- Email: [email protected]
The identification and characterization of a differentially expressed locus in Helicobacter pylori using arbitrarily primed PCR of RNA
A968 AGA ABSTRACTS
GASTROENTEROLOGYVol. 114, No. 4
G3966
WHY DO PATIENTS WITH INFLAMMATORY BOWEL DISEASE STOP AZATHIOPRINE AND WHAT HAPPENS TO THEM? M.T.Donnelly, D.R Davies, M.J.Carter, C.Gummer, A.J.Lobo. The Gastroenterology and Liver Unit, The Royal Hallamshire Hospital, Sheffield S 10 2JF, UK. Introduction. Azathioprine has become a mainstay in the treatment of inflammatory bowel disease, particularly in those patients with steroid dependent or severe disease. Aims. To discover the reason for and consequences of azathioprine discontinuation in patients with inflammatory bowel disease. Methods. A prospective audit of patients treated with azathioprine utilising a specialist nurse-managed computer database. Results. A total of 266 IBD patients were treated with azathioprine between November 1995 and October 1997. 66 (25%)of these patients stopped azathioprine for reasons shown below: Reason Blood count abnormalities Nausea Influenza symptoms Lack of efficacy Non-compliance Others
Number 20 11 11 8 9 7
% 30 17 17 12 14 11
Of these 66 patients, current follow-up data is available on 37 (56%) with a mean follow-up of 11 months. The outcome in these 37 patients is shown below: Outcome Steroid dependent Surgery Tolerating lower dose Controlled without steroids Died (unrelated)
Number 15 8 5 7 2
% 41 22 14 18 5
Discussion 16% of patients stopped azathioprine because of blood count abnormalities or side effects. Over 60% of patients who need to stop azathioprine will need surgery or are still steroid dependent at a mean follow-up of 11 months • G3967 EVIDENCE FOR HELICOBACTER PYLORI SPECIFIC IgG SECRETION BY THE HUMAN GASTRIC MUCOSA. G. Dorta P. Pescatore, P. Wiesel, N. Porta, E. Saraga*, P Michetti +, AL Blum and I Cnrth~sy-Theulaz. Gastroenterology, CHUV, Lausanne and *Pathology Institute, Lausanne, Switzerland, +Beth Israel Deaconess Med. Center, Boston, MA. Background: Helicobacter pylori (Hp) infection induces both a systemic and mucosal humoral response. The aim of this study was to compare the Hp specific antibody titers present locally at the gastric mucosa to those found in gastric juice and in serum. Methods: Ten asymptomatic volunteers (mean 32 years range 27-43) presenting positive breath tests underwent gastroscopy. Hp positivity was confirmed by rapid urease test (RUT) and/or histology. Mucosal antibody secretion was sampled by absorbant wicks mounted on an endoscopic catheter and applied directly against the gastric mucosa; gastric juice was harvested by aspiration through a sampling catheter. Serial dilutions of wicks and secretion samples were analysed in triplicates by ELISA on plates coated with Hp sonicates for presence of both specific and total IgAs and IgGs; reciprocal dilutions of Hp antibodies (i.e. the log10 of end point dilution values higher than 4x values of Helicobacter negative patients' dilutions) were performed and expressed per ~tg of total IgG or IgA antibodies. Results: Table shows Hp specific IgA and IgG antibody titers N = 10
Hp IgAs/pg IgAs Hp IgGs pg IgGs
Gastric mucosa 2+2 9 -+ 23
Gastric juice 6 -+ 8 0 -+ 1
p* 0.002 0.002 * Students t-test
Both Hp specific IgAs and IgGs were detected on the surface of the gastric mucosa. Blood contamination of the mucosal and luminal samples was ruled out by analysis of free hemoglobin. While titers of specific IgGs were higher than those of IgAs in the vicinity of the gastric mucosa, specific IgGs were barely detectable in gastric secretions, suggesting that IgGs are rapidly degraded in gastric juice, in contrast to IgAs. There was no correlation between titers of specific antibody subclasses present in mucosal secretions and in serum. Conclusions: Antibody responses to Hp measured in serum and in gastric juice do not reflect the local response at the gastric mucosa. The local detection of high titers of IgGs suggests that the gastric mucosa is able to transport IgGs but these antibodies seem to be rapidly degraded in gastric juice. Specific IgAs are either more stable and/or they could also come from the salivary glands. The high variability of juxta-mucosal IgGs might be a marker for the course of infection. Supported by Swiss Cancer Research n ° 387-9-1996
• G3968
