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Ige receptor affects fecal flora, bacterial translocation and intestinal inflammation
GASTROENTEROLOGY Vol. 118, No.4
A694 AGA ABSTRACTS
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3810
THE HIDDEN BURDEN OF FECAL INCONTINENCE AND ITS ASSOCIATED RISK FACTORS: A POPULATION-BASED STUDY. Jamshid S. Kalantar, Stuart Howell, Nicholas J. Talley, Univ of Sydney, Penrith, Australia. Fecal incontinence (Fl) represents a significant social and public health problem. Patients suffering from fecal incontinence may be reluctant to seek medical advice and its true prevalence remains uncertain. Studies of potential risk factors have been conflicting. The aim of this study was to determine the prevalence of fecal incontinence in the community and identify possible risk factors. Methods: A standard questionnaire was mailed to a gender-stratified sample of 990 subjects, who were randomly selected from the electoral rolls of Lindsay (an electoral district in western Sydney, Australia). A response rate of 68% was achieved. Results: The prevalence of solid and liquid fecal incontinence (defined by ever having any stool leakage over the past 12 months) was 2% and 9%, respectively. The mean age of subjects with FI was 53 years (median: 50 years) and 55% of subjects with Fl were female; 18% reported Fl at least weekly, and 18% reported that they required a change of underwear at least once per week because of FI. After adjustment for age, Fl was significantly associated with laxative use (p<0.OO3), injury or tear to perineal area (p<0.03), anal surgery (p<0.OOO2), feeling of incomplete defecation (p
EXPRESSION OF A NOVEL SUPEROXIDE-GENERATING, TRANSFORMATION- ASSOCIATED OXIDASE (MOXI) IN GASTRIC PIT CELLS. Kazuhito Rokutan, Shigetada Teshima, Tsukasa Kawahara, Tomoko Kawai, Takeshi Nikawa, Hiromu Kutsumi, Sch of Medicine, Univ of Tokushima, Tokushima, Japan; Kyoto First Red Cross Hosp, Kyoto, Japan.
3809 IGE RECEPTOR AFFECTS FECAL FLORA, BACTERIAL TRANSLOCATION AND INTESTINAL INFLAMMATION. Sophie Nutten, David Dombrowicz, Christelle Neut, Jean P. Papin, Gerard Torpier, Jean F. Colombel, Monique Capron, Pierre Desreumaux, Lab de Research sur les MICI, CHU de Lille, Lille, France; INSERM 167, Inst Pasteur de Lille, Lille, France; Lab de Bacteriology, Faculty de Pharmacie, Lille, France. Background: Colitis in TCR a -1- mice and early ileal lesions of Crohn's disease depend on the presence of luminal bacteria and are associated with a mucosal Th2 immune response characterized by IL-4 synthesis and production of IgE and its high-affinity receptor (FcERI)(I, 2). Aim: to evaluate the role of mucosal FCERIexpression in the control of bacterial translocation and of intestinal inflammation. Methods: The endogenous flora, rate of bacterial translocation and course of TNBS-induced colitis were analyzed in various mouse strains expressing FCER! with different cellular distributions: wild-type (WT) animals expressing FCERI on mast cells and basophils; Fcekl-deficient (FcERIa-I-) animals and hFcERIaTg animals additionally expressing a humanized FCERIon macrophages, eosinophils, Langerhans cells, as in humans. Results: No evidence of spontaneous colitis was observed in any of the 3 strains. As compared with WT and FCERIa-l- animals, hFcERIaTg mice were characterized by: I) a 5000 fold increase in Bacteroides count in fecal flora; 2) a 30 fold increase in bacterial translocation towards mesenteric lymph nodes mainly involving Lactobacillus; 3) an increased IL-4 colonic expression. The outcome of TNBS-induced colitis was dramatically different in the 3 strains: while hFcERIaTg exhibited a more pronounced inflammation than WT animals, FCERI-I- animals were completely protected from colitis. Conclusion: These results suggest that the expression of FCER! and of IL-4 in the mucosa affects the variety of endogenous flora and bacterial translocation. FCER! expression on eosinophil and macrophages rendered mice more susceptible to TNBS-induced colitis while its lack of expression prevented the colitis. (I): Mizoguchi et al. Gastroenterology 1999; 116: 320. (2) Desreumaux et al. Gastroenterology 1997; 113: 118. Supported in part by Association Francois Aupetit.
