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Cryptosporidium parvum soluble phospholipase activity associated with host cell invasion
April 2000
AGAA817
4321
4323
ANTIBIOTIC THERAPY ATTENUATES COLITIS BY ALTERING ADHERENT BACTERIAL LEVELS AND PROFILES IN IL-I0'\' MICE. Karen L. Madsen, Jason S. Doyle, Michele M. Tavernini, Lawrence D.
CRYPTOSPORIDIUM PARVUM SOLUBLE PHOSPHOLIPASE ACTIVITY ASSOCIATED WITH HOST CELL INVASION. Richard C. Pollok, Vincent McDonald, Louise Maher, Michael 1. Farthing,
Jewell, Robert P. Rennie, Richard N. Fedorak, Univ of Alberta, Edmonton, AB, Canada. IL-lO'\' mice, raised under germ-free conditions, do not develop colitis, implying a role for bacteria in the inflammation. We have previously shown that both prior to, and after the development of inflammation, IL-IO'\' mice demonstrate altered levels of colonic mucosal adherent and translocated bacteria in addition to differences in bacterial species. Aim: This study was designed to use antiobiotic therapy to determine the association of specific bacterial species with colitis in IL-lO'\' mice. Methods: IL-lO'\' mice were treated with: (1) ciprofloxacin (42 mg/kg/g/d; Cipro), or (2) neomycin (85 mg/kg/d) and metronidazole (145 mg/kg/d) (NM), either from 0-4 wks or from 8-12 weeks of age. Colons were harvested for histologic scoring, bacterial culture, and measurement of adherent and translocated bacterial levels. Results: Treatment of IL-lO'\' mice with either antibiotic therapy during the neonatal period reduced total levels of adherent mucosal and translocated bacteria and prevented the development of colitis at 4 wks of age. 8 wks after removal of the antibiotics, those mice which had received antibiotics during the neonatal period continued to show significantly (p
4322 CELLULAR MECHANISMS OF INTERFERON·r MEDIATED INHIBITION OF CRYPTQSPORIDIUM PARVUM INFECTION. Richard C. Pollok, Michael 1. Farthing, Mona Bajaj-Elliott, Vincent MeDonald, St Bartholomew's and The Royal London Sch of Medicine, London, United Kingdom. Introduction Interferon "'/ (IFN "'/) plays a key role in the control of Cparvum infection. We have previously shown IFN y may inhibit Cparvum infection of human intestinal cell lines. In this study we have examined the role of the IFN y receptor (IFN yR) and subsequent cellular metabolism in mediating this action. Methods As previously described, HT29, Caco 2 transformed cells or H4 primary human intestinal cells grown on coverslips were infected with parasites and infection quantified by immunofluorescence assay (IFA) or Giernsa staining. The differential inhibitory effect of IFN 'Y was determined in each cell line. Parasite attachment to paraformaldhyde-fixed HT29 cells was quantified by IFA and compared with unfixed cells after 1.5h incubation. IFN yR expression was determined by immunoblotting using an antibody specific for the IFN yR. The action of the specific Janus kinase 2 inhibitor tyrophostin B42 (1-200pM), exogenous tryptophan, and ferrous sulphate (12.5-200p,M) on IFN y inhibition was also determined in separate experiments. Results IFN y inhibits infection in HT 29 cells and to a lesser extent Caco 2 cells but failed to inhibit infection in H4 cells. This difference was reflected in IFN yR expression in the respective cell lines, with no detectable expression in H4 cells. IFN y failed to inhibit Cparvum attachment but inhibited invasion by 40.4% ± 3.4, t test p<0.OO3)in unfixed cells. Both B42 and ferrous sulphate partially abrogated the inhibitory effect of IFN 'Y in a dose dependent fashion (maximal inhibition 41.6% ± 3.6, ANaVA p
