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CpG motifs in plasmid DNA exacerbate inflammation in experimental celltis
monoassociation alone is sufficient to lead to an intestinal inflammation in susceptible IL-IO deficient mice, despite the fact that loss of tolerance occurs. Further experiments employing other bacterialstrains as well as combinations of strains will haveto be performedto establish whether individual bacteria can activate an inflammatory response in the intestine of these mice, resulting in harmful damage of intestinal tissue. 2633 Decrease of Interleukin-7-inducad Apoptosis of IL-7 Receptor* Intestinal Mucosal Lymphocytes in Chronic Inflamaed Colonic Mucosa of Ulcerative Colitis Motomi Yamazaki,GraduateSch, Tokyo Medical and Dental Univ, Tokyo Japan; Tomoharu Yajima, Keio cancer Ctr, Tokyo Japan; Ryuichi Okamoto, Shigero Ohshima, Takanori Kanai, Graduate Sch, Tokyo Medical and Dental Univ, Tokyo Japan; KazutakaKoganei,Tsuneo Fukushima, Yokohama city Hosp, KanagawaJapan; Toshifumi Hibi, Keio cancer CtT, Tokyo Japan; Mamoru Watanabe,GraduateSch, Tokyo Medical and Dental Univ, Tokyo Japan BACKGROUND:We have demonstrated that intestinal epithelial cells, especially goblet cells produce interleukin-7 (IL-7) and IL-7 serves as a regulatoryfactor for proliferation of mncosal lymphocytes. Recent studies demonstrated that intestinal epithelial cell~erived IL-7 plays a crucial role in the organizationof mucosat lymphoid tissues and regulatingthe normal immune response in the intestinal mucosa. Here we investigated the dysregulation of mucossl 11--7/ IL-7 receptor (IL-7R)-mediatedimmune responsesat chronic inflammatory sites in the colonic mucosa of ulcerative colitis (UC). METHODS:Expression of IL-7, IL-7R and Fas in colonic mucosa was determined by using RT-PCR, immunohistochemistry, and flow cytometry. The functional activity of IL-7R was assessed by the ability of recombinant IL-7 to stimulate the growth of intestinal mucosal lymphocytes. Apoptotic cells were defected with Annexin V by flow cytometry, and terminal deoxynucleotidyl transferase-mediated dUTP-blotin nick end labelingassay. RESULTS:The expressionof IL-7 mRNAand I L-7 protein was notablydecreased in the inflamedcolonic mucosaof UCat chronic active stage.In contrast, IL-7 mRNAexpression was varied during remission stage. IL-7R- lymphocyteswere remarkably infiltrated in lamina propria of the chronic inflamed mucosa of UC. IL-7R*IL-2R + cells were increasedin isolated lamina propria lymphocytes (LPLs). The intensity of Fas expression was increased in LPLs from inflamed mucosa. IL-7 inhibits the proliferation of LPLs from the chronic inflamed mucosa of UC. IL-7 significantly reduced the expression of IL-7R and enhanced apoptosis induced by anti-Fas mAb in LPLs. IL-7 significantly increased the expression of IL-2R and the intensity of Fas expression on LPLs from inflamed mucosa. MoreOver,apoptotic LPLs were hardly detectedin the chronic inflamed UC mucose, but abundant in the colonic mucose of UC at remission stage. This increase of apoptotic LPLs was correlated with the recovery of IL-7 protein expression in the colonic mucoca. CONCLUSION: Downregulation of IL-7 expression may lead to the decreaseof Fas-mediatedapoptosis of activated IL-7R expressing mucosal lymphocytes in the chronic inflamed mucosa of UC. Therefore, locally-produced IL7 may maintain the normal mucosal immune responses.
