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Virulence factors of escherichia coli strains isolated from ileal mucosa in Crohn's disease (CD)
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
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0,5 11100
1/4(]0
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*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
April
1998
undescribed adhesins, and in which 24% of strains produced an a-hemolysin was defined. Adhesive ability and synthesis of a-cytotoxin would enable these bacteria to participate in the inflammatory process. • G3931 RECOMBINANT FACTOR XIII AND EXPERIMENTAL COLITIS IN RATS: IMMUNOHISTOCHEMICAL EVIDENCE FOR TISSUE ENZYME LOCATION. G. D'ar~,enio*, V. Cosenza*, A. Grnssmann§, K.H. Sprugel§, N. Della Valle*, G. Mazzacca*, C.E. Hart§ And P. D. Bishop§. *Gastrointestinal Unit, School of Medicine, Federico II University, Naples, Italy. ~Zymogenetics, Inc., Seattle, WA 98102 USA. Backeround&Aims: We recently showed that coagulation Factor XIII (FXIII), a circulating form of transglutaminase involved in tissue repair, is reduced in serum of patients with inflammatory bowel diseases (IBD) and there is a significant inverse correlation of FXIII levels with clinical severity. In the present study we investigated the effects of a recombinant FXIII (rFXIII) intravenous treatment in rat colitis on both acute and established lesions. Moreover we investigated tissue location of rFXIIL Methods: Colitis was induced by colonic instillation of 2,4,6-trinitrobenzenesulfonic acid in ethanol. The effects of 10 days rFXIII administration either immediately (developing lesions) or 7 days after (established lesions) colitis induction, were assessed vs both positive (Mesalamine), and negative (BSA) controls. At the end of treatment or 18 days after treatment was discontinued, the severity of lesions was determined by colon weight, macroscopic and histologic scores and transglutaminase activity was measured in both serum and colon tissues. Immunohistochemistry was performed to localize rFXIII in colonic mucosa using a specific anti-human FXIIIa antibody recognizing rFXIII but not the rat endogenous enzyme. Results: Lesion severity inversely correlated with transglutaminase levels. Consistent with these observations, rFXIII treatment restored serum and tissue transglutaminase levels and significantly reduced lesion severity in both developing (p<0.05 vs BSA) and established lesions (p<0.05 vs BSA and Mesalamine). Additionally, rFXIII treatment was efficacious for at least 18 days after treatment was discontinued. The immunohistochemistry assessment showed a localization of rFXIII to the extracellular matrix of damaged area where several proteins (i.e. fibronectin) suitable substrates for transglutaminases are present. Conclusions: These data show the efficacy of rFXIII replacement therapy in both acute and chronic phases of experimental colitis suggesting a key role of FXIII in colonic mucosal healing. We suggest that the therapeutic efficacy of rFXIII in human IBD should be investigated.
• G3932 SERUM TRANSGLUTAMINASE CORRELATES WITH ENDOSCOPICAL AND HISTOPATHOLOGICAL GRADING IN PATIENTS WITH ULCERATIVE COLITIS. Gruppo Italiano per lo Studio del Colon e del Retto (GISC)* Steering committee: G. D'Argenio, V. Cosenza, N. Della Valle, F. De Riffs and G. Mazzacca. Gastrointestinal Unit, School of Medicine, Federico II University, Naples, Italy. *GISC partecipants: G. Riegler, F. Morace (Napoli); R. D'Inc~, A. D'Odorico (Padova); P. Paoluzi, M.C. Di Paolo (Roma); V. Annese, G. Lombardi (S. Giovanni Rotondo); C. Mansi (Genova); R. Caprilli, O. Taddei (L'Aquila). Background: The clinical activity of inflammatory bowel diseases (IBD) is based on clinical and laboratory evaluations which do not always reflect the pathogenic processes taking place in the intestine. There is evidence that Factor XIIIa, a circulating form of transglutaminase (TG), plays a key role in intestinal mucosal repair. We previously found that TG levels are decreased in serum of patients with IBD and demonstrated in a rat model of chronic colitis that serum TG is closely related to the severity of intestinal damage. Aims: The aim of this study was to examine whether serum TG levels are related to the clinical activity index in patients with ulcerative colitis (UC). We also compared serum TG with endoscopic and histologic indices. Patients/Methods: In 234 patients with UC serum TG activity was assayed by a radioenzymatic method and clinical activity index (CAI) was measured acording to modified Rachmilewitz's criteria (BMJ 1989). In 73 patients, endoscopic and histologic indices were also evaluated according to Harig et al (N Eng J Med 1989) and Scheppac et al (Gastroenterology 1992), respectively. Results: Clinical, endoscopic and histologic scores correlated with each other; overall, histology showed a very good correlation with endoscopic indices (r = 0.81; p<0.001). Serum TG levels were significantly correlated with the CAI scoring (r = -0.58; p<0.01); moreover, serum TG showed the best correlation with endoscopic (r = -0.65; p<0.01) and histologic (r = -0.67; p<0.001) scores. Conclusions: These results suggest that TG assay may prove useful in management of UC as a new serological non-invasive indicator of intestinal mucosal status.