St Bartholomew's and The Royal London Sch of Medicine, London, United Kingdom. Introduction There is increasing evidence that microbial phospholipase (PL) activity plays an important role in the virulence of a range of pathogens. In preliminary work we have shown that Cparvum infection in Caco-2 cells is inhibited by the specific PL inhibitor p-bromophenacyl bromide (P-BPB). Using an established in vitro model we have further characterised the role of PL in Cparvum infection. Methods As previously described, HT29 and Caco 2 cells grown on coverslips were infected with Cparvum and infection quantified by immunofluorescence assay. The inhibitory effect of the PL inhibitor p-BPB (0.1 -lO/LM) on infection was compared in the cell lines. The action of anti-PLA antibodies (50-1000/Lg/ mL), and exogenous PLA (O.1-lO/Lg/mL) on infection was subsequently determined in HT29 cells. Soluble PLAz activity in sonicated Cparvum Iysates was quantified using a substrate based colorimetric microassay (Assay Designs Inc.). Activity was compared with sonicated Helicobacter pylori lysates, a pathogen known to produce PLAz. Results Dose dependent inhibition by p-BPB occured in Caco 2 and HT29 cell lines, with a maximal inhibition of 72.9 % ± 5.0 and 39.2 % ± 2.9 of control respectively (ANaVA p
4324 INFECTION BY ENTEROAGGREGATIVE ESCHERICHIA COLI ALTERS TRANSEPITHELIAL RESISTANCE OF INTESTINAL EPITHELIAL CELLS IN VITRO. Safak Reka, Donald P. Kotler, Emilia Mia Sordillo, State Univ of New York at Brooklyn, Brooklyn, NY; St Luke's-Roosevelt Hosp Ctr, New York, NY. Background: Infection with enteroaggregative E. coli (EAEC) causes diarrhea in infants, immunocompromised individuals, and travelers. Another diarrhea-causing bacterium, enteropathogenic E.coli (EPEC), has been reported to decrease transepithelial resistance (TER) across polarized intestinal cell monolayers, but an effect of EAEC has not been reported. Aim: To evaluate changes in TER during infection with EAEC, compared to EPEC and to diffusely adherent (DA) and nonadherent E.coli (NA) strains. Methods: T84 monolayers were grown on collagen-coated Transwells with 3 micron pore size and 0.3 sq ern area in DMEMlF12 medium with 5% FCS without antibiotics until TER exceeded 1000 ohms.sq em. Monolayers were incubated with medium alone (control), or inoculated with a suspension of E. coli made from an 18-24 h subculture on trypticase soy agar plus 5% sheep blood. All bacterial strains had been passed between 2 and 8 times from frozen stocks. TER was measured at baseline, 1,3,5, and 7 h after inoculation. Results: Infection with EAEC decreased TER within 1 h compared to control incubation (p
AGAA817
4321
4323
ANTIBIOTIC THERAPY ATTENUATES COLITIS BY ALTERING ADHERENT BACTERIAL LEVELS AND PROFILES IN IL-I0'\' MICE. Karen L. Madsen, Jason S. Doyle, Michele M. Tavernini, Lawrence D.
CRYPTOSPORIDIUM PARVUM SOLUBLE PHOSPHOLIPASE ACTIVITY ASSOCIATED WITH HOST CELL INVASION. Richard C. Pollok, Vincent McDonald, Louise Maher, Michael 1. Farthing,
Jewell, Robert P. Rennie, Richard N. Fedorak, Univ of Alberta, Edmonton, AB, Canada. IL-lO'\' mice, raised under germ-free conditions, do not develop colitis, implying a role for bacteria in the inflammation. We have previously shown that both prior to, and after the development of inflammation, IL-IO'\' mice demonstrate altered levels of colonic mucosal adherent and translocated bacteria in addition to differences in bacterial species. Aim: This study was designed to use antiobiotic therapy to determine the association of specific bacterial species with colitis in IL-lO'\' mice. Methods: IL-lO'\' mice were treated with: (1) ciprofloxacin (42 mg/kg/g/d; Cipro), or (2) neomycin (85 mg/kg/d) and metronidazole (145 mg/kg/d) (NM), either from 0-4 wks or from 8-12 weeks of age. Colons were harvested for histologic scoring, bacterial culture, and measurement of adherent and translocated bacterial levels. Results: Treatment of IL-lO'\' mice with either antibiotic therapy during the neonatal period reduced total levels of adherent mucosal and translocated bacteria and prevented the development of colitis at 4 wks of age. 8 wks after removal of the antibiotics, those mice which had received antibiotics during the neonatal period continued to show significantly (p