ENHANCEMENT OF INTESTINAL EPITHELIAL REPAIR BY A HUMAN GLUCAGON-LIKE PEPTIDE-2 ANALOGUE, h[GIy2]-GLP-2, IN A MURINE MODEL OF ULCERATIVE COLITIS. Daniel. J. Drucker, L. DeForest, B. Yusta, and P. L. Brubaker, Departments of Medicine and Physiology, The Toronto Hospital, University of Toronto, Toronto, Canada. The pathology of Crohn's disease and ulcerative colitis is characterized by chronic inflammation and destruction of the gastrointestinal epithelium. Although suppression of inflammatory mediators is an important component of current disease therapeutics, strategies for enhancing repair and regeneration of the compromised intestinal epithelium have not been widely explored. The demonstration that a peptide hormone secreted by the intestinal epithelium, glucagon-like peptide-2 (GLP-2) is a potent endogenous stimulator of intestinal epithelial proliferation in both the small and large intestine prompted studies of the therapeutic efficacy of GLP-2 in mice with dextran sulfate (DS)-induced colitis. Mice were given DS in drinking water, with concomitant subcutaneous administration of saline or h[GIy2]-GLP-2 for 10 days. All mice developed bloody diarrhea by day 5; saline-treated mice were lethargic, and lost over 20% of body weight by day 10. In contrast, mice treated with h[Gly2]-GLP-2 appeared well and lost only 2% of body weight (p<.05). Analysis of intestinal histology demonstrated extensive epithelial inflammation and disruption of mucosal integrity in saline-treated mice with DS-colitis. Mice treated with h[Gly2]-GLP-2 exhibited improved histological indices of disease activity, including significant increases in total mucosal area and mucosal integrity and a reduced colitis activity score (p<.05). Furthermore, the expression, in colon, of inflammation-associated cytokines such as interleukin-1 was activated in mice with colitis and suppressed in mice treated with h[Gly2]-GLP-2. As GLP-2 is produced in the enteroendocrine cells of the GI tract and its secretion is upregulated in response to intestinal injury, our observations suggest that enhancing normal mechanisms used to regulate intestinal proliferation may be a useful adjunct for healing of mucosa in the presence of active intestinal inflammation. This work was supported by and D. Drucker is a consultant to Allelix Biopharmaceuticals Inc. • G3969
PHENOTYPIC CHANGES OF INTRAEPTIHELIAL LYMPHOCYTES (IELS) IN INTESTINAL LESIONS OF CROHN'S DISEASE (CD). S. DubucquoP, B. Meresse 1,2, B. Tourvieille-Philibert 1, D. Koslowski 2, L. Gambier 3, P. Desreumaux 2, J.F. Colombel 2, J.P. Dessaint I. 1 Laboratoire d'Immunologie; 2 Laboratoire de Recherche sur les MICI (CRI4U004B); 3 Service de Chirurgie Adulte, CHRU Lille, France. Background: Mucosal lesions in CD could be related to local immune
dysregulation. IELs, localized at the interface between digestive mucosa and intestinal antigens, could play a critical role in the induction and the persistance of tissue lesions in CD. Shorten telomeres have been associated with replicative senescence in memory (CD45RO) lymphocytes from elderly subjects, and in CD8+CD28- from HIV patients(D. The aim of the study was to compare purified IELs collected after surgical resection, in healthy and altered mucosa from CD patients. Methods: IELs were purified, using percoll gradients as previously described. Phenotypic characteristics of IELs were analyzed by flow cytometry in 10 patients. The telomere length of IELs was analyzed by Southern blot and molecular hybridization with a specific probe (A2TC3), to compare to the telomere length from both lamina propria lymphocytes (LPLs) and blood lymphocytes (PBLs) in 3 patients. Results: IELs were predominantly CD3÷ CD8 ÷ in both healthy and altered mucosa (73.6% vs 71.9%, respectively). In healthy ileal mucosa, CD3÷ CD8 ÷ IELs did not express the CD28 surface marker (CD8 ÷ CD28 ÷ = 9.8% ( -+ 5.3)), and exhibited shorter telomeres than LPLs or PBLs. On the other hand, in altered mucosa. CD3÷ CD8÷ IELs expressed the CD28 marker (CD8 ÷ CD28 ÷ = 53.2 ( - 25.0%)), and exhibited longer telomeres than in healthy mucosa. Conclusion: In normal ileal mucosa, CD3 ÷ CD8÷ CD28- IELs, with short telomeres might exhibit a replicative senescence resulting from a previous cell proliferation triggered by repeated contacts with intestinal bacterial flora. The phenotypic changes of IELs in altered mucosa, CD3÷/CD8÷/CD28÷, with longer telomeres could reflect the recruitment of T cells in response to persistent abormal antigenic stimulation. A relation between such cell recruitment and local modification of immune response is under investigation. 1 Monteiro J. et al. Shortened telomeres in clonally expanded CD28-CD8+ T cells imply a replicative history that is distinct from their CD28÷CD8+ counterparts. [l] J Immunol 1996; 156: 3587-90. • G3970 THE IDENTIHCATION AND CHARACTERIZATION OF A DIFFERENTIALLY EXPRESSED LOCUS IN HELICOBACTER PYLORI USING ARBITRARILY PRIMED PCR OF RNA. W. G. Dundon. D. G. Marshall, C. J. Smyth, Microbiology Dept., Trinity College, Dublin, Ireland. C. A. O'Morain, Gastroenterology Dept., Meath/Adelaide Hospitals, Dublin, Ireland.
