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Immunomodulatory effect of lactic acid bacteria on human colonic mucosa
April 2000
AGAA99
668
670
THE HEUCOBACTER PYLORI VACUOLATING CYTOTOXIN (VACA) DISRUPTS THE EPITHELIAL CELL CYTOSKELETON IN A STRAIN AND CELL TYPE SPECIFIC MANNER. 1. R. Bebb, T. 1. Baldwin, D. P. Letley, T. Gray, Y. Mahida, 1. C. Atherton, Div Gastro & Inst Infect & Imm Univ Hosp, Nottingham, United Kingdom; Inst Infect & Imm Univ Hosp, Nottingham, United Kingdom; Em Unit, Univ Hosp, Nottingham, United Kingdom. Helicobacter pylori produces a cytotoxin, VacA, some forms of which induce vacuolation in eukaryotic cell lines. However, both vacuolating and non-vacuolating strains of H.pylori may be associated with disease, implying that features other than vacuolating activity are important. We have previously described a cell-rounding effect induced by a rhesus monkeyderived H.pylori strain, M99, and shown that the effect is dependent on VacA. We aimed to assess the mechanisms underlying this effect, and whether human strains induced similar changes. Methods:48 hour cultures of non-vacuolating strains (M99,1116,Tx30a) and a vacuolating strain (60190) of H.pylori were concentrated 20-fold by ultrafiltration and added in serial doubling dilutions to subconfluent epithelial cell lines (Hel.a, human cervical; RK-13, rabbit kidney; AGS, human gastric). Final concentrations causing SO% and 80% of cells to round were recorded. All experiments were performed 3 to 8 times. Selected cultures were processed for electron microscopy or mc phalloidin staining. Statistical differences in rounding effects of strains and susceptibility of cell lines were assessed by Wilcoxon's signed rank test. Results: Culture supernatants (CS) from all H.pylori strains caused rounding in at least one cell line, but strains differed in potency, with M99>60190>1116>Tx30a (pRK13 and HeLa, p
CHARACTERISATION OF STRAIN DEPENDENT CAMPYLOBACTER JEJUNI PATHOGENICITY. Joanne L. Brough, Johannes H. Beltinger, John P. Thomley, William A. Stack, Robin C. Spiller, Chris J. Hawkey, Univ Hosp Nottingham, Nottingham, United Kingdom. INTRODUCTION: Diarrhoeal illnesses due to Campytobacter jejuni are common but their pathogenesis is very poorly understood. We have previously shown that the effect of C. jejuni on human colonic epithelial cells is strain dependent. We now report that all strains tested produce an enterotoxin whilst some, more invasive strains, also produce a cytotoxin that destroys barrier function. METHODS: HCA7 cells were grown to confluent monolayers on Transwell filters. C. jejuni broths were concentrated using Centriprep spin columns and incubated with the monolayers. Cytotoxicity was also assessed with HEP2 and VERO cell lines. CHO-KI cells were used to assess enterotoxic activity. Strain dependent invasion of HCA7 cells was measured by a modified gentamicin protection assay and barrier function by electrical resistance (EVOHM) and 3H-mannitol and horseradish peroxidase (HRP) fluxes. C. jejuni translocation was assessed by colony counts from basolateral media. RESULTS: Of 18 clinical strains tested, all translocated HCA7 monolayers and all caused elongation of CHO-KI cells (enterotoxin assay). Five strains additionally caused rounding of HEP2 and VERO cells (cytotoxin assay). These S strains caused HCA7 epithelial cell disru~tion, assessed by the fall in resistance of 134±60cm2 vs 321 ±SOcm (control, p3.S bacteria per cell vs 0.OOO2±0.OOOO3 bacteria per cell), causing epithelial cell loss (1.4±0.1S x 104 vs S3± I.S x 104 cells remaining at 6 hours, p
