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Bacteroides fragllis entertoxin induces c-Myc expression and cellular proliferation
increased 5-fold for Na+/K+ ATPaseand 2.2-fold for ,81integrin. Confocal microscopy confirmed protein redistribution. To assess whether apically-situated,81 integrin contributes to EPEC-inducedfunctional consequenceson host cells, the AP surface of ]84 monolayerswas exposedto antibody (Ab) against ,81, Na+/K* ATPase, or non-immune serum and the effect on transepithelial electrical resistance (TER) was determined. Neither non-immune serum nor Na+/K+ ATPaseAb protected TER, ,81 Ab afforded no protection at 2 or 4h, but by 6h TER decreased only 25_4% for EPEC+~ Ab vs 45+-5% for EPECalone (p=O.Of8; n=5 exps of triplicates). Interestingly,the drop in TER was identicalfor EPECalone at 4h and EPEC+,81 Ab at 6h suggesting that the later decrease may be ~-mediated. Deletion of the intimin receptor Tir, abolished the effect on TER (2_+4% increase at 6h). We conclude that EPEC disruption of TJs impacts both barrier function and cell polarity and that accessibility to key 8L proteins such as ,81 (ntegitn accentuates physiological perturbations. We speculatethat Tir/intimin interactionsare necessaryto initiate effects on TJs, allowing/TI integrin to migrate to the AP membrane, bind intimin and potentiate disruption of the TJ barrier.
1675 A Search For Mycobacterium Paratuberculosis By PCR In Crohn's Granulomas Isolated Laser Capture Microdissection Paul Ryan, National Univ of Ireland, Cork, Cork Ireland; Michael W. Bennett, Simon Aarons, Gary Lee, Joe O'Connell, John K. Collins, Gerald C. O'Sullivan, Fergus Shanahan, National gniv of Ireland, Cork Iretand Background: The role of Mycobacterium paratuberculosis in the pathogenesis of Crohn's disease is controversial. Interpretation of results has been confounded by possible tissue contamination with mycobacteriafrom the intestinal lumen. Isolation of subepithelialgranulomas by laser capture microdissection (LCM) may circumvent this. It also seems reasonable to predict that etiologically relevant infectious agents should be present within granulomas. We used LCM and PCR to address this. Methods: Archival formalin-fixed, paraffin-embedded tissue from patients with confirmed Crohn's disease was used. DNA from granulomas was isolated along with that of full thickness samples. Positive controls included purified /14. paratuberculosis isolates and granulomasisolatedfrom a bull infected with M. paratuberculosis. Negativecontrols included sarcoid and foreign body granulomas. The potential adverse effects of formalin fixation and that of the LCM techniqueon the amplification of mycobactedal DNA segment~ were controlled by comparative amplification of similar lengths of the APC gene from the same samples. M. paratuberculosis was tested for by nested PCRamplification of a 413bp and then a 326bp internal fragment of the IS900 gene. In addition, nested PCR amplification of a 193bp and 155bp fragment was performed. A 4gObpand a 135bp segment of the APC gene was also amplified. Results: Amplification of the larger segment of the APC gene was inconsistent in microdissected samples but amplification of the smaller segment of the APC gene was positive in all samples. Nested PCR for the larger Isgoo fragment was positive in 1 of 8 full thickness samplesfrom Crohn's diseaseand in 0/8 of the corresponding isolated granulomas. Amplification of the smaller segment was positive in 2/8 full thickness samples and in 2/8 of the granulomas tested to date. Conclusions: (1) LCM of Crohn's granulomas can be used to search by for infectious agents by PCR, but formalin fixation requires that relatively short DNA segments be amplified; (2) With this technique, we cannot exclude a role M. paratuberculosis in a minority of cases of granulomatous Crohn's disease.
