Superinfection with helicobacter hepaticus does not alter the natural course of colitis in IL-10 deficient mice

GASTROENTEROLOGYVol. 114, No. 4

A95S AGA ABSTRACTS • G3927 SUPERINFECTION WITH HELICOBACTER HEPATICUS DOES NOT ALTER THE NATURAL COURSE OF COLITIS IN IL-10 DEFICIENT MICE. S. J. Czinn, B. M. Zagorski, D. M. Spencer, C Garhart, A. D. Levine. Case Western Reserve University, Cleveland, Ohio, USA. IL-10 deficient mice (IL-10 -/-) spontaneously develop colitis when housed in microisolators (SPF housing). The severity and rate of disease onset is aggravated when these mice are maintained in conventional housing, suggesting that gastrointestinal flora contribute to the inflammation. Recent reports have implicated intestinal Helicobacter species in the development of colitis in immunodeficient mice. We investigated whether H. hepaticus infection can promote the development of colitis in IL-10 "/" mice. Clinical signs of colitis in SPF-housed IL-10 "/- mice on a C57BL/10 background (weight loss, blood in the stool) are observed at 12 weeks of age and histological inflammation (crypt abscesses, ulcerations, skip lesions, transmural infiltration, epithelial hyperplasia) is maximal at 18-20 weeks. IL-104- were superinfected with 107 bacteria by oral gavage at 12 weeks of age and examined for occult blood in the feces, Helicobacter specific serum antibody titers and microscopic inflammation at 24 weeks. All naive and superinfected mice had evidence of fecal blood. Rectal prolapse was observed in 50% of the animals in both groups. Histological scoring for tissue damage and degree of inflammation demonstrated severe focal inflammation in all animals. No significant differences were noted between groups. These data demonstrate that superinfection with H. hepaticus does not increase the rate of disease onset, exacerbate the severity of inflammation, or modify the course of the colitis. It remains to be determined whether intestinal Helicobacter species play a role in the initiation or propagation Of colitis. • G3928 ANTI-GAL ANTIBODIES IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE. M.D'Alessandro. P.Mariani, A.Bachetoni, D.Lomanto, P.Mazzocchi, V.Speranza. Clinica Chirurgica II, University of "La Sapienza" Rome, Italy. Background: Anti-a-l,3-galactosyl (anti-Gal) is an ubiquitous natural human serum antibody that binds to terminal galactose-a 1,3-galactose (a-galactosyl) residues, not expressed on human cells. Human anti-Gal antibody production seems to be constantly stimulated by bacterial strains of the gastrointestinal flora that express a-galactosyl epitopes and it may contribute to defense against infectious organism. Several studies suggested that anti-Gal may be associated with autoimmune, inflammatory and parasitic disorders. Aim: To determine the seroprevalence of anti-Gal antibodies in sera from patients with Crohn's disease (CD) and ulcerative colitis (UC) and to postulate a relationship with these disorders. Methods: Sera were obtained from 50 pts with CD, 30 pts with UC and 25 healthy controls. An ELISA assay was performed to analyse circulating antibodies against the disaccharide Gall-a3Gal coupled to human serum albumin (IsoSep-Sweden). Plates were coated with the antigen (1 mg/ml). Human sera, serially diluted in PBS-Tween, were added to the antigen coated wells. After incubation with AKP-conjugate rabbit anti-human Ig (lgGIgAIgM) antibody (Dako), bound reactants were detected with p-NPP. The results have been reported as absolute absorbance (405nM). The detection of immunoglobulin class were performed in a similar way with sera diluted 1:100 and addition of AKP-conjugated rabbit antihuman IgG,IgA and IgM. Results: Fig. 1 shows a titration curve of reactivity of sera from pts with MC, UC and normal controls. The mean + SD absorbance value of UC and strongly of CD were significantly higher than controls. Tab. 1 shows the immunoglobulin class distribution: serum IgG, IgA and IgM anti-Gal antibodies were detected at a significantly increased prevalence in CD pts compared with controls. In UC pts an increase of IgA and IgM but not IgG was observed. Fig. 1: Reciprocal of serum dilution Z5

1,5 "~ 1

* v--o.001

[ ' ~ - ~

T

i

....

:~(~m~

0,5 11100

1/4(]0

Tab. 1 Ig class distribution Op < 0.001

*p < 0.03

CD UC Ctrl

1/200

Pts 25 10 10

IgG . IgA 1.18 _+,75* 0.32 - .24 ° 0.79 -+ .51 0.23 --- .18" 0.71 -+ .48 0.07 -+ .05