Helicobacter pylori is a pathogenic bacterium which displays extreme diversity at the genetic level. To date several genes have been identified that
April 1998
are expressed in only a certain proportion of strains, some of them being correlated with the pathogenicity of the bacterium. The aim of the present study was to identify other genes that are differentially expressed between clinical isolates of H. pylori. Using a modification of a technique for arbitrarily primed PCR of RNA, a eDNA fragment of 187 bp (designated del for differentially _expressed locus) was identified that was expressed in only one of a pair of clinical isolates tested. The fragment was purified, cloned and sequenced. An initial search of the databases showed no sequence similarity with any other known genes or gene-products. Inverse PCR was used to obtain further nucleotide sequence information surrounding the del locus. Comparison of the nucleotide sequence of del with the recently released genome sequence of H. pylori identified the del locus as a hypothetical protein (HP0945). Comparison of the nucleotide sequences between a del positive and del negative isolate showed that the locus is located between two transfer RNAs in H. pylori. The % G+C content of del suggests that it has been acquired by horizontal transfer. A DNA probe derived from del hybridized with 27 (46.5%) of 57 clinical 1-1. pylori isolates tested. The cagA/CagA and vacAIVacA genotype and phenotype of 42 of these isolates was determined. No correlation between the presence or absence of del and cagAICagA or vacA/VacA status in these strains was seen. G3971 HT-29 HUMAN INTESTINAL EPITHELIAL CELLS EXPRESS mRNA AND PROTEIN FOR TUMOR NECROSIS FACTOR RECEPTORS I AND II, M. B. Dwinell L. Eckmann, and M.F. Kagnoff, Laboratory of Mucosal Immunology, Department of Medicine, University of California, San Diego, La Jolla, CA. HT-29 cells rapidly secrete proinflammatory cytokines and later undergo apoptosis in response to tumor necrosis factor (TNF) t~ stimulation. The biologic activities of TNFct are mediated through two distinct receptors, tumor necrosis factor receptor (TNFR) I and TNFRII. We asked which types of TNFR are expressed on intestinal epithelial cells, and if expression of those receptors is regulated by proinflammatory cytokines. Methods: mRNA and cell surface expression of TNFRI and TNFRII in HT-29 cells were assessed by RT-PCR and flow cytometry. Soluble TNFR I and II in culture supematants were measured by ELISA. To assess possible regulated expression of the TNFR's, cells were stimulated with IL-l~x and interferon-`/. Results: HT-29 cells constitutively expressed TNFRI and TNFRII mRNA and surface protein. Interferon-`/ and IL-l~t did not alter TNFRI or TNFRII expression, but increased the release of soluble TNFRI. Soluble TNFRII was not detectable in culture supernatants of unstimulated or agonist stimulated HT-29 cells. Blocking TNFct stimulation with antibodies against TNFRI inhibited IL-8 secretion by 90%. Conclusion: Both TNFRI and TNFRII are expressed on HT-29 colonic epithelial ceils, and their expression is not regulated by IL-lct or interferon-'/. TNFRI plays a major role in signaling increased IL-8 production in these cells and TNFRI is released in soluble form in response to agonist stimulation. Supported by NIH grant DK35108, NIH training grant DK07202, and a Crohn's and Colitis Foundation Award. • G3972 PHARMACOGENETICS OF 5-ASA METABOLISM IN IBD PATIENTS: PRELIMINARY REPORT OF VARIATION IN NACETYLTRANSFERASE 1 GENE POLYMORPHISMS AND 5-ASA METABOLISM RATES IN MUCOSAL BIOPSIES. J. Easterbrook, G. Armstrong, T.M. Bayless, M.L. Harris and M.H. Montrose. Dept. of Medicine, Johns Hopkins Univ., Baltimore, MD. 5-Aminosalicylate (5-ASA) is the most commonly prescribed therapy for inflammatory bowel diseases (IBDs) such as ulcerative colitis (UC) and Crohn's disease (CD). The aim of this study was to examine the correlation between variations in the colonic mucosal N-acetyl transferase 1 enzyme (NAT1) which metabolizes 5-ASA, and patient response to therapy. Polymorphisms have been identified in the NAT1 gene, but no clinical evidence of the impact of these polymorphisms on patient response to 5-ASA therapy has been described. Methods. IBD patients were stratified as normal or poor responders to 5-ASA by physician assessment. Colonic biopsy samples taken from patients during routine endoscopy were homogenized and the resultant supematant measured for NAT1 activity using a fluorescent assay of N-acetyl-5-ASA production (excitation at 314 nm and emission 440 nm). Results. The 5-ASA acetylation rate in IBD patients was significantly lower in poor responders (3.05 +_ 1. I 1 nmol N-acetyl 5-ASA/min/~tg protein, n=5) than in normal responders (6.16 _+0.85, n=12: p=0.058, student t-test). No other significant differences in N-acetyl 5-ASA production were identified when patients were classified according to disease status (active vs, quiescent disease) or diagnosis (UC vs. CD) (p=3.26, ANOVA). Sequencing of genomic DNA from 17 patients (including 5 poor responders) identified one poor responder heterozygous for a known NAT1 polymorphism (NATI*17), and two other poor responders that have a common polymorphism (757 Ile->Leu) that has not been described previously and is NOT in the 12 patients who are normal responders to 5-ASA therapy. Conclusion. Patient response to 5-ASA therapy may correlate with NAT1 enzyme activity, although further studies
Immunology, Microbiology, and Inflammatory Disorders A969
will be needed to identify recurrent changes in the NAT1 gene which are specific for the responder populations. This research was funded by the CCFA. G3973 CO-COLONIZATION AND IN VIVO INTERACTION BETWEEN STRAINS OF HELICOBACTER PYLORI. K.A. Eaton, S. Krakowka, Ohio State University, Columbus, OH; and D.E. Berg, Washington University School of Medicine, St. Louis, MO.