d~sease (CD) and is the target of a disease-specific antibody response. In this study, we characterize the properties of the 12 organism in the mouse system. METHODS: 12 was localized in murine tissues by PCR analysis. The murine immune responseto a recombinant 12 antigen was analyzedby cytokine ELISA, and T cell proliferation assays.V/3 expressionon the 12 responding cells was analyzed by flow cytometry. RESULTS:The 12 gene is present in the small and large intestine of normal C57BL/6J mice reared in specific pathogen free (SPF) mice, but absent in defined flora conditions. 12 protein induces a strong proliferative and cytokine response for both murine naive and memory CD4÷ T cell populations, derived from normal mice (C57BL/6J). The 12 response was dependent on MHC class II-mediated recognition, but did not require antigen processing basedon paraformaldehydefixation experiments. Among the responding T cells, selectively recruitment was predominant in the TCR V,8 5.1, 5.2 subpopulation. CONCLUSIONS:These results present several lines of evidence that 12 is a T cell superantigen. The lesionat association of 12 suggests that colonization with the putative 12-expressingmicroorganism may provide a local superantigenic stimulus pertinent to the pathogenesisof Crohn's disease.Supported by NIH DK46763and the Crohn's and Colitis Foundation of America.
AntI-TNF Treatment Restores the Gut Barrier in Crohn's Disease Peter SuenaerL Veerle Buiteel, Liesbath Lemmens, Maja Noman, Benny Geypens,Gert Van Assche, Paul Rutgeorts, Univ Hosp Gasthuisberg, Leuven Belgium Introduction: Tumor necrosis factor (TNF) plays a key role in the modulation of the gut barrier as evidenced in vitroL Since the favourable clinical, endoscopic, histological and immunoflistnebernical effects of the anti-TNF chimeric monoclonal antibodies (infliximab, Remicade~) in Crohn's disease (CD) patients, we aimed to assess the effect of anti-TNF on intestinal permeabilityin vivo. Methods: 23 patients (14 women) with CD underwent intestinal permeability testing 2 days before and 4 weeks after the first infusion of infliximab (5rag/ kg). 31 healthy non-smoker volunteers (18 women) underwent the same permeability test using 50 p.Ci of 5~Cr-EDTAin 160 mL of Nutridrink® (Nutricia, Belgium) after an overnight fast. Urine aliquots were counted for radioactivity by a ,8-liquid scintillation counter. Clinical scores were recorded by means of Crohn's diseaseactivity index (CDAI) and CRP respectively 7 days and 1 day before and 4 weeks after the first infusion. Results: The mean small bowel and whole gut permeability before infliximab was significantly higher in CD patients than in control suti~cts (* p
2634 CpG MotHs in Plasmid DNA Exacerbate Inflammation In F.xpedmeMal CntlUs Kyoko Katakura, Yukio Sato, Naoto Sato, Hitoshi Oyama,Takuto Hikichi, Ryoji Yoshloka, Hiroshi Orikasa, Atsushi Irisewa, Mitsuru Saka, Katsutoshi Obera, Reiji Kasukawa, Fukushima Medical Univ Sch of Medicine, Fukushima Japan Inflammatory bowel diseases (IBDs) are associated with an increased production of a range of proinflammatory cytokines including tumor necrosis factor-a (TNF-a)that contribute to the pathogenesis of intestinal inflammation. A novel therapeutic approach is to down-regulate the action of these cytokines by transfer of plasmid DNA (pDNA) encoding intedeukin-lO (IL10) that suppresses their inflammatory action into the affected colon since IL-IO might be expressed for long period. However, preliminary experiments showed that intracolonic administration of pDNAencoding IL-lO (pACB-IL-IO) exacerbatesexperimentalcolitis induced by the hapten reagent2, 4, 6-trinitrobenzenesuffonic acid (TNBS). Since pDNA has immunostimulatory DNA sequences (ISSs) with unmethylated CpG motifs that were demonstratedto stimulate the production of proinflammatory cytokines including TNF-a by macrophages, it is possible that pDNA exacerbatesTNBS colitis through its contents of ISSs. To test this hypothesis, pACB or pACB treated with CpG methylase mixed with cationic liposomes, or liposome alone was inoculated into the colon of Wister rats using a double-balloon catheter, and 24 hours later, TNBS was administered per rectum. Seven days later, rats were killed for assessmentof macroscopicand microscopic findings and TNF-a production of the affected colon. Macroscopic and microscopic damage scores for the pACB group were significantly higher than those in the control (liposome alone) group (p < 0.01 by ANOVA),and methylation of pACB reduced both damage scores similar to those in the control group. The TNF-a concentrations in inflamed colonic tissues were higher in the pACBgroup than in the control group (p < 0.05), and methylationof pACBreducedthe TNF-aconcentrationsto 30% of those in the pACBgroup. Sincemethylationof CpG motifs in ISSsabolishestheir immunostimulatory activity, these results show that CpG motifs in pDNA may exacerbateinflammation in experimental colitis. Therefore the pDNA for the patients with IBD should be designed to lack these ISSs.
