) (p
1796 CHARACTERIZATION OF GENOMIC AND FUNCTIONAL VARIATION THROUGHOUT THE TNF GENE IN PATIENTS WITH ran Denise K. Bonen, Richard Ramos, Sanggyu Lee, Sarah Corradino, Heidi M. Britton, Barbara S. Kirschner, Steven R. Brant, Stephen B. Hanauer, Judy H. Cho, Univ of Chicago, Chicago, IL; Johns Hopkins Univ Sch of Medicine, Baltimore, MD. Background: Deletion of the AU-rich region in the 3' UTR of the tumor necrosis factor (TNF) gene increases expression in mice and results in an

April 2000

IBD phenotype. Linkage has been observed in IBD patients at chr6p and case control studies have demonstrated associations in the TNF promoter. Aim: To characterize genomic variation throughout the TNF gene in IBD patients and assess the functional impact of suggestive genetic variants. Methods: The TNF gene was sequenced in 12 unrelated IBD patients from families with greatest evidence for linkage at chr6p. Single nucleotide polymorphisms (SNPs) were typed in 135 multiply affected families and studied using the transmission/disequilibrium test (TOT). Allele-specific binding was tested with electromobility shift assays (EMSAs). Results: Promoter variants at -1031, -863, -308 and -238 were identified. The -857 variant reported in the Japanese IBD population was not observed. Two intronic variants were identified, SNPintrona and SNPintronb' No SNPs were present in coding or 3' -untranslated regions. The single locus TDT tests for linkage showed neutral or inverse transmission for the less common -863 and -308 A alleles. No significant linkage was observed with the less common A alleles at -1031 (62 transmissions, 51 non-transmissions) or -238 (17 transmissions, 13 non-transmissions). The -238 variant was in complete and significant (p = 1.3 x 10-6 ) linkage disequilibrium with SNPintrona and SNPintronb' respectively. The (C-A-G), (-1031, -238, SNP intronb] haplotype demonstrated evidence for association by TDT (using a single, randomly selected affected from 192 multiplex families), with 15 transmissions (both CD and UC) and 5 non-transmissions (p= 0.03, uncorrected). The -238 variant is contained within a known repressor element. EMSAs were performed using allele-specific oligos in this region incubated with nuclear extracts from U937 mononuclear cells stimulated with phorbol myristyl acetate. Three specific DNA-protein complexes were observed, but no allele-specific binding was present. Conclusions: No single TNF promoter polymorphism by itself is significantly associated with IBD as assessed by the TDT test in multiply affected families. Nominal evidence for association with the 3 locus, C-A-G (-1031, -238, SNPintronb) haplotype is observed. Linkage disequilibrium patterns throughout the TNF gene are complex, reflecting selection patterns throughout evolution. Consideration of haplotypes for both genetic and functional studies is required.

1797 LIMITED GENETIC EVIDENCE FOR MDRI AS A CANDIDATE GENE FOR INFLAMMATORY BOWEL DISEASE. Steven R. Brant, Richard Ramos, Denise K. Bonen, Stuart M. Lubinski, Geoffrey Ravenhill, Patrick M. Rohal, Sinda Lee, Judy H. Cho, Johns Hopkins Univ, Baltimore, MD; Univ of Chicago, Chicago, IL. Introduction: The multidrug resistance gene MORI is expressed in intestinal epithelial cells and lymphoid cells. Recently, MORI knock-out mice have been shown to develop spontaneous colitis when maintained in pathogen-free conditions (Panwala et al., J Irnmunol, 1998). The human MDRI gene maps to chromosome 7q, and thus is a candidate gene for the 7q putative inflammatory bowel disease (IBD)locus identifiedby Satsangi et al., Nat Gen, 1996. Linkage analysis in sibling pair pedigrees from our previous reported genome screen (Cho et al., PNAS, 1998) was maximal at 91 cM on chromosome 7q (Mlod = 2.81, Z=3.6, p=1.59 x 10-4), between markers D7S2204 and D7S820 and within 10 cM of the MORI gene (Brant & Cho, unpublished results).We thus evaluated MORI as a candidate gene. Methods: The genomic sequences of the 27 MOR I coding exons, containing over 4000 bp, and > 20 bp of each flanking intron sequence were determined by PCR amplification with MORI specific primers, PCR product purification and then fluorescent sequencing, using the DNA purified from 18 individuals with IBD and two controls. These individuals were probands from pedigrees having maximal linkage evidence to the MDRI mapping location in our genome screen. Two polymorphisms were genotyped in 169 multiplex IBD pedigrees (36% Ashkenazi Jewish) and in 20 additional pedigrees with DNA samples obtained from both parents and a single affected child. 103 pedigrees were Crohn' s, 24 were UC and 62 were mixed. Tests for linkage using Genehunter-Plus and linkage disequilibrium (TOn by Chi-Square analysis were performed. Results: Six polymorphisms were identified.Two resulted in amino acid changes. Addition of these two amino acid polyrnorphisms did not significantly alter linkage evidence. TOT was minimally significant (p= 0.029). No specific association was observed with either Crohn's disease or ulcerative colitis, or with Ashkenazi Jewish ethnicity. Conclusion:There is preliminary evidence that MDRI may have coding changes specific to lBD. Genotyping of additional pedigrees and functional studies will be necessary for more conclusive evidence.