Purpose: To determine 1) the ability of H. pylori strains to co-colonize and compete in vivo, and 2) the frequency of genetic transformation between co-colonizing strains. Methods: Two different combinations of strains, 26695vacAf2aph3'-Ill (kanamycin resistant) + N6virB11f2CAT (chloramphenicol resistant), and 26695vacAf2aph3'-lll +26695virB11~CAT were co-cultured in vitro and in vivo in germ-free piglets. Piglets were orally inoculated with either the individual strains or the mixture (equal numbers of the 2 strains), and their stomachs were cultured 3, 6, and 12 weeks after inoculation. Results: In vitro, all strains grew equally well in co-culture and both mixtures resulted in transformants (double mutants) after 24 hours. Transformation within strain 26695 (approximately 10-7 transformants/cfu) was 10-fold greater than transformation between the 2 strains. In vivo, 5/6 piglets given both 26695vacAD.aph3'-llI and N6virB11f2CAT were colonized only with 26695vacAf~ph3'-llL The single piglet colonized with both strains remained co-colonized for the duration of the experiment. In contrast, all 6 piglets given 26695vacAD.aph3'-lll + 26695virBllf2CAT were colonized with both mutants. By 3 months after inoculation, 4/5 piglets remained colonized, 3 with both strains. In all piglets colonized with two strains, approximately 1/2 the colonized sites contained both strains. Double mutants were not recovered from any piglet at any time interval, but co-culture of biopsies containing both strains resulted in transformation in vitro after 5 days in culture. Conclusions: 1. H. pylori strains are able to co-colonize piglets. 2. Strain 26695 is able to out-compete N6 in most cases. 3. Neither vacA nor virBll is necessary for colonization and neither confers a selective advantage for colonization. 4. Natural transformation occurs rapidly in vitro, but does not occur in vivo even after co-colonization for 3 months. Thus, transformation in vivo is an unlikely mechanism for development of the marked heterogeneity of H. pylori in nature. • G3974 THE ROLE OF MOUSE STRAIN AND NORMAL FLORA IN THE RESPONSE OF MICE TO CHOLERA TOXIN AND HELICOBACTER VACCINATION. K.A. Eaton. SJ. Danon, S. Krakowka; Ohio State University, Columbus, OH. Purpose: To determine if mouse strain or absence of normal flora determines the response of mice to cholera toxin used as an oral adjuvant for helicobacter vaccination. Methods: Six-12 week old germ-free C57BL/6 mice and conventional BALB/c mice were orally vaccinated 4 times at weekly intervals, challenged with live Helicobacterfelis 2 weeks later, and killed 6 weeks after challenge Germ-free contact controls were not challenged with live bacteria, but were kept in the same isolators (but not the same cages) as infected mice. Vaccination groups were: cholera toxin (CT) + H. felis sonicate, sonicate alone, CT alone, and sterile saline. Bacterial colonization was quantified, and severity of gastritis was determined by the percent of microscopic fields which contained neutrophilic inflammation, displacement of glands by inflammatory infiltrate, and/or epithelial metaplasia. Results: Bacteria were recovered from inoculated and contact control groups, but not from uninfected controls. In conventional BALB/c mice H. felis infection in the absence of vaccination did not cause gastritis. Vaccination with either CT alone or CT + sonicate suppressed colonization in inoculated mice and caused severe gastritis in inoculated mice and moderate gastritis in uninfected mice. In germ-free C57BL/6 inoculated and contact control mice infection caused severe gastritis. Vaccination with CT + sonicate but not with CT alone or sonicate alone suppressed colonization, but CT vaccination did not affect severity of gastritis. CT did not cause gastritis in uninfected germfree C57BL/6 mice. Cunclusions: 1. H. fells is easily transmitted between germ-free mice by incidental contact. 2. CT is an effective oral adjuvant for helicobacter vaccination in both conventional BALB/c and germ-free C57BL/6 mice. 3. CT alone suppresses colonization in conventional but not germ-free mice. 4. In conventional BALB/c mice CT causes gastritis even in the absence of bacterial colonization, and enhances severity of gastritis in inoculated mice. 5. Cross-reacting enteric bacteria may promote gastritis and immunity to gastric helicobacter in conventional mice treated with oral CT.