2635 The Crohn's Disease-Associated Bacterial Protein, 12, Is a Novel Enteric T Cell Superantigen Harnisha N. Dalwadi, Bo Wei, UCLA, Los Angeles, CA; Mitchell Kronenberg,La Julia Institute of Allergy and Immunology, La Julia, CA; Christopher L. Sutton, Santaros, Inc, San Digeo, CA; Jonathan Braun, UCLA, Los Angeles, CA BACKGROUND:The aberrant T cell response to enteric bacteria plays an important role in the pathogenesisof inflammatory bowel disease (IBD). However,the identity of the relevant microbial antigens and the etiology of the colitigenic T cell response are unresolved. We recently isolated a microbial gene (12), a Pseudomonas homologue of the tetR bacterial transcription factor family, which is specifically localizedto mucosat lesions in human Crohn's
A-518
%¢Iowrlc~m~
O-6h median(IQR)
0-24h rnedian(IQR)
C e ~ values Before am~-TNF /diet antJ-TNF
1.12 (0.85- 1.58) 1.63 (1.06- 207) • 1.04 (0 74- 1.54) .1:
2.28 (1.88- 2.86) 3.27 (2.40- 4.38) * 2.42 (2.03 - 2.80) ~t
~7 Survival and Multiplication of Adherent-lnvasive Escherichia coli Strains Isolated from Patients with Crohn's Disease (CD) in the J774 Murine Macrophage-like Cell Line Anne-Use Glasser, Jerome Boudeau,Nicolas Bamich, Universife d'Auvergne, Clermont Ferran France; Jean-Frederic Colombel, Gastroenterologie,CHRU, Lille France; Arleffe Darfeuille-Michaud, Universite d'Auvergne, Clermont Ferran France BACKGROUND:The characteristic pathologic elements of Crohn's disease (CD) suggest the involvement of invasive bacteria. Previous studies revealed a high prevalenceof invasive E. coil in patients with CD, belonging to a new pathogenic E. coli group designated AtEC for adherent-invasive E. coll. AIM: To analysethe behavior of these invasive strains within the J774 macrophage-likecell line. METHODS:Bacterialuptake,survival and multiplication abilities of 15 invasive E. coil strains from CD patients and 6 from controls were tested in vitro using the gentamicin protection assay with infected J774 macrophage-tike cells. Assays were performed from 1 h to 48 h postinfectlon and visualized using transmission electronic microscopy. Cell death was assessed by lactate dehydrogenase (LDH) release and DNA fragmentation. Releaseof prointiammatory cytokines was quantified by ELISA. RESULTS:The AIEC prototype strain LF82 highly replicated within non-stimulated or LPS-prestimulated macrophages until 48 hours postinfection, in contrast to the K-12 C600 strain, which was rapidly degraded after phagocytosis. Transmission electron micrographs of LF82-infected macrophages showed that at 24 and 48 hours postinfection, infected macrophagesharbored large vacuoles containing numerous bacteria, as a result of the fusion of several vacuoles observed from 8 hours postinfection. Infected macrophagesshowed no sign of cell death as determined by LDH release and DNA analysis; they did not release 11-1,8 but secreted high amounts of TNFez. One hundred percent of the AIEC strains tested were able to replicate extensivelywithin the J774 macrophagesafter phagocytosis,versus 0% of the invasivestrains isolated from controls. CONCLUSION:The AIEC strains have not only the ability to adhere to and to invadeintestinal epithelial cells, but are also able to highly replicatewithin macrophages without inducing cell death. This last property could be related to granuloma formation, one of the hallmarks of CD lesions.