669 IMMUNOMODULATORY EFFECT OF LACTIC ACID BACTE· RIA ON HUMAN COLONIC MUCOSA. Natalia Borruel, Francese Casellas, Maria Antolin, Antonia Andreu, Francisco Guarner, Juan-Ramon Malagelada, Digest System Research Unit Hosp Gen Vall d'Hebron, Barcelona, Spain; Hosp G Vall d'Hebron, Barcelona, Spain; Microbiological Service Hosp Gen Vall d'Hebron, Barcelona, Spain. Lactobacilli (LB) are part of the normal human colonic microflora. Recent studies suggest that certain strains of LB may prevent the developement of colitis in animal models. However, mechanisms by which LB interact with the intestinal mucosa remain largely unknown. We hypothesized that LB regulate cytokine sinthesis. Our aim was to assess cytokine production by human colonic mucosal in response to LB. Thus, mucosal explants(20-30 mg)of 6 normal human colons obtained from colectomy, were immediately cultured at 37 "C with 0iC02 9S%/S% in RPMI 1640 plus penicillin G. Fourteen mucosal explants from each colon were cultured with 107 viable bacteria/ml of non-pathogen E. Coli, 3 different strains of L. jensenii and 2 strains of L. erispatus. After 24 hours culture, TNF-a, IL-8 and IL-1O concentration in supernatants were measured by ELISA. Tissue viability was always confirmed by LDH release into culture medium. RESULTS: Viability was similar in all incubation wells ( 60 - 70%). Cytokine concentrations are expresssed corrected by mg of protein, and shown in the table. (* =p
Control E.Coli l.jensenll1 l.jensenil2 lJensenll3 l.crispalus 1 l.crispalusr2
TNF.(l lpg)
Il·8 (ng)
Il·10(pg)
13.5 ± 0.9 87.8 ± 13.2 * 29.9 ± 4.7 12.4±1.4 231± 4.8 22.1 ±4.8 32.2 + 7
254.0 ± 55.3 449.9 ± 94.1 183.4 ± 25.4 1202± 20.4 * 114.7 ± 25.9 * 116.5±13.2* 123.1 ± 24.0 *
27.4 ± 7. 103.7 ± 23.6 * 57.7±10.7* 35.6 ± 7.5 24.6 ± 5.3 26.9 ± 4.6 29.9 ± 7.3
671 RECOMBINANT ADENOVIRUS SEROTYPE SWITCHING FOR ANALYSIS OF EFFICIENCY AND ROUTE OF INTESTINAL CELL ENTRY. Joel R. Chamberlain, Micheal H. Shaw, John M. Hilfinger, Ellen M. Zimmerman, Blake J. Roessler, Univ of Michigan, Ann Arbor, MI; TSRL, Inc, Ann Arbor, MI. Our ultimate goal is to develop adenoviral vectors to transfer genes to the intestinal epithelium via the lumenal route. Studies to date have demonstrated a limited ability of Ad S, the most widely used serotype for gene therapy applications, to transfect the intestinal epithelium. In order to enhance adenoviral cell entry into the intestinal epithelium and explore mechanisms of viral-epithelial cell interactions, we are using recombinant DNA techniques to manipulate the adenoviral fiber protein. Adenovirus serotype S (AdS) contacts cells by means of viral fiber protein and Coxsackie and Adenovirus receptor (CAR) interaction. We have switched the fiber protein of the easily propagated AdS serotype with the fiber protein from Ad41, the enterotropic serotype, to explore the pathway of intestinal cell entry. Inhibition of CAR-dependent cell entry does not appear to significantly inhibit Ad41 transduction in vitro, indicating an alternate route for virus internalization (Roelvink et at. J. Virol., 72:7909-15). AdS:4l pseudotyping was accomplished using bacterial DNA recombination in recombination competent E. coli by the method of Chartier et at. (1. Virol., 70:480S-IO). Two plasmids were prepared for transformation and recombination in E. coli: I) a plasmid containing the entire AdS genome was modified to contain a unique restriction endonuclease site in the AdS fiber gene and 2) a routine cloning vector was used to construct DNA sequences containing each of the two Ad41 fiber genes flanked by AdS S' and 3' fiber gene regions. Simultaneous transformation of the linearized AdS plasmid and each of the Ad41 fiber gene DNA fragments yielded recombinants positive for Ad41 fiber sequences. PCR analysis indicated that approximately 70 percent of a subset of the 110 bacterial colonies obtained tested positive for the recombination event intended to both deleted the AdS fiber gene and insert the Ad41 fiber genes. The pseudotyped viruses will be a valuable tool for exploring epithelial cell entry pathways and wil be tested for altered intestinal tropism necessary for gene therapy applications.