GASTROENTEROLOGYVol. 114, No. 4
G3966
WHY DO PATIENTS WITH INFLAMMATORY BOWEL DISEASE STOP AZATHIOPRINE AND WHAT HAPPENS TO THEM? M.T.Donnelly, D.R Davies, M.J.Carter, C.Gummer, A.J.Lobo. The Gastroenterology and Liver Unit, The Royal Hallamshire Hospital, Sheffield S 10 2JF, UK. Introduction. Azathioprine has become a mainstay in the treatment of inflammatory bowel disease, particularly in those patients with steroid dependent or severe disease. Aims. To discover the reason for and consequences of azathioprine discontinuation in patients with inflammatory bowel disease. Methods. A prospective audit of patients treated with azathioprine utilising a specialist nurse-managed computer database. Results. A total of 266 IBD patients were treated with azathioprine between November 1995 and October 1997. 66 (25%)of these patients stopped azathioprine for reasons shown below: Reason Blood count abnormalities Nausea Influenza symptoms Lack of efficacy Non-compliance Others
Number 20 11 11 8 9 7
% 30 17 17 12 14 11
Of these 66 patients, current follow-up data is available on 37 (56%) with a mean follow-up of 11 months. The outcome in these 37 patients is shown below: Outcome Steroid dependent Surgery Tolerating lower dose Controlled without steroids Died (unrelated)
Number 15 8 5 7 2
% 41 22 14 18 5
Discussion 16% of patients stopped azathioprine because of blood count abnormalities or side effects. Over 60% of patients who need to stop azathioprine will need surgery or are still steroid dependent at a mean follow-up of 11 months • G3967 EVIDENCE FOR HELICOBACTER PYLORI SPECIFIC IgG SECRETION BY THE HUMAN GASTRIC MUCOSA. G. Dorta P. Pescatore, P. Wiesel, N. Porta, E. Saraga*, P Michetti +, AL Blum and I Cnrth~sy-Theulaz. Gastroenterology, CHUV, Lausanne and *Pathology Institute, Lausanne, Switzerland, +Beth Israel Deaconess Med. Center, Boston, MA. Background: Helicobacter pylori (Hp) infection induces both a systemic and mucosal humoral response. The aim of this study was to compare the Hp specific antibody titers present locally at the gastric mucosa to those found in gastric juice and in serum. Methods: Ten asymptomatic volunteers (mean 32 years range 27-43) presenting positive breath tests underwent gastroscopy. Hp positivity was confirmed by rapid urease test (RUT) and/or histology. Mucosal antibody secretion was sampled by absorbant wicks mounted on an endoscopic catheter and applied directly against the gastric mucosa; gastric juice was harvested by aspiration through a sampling catheter. Serial dilutions of wicks and secretion samples were analysed in triplicates by ELISA on plates coated with Hp sonicates for presence of both specific and total IgAs and IgGs; reciprocal dilutions of Hp antibodies (i.e. the log10 of end point dilution values higher than 4x values of Helicobacter negative patients' dilutions) were performed and expressed per ~tg of total IgG or IgA antibodies. Results: Table shows Hp specific IgA and IgG antibody titers N = 10
Hp IgAs/pg IgAs Hp IgGs pg IgGs
Gastric mucosa 2+2 9 -+ 23
Gastric juice 6 -+ 8 0 -+ 1
p* 0.002 0.002 * Students t-test
Both Hp specific IgAs and IgGs were detected on the surface of the gastric mucosa. Blood contamination of the mucosal and luminal samples was ruled out by analysis of free hemoglobin. While titers of specific IgGs were higher than those of IgAs in the vicinity of the gastric mucosa, specific IgGs were barely detectable in gastric secretions, suggesting that IgGs are rapidly degraded in gastric juice, in contrast to IgAs. There was no correlation between titers of specific antibody subclasses present in mucosal secretions and in serum. Conclusions: Antibody responses to Hp measured in serum and in gastric juice do not reflect the local response at the gastric mucosa. The local detection of high titers of IgGs suggests that the gastric mucosa is able to transport IgGs but these antibodies seem to be rapidly degraded in gastric juice. Specific IgAs are either more stable and/or they could also come from the salivary glands. The high variability of juxta-mucosal IgGs might be a marker for the course of infection. Supported by Swiss Cancer Research n ° 387-9-1996
• G3968