1678 Molecular Mechanism of CryptosporidiumParvum Invasion of Biliary Epithelia Xian-Ming Chen, 8ing Q. Huang, Patrick L Splinter, Mark A. McNiven, Nicholas F. Larusso, Mayo Medical Sch, Clin and Fdn, Rochester, MN Cryptosporidium parvum (CP) opportunistically infects intestinal and biliary epithelia causing considerable morbidity especially in patients with AIDS. To begin to explore the molecular mechanisms by which CP invadesepithelialcells, we previouslydemonstratedthat CP induces actin rearrangement at the epithelial cell-parasite interface using an in vitro model of CP infection of cultured human biliary epithelial cells (Hepstology28;906,1998 and Gastroenterology 118;368,2000). Since recent studies have demonstratedthat pp6Oc-src (a cytoskeletonassociated protein tyrosine kinase) and small GTP-binding proteins of the Rho family are involved in actin reorganization,we tested the HYPOTHESISthat CP stimulates pp6Oc-src and Rho GTP-bindingproteins causingactin rearrangementand epithelialcell invasion. METHODS: Bile duct epithelial cells (i.e., cholangiocytes) derived from normal human liver and immortalized by SM40transformation were exposedto freshly excysted CP sporozoites, and activation of pp6Oc-src, Rho GTP-bindingproteins and actin associatedproteins assessed by morphologicaland molecularapproaches.RESULTS:By immunoconfocal microscopy, pp6Ocsrc in cholangiocytes was found to be recruited to submembranous actin filaments at the parasite-cholangiocyteinterface during microbial invasion. Of the Rho GTP-bindingproteins, Rho and cdc42, hut not Rac, accumulated in cholangiocytesdirectly adjacentto the invading CP organism. Cortactin,an actin-binding protein previously identifiedas a substratefor ppBOcsrc, also showed strong staining within cholangiocytesat the parasite-cholangiocyteinterface by both confocal and immunoelectron microscopy. Moreover, inhibition of pp6Oc-src by a selective inhibitor (i.e., PP2) or by transfection of cholangiocyteswith a dominant negative mutant of pp6Oc-src, completely blocked CP invasion of cholangiocytes.CONCLUSIONS:CP invasion of cholangiocytes involves accumulation of cholangiocyte cortactin and Rho GTPbinding proteins and rearrangementof cholangiocyteactin at the parasite-cholangiocyteinterface, a process requiring recruitment and kinase activation of pp6Oc-src.
1676 Enterohaemorrhagic Escherichia coil (EHEC) Infection Induce IL-8 Productionvia MAP Kinases Activation in T84 Cells. Stephanie Dahan, Patrick Rampal, Dorota Czerucka, Univ Nice-SophiaAntipolis, Nice France BACKGROUND:TheShiga-liketoxin producing Escherichia coil (EHEC) infection is associated with bloody diarrhea and can lead to systemic complications including the haemolytic uremic syndrome (HUS). The high level of proinflammatory cytokine IL-8 was an indicator of risk for developing HUS (Cytokine, 2000, 12-6, pp822-827). Previously, we reported that enteropathogenic Escherichia co/i (EPEC) infection induced MAP kinases activation in intestinal cells. Thesesignaling pathwayswere implicated in invasion processand cytokinesexpression. AIM: The purpose of this study was to demonstrate the ability of EHECto activate host cell signal transduction pathways that lead to the production of proinflammatory cytokines such as IL-8. METHODS:T84 cells were infected for various times with the EDL931 EHECstrain (100 bacteria/cell). Prior infection, cells were serum deprived for 12 hours. The whole cell lysates (WCL) were analysed by western blots (WB) that were performed with antibodies toward the phosphorylatedforms of different members of MAPK family (ERK1/2, P3B and JNK). The IL-8 concentration of the culture supernatants was determinated by ELISA. When indicated, cells were treated with highly specific MEK1/2 MAPKK inhibitor, U0126 (IO/~M), or P38 MAPK inhibitor, SB203580 (IO~M), one hour before bacterial infection and during infection. RESULTS: Kinetic studies revealed that 3 hours after infection, EHEC induced activation of the three families of MAPK (ERK1/2, P38 and JNK) in T84 cells. EHECinfection of cultured intestinal cells results in time-dependant production of IL-8 that was dominant after 6 hours of infection (13.6 +-2.9 ng/mt vs 0.9 +-0.09 ng/ml in control cells). Pretreatment of cells with the highly specific MEK1/2 inhibitor U0126 or P38 inhibitor SB203580 lowered by about 70 percent the EHEC-induced IL-8 production (3.6 +-1.1 ng/ml and 4.5 +-1.1 ng/ ml respectivelyp-~O.O5vs EHECalone infected cells), CONCLUSIONS:EHECinduce activation of ERKI/2, P38 and JNK in infected T84 cells. We show that ERK1/2 and P38 MAPK are directly involved in IL-8 production. The implication of JNK in the physiopathologyand the nuclear responsesof EHECinfection are under current investigation.The researchwas funded by Laboratoires BIOCODEX,France.