AGAA99
668
670
THE HEUCOBACTER PYLORI VACUOLATING CYTOTOXIN (VACA) DISRUPTS THE EPITHELIAL CELL CYTOSKELETON IN A STRAIN AND CELL TYPE SPECIFIC MANNER. 1. R. Bebb, T. 1. Baldwin, D. P. Letley, T. Gray, Y. Mahida, 1. C. Atherton, Div Gastro & Inst Infect & Imm Univ Hosp, Nottingham, United Kingdom; Inst Infect & Imm Univ Hosp, Nottingham, United Kingdom; Em Unit, Univ Hosp, Nottingham, United Kingdom. Helicobacter pylori produces a cytotoxin, VacA, some forms of which induce vacuolation in eukaryotic cell lines. However, both vacuolating and non-vacuolating strains of H.pylori may be associated with disease, implying that features other than vacuolating activity are important. We have previously described a cell-rounding effect induced by a rhesus monkeyderived H.pylori strain, M99, and shown that the effect is dependent on VacA. We aimed to assess the mechanisms underlying this effect, and whether human strains induced similar changes. Methods:48 hour cultures of non-vacuolating strains (M99,1116,Tx30a) and a vacuolating strain (60190) of H.pylori were concentrated 20-fold by ultrafiltration and added in serial doubling dilutions to subconfluent epithelial cell lines (Hel.a, human cervical; RK-13, rabbit kidney; AGS, human gastric). Final concentrations causing SO% and 80% of cells to round were recorded. All experiments were performed 3 to 8 times. Selected cultures were processed for electron microscopy or mc phalloidin staining. Statistical differences in rounding effects of strains and susceptibility of cell lines were assessed by Wilcoxon's signed rank test. Results: Culture supernatants (CS) from all H.pylori strains caused rounding in at least one cell line, but strains differed in potency, with M99>60190>1116>Tx30a (p
CHARACTERISATION OF STRAIN DEPENDENT CAMPYLOBACTER JEJUNI PATHOGENICITY. Joanne L. Brough, Johannes H. Beltinger, John P. Thomley, William A. Stack, Robin C. Spiller, Chris J. Hawkey, Univ Hosp Nottingham, Nottingham, United Kingdom. INTRODUCTION: Diarrhoeal illnesses due to Campytobacter jejuni are common but their pathogenesis is very poorly understood. We have previously shown that the effect of C. jejuni on human colonic epithelial cells is strain dependent. We now report that all strains tested produce an enterotoxin whilst some, more invasive strains, also produce a cytotoxin that destroys barrier function. METHODS: HCA7 cells were grown to confluent monolayers on Transwell filters. C. jejuni broths were concentrated using Centriprep spin columns and incubated with the monolayers. Cytotoxicity was also assessed with HEP2 and VERO cell lines. CHO-KI cells were used to assess enterotoxic activity. Strain dependent invasion of HCA7 cells was measured by a modified gentamicin protection assay and barrier function by electrical resistance (EVOHM) and 3H-mannitol and horseradish peroxidase (HRP) fluxes. C. jejuni translocation was assessed by colony counts from basolateral media. RESULTS: Of 18 clinical strains tested, all translocated HCA7 monolayers and all caused elongation of CHO-KI cells (enterotoxin assay). Five strains additionally caused rounding of HEP2 and VERO cells (cytotoxin assay). These S strains caused HCA7 epithelial cell disru~tion, assessed by the fall in resistance of 134±60cm2 vs 321 ±SOcm (control, p