ENHANCEMENT OF INTESTINAL EPITHELIAL REPAIR BY A HUMAN GLUCAGON-LIKE PEPTIDE-2 ANALOGUE, h[GIy2]-GLP-2, IN A MURINE MODEL OF ULCERATIVE COLITIS. Daniel. J. Drucker, L. DeForest, B. Yusta, and P. L. Brubaker, Departments of Medicine and Physiology, The Toronto Hospital, University of Toronto, Toronto, Canada. The pathology of Crohn's disease and ulcerative colitis is characterized by chronic inflammation and destruction of the gastrointestinal epithelium. Although suppression of inflammatory mediators is an important component of current disease therapeutics, strategies for enhancing repair and regeneration of the compromised intestinal epithelium have not been widely explored. The demonstration that a peptide hormone secreted by the intestinal epithelium, glucagon-like peptide-2 (GLP-2) is a potent endogenous stimulator of intestinal epithelial proliferation in both the small and large intestine prompted studies of the therapeutic efficacy of GLP-2 in mice with dextran sulfate (DS)-induced colitis. Mice were given DS in drinking water, with concomitant subcutaneous administration of saline or h[GIy2]-GLP-2 for 10 days. All mice developed bloody diarrhea by day 5; saline-treated mice were lethargic, and lost over 20% of body weight by day 10. In contrast, mice treated with h[Gly2]-GLP-2 appeared well and lost only 2% of body weight (p<.05). Analysis of intestinal histology demonstrated extensive epithelial inflammation and disruption of mucosal integrity in saline-treated mice with DS-colitis. Mice treated with h[Gly2]-GLP-2 exhibited improved histological indices of disease activity, including significant increases in total mucosal area and mucosal integrity and a reduced colitis activity score (p<.05). Furthermore, the expression, in colon, of inflammation-associated cytokines such as interleukin-1 was activated in mice with colitis and suppressed in mice treated with h[Gly2]-GLP-2. As GLP-2 is produced in the enteroendocrine cells of the GI tract and its secretion is upregulated in response to intestinal injury, our observations suggest that enhancing normal mechanisms used to regulate intestinal proliferation may be a useful adjunct for healing of mucosa in the presence of active intestinal inflammation. This work was supported by and D. Drucker is a consultant to Allelix Biopharmaceuticals Inc. • G3969
PHENOTYPIC CHANGES OF INTRAEPTIHELIAL LYMPHOCYTES (IELS) IN INTESTINAL LESIONS OF CROHN'S DISEASE (CD). S. DubucquoP, B. Meresse 1,2, B. Tourvieille-Philibert 1, D. Koslowski 2, L. Gambier 3, P. Desreumaux 2, J.F. Colombel 2, J.P. Dessaint I. 1 Laboratoire d'Immunologie; 2 Laboratoire de Recherche sur les MICI (CRI4U004B); 3 Service de Chirurgie Adulte, CHRU Lille, France. Background: Mucosal lesions in CD could be related to local immune
dysregulation. IELs, localized at the interface between digestive mucosa and intestinal antigens, could play a critical role in the induction and the persistance of tissue lesions in CD. Shorten telomeres have been associated with replicative senescence in memory (CD45RO) lymphocytes from elderly subjects, and in CD8+CD28- from HIV patients(D. The aim of the study was to compare purified IELs collected after surgical resection, in healthy and altered mucosa from CD patients. Methods: IELs were purified, using percoll gradients as previously described. Phenotypic characteristics of IELs were analyzed by flow cytometry in 10 patients. The telomere length of IELs was analyzed by Southern blot and molecular hybridization with a specific probe (A2TC3), to compare to the telomere length from both lamina propria lymphocytes (LPLs) and blood lymphocytes (PBLs) in 3 patients. Results: IELs were predominantly CD3÷ CD8 ÷ in both healthy and altered mucosa (73.6% vs 71.9%, respectively). In healthy ileal mucosa, CD3÷ CD8 ÷ IELs did not express the CD28 surface marker (CD8 ÷ CD28 ÷ = 9.8% ( -+ 5.3)), and exhibited shorter telomeres than LPLs or PBLs. On the other hand, in altered mucosa. CD3÷ CD8÷ IELs expressed the CD28 marker (CD8 ÷ CD28 ÷ = 53.2 ( - 25.0%)), and exhibited longer telomeres than in healthy mucosa. Conclusion: In normal ileal mucosa, CD3 ÷ CD8÷ CD28- IELs, with short telomeres might exhibit a replicative senescence resulting from a previous cell proliferation triggered by repeated contacts with intestinal bacterial flora. The phenotypic changes of IELs in altered mucosa, CD3÷/CD8÷/CD28÷, with longer telomeres could reflect the recruitment of T cells in response to persistent abormal antigenic stimulation. A relation between such cell recruitment and local modification of immune response is under investigation. 1 Monteiro J. et al. Shortened telomeres in clonally expanded CD28-CD8+ T cells imply a replicative history that is distinct from their CD28÷CD8+ counterparts. [l] J Immunol 1996; 156: 3587-90. • G3970 THE IDENTIHCATION AND CHARACTERIZATION OF A DIFFERENTIALLY EXPRESSED LOCUS IN HELICOBACTER PYLORI USING ARBITRARILY PRIMED PCR OF RNA. W. G. Dundon. D. G. Marshall, C. J. Smyth, Microbiology Dept., Trinity College, Dublin, Ireland. C. A. O'Morain, Gastroenterology Dept., Meath/Adelaide Hospitals, Dublin, Ireland.