d~sease (CD) and is the target of a disease-specific antibody response. In this study, we characterize the properties of the 12 organism in the mouse system. METHODS: 12 was localized in murine tissues by PCR analysis. The murine immune responseto a recombinant 12 antigen was analyzedby cytokine ELISA, and T cell proliferation assays.V/3 expressionon the 12 responding cells was analyzed by flow cytometry. RESULTS:The 12 gene is present in the small and large intestine of normal C57BL/6J mice reared in specific pathogen free (SPF) mice, but absent in defined flora conditions. 12 protein induces a strong proliferative and cytokine response for both murine naive and memory CD4÷ T cell populations, derived from normal mice (C57BL/6J). The 12 response was dependent on MHC class II-mediated recognition, but did not require antigen processing basedon paraformaldehydefixation experiments. Among the responding T cells, selectively recruitment was predominant in the TCR V,8 5.1, 5.2 subpopulation. CONCLUSIONS:These results present several lines of evidence that 12 is a T cell superantigen. The lesionat association of 12 suggests that colonization with the putative 12-expressingmicroorganism may provide a local superantigenic stimulus pertinent to the pathogenesisof Crohn's disease.Supported by NIH DK46763and the Crohn's and Colitis Foundation of America.
AntI-TNF Treatment Restores the Gut Barrier in Crohn's Disease Peter SuenaerL Veerle Buiteel, Liesbath Lemmens, Maja Noman, Benny Geypens,Gert Van Assche, Paul Rutgeorts, Univ Hosp Gasthuisberg, Leuven Belgium Introduction: Tumor necrosis factor (TNF) plays a key role in the modulation of the gut barrier as evidenced in vitroL Since the favourable clinical, endoscopic, histological and immunoflistnebernical effects of the anti-TNF chimeric monoclonal antibodies (infliximab, Remicade~) in Crohn's disease (CD) patients, we aimed to assess the effect of anti-TNF on intestinal permeabilityin vivo. Methods: 23 patients (14 women) with CD underwent intestinal permeability testing 2 days before and 4 weeks after the first infusion of infliximab (5rag/ kg). 31 healthy non-smoker volunteers (18 women) underwent the same permeability test using 50 p.Ci of 5~Cr-EDTAin 160 mL of Nutridrink® (Nutricia, Belgium) after an overnight fast. Urine aliquots were counted for radioactivity by a ,8-liquid scintillation counter. Clinical scores were recorded by means of Crohn's diseaseactivity index (CDAI) and CRP respectively 7 days and 1 day before and 4 weeks after the first infusion. Results: The mean small bowel and whole gut permeability before infliximab was significantly higher in CD patients than in control suti~cts (* p
2634 CpG MotHs in Plasmid DNA Exacerbate Inflammation In F.xpedmeMal CntlUs Kyoko Katakura, Yukio Sato, Naoto Sato, Hitoshi Oyama,Takuto Hikichi, Ryoji Yoshloka, Hiroshi Orikasa, Atsushi Irisewa, Mitsuru Saka, Katsutoshi Obera, Reiji Kasukawa, Fukushima Medical Univ Sch of Medicine, Fukushima Japan Inflammatory bowel diseases (IBDs) are associated with an increased production of a range of proinflammatory cytokines including tumor necrosis factor-a (TNF-a)that contribute to the pathogenesis of intestinal inflammation. A novel therapeutic approach is to down-regulate the action of these cytokines by transfer of plasmid DNA (pDNA) encoding intedeukin-lO (IL10) that suppresses their inflammatory action into the affected colon since IL-IO might be expressed for long period. However, preliminary experiments showed that intracolonic administration of pDNAencoding IL-lO (pACB-IL-IO) exacerbatesexperimentalcolitis induced by the hapten reagent2, 4, 6-trinitrobenzenesuffonic acid (TNBS). Since pDNA has immunostimulatory DNA sequences (ISSs) with unmethylated CpG motifs that were demonstratedto stimulate the production of proinflammatory cytokines including TNF-a by macrophages, it is possible that pDNA exacerbatesTNBS colitis through its contents of ISSs. To test this hypothesis, pACB or pACB treated with CpG methylase mixed with cationic liposomes, or liposome alone was inoculated into the colon of Wister rats using a double-balloon catheter, and 24 hours later, TNBS was administered per rectum. Seven days later, rats were killed for assessmentof macroscopicand microscopic findings and TNF-a production of the affected colon. Macroscopic and microscopic damage scores for the pACB group were significantly higher than those in the control (liposome alone) group (p < 0.01 by ANOVA),and methylation of pACB reduced both damage scores similar to those in the control group. The TNF-a concentrations in inflamed colonic tissues were higher in the pACBgroup than in the control group (p < 0.05), and methylationof pACBreducedthe TNF-aconcentrationsto 30% of those in the pACBgroup. Sincemethylationof CpG motifs in ISSsabolishestheir immunostimulatory activity, these results show that CpG motifs in pDNA may exacerbateinflammation in experimental colitis. Therefore the pDNA for the patients with IBD should be designed to lack these ISSs.