• G3933 COMPARISON OF MAGNETIC RESONANCE IMAGING WITH SUPER PARAMAGNETIC ORAL CONTRAST AND ENDOSCOPY IN THE EVALUATION OF EXTENT AND ACTIVITY OF ULCERATIVE COLITIS. D'Arienzo A, Belfiore G*, Scaglione G, Vicinanza G, Imbriaco M*, Manguso F, Bennato R, Romano M*, Sanges M, Mazzacca G. Unit of Gastroenterology, *Department of Biomorphological and Functional Sciences, University "Federico II", Naples, Italy. Magnetic Resonance Imaging (MRI) has not been extensively used in the evaluation of patients with Ulcerative Colitis (UC). This has been largely due
Inununology, Microbiology, and Inflanmlatory Disorders A959
to artifacts caused by respiration and bowel peristalsis, prolonged imaging times and poor contrast resolution. The introduction of new oral contrast agents which enhance image quality prompted us to evaluate the usefulness of MRI in the management of the disease. Fourteen consecutive patients affected by UC (M/F: 9/5; mean age -+SD: 37 -+ 14) entered the study. All of them had, in the past, a histological diagnosis of pancolitis and were in clinic activity at the admission. Both pancolonoscopy and MRI were performed in all patients within a 7-day period. In each patient clinical activity index (CAI) according to Rachmilewitz, extent and endoscopic activity of the disease were assessed. A clinic-endoscopic activity index was then calculated. MRI was performed with a 0.5 T super conductive magnet (GE system), using a body coil. Axial and coronal T1 and T2-weighted images were acquired two hours after the oral administration of 900 ml of a negative super paramagnetic contrast agent (AMI-121-Lumirem) and following anticholinergic medication. Length of affected bowel segments was evaluated. To determine the degree (mild, moderate or severe) of bowel inflammation, wall thickness (mm) and intensity of perivisceral fat signal on magnetic resonance images were measured. An MRI activity index was calculated by combining these two parameters. To compare the endoscopic and MRI extent of the disease, Fisher's exact test (Table) was applied (p=0.01). In 3 patients MRI documented more extensive bowel involvement. A significative correlation was observed between MRI and clinic-endoscopic activity (Figure). In 7 cases a complete absence of the haustra, possibly related to the duration of the disease, was evidenced. ,,~
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In conclusion, our preliminary results indicate that super paramagnetic contrast agent MRI is at least as useful as endoscopy in determining the real extent of UC. In addition, transmural assessment and multiplane images allow a better evaluation of the severity of the disease. Finally, the lack of invasiveness represents an attractive feature of MRI. • G3934 THE MOLECULAR SPECIFICITY OF TROPOMYOSIN-REACTIVE AUTOANTIBODIES IN ULCERATIVE COLITIS. A. Dasgupta. X. Geng, K. Ramaswamy, K. Kesari, K.M. Das. UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. Anti-tropomyosin autoantibodies are found in ulcerative colitis (UC) (Gastroenterology 1995; 109:3) and in experimental colitis in TCR knockout mice (J Exp Med 1996; 183:847). In the colonic epithelium, human tropomyosin isoform 5 (hTM5) is the major tropomyosin isoform and is expressed on the cell surface in association with a colon epithelium-specific protein (Gastroenterology 1997;112:A955). Earlier, using ELISA, we had determined the prevalence of antihuman tropomyosin isoform-5 (hTM5) antibodies in a large number of UC sera. However, neither UC sera nor immunoglobulins (IgG) purified from UC sera reacted with recombinant hTM5 (rhTM5) in the Western blot analysis. Because of these findings, the true identity of the antigen recognized by the autoantibodies could not be resolved. To examine the molecular specificity of these autoantibodies, we purified serum IgG by immunoaffinity chromatography on a rhTM5Sepharose column. For this purpose, hTM5 was purified to homogeneity from a bacterially expressed hTM5 eDNA clone. The purity and immunoreactivity of hTM5 was examined by SDS-PAGE, Western blot analysis, and ELISA using two anti-hTM5 monoclonal antibodies, CG3 and LC1. To