Helicobacter pylori is a pathogenic bacterium which displays extreme diversity at the genetic level. To date several genes have been identified that
April 1998
are expressed in only a certain proportion of strains, some of them being correlated with the pathogenicity of the bacterium. The aim of the present study was to identify other genes that are differentially expressed between clinical isolates of H. pylori. Using a modification of a technique for arbitrarily primed PCR of RNA, a eDNA fragment of 187 bp (designated del for differentially _expressed locus) was identified that was expressed in only one of a pair of clinical isolates tested. The fragment was purified, cloned and sequenced. An initial search of the databases showed no sequence similarity with any other known genes or gene-products. Inverse PCR was used to obtain further nucleotide sequence information surrounding the del locus. Comparison of the nucleotide sequence of del with the recently released genome sequence of H. pylori identified the del locus as a hypothetical protein (HP0945). Comparison of the nucleotide sequences between a del positive and del negative isolate showed that the locus is located between two transfer RNAs in H. pylori. The % G+C content of del suggests that it has been acquired by horizontal transfer. A DNA probe derived from del hybridized with 27 (46.5%) of 57 clinical 1-1. pylori isolates tested. The cagA/CagA and vacAIVacA genotype and phenotype of 42 of these isolates was determined. No correlation between the presence or absence of del and cagAICagA or vacA/VacA status in these strains was seen. G3971 HT-29 HUMAN INTESTINAL EPITHELIAL CELLS EXPRESS mRNA AND PROTEIN FOR TUMOR NECROSIS FACTOR RECEPTORS I AND II, M. B. Dwinell L. Eckmann, and M.F. Kagnoff, Laboratory of Mucosal Immunology, Department of Medicine, University of California, San Diego, La Jolla, CA. HT-29 cells rapidly secrete proinflammatory cytokines and later undergo apoptosis in response to tumor necrosis factor (TNF) t~ stimulation. The biologic activities of TNFct are mediated through two distinct receptors, tumor necrosis factor receptor (TNFR) I and TNFRII. We asked which types of TNFR are expressed on intestinal epithelial cells, and if expression of those receptors is regulated by proinflammatory cytokines. Methods: mRNA and cell surface expression of TNFRI and TNFRII in HT-29 cells were assessed by RT-PCR and flow cytometry. Soluble TNFR I and II in culture supematants were measured by ELISA. To assess possible regulated expression of the TNFR's, cells were stimulated with IL-l~x and interferon-`/. Results: HT-29 cells constitutively expressed TNFRI and TNFRII mRNA and surface protein. Interferon-`/ and IL-l~t did not alter TNFRI or TNFRII expression, but increased the release of soluble TNFRI. Soluble TNFRII was not detectable in culture supernatants of unstimulated or agonist stimulated HT-29 cells. Blocking TNFct stimulation with antibodies against TNFRI inhibited IL-8 secretion by 90%. Conclusion: Both TNFRI and TNFRII are expressed on HT-29 colonic epithelial ceils, and their expression is not regulated by IL-lct or interferon-'/. TNFRI plays a major role in signaling increased IL-8 production in these cells and TNFRI is released in soluble form in response to agonist stimulation. Supported by NIH grant DK35108, NIH training grant DK07202, and a Crohn's and Colitis Foundation Award. • G3972 PHARMACOGENETICS OF 5-ASA METABOLISM IN IBD PATIENTS: PRELIMINARY REPORT OF VARIATION IN NACETYLTRANSFERASE 1 GENE POLYMORPHISMS AND 5-ASA METABOLISM RATES IN MUCOSAL BIOPSIES. J. Easterbrook, G. Armstrong, T.M. Bayless, M.L. Harris and M.H. Montrose. Dept. of Medicine, Johns Hopkins Univ., Baltimore, MD. 5-Aminosalicylate (5-ASA) is the most commonly prescribed therapy for inflammatory bowel diseases (IBDs) such as ulcerative colitis (UC) and Crohn's disease (CD). The aim of this study was to examine the correlation between variations in the colonic mucosal N-acetyl transferase 1 enzyme (NAT1) which metabolizes 5-ASA, and patient response to therapy. Polymorphisms have been identified in the NAT1 gene, but no clinical evidence of the impact of these polymorphisms on patient response to 5-ASA therapy has been described. Methods. IBD patients were stratified as normal or poor responders to 5-ASA by physician assessment. Colonic biopsy samples taken from patients during routine endoscopy were homogenized and the resultant supematant measured for NAT1 activity using a fluorescent assay of N-acetyl-5-ASA production (excitation at 314 nm and emission 440 nm). Results. The 5-ASA acetylation rate in IBD patients was significantly lower in poor responders (3.05 +_ 1. I 1 nmol N-acetyl 5-ASA/min/~tg protein, n=5) than in normal responders (6.16 _+0.85, n=12: p=0.058, student t-test). No other significant differences in N-acetyl 5-ASA production were identified when patients were classified according to disease status (active vs, quiescent disease) or diagnosis (UC vs. CD) (p=3.26, ANOVA). Sequencing of genomic DNA from 17 patients (including 5 poor responders) identified one poor responder heterozygous for a known NAT1 polymorphism (NATI*17), and two other poor responders that have a common polymorphism (757 Ile->Leu) that has not been described previously and is NOT in the 12 patients who are normal responders to 5-ASA therapy. Conclusion. Patient response to 5-ASA therapy may correlate with NAT1 enzyme activity, although further studies
Immunology, Microbiology, and Inflammatory Disorders A969
will be needed to identify recurrent changes in the NAT1 gene which are specific for the responder populations. This research was funded by the CCFA. G3973 CO-COLONIZATION AND IN VIVO INTERACTION BETWEEN STRAINS OF HELICOBACTER PYLORI. K.A. Eaton, S. Krakowka, Ohio State University, Columbus, OH; and D.E. Berg, Washington University School of Medicine, St. Louis, MO.