Background & Aims: Recently, a homolog of the phagocyte NADPH oxidase subunit (gp91-phox) called a superoxide-generating mitogenic oxidase (Mox l) was molecularly identified (Nature 1999; 401:79-82). This novel gene product is expressed in distinct types of nonphagocytic cells possessing superoxide-generating activity, and overexpression of Mox l was shown to promote disregulated growth of cancer cells in vitro and in vivo. We already demonstrated that primary cultures of guinea pig gastric pit cells secrete abundant superoxide anion (Oi) (Gastroenterology 1998; 115:1186-96). We report here that pit cells express Moxl and all of the other essential components for the phagocyte NADPH oxidase. Furthermore, we found that Helicobacter pylori (H. pylori) infection specifically up-regulated the Mox l expression in gastric pit cells both in vivo and in vitro. Methods: 0i release was measured as the SOD-inhibitable reduction of ferricytochrome c. A primer set was designed to specifically amplify the MoxI transcript, but not gp91-phox, and the RT-PCR product (600 bp) was used for detection of cells expressing the mox l mRNA in human biopsy specimens by in situ hybridization and for measurement of the transcript level in cultured pit cells by Northern blotting. The levels of mox l and NADPH oxidase components (p67-, p47-, p40, and p22-phoxes) and cells containing these components were analyzed by Western blotting and immunohistochemistry, respectively. Results & Conclusions: Pit cells cultured in LPS-free conditions constitutively expressed mox l, but not gp91phox, and all of the other essential components for phagocyte NADPH oxidase, and they spontaneously secreted 0iat 10 nmoUmg proteinlh. Confocal laser scanning microscopy showed that significant amounts of p67- and p47-phox were constitutively localized along the plasma membrane, which may explain the spontaneous Oirelease by the cells. H. pylori LPS induced Mox l, p67- and p22-phoxes and markedly increased 02production by the cells more than lO-fold. Among these components, the magnitude of p67-phox expression consistently correlated with the upregulation of 0i production. In situ hybridization and immunohistchemistry demonstrated that Mox l was expressed in pit cells of human gastric mucosa infected with H. pylori, but not in normal gastric mucosa. These observations suggest an important role of the unique NADPH oxidase in the pathogenesis of disregulated cell growth in association with H. pylori infection.
3811 FUNCTIONAL TRANSFER OF GENES FROM BACTERIA TO INTESTINAL EPITHELIAL CELLS. Richard James Aspinall, Georges Vassaux, Nicholas Lemoine, Imperial Coll Sch Of Medicine, London, United Kingdom. INTRODUCTION It has previously been demonstrated that bacteria can transfer genes to other unrelated bacteria, yeasts and plants. Recently, the first report of in vitro gene transfer from bacteria to mammalian cells was published I, showing subsequent expression of a reporter gene in COS-I, HeLa and CHO cell lines. Given the exposure of gastrointestinal epithelia to the endogenous bacterial flora, we wished to test whether such gene transfer was possible in marnrnalian intestinal cells. METHODS We used an auxotrophic, diaminopimelate (dapr-dependent strain of E.Coli , BM27 10, (a generous gift from Dr C Grillot-Courvalin of the Insitut Pasteur, Paris) engineered to express the Yersinia pseudotuberculosis invasin gene, the listeria monocytogenes listeriolysin 0 hly gene and also carrying the pEGFP-CI plasmid expressing GFP under eukaryotic promoter control. The mammalian cell lines studied were COS-I, CAC02, CMT93, HepG2, SKBR3, 293 and MKN45. 105 Cultured cells were incubated for 2 hours with BM2710 at a range of doses from 5x104 to 5xl09 bacteria in DMEM medium supplemented with dap. Cells were then washed three times with DMEM and incubated for 48 hours in complete medium containing gentamicin. After 48 hours, the cells were trypsinised, washed in PBS, resuspended in medium and anlaysed for GFP expression by FACS analysis. RESULTS Transfer and expression of the GFP plasmid was best demonstrated in the cell lines COS-I, 293, CAC02 and HepG2 in a dose-dependent manner, with up to 12% of cells showing positivity. A lesser effect was seen only at the highest doses in CMT93 cells. Repeated experiments on confluent marnrnalian cell preparations suggested transfer was more effective in actively dividing cell populations. CONCLUSIONS Auxotrophic E.Coli engineered to express the hly and invasin genes may be a suitable vehicle for delivering plasmid DNA to intestinal epithelia. We are currently exploring this method of gene delivery in murine models in vivo. 'Grillot-Courvalin C, Goussard S, Huetz F, et al. Nature Biotechnology 1998; 16: 862-866