2635 The Crohn's Disease-Associated Bacterial Protein, 12, Is a Novel Enteric T Cell Superantigen Harnisha N. Dalwadi, Bo Wei, UCLA, Los Angeles, CA; Mitchell Kronenberg,La Julia Institute of Allergy and Immunology, La Julia, CA; Christopher L. Sutton, Santaros, Inc, San Digeo, CA; Jonathan Braun, UCLA, Los Angeles, CA BACKGROUND:The aberrant T cell response to enteric bacteria plays an important role in the pathogenesisof inflammatory bowel disease (IBD). However,the identity of the relevant microbial antigens and the etiology of the colitigenic T cell response are unresolved. We recently isolated a microbial gene (12), a Pseudomonas homologue of the tetR bacterial transcription factor family, which is specifically localizedto mucosat lesions in human Crohn's
A-518
%¢Iowrlc~m~
O-6h median(IQR)
0-24h rnedian(IQR)
C e ~ values Before am~-TNF /diet antJ-TNF
1.12 (0.85- 1.58) 1.63 (1.06- 207) • 1.04 (0 74- 1.54) .1:
2.28 (1.88- 2.86) 3.27 (2.40- 4.38) * 2.42 (2.03 - 2.80) ~t
~7 Survival and Multiplication of Adherent-lnvasive Escherichia coli Strains Isolated from Patients with Crohn's Disease (CD) in the J774 Murine Macrophage-like Cell Line Anne-Use Glasser, Jerome Boudeau,Nicolas Bamich, Universife d'Auvergne, Clermont Ferran France; Jean-Frederic Colombel, Gastroenterologie,CHRU, Lille France; Arleffe Darfeuille-Michaud, Universite d'Auvergne, Clermont Ferran France BACKGROUND:The characteristic pathologic elements of Crohn's disease (CD) suggest the involvement of invasive bacteria. Previous studies revealed a high prevalenceof invasive E. coil in patients with CD, belonging to a new pathogenic E. coli group designated AtEC for adherent-invasive E. coll. AIM: To analysethe behavior of these invasive strains within the J774 macrophage-likecell line. METHODS:Bacterialuptake,survival and multiplication abilities of 15 invasive E. coil strains from CD patients and 6 from controls were tested in vitro using the gentamicin protection assay with infected J774 macrophage-tike cells. Assays were performed from 1 h to 48 h postinfectlon and visualized using transmission electronic microscopy. Cell death was assessed by lactate dehydrogenase (LDH) release and DNA fragmentation. Releaseof prointiammatory cytokines was quantified by ELISA. RESULTS:The AIEC prototype strain LF82 highly replicated within non-stimulated or LPS-prestimulated macrophages until 48 hours postinfection, in contrast to the K-12 C600 strain, which was rapidly degraded after phagocytosis. Transmission electron micrographs of LF82-infected macrophages showed that at 24 and 48 hours postinfection, infected macrophagesharbored large vacuoles containing numerous bacteria, as a result of the fusion of several vacuoles observed from 8 hours postinfection. Infected macrophagesshowed no sign of cell death as determined by LDH release and DNA analysis; they did not release 11-1,8 but secreted high amounts of TNFez. One hundred percent of the AIEC strains tested were able to replicate extensivelywithin the J774 macrophagesafter phagocytosis,versus 0% of the invasivestrains isolated from controls. CONCLUSION:The AIEC strains have not only the ability to adhere to and to invadeintestinal epithelial cells, but are also able to highly replicatewithin macrophages without inducing cell death. This last property could be related to granuloma formation, one of the hallmarks of CD lesions.