Purpose: To determine 1) the ability of H. pylori strains to co-colonize and compete in vivo, and 2) the frequency of genetic transformation between co-colonizing strains. Methods: Two different combinations of strains, 26695vacAf2aph3'-Ill (kanamycin resistant) + N6virB11f2CAT (chloramphenicol resistant), and 26695vacAf2aph3'-lll +26695virB11~CAT were co-cultured in vitro and in vivo in germ-free piglets. Piglets were orally inoculated with either the individual strains or the mixture (equal numbers of the 2 strains), and their stomachs were cultured 3, 6, and 12 weeks after inoculation. Results: In vitro, all strains grew equally well in co-culture and both mixtures resulted in transformants (double mutants) after 24 hours. Transformation within strain 26695 (approximately 10-7 transformants/cfu) was 10-fold greater than transformation between the 2 strains. In vivo, 5/6 piglets given both 26695vacAD.aph3'-llI and N6virB11f2CAT were colonized only with 26695vacAf~ph3'-llL The single piglet colonized with both strains remained co-colonized for the duration of the experiment. In contrast, all 6 piglets given 26695vacAD.aph3'-lll + 26695virBllf2CAT were colonized with both mutants. By 3 months after inoculation, 4/5 piglets remained colonized, 3 with both strains. In all piglets colonized with two strains, approximately 1/2 the colonized sites contained both strains. Double mutants were not recovered from any piglet at any time interval, but co-culture of biopsies containing both strains resulted in transformation in vitro after 5 days in culture. Conclusions: 1. H. pylori strains are able to co-colonize piglets. 2. Strain 26695 is able to out-compete N6 in most cases. 3. Neither vacA nor virBll is necessary for colonization and neither confers a selective advantage for colonization. 4. Natural transformation occurs rapidly in vitro, but does not occur in vivo even after co-colonization for 3 months. Thus, transformation in vivo is an unlikely mechanism for development of the marked heterogeneity of H. pylori in nature. • G3974 THE ROLE OF MOUSE STRAIN AND NORMAL FLORA IN THE RESPONSE OF MICE TO CHOLERA TOXIN AND HELICOBACTER VACCINATION. K.A. Eaton. SJ. Danon, S. Krakowka; Ohio State University, Columbus, OH. Purpose: To determine if mouse strain or absence of normal flora determines the response of mice to cholera toxin used as an oral adjuvant for helicobacter vaccination. Methods: Six-12 week old germ-free C57BL/6 mice and conventional BALB/c mice were orally vaccinated 4 times at weekly intervals, challenged with live Helicobacterfelis 2 weeks later, and killed 6 weeks after challenge Germ-free contact controls were not challenged with live bacteria, but were kept in the same isolators (but not the same cages) as infected mice. Vaccination groups were: cholera toxin (CT) + H. felis sonicate, sonicate alone, CT alone, and sterile saline. Bacterial colonization was quantified, and severity of gastritis was determined by the percent of microscopic fields which contained neutrophilic inflammation, displacement of glands by inflammatory infiltrate, and/or epithelial metaplasia. Results: Bacteria were recovered from inoculated and contact control groups, but not from uninfected controls. In conventional BALB/c mice H. felis infection in the absence of vaccination did not cause gastritis. Vaccination with either CT alone or CT + sonicate suppressed colonization in inoculated mice and caused severe gastritis in inoculated mice and moderate gastritis in uninfected mice. In germ-free C57BL/6 inoculated and contact control mice infection caused severe gastritis. Vaccination with CT + sonicate but not with CT alone or sonicate alone suppressed colonization, but CT vaccination did not affect severity of gastritis. CT did not cause gastritis in uninfected germfree C57BL/6 mice. Cunclusions: 1. H. fells is easily transmitted between germ-free mice by incidental contact. 2. CT is an effective oral adjuvant for helicobacter vaccination in both conventional BALB/c and germ-free C57BL/6 mice. 3. CT alone suppresses colonization in conventional but not germ-free mice. 4. In conventional BALB/c mice CT causes gastritis even in the absence of bacterial colonization, and enhances severity of gastritis in inoculated mice. 5. Cross-reacting enteric bacteria may promote gastritis and immunity to gastric helicobacter in conventional mice treated with oral CT.