1798 SEX SPECIFIC ASSOCIATION OF OSTEOPOROSIS WITH VITA· MIN D RECEPTOR POLYMORPHISM (FOKI).

AGAA831

statistically significant association between the genotype and the bone mineral density in female patients with inflammatory bowel disease. Male IBD patients with the ff genotype had an significantly (1 SD) lower bone mineral density of the lumbar spine compared with the FF group (p=0.037). None of the men with the FF-genotype had osteoporosis. Conclusions: The FokI polymorphism seems to be a genetic risk factor for developing osteoporosis in male patients with inflammatory bowel disease

1799 PASSIVE SMOKING IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE - AN ISRAELI MULTICENTER CASE CONTROLSTUDY. Rami Eliakim, Shimon Reif, Alexandra Lavy, Daniel Keter, Shmuel Odes, Aharon Halak, Efrattt Broide, Yaron Niv, Yishai Ron, Julian Paz, Alexander Fich, Yael Villa, Tuvia Gilet, Hadassah Univ Hosp, Jerusalem, Israel; Rambam Hosp, Israel; Kaplan Hosp; Tel-Aviv Sourasky Med Ctr; Assaf Harofeh, Zerifin, Israel; Beilinson Hosp, Petach Tikva, Israel; Wolfson Med Ctr, Holon, Israel; Shaare Zedek Hosp, Jerusalem, Israel; Soroka Hosp, Beersheba, Israel. Background: The association between smoking and inflarumatory bowel disease (IBD) is well-established. There are no large scale studies of passive smoking in inflammatory bowel disease (IBD) Aim: To study the passive smoking exposure of Jewish IBD patients in Israel in a large scale multicenter study. Methods: Patients with established IBD aged 18-70 years, were interviewed. Two controls, clinic and neighborhood, matched by age, sex, community group, and education, were sought. Results: 534 patients [273 ulcerative colitis (UC) and 261 Crohn's disease (CD»), 478 clinic controls and 430 community controls were interviewed. There were no significant differences in the passive smoking habits between IBD patients and their controls. 51% of UC patients, 50% of the clinic controls and 58% of the community controls were exposed to passive smoking at home (NS); similar results were found among crn patients. When a quantitative exposure index was used UC patients were significantly less exposed to passive smoking than their community controls (7.46 :!:8.40 versus 9.36 :!: 9.46, n=229, p<0.031). No differences in exposure to passive smoking were found when DC patients who had never smoked were compared with their controls. Conclusion: There is a lack of association between passive smoking and IBD in Jewish patients in Israel. When a quantitative exposure index was used DC patients were found to be less exposed to passive smoking than their community controls.

1800 IS THERE GENETIC ANTICIPATION IN mD? A POPULATIONBASED STUDY. Elisa M. Faybush, James F. Blanchard, Patricia Rawsthorne, Charles N. Bernstein, Univ of Manitoba, Winnipeg, MB, Canada. Background: Case ascertainment bias may be responsible for the observation from tertiary referral centers that affected offspring of parents with IBD are diagnosed at a younger age. We aimed to determine if genetic anticipation existed in a population-based database. Methods: In creating the population-based University of Manitoba IBD Database (1996) we collected questionnaires from 2759 subjects. We contacted those subjects who reported having family members (first or second degree) with IBD and their family members for verification of diagnosis & age at diagnosis (AAD). Results: Of 475 subjects reporting positive family histories, 300 (63%) were reachable by phone (161 Crohn s (CD) and 139 (UC). There were 153 cases with 2: 1 1 relative, and 179 cases with 2: 1 2 relative. There were no gender specific trends for either older or younger members of pairings & no differences for UC vs CD. Mean age of onset in familial cases=33.4 :!: 14.3y, non-familial cases=34.1 :!: 15.3y (p=0.49). A similar number of familial cases had appendectomies prior to disease diagnosis for UC (11.5%) as for CD (14.9%) (p=0.49). Conclusions: In a populationbased database we found that anticipation for AAD existed similarly for CD & UC, and there were no gender differences for pairings. There is a similar effect examining pairs of child-parent or child-aunt/uncle & a near doubling of effect when examining child-grandparent. Familial cases on average do not have lower AAD than non-familial cases. It is likely that the anticipation in years for AAD reflects an environmental factor associated with a period of increased incidence. Furthermore, if appendectomy is protective in UC it may only be true in non genetically-linked cases. 0

0

Nicole Bregenzer, Schaeffler Andreas, Dept ofInternal Medicine I, Univ of Regensburg, Regensburg, Germany. Background & Aims: We examined the association of bone mineral density of patients with inflammatory bowel disease with a polymorphism in the gene encoding for the vitamin D receptor. The thymine/cytosine (T/C) polymorphism in the first of two start codons can be defined by a restriction fragment length polymorphism using the restriction endonuclease FokI. Vitamin D receptor alleles containing this polymorphism were denoted by f and alleles lacking the site by F. Methods: The FokI genotype was determined in 138 Caucasian patients with inflammatory bowel disease, 62 were men and 76 women, 92 had Crohn's disease and 46 ulcerative colitis. Bone mineral density of the lumbar spine and the femoral neck was measured by dual-energy X-ray absorptiometry. Results: There was no

Ox inolder case

UC +CD (n=68) UC +CD (n=72) UC+CD(20) data inmean yrs

ParentAAD

Offspring AAD

Anticipation

45.3

28,9

16,4

Aunt/uncle AAD

Nlecelneph AAD

45.0

26.8

Grnomlgdad AAD 62,5

GchlldAAD

22.9

182 29.6