1679
Bacteroides Fragilis Entedoxin Induces c-Myc Expression And Cellular Proliferation Shaoguang Wu, Johns Hopkins Univ, Baltimore, MD; Pat J. Morin, National Institute on Aging, Baltimore, MD; Djik Maouyo, Cynthia L Sears, Johns Hopkins Univ, Baltimore, MD BACKGROUND:EnterotoxigenicBacteroides fragi/is (ETBF) are associatedwith diarrheal diseases in children, adults, and livestock. The pathogenicity of ETBFis ascribed to a heat-labile -20 kD metafloproteasetoxin (B. fragflistoxin,BFT). 8FT stimulates secretion in ligated lamb intestinal segments,alters cell morphologyand increasesthe permeabilityof intestinalepithelial cell monolayers and human colonic mucosa in vitro. We have previously demonstrated that BFT specifically cleaves E-cadherin resulting in a complete loss of cellular E-cadherin. Oownregulation of E-cadherin on epithelial tumor cells has been associated with enhanced metastatic potential of these cells. ~catenin, a portion of which is bound to E-cadherin, is known to trigger the Wnt signal transduction pathway,an important pathwayin tumorigenesis. The objective of this study was to evaluate if the cleavageand temporary downregulation of E-cadherin due to BFT treatment triggers ,8-catenin signaling in a cloned intestinal cell line, HT29/C1. METHODSAND RESULTS: Using confocal immunofluorascent microscopy, ,8catecin staining localizedmainly at the cell membrane in control HT2g/C1 cells. After 3 hours of BFT treatment, ~satenin staining was detected diffusely in the cytoplasm and nuclei. Accumulation of ~catenin in HT29/C1 cell nuclei persistedfor > 24 hours after BFTtreatment. To evaluatethe functional consequencesof ~-catenin nucleartranslocation, c-myctranscription and translation was evaluatedby ribonucleaseprotection assay (RPA) and Western Blot. RPA analyses indicated that c-myc mRNA increased as early as 6 hours after BFT treatment of HT29/C1 cells. Western blot analyses revealed a significant increase in c-Myc protein 7 to 24 hours after DR treatment when compared to control cells (P
1677 EPEC Infection Allows Migration of .81 Integrin to Apical Membrane Which Contributes to Functional Perturbations Gall Hecht, Athanasia Koutsouris, Michelle M. Muza, Univ of Illinois at Chicago, Chicago, IL Enteropathogenic=Ecoil (EPEC) disrupts host intestinal epithelial tight junctions (TJs) which serve as paracellular barriers and maintain the polar distribution of membrane proteins. In polarizedcells, ,81 integrin and Na+/K* ATPaseare restrictedto the basolateral(BL) membrane. The outer membrane EPEC protein intimin can bind to both the EPECtranslocated intimin receptor(Tir) and ,81 integrin. Restrictionof,81 integrin to the BL domain precludesopportunity for interaction. We have reported that EPEC perturbs the TJ barrier but have not examined the impact on cell polarity. We hypothesizethat EPEC-induceddisruption of TJs allows BL proteins, like ,81 integrin, to migrate to the apical (AP) membrane of host cells thus allowing interaction with intimin. The aim of this study was to determine if EPECperturbs the polar distribution of BL proteins and if such redistribution contributes to pathogenesis.Cultured human intestinal epithelial cells, T84, and EPECE2348/69 were used. The effect of EPECon the polar distribution of select BL proteins was determined by labeling AP or BL surfaces of T84 monolayerswith biotin, immunoprecipitating Na+/K÷ ATPaseor/TI from the AP and BL fractions, and quantifying by streptavidin immunoblot. After 6h of infection, the AP:BL ratio
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1675 A Search For Mycobacterium Paratuberculosis By PCR In Crohn's Granulomas Isolated Laser Capture Microdissection Paul Ryan, National Univ of Ireland, Cork, Cork Ireland; Michael W. Bennett, Simon Aarons, Gary Lee, Joe O'Connell, John K. Collins, Gerald C. O'Sullivan, Fergus Shanahan, National gniv of Ireland, Cork Iretand Background: The role of Mycobacterium paratuberculosis in the pathogenesis of Crohn's disease is controversial. Interpretation of results has been confounded by possible tissue contamination with mycobacteriafrom the intestinal lumen. Isolation of subepithelialgranulomas by laser capture microdissection (LCM) may circumvent this. It also seems reasonable to predict that etiologically relevant infectious agents should be present within granulomas. We used LCM and PCR to address this. Methods: Archival formalin-fixed, paraffin-embedded tissue from patients with confirmed Crohn's disease was used. DNA from granulomas was isolated along with that of full thickness samples. Positive controls included purified /14. paratuberculosis isolates and granulomasisolatedfrom a bull infected with M. paratuberculosis. Negativecontrols included sarcoid and foreign body granulomas. The potential adverse effects of formalin fixation and that of the LCM techniqueon the amplification of mycobactedal DNA segment~ were controlled by comparative amplification of similar lengths of the APC gene from the same samples. M. paratuberculosis was tested for by nested PCRamplification of a 413bp and then a 326bp internal fragment of the IS900 gene. In addition, nested PCR amplification of a 193bp and 155bp fragment was performed. A 4gObpand a 135bp segment of the APC gene was also amplified. Results: Amplification of the larger segment of the APC gene was inconsistent in microdissected samples but amplification of the smaller segment of the APC gene was positive in all samples. Nested PCR for the larger Isgoo fragment was positive in 1 of 8 