A694 AGA ABSTRACTS
3795
3810
THE HIDDEN BURDEN OF FECAL INCONTINENCE AND ITS ASSOCIATED RISK FACTORS: A POPULATION-BASED STUDY. Jamshid S. Kalantar, Stuart Howell, Nicholas J. Talley, Univ of Sydney, Penrith, Australia. Fecal incontinence (Fl) represents a significant social and public health problem. Patients suffering from fecal incontinence may be reluctant to seek medical advice and its true prevalence remains uncertain. Studies of potential risk factors have been conflicting. The aim of this study was to determine the prevalence of fecal incontinence in the community and identify possible risk factors. Methods: A standard questionnaire was mailed to a gender-stratified sample of 990 subjects, who were randomly selected from the electoral rolls of Lindsay (an electoral district in western Sydney, Australia). A response rate of 68% was achieved. Results: The prevalence of solid and liquid fecal incontinence (defined by ever having any stool leakage over the past 12 months) was 2% and 9%, respectively. The mean age of subjects with FI was 53 years (median: 50 years) and 55% of subjects with Fl were female; 18% reported Fl at least weekly, and 18% reported that they required a change of underwear at least once per week because of FI. After adjustment for age, Fl was significantly associated with laxative use (p<0.OO3), injury or tear to perineal area (p<0.03), anal surgery (p<0.OOO2), feeling of incomplete defecation (p
EXPRESSION OF A NOVEL SUPEROXIDE-GENERATING, TRANSFORMATION- ASSOCIATED OXIDASE (MOXI) IN GASTRIC PIT CELLS. Kazuhito Rokutan, Shigetada Teshima, Tsukasa Kawahara, Tomoko Kawai, Takeshi Nikawa, Hiromu Kutsumi, Sch of Medicine, Univ of Tokushima, Tokushima, Japan; Kyoto First Red Cross Hosp, Kyoto, Japan.
3809 IGE RECEPTOR AFFECTS FECAL FLORA, BACTERIAL TRANSLOCATION AND INTESTINAL INFLAMMATION. Sophie Nutten, David Dombrowicz, Christelle Neut, Jean P. Papin, Gerard Torpier, Jean F. Colombel, Monique Capron, Pierre Desreumaux, Lab de Research sur les MICI, CHU de Lille, Lille, France; INSERM 167, Inst Pasteur de Lille, Lille, France; Lab de Bacteriology, Faculty de Pharmacie, Lille, France. Background: Colitis in TCR a -1- mice and early ileal lesions of Crohn's disease depend on the presence of luminal bacteria and are associated with a mucosal Th2 immune response characterized by IL-4 synthesis and production of IgE and its high-affinity receptor (FcERI)(I, 2). Aim: to evaluate the role of mucosal FCERIexpression in the control of bacterial translocation and of intestinal inflammation. Methods: The endogenous flora, rate of bacterial translocation and course of TNBS-induced colitis were analyzed in various mouse strains expressing FCER! with different cellular distributions: wild-type (WT) animals expressing FCERI on mast cells and basophils; Fcekl-deficient (FcERIa-I-) animals and hFcERIaTg animals additionally expressing a humanized FCERIon macrophages, eosinophils, Langerhans cells, as in humans. Results: No evidence of spontaneous colitis was observed in any of the 3 strains. As compared with WT and FCERIa-l- animals, hFcERIaTg mice were characterized by: I) a 5000 fold increase in Bacteroides count in fecal flora; 2) a 30 fold increase in bacterial translocation towards mesenteric lymph nodes mainly involving Lactobacillus; 3) an increased IL-4 colonic expression. The outcome of TNBS-induced colitis was dramatically different in the 3 strains: while hFcERIaTg exhibited a more pronounced inflammation than WT animals, FCERI-I- animals were completely protected from colitis. Conclusion: These results suggest that the expression of FCER! and of IL-4 in the mucosa affects the variety of endogenous flora and bacterial translocation. FCER! expression on eosinophil and macrophages rendered mice more susceptible to TNBS-induced colitis while its lack of expression prevented the colitis. (I): Mizoguchi et al. Gastroenterology 1999; 116: 320. (2) Desreumaux et al. Gastroenterology 1997; 113: 118. Supported in part by Association Francois Aupetit.