prepare the immunoaffinity column, the purified hTM5 was immobilized on CNBractivated Sepharose 4B. Purified serum IgG from UC (n=2), Crohn's disease (CD) (n=2), and normal subject (n=2) was individually passed through the hTM5-Sepharose column. Bound IgG was eluted with glycine-HCl buffer (pH2.7). The reactivity of bound IgG with rhTM5 was determined by ELISA and Western blot analyses. In addition, a cytofluorimetric analysis was performed to examine the ability of these antibodies to bind to a colon cancer cell line, T84, which expresses hTM5 on the cell surface. The immunoaffinity-purified antibodies from UC reacted strongly with rhTM5 in both ELISA and Western blot analyses and showed a clear staining of the T84 cell surface in the cytofluorimetry. Although a small amount of IgG, from CD and normal subjects, could bind to the rhTM5-Sepharose column, these IgG did not react with rhTM5 or T84 ceils, suggesting the nonspecific binding of some IgG molecules to the column. Taken together, these data demonstrate the molecular specificity of antitropomyosin autoantibodies in UC and their ability to bind to the colon epithelial cells.
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
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....
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0,5 11100
1/4(]0
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*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
April
1998
undescribed adhesins, and in which 24% of strains produced an a-hemolysin was defined. Adhesive ability and synthesis of a-cytotoxin would enable these bacteria to participate in the inflammatory process. • G3931 RECOMBINANT FACTOR XIII AND EXPERIMENTAL COLITIS IN RATS: IMMUNOHISTOCHEMICAL EVIDENCE FOR TISSUE ENZYME LOCATION. G. D'ar~,enio*, V. Cosenza*, A. Grnssmann§, K.H. Sprugel§, N. Della Valle*, G. Mazzacca*, C.E. Hart§ And P. D. Bishop§. *Gastrointestinal Unit, School of Medicine, Federico II University, Naples, Italy. ~Zymogenetics, Inc., Seattle, WA 98102 USA. Backeround&Aims: We recently showed that coagulation Factor XIII (FXIII), a circulating form of transglutaminase involved in tissue repair, is reduced in serum of patients with inflammatory bowel diseases (IBD) and there is a significant inverse correlation of FXIII levels with clinical severity. In the present study we investigated the effects of a recombinant FXIII (rFXIII) intravenous treatment in rat colitis on both acute and established lesions. Moreover we investigated tissue location of rFXIIL Methods: Colitis was induced by colonic instillation of 2,4,6-trinitrobenzenesulfonic acid in ethanol. The effects of 10 days rFXIII administration either immediately (developing lesions) or 7 days after (established lesions) colitis induction, were assessed vs both positive (Mesalamine), and negative (BSA) controls. At the end of treatment or 18 days after treatment was discontinued, the severity of lesions was determined by colon weight, macroscopic and histologic scores and transglutaminase activity was measured in both serum and colon tissues. Immunohistochemistry was performed to localize rFXIII in colonic mucosa using a specific anti-human FXIIIa antibody recognizing rFXIII but not the rat endogenous enzyme. Results: Lesion severity inversely correlated with transglutaminase levels. Consistent with these observations, rFXIII treatment restored serum and tissue transglutaminase levels and significantly reduced lesion severity in both developing (p<0.05 vs BSA) and established lesions (p<0.05 vs BSA and Mesalamine). Additionally, rFXIII treatment was efficacious for at least 18 days after treatment was discontinued. The immunohistochemistry assessment showed a localization of rFXIII to the extracellular matrix of damaged area where several proteins (i.e. fibronectin) suitable substrates for transglutaminases are present. Conclusions: These data show the efficacy of rFXIII replacement therapy in both acute and chronic phases of experimental colitis suggesting a key role of FXIII in colonic mucosal healing. We suggest that the therapeutic efficacy of rFXIII in human IBD should be investigated.