405 nM IgM 0.45 -+ .26* 0.40 -+.28 0.25 -+ .14

Conclusions: Pts with IBD and particularly with CD show increased levels of circulating antibodies against the Gal-al-3Gal epitopes. We can hypothesize an increase of the immune response to luminal bacterial content due to the altered intestinal permeability and to continous exposure to microbial antigens. The presence of elevated levels of IgA anti-Gal antibodies suggests a gut mucosal sensitization associated with chronic antigen exposure. This is

consistent with a loss of tolerance to the normal flora of the gut that could trigger a chronic inflammatory response, typical of CD. G3929 ISOGENIC FLAGELLA MUTANTS OF HELICOBACTER PYLORI: CANDIDATES FOR AN ATTENUATED VACCINE? Steohen J. Danon and Kathryn A. Eaton. Department of Veterinary Biosciences, :The Ohio State University, Columbus, OH. The purpose of this study was to 1) createflaA- andflaB- isogenic mutants of Helicobacter pylori strain SS1, a mouse-adapted human isolate, 2) to investigate the colonisation pattem of the mutants in vivo, and 3) to investigate the potential of these mutants as candidates for an attenuated vaccine of H. pylori. Methods: SSlflaA::km was created by electroporation with genomic DNA from N6flaA::km. SSlflaB::km was created by natural transformation with DNA from N6flaB::km. Resulting flaA and t a B mutants were confirmed by growth on blood agar containing 20pg/ml kanamycin and by PCR amplification of the flaA and t a B genes. Germ-free C57BL/6 mice were orally inoculated with 2 doses of either 109 cfu/ml SSlflaA::km (n=4) or SSlflaB::km (n=5). Two animals from each group were killed 4 weeks after inoculation. At 5 weeks after inoculation, 2 animals from each group, and 2 uninfected control animals, were inoculated with 109 cfu/ml SS 1 wildtype. All animals were killed 2 weeks later. At sacrifice serum was collected for ELISA, half the stomach was homogenised for quantitative culture, and half the stomach was used for urease and histologic examination. Results: Table 1 Colonisation of germ-free mice with flagellar mutants ofH. pylori. Inoculum number 1

SS lflaA::km SSlflaA::km SSlflaB::km

SSlflaB::km

Inoculum na Sacrifice number 2 interval (weeks)

Titre IgGb

Culture (mutant) cfu/g stomach

2 2 2 3 2

1:640 1:320 1:320 1:2560 1:160

0 0 5.5 x 106 3.4 x 10s

SS1 SS1 SS1

4 7 4 7 2

Culture (nonselective) cfu/g stomach 1.9 x 106 6.2 x 105 1.02 x 106

(a) n=number of animals per group (b) Positive control sera IgG titre 1:10,240, negative control sera IgG titre
Conclusion: SSlflaA::km does not colonise mice but does induce a humoral immune response which decreases over time. In contrast, SSlflaB::km does colonise the stomach. The immunity induced by both mutants was ineffective at preventing colonisation of the wildtype. However, ability of a noncolonizing mutant to induce humoral immunity suggests that this mutant might be a candidate for an attenuated vaccine. • G3930 VIRULENCE FACTORS OF ESCHERICHIA COLI STRAINS ISOLATED FROM ILEAL MUCOSA IN CROHN'S DISEASE (CD). A Darfeuille-Michaud, C Neut, E Lederman, N Barnich, P Di Martino, P Desreumaux, L Gambiez, A Cortot, B Joly, JF Colombel. Laboratoires de Bact6riologie, Facult6s de Pharmacie, Clermont-Ferrand et Lille; Laboratoire de Recherche sur les MICI (CR14U004B); Service de Chirurgie Adulte. CHU Lille, France. Background: Experimental and clinical data incriminate bacterial flora in the initiation and perpetuation of the inflammatory process in CD. Among the flora, a particular role for E.coli has been suspected. Aim : To characterize adherence properties and other virulence factors of E.coli strains associated with the ileal mucosa of patients with CD. Patients/Methods : Ileal biopsies were analyzed from 50 patients with CD and 13 controls. In 20 patients, E.coli strains were recovered from surgically resected lesions (chronic CD ileal lesions). In a second group of 30 patients, E.coli strains were recovered from biopsies of neoterminal ileum of patients who had an ileocolectomy. 19/30 patients had an endoscopic recurrence (early CD ileal lesions) according to Rutgeert's criteria and 11/30 had no endoscopic recurrence (healthy ileum). Ileal biopsies were also studied in 13 controls. Adhesive properties for Caco-2 cells were studied and the presence of other associated virulence factors was determined using DNA hybridization and PCR experiments. Results: Number of strains adhering to Caco-2 cells. Ileal Chronic CD ileal Early CD ileal Healthy Controls Biopsies lesions (n=20) lesions (n=19) ileum (n=l 1) (n=13) N ° of strains 11/13 (85%)* 16/19 (84%)* 4/7 (57%) 3/9 (33%) * : p<0.02 vs controls. Seven strains (22%) from patients with CD harbored genes encoding a Pap adhesin. The adhesion was mediated by hitherto unknown adhesive factors in 22 (73%) of the adhering strains. 24% of them induced a cytolytic effect by synthesis of an a-hemolysin. None of the E.coli strains harbored any of the virulence factor-encoding genes described for E.coli involved in enteric diseases. Conclusion : Early and chronic ileal lesions of CD are often colonized by E.coli strains devoid of the virulence genes described for E.coli involved in enteric diseases. A potentially new E.coli pathovar with as yet