Background & Aims: Recently, a homolog of the phagocyte NADPH oxidase subunit (gp91-phox) called a superoxide-generating mitogenic oxidase (Mox l) was molecularly identified (Nature 1999; 401:79-82). This novel gene product is expressed in distinct types of nonphagocytic cells possessing superoxide-generating activity, and overexpression of Mox l was shown to promote disregulated growth of cancer cells in vitro and in vivo. We already demonstrated that primary cultures of guinea pig gastric pit cells secrete abundant superoxide anion (Oi) (Gastroenterology 1998; 115:1186-96). We report here that pit cells express Moxl and all of the other essential components for the phagocyte NADPH oxidase. Furthermore, we found that Helicobacter pylori (H. pylori) infection specifically up-regulated the Mox l expression in gastric pit cells both in vivo and in vitro. Methods: 0i release was measured as the SOD-inhibitable reduction of ferricytochrome c. A primer set was designed to specifically amplify the MoxI transcript, but not gp91-phox, and the RT-PCR product (600 bp) was used for detection of cells expressing the mox l mRNA in human biopsy specimens by in situ hybridization and for measurement of the transcript level in cultured pit cells by Northern blotting. The levels of mox l and NADPH oxidase components (p67-, p47-, p40, and p22-phoxes) and cells containing these components were analyzed by Western blotting and immunohistochemistry, respectively. Results & Conclusions: Pit cells cultured in LPS-free conditions constitutively expressed mox l, but not gp91phox, and all of the other essential components for phagocyte NADPH oxidase, and they spontaneously secreted 0iat 10 nmoUmg proteinlh. Confocal laser scanning microscopy showed that significant amounts of p67- and p47-phox were constitutively localized along the plasma membrane, which may explain the spontaneous Oirelease by the cells. H. pylori LPS induced Mox l, p67- and p22-phoxes and markedly increased 02production by the cells more than lO-fold. Among these components, the magnitude of p67-phox expression consistently correlated with the upregulation of 0i production. In situ hybridization and immunohistchemistry demonstrated that Mox l was expressed in pit cells of human gastric mucosa infected with H. pylori, but not in normal gastric mucosa. These observations suggest an important role of the unique NADPH oxidase in the pathogenesis of disregulated cell growth in association with H. pylori infection.
3811 FUNCTIONAL TRANSFER OF GENES FROM BACTERIA TO INTESTINAL EPITHELIAL CELLS. Richard James Aspinall, Georges Vassaux, Nicholas Lemoine, Imperial Coll Sch Of Medicine, London, United Kingdom. INTRODUCTION It has previously been demonstrated that bacteria can transfer genes to other unrelated bacteria, yeasts and plants. Recently, the first report of in vitro gene transfer from bacteria to mammalian cells was published I, showing subsequent expression of a reporter gene in COS-I, HeLa and CHO cell lines. Given the exposure of gastrointestinal epithelia to the endogenous bacterial flora, we wished to test whether such gene transfer was possible in marnrnalian intestinal cells. METHODS We used an auxotrophic, diaminopimelate (dapr-dependent strain of E.Coli , BM27 10, (a generous gift from Dr C Grillot-Courvalin of the Insitut Pasteur, Paris) engineered to express the Yersinia pseudotuberculosis invasin gene, the listeria monocytogenes listeriolysin 0 hly gene and also carrying the pEGFP-CI plasmid expressing GFP under eukaryotic promoter control. The mammalian cell lines studied were COS-I, CAC02, CMT93, HepG2, SKBR3, 293 and MKN45. 105 Cultured cells were incubated for 2 hours with BM2710 at a range of doses from 5x104 to 5xl09 bacteria in DMEM medium supplemented with dap. Cells were then washed three times with DMEM and incubated for 48 hours in complete medium containing gentamicin. After 48 hours, the cells were trypsinised, washed in PBS, resuspended in medium and anlaysed for GFP expression by FACS analysis. RESULTS Transfer and expression of the GFP plasmid was best demonstrated in the cell lines COS-I, 293, CAC02 and HepG2 in a dose-dependent manner, with up to 12% of cells showing positivity. A lesser effect was seen only at the highest doses in CMT93 cells. Repeated experiments on confluent marnrnalian cell preparations suggested transfer was more effective in actively dividing cell populations. CONCLUSIONS Auxotrophic E.Coli engineered to express the hly and invasin genes may be a suitable vehicle for delivering plasmid DNA to intestinal epithelia. We are currently exploring this method of gene delivery in murine models in vivo. 'Grillot-Courvalin C, Goussard S, Huetz F, et al. Nature Biotechnology 1998; 16: 862-866