• G3932 SERUM TRANSGLUTAMINASE CORRELATES WITH ENDOSCOPICAL AND HISTOPATHOLOGICAL GRADING IN PATIENTS WITH ULCERATIVE COLITIS. Gruppo Italiano per lo Studio del Colon e del Retto (GISC)* Steering committee: G. D'Argenio, V. Cosenza, N. Della Valle, F. De Riffs and G. Mazzacca. Gastrointestinal Unit, School of Medicine, Federico II University, Naples, Italy. *GISC partecipants: G. Riegler, F. Morace (Napoli); R. D'Inc~, A. D'Odorico (Padova); P. Paoluzi, M.C. Di Paolo (Roma); V. Annese, G. Lombardi (S. Giovanni Rotondo); C. Mansi (Genova); R. Caprilli, O. Taddei (L'Aquila). Background: The clinical activity of inflammatory bowel diseases (IBD) is based on clinical and laboratory evaluations which do not always reflect the pathogenic processes taking place in the intestine. There is evidence that Factor XIIIa, a circulating form of transglutaminase (TG), plays a key role in intestinal mucosal repair. We previously found that TG levels are decreased in serum of patients with IBD and demonstrated in a rat model of chronic colitis that serum TG is closely related to the severity of intestinal damage. Aims: The aim of this study was to examine whether serum TG levels are related to the clinical activity index in patients with ulcerative colitis (UC). We also compared serum TG with endoscopic and histologic indices. Patients/Methods: In 234 patients with UC serum TG activity was assayed by a radioenzymatic method and clinical activity index (CAI) was measured acording to modified Rachmilewitz's criteria (BMJ 1989). In 73 patients, endoscopic and histologic indices were also evaluated according to Harig et al (N Eng J Med 1989) and Scheppac et al (Gastroenterology 1992), respectively. Results: Clinical, endoscopic and histologic scores correlated with each other; overall, histology showed a very good correlation with endoscopic indices (r = 0.81; p<0.001). Serum TG levels were significantly correlated with the CAI scoring (r = -0.58; p<0.01); moreover, serum TG showed the best correlation with endoscopic (r = -0.65; p<0.01) and histologic (r = -0.67; p<0.001) scores. Conclusions: These results suggest that TG assay may prove useful in management of UC as a new serological non-invasive indicator of intestinal mucosal status.