669 IMMUNOMODULATORY EFFECT OF LACTIC ACID BACTE· RIA ON HUMAN COLONIC MUCOSA. Natalia Borruel, Francese Casellas, Maria Antolin, Antonia Andreu, Francisco Guarner, Juan-Ramon Malagelada, Digest System Research Unit Hosp Gen Vall d'Hebron, Barcelona, Spain; Hosp G Vall d'Hebron, Barcelona, Spain; Microbiological Service Hosp Gen Vall d'Hebron, Barcelona, Spain. Lactobacilli (LB) are part of the normal human colonic microflora. Recent studies suggest that certain strains of LB may prevent the developement of colitis in animal models. However, mechanisms by which LB interact with the intestinal mucosa remain largely unknown. We hypothesized that LB regulate cytokine sinthesis. Our aim was to assess cytokine production by human colonic mucosal in response to LB. Thus, mucosal explants(20-30 mg)of 6 normal human colons obtained from colectomy, were immediately cultured at 37 "C with 0iC02 9S%/S% in RPMI 1640 plus penicillin G. Fourteen mucosal explants from each colon were cultured with 107 viable bacteria/ml of non-pathogen E. Coli, 3 different strains of L. jensenii and 2 strains of L. erispatus. After 24 hours culture, TNF-a, IL-8 and IL-1O concentration in supernatants were measured by ELISA. Tissue viability was always confirmed by LDH release into culture medium. RESULTS: Viability was similar in all incubation wells ( 60 - 70%). Cytokine concentrations are expresssed corrected by mg of protein, and shown in the table. (* =p
Control E.Coli l.jensenll1 l.jensenil2 lJensenll3 l.crispalus 1 l.crispalusr2
TNF.(l lpg)
Il·8 (ng)
Il·10(pg)
13.5 ± 0.9 87.8 ± 13.2 * 29.9 ± 4.7 12.4±1.4 231± 4.8 22.1 ±4.8 32.2 + 7
254.0 ± 55.3 449.9 ± 94.1 183.4 ± 25.4 1202± 20.4 * 114.7 ± 25.9 * 116.5±13.2* 123.1 ± 24.0 *
27.4 ± 7. 103.7 ± 23.6 * 57.7±10.7* 35.6 ± 7.5 24.6 ± 5.3 26.9 ± 4.6 29.9 ± 7.3
671 RECOMBINANT ADENOVIRUS SEROTYPE SWITCHING FOR ANALYSIS OF EFFICIENCY AND ROUTE OF INTESTINAL CELL ENTRY. Joel R. Chamberlain, Micheal H. Shaw, John M. Hilfinger, Ellen M. Zimmerman, Blake J. Roessler, Univ of Michigan, Ann Arbor, MI; TSRL, Inc, Ann Arbor, MI. Our ultimate goal is to develop adenoviral vectors to transfer genes to the intestinal epithelium via the lumenal route. Studies to date have demonstrated a limited ability of Ad S, the most widely used serotype for gene therapy applications, to transfect the intestinal epithelium. In order to enhance adenoviral cell entry into the intestinal epithelium and explore mechanisms of viral-epithelial cell interactions, we are using recombinant DNA techniques to manipulate the adenoviral fiber protein. Adenovirus serotype S (AdS) contacts cells by means of viral fiber protein and Coxsackie and Adenovirus receptor (CAR) interaction. We have switched the fiber protein of the easily propagated AdS serotype with the fiber protein from Ad41, the enterotropic serotype, to explore the pathway of intestinal cell entry. Inhibition of CAR-dependent cell entry does not appear to significantly inhibit Ad41 transduction in vitro, indicating an alternate route for virus internalization (Roelvink et at. J. Virol., 72:7909-15). AdS:4l pseudotyping was accomplished using bacterial DNA recombination in recombination competent E. coli by the method of Chartier et at. (1. Virol., 70:480S-IO). Two plasmids were prepared for transformation and recombination in E. coli: I) a plasmid containing the entire AdS genome was modified to contain a unique restriction endonuclease site in the AdS fiber gene and 2) a routine cloning vector was used to construct DNA sequences containing each of the two Ad41 fiber genes flanked by AdS S' and 3' fiber gene regions. Simultaneous transformation of the linearized AdS plasmid and each of the Ad41 fiber gene DNA fragments yielded recombinants positive for Ad41 fiber sequences. PCR analysis indicated that approximately 70 percent of a subset of the 110 bacterial colonies obtained tested positive for the recombination event intended to both deleted the AdS fiber gene and insert the Ad41 fiber genes. The pseudotyped viruses will be a valuable tool for exploring epithelial cell entry pathways and wil be tested for altered intestinal tropism necessary for gene therapy applications.