4322 CELLULAR MECHANISMS OF INTERFERON·r MEDIATED INHIBITION OF CRYPTQSPORIDIUM PARVUM INFECTION. Richard C. Pollok, Michael 1. Farthing, Mona Bajaj-Elliott, Vincent MeDonald, St Bartholomew's and The Royal London Sch of Medicine, London, United Kingdom. Introduction Interferon "'/ (IFN "'/) plays a key role in the control of Cparvum infection. We have previously shown IFN y may inhibit Cparvum infection of human intestinal cell lines. In this study we have examined the role of the IFN y receptor (IFN yR) and subsequent cellular metabolism in mediating this action. Methods As previously described, HT29, Caco 2 transformed cells or H4 primary human intestinal cells grown on coverslips were infected with parasites and infection quantified by immunofluorescence assay (IFA) or Giernsa staining. The differential inhibitory effect of IFN 'Y was determined in each cell line. Parasite attachment to paraformaldhyde-fixed HT29 cells was quantified by IFA and compared with unfixed cells after 1.5h incubation. IFN yR expression was determined by immunoblotting using an antibody specific for the IFN yR. The action of the specific Janus kinase 2 inhibitor tyrophostin B42 (1-200pM), exogenous tryptophan, and ferrous sulphate (12.5-200p,M) on IFN y inhibition was also determined in separate experiments. Results IFN y inhibits infection in HT 29 cells and to a lesser extent Caco 2 cells but failed to inhibit infection in H4 cells. This difference was reflected in IFN yR expression in the respective cell lines, with no detectable expression in H4 cells. IFN y failed to inhibit Cparvum attachment but inhibited invasion by 40.4% ± 3.4, t test p<0.OO3)in unfixed cells. Both B42 and ferrous sulphate partially abrogated the inhibitory effect of IFN 'Y in a dose dependent fashion (maximal inhibition 41.6% ± 3.6, ANaVA p
St Bartholomew's and The Royal London Sch of Medicine, London, United Kingdom. Introduction There is increasing evidence that microbial phospholipase (PL) activity plays an important role in the virulence of a range of pathogens. In preliminary work we have shown that Cparvum infection in Caco-2 cells is inhibited by the specific PL inhibitor p-bromophenacyl bromide (P-BPB). Using an established in vitro model we have further characterised the role of PL in Cparvum infection. Methods As previously described, HT29 and Caco 2 cells grown on coverslips were infected with Cparvum and infection quantified by immunofluorescence assay. The inhibitory effect of the PL inhibitor p-BPB (0.1 -lO/LM) on infection was compared in the cell lines. The action of anti-PLA antibodies (50-1000/Lg/ mL), and exogenous PLA (O.1-lO/Lg/mL) on infection was subsequently determined in HT29 cells. Soluble PLAz activity in sonicated Cparvum Iysates was quantified using a substrate based colorimetric microassay (Assay Designs Inc.). Activity was compared with sonicated Helicobacter pylori lysates, a pathogen known to produce PLAz. Results Dose dependent inhibition by p-BPB occured in Caco 2 and HT29 cell lines, with a maximal inhibition of 72.9 % ± 5.0 and 39.2 % ± 2.9 of control respectively (ANaVA p
4324 INFECTION BY ENTEROAGGREGATIVE ESCHERICHIA COLI ALTERS TRANSEPITHELIAL RESISTANCE OF INTESTINAL EPITHELIAL CELLS IN VITRO. Safak Reka, Donald P. Kotler, Emilia Mia Sordillo, State Univ of New York at Brooklyn, Brooklyn, NY; St Luke's-Roosevelt Hosp Ctr, New York, NY. Background: Infection with enteroaggregative E. coli (EAEC) causes diarrhea in infants, immunocompromised individuals, and travelers. Another diarrhea-causing bacterium, enteropathogenic E.coli (EPEC), has been reported to decrease transepithelial resistance (TER) across polarized intestinal cell monolayers, but an effect of EAEC has not been reported. Aim: To evaluate changes in TER during infection with EAEC, compared to EPEC and to diffusely adherent (DA) and nonadherent E.coli (NA) strains. Methods: T84 monolayers were grown on collagen-coated Transwells with 3 micron pore size and 0.3 sq ern area in DMEMlF12 medium with 5% FCS without antibiotics until TER exceeded 1000 ohms.sq em. Monolayers were incubated with medium alone (control), or inoculated with a suspension of E. coli made from an 18-24 h subculture on trypticase soy agar plus 5% sheep blood. All bacterial strains had been passed between 2 and 8 times from frozen stocks. TER was measured at baseline, 1,3,5, and 7 h after inoculation. Results: Infection with EAEC decreased TER within 1 h compared to control incubation (p