• G3933 COMPARISON OF MAGNETIC RESONANCE IMAGING WITH SUPER PARAMAGNETIC ORAL CONTRAST AND ENDOSCOPY IN THE EVALUATION OF EXTENT AND ACTIVITY OF ULCERATIVE COLITIS. D'Arienzo A, Belfiore G*, Scaglione G, Vicinanza G, Imbriaco M*, Manguso F, Bennato R, Romano M*, Sanges M, Mazzacca G. Unit of Gastroenterology, *Department of Biomorphological and Functional Sciences, University "Federico II", Naples, Italy. Magnetic Resonance Imaging (MRI) has not been extensively used in the evaluation of patients with Ulcerative Colitis (UC). This has been largely due
Inununology, Microbiology, and Inflanmlatory Disorders A959
to artifacts caused by respiration and bowel peristalsis, prolonged imaging times and poor contrast resolution. The introduction of new oral contrast agents which enhance image quality prompted us to evaluate the usefulness of MRI in the management of the disease. Fourteen consecutive patients affected by UC (M/F: 9/5; mean age -+SD: 37 -+ 14) entered the study. All of them had, in the past, a histological diagnosis of pancolitis and were in clinic activity at the admission. Both pancolonoscopy and MRI were performed in all patients within a 7-day period. In each patient clinical activity index (CAI) according to Rachmilewitz, extent and endoscopic activity of the disease were assessed. A clinic-endoscopic activity index was then calculated. MRI was performed with a 0.5 T super conductive magnet (GE system), using a body coil. Axial and coronal T1 and T2-weighted images were acquired two hours after the oral administration of 900 ml of a negative super paramagnetic contrast agent (AMI-121-Lumirem) and following anticholinergic medication. Length of affected bowel segments was evaluated. To determine the degree (mild, moderate or severe) of bowel inflammation, wall thickness (mm) and intensity of perivisceral fat signal on magnetic resonance images were measured. An MRI activity index was calculated by combining these two parameters. To compare the endoscopic and MRI extent of the disease, Fisher's exact test (Table) was applied (p=0.01). In 3 patients MRI documented more extensive bowel involvement. A significative correlation was observed between MRI and clinic-endoscopic activity (Figure). In 7 cases a complete absence of the haustra, possibly related to the duration of the disease, was evidenced. ,,~
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In conclusion, our preliminary results indicate that super paramagnetic contrast agent MRI is at least as useful as endoscopy in determining the real extent of UC. In addition, transmural assessment and multiplane images allow a better evaluation of the severity of the disease. Finally, the lack of invasiveness represents an attractive feature of MRI. • G3934 THE MOLECULAR SPECIFICITY OF TROPOMYOSIN-REACTIVE AUTOANTIBODIES IN ULCERATIVE COLITIS. A. Dasgupta. X. Geng, K. Ramaswamy, K. Kesari, K.M. Das. UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. Anti-tropomyosin autoantibodies are found in ulcerative colitis (UC) (Gastroenterology 1995; 109:3) and in experimental colitis in TCR knockout mice (J Exp Med 1996; 183:847). In the colonic epithelium, human tropomyosin isoform 5 (hTM5) is the major tropomyosin isoform and is expressed on the cell surface in association with a colon epithelium-specific protein (Gastroenterology 1997;112:A955). Earlier, using ELISA, we had determined the prevalence of antihuman tropomyosin isoform-5 (hTM5) antibodies in a large number of UC sera. However, neither UC sera nor immunoglobulins (IgG) purified from UC sera reacted with recombinant hTM5 (rhTM5) in the Western blot analysis. Because of these findings, the true identity of the antigen recognized by the autoantibodies could not be resolved. To examine the molecular specificity of these autoantibodies, we purified serum IgG by immunoaffinity chromatography on a rhTM5Sepharose column. For this purpose, hTM5 was purified to homogeneity from a bacterially expressed hTM5 eDNA clone. The purity and immunoreactivity of hTM5 was examined by SDS-PAGE, Western blot analysis, and ELISA using two anti-hTM5 monoclonal antibodies, CG3 and LC1. To prepare the immunoaffinity column, the purified hTM5 was immobilized on CNBractivated Sepharose 4B. Purified serum IgG from UC (n=2), Crohn's disease (CD) (n=2), and normal subject (n=2) was individually passed through the hTM5-Sepharose column. Bound IgG was eluted with glycine-HCl buffer (pH2.7). The reactivity of bound IgG with rhTM5 was determined by ELISA and Western blot analyses. In addition, a cytofluorimetric analysis was performed to examine the ability of these antibodies to bind to a colon cancer cell line, T84, which expresses hTM5 on the cell surface. The immunoaffinity-purified antibodies from UC reacted strongly with rhTM5 in both ELISA and Western blot analyses and showed a clear staining of the T84 cell surface in the cytofluorimetry. Although a small amount of IgG, from CD and normal subjects, could bind to the rhTM5-Sepharose column, these IgG did not react with rhTM5 or T84 ceils, suggesting the nonspecific binding of some IgG molecules to the column. Taken together, these data demonstrate the molecular specificity of antitropomyosin autoantibodies in UC and their ability to bind to the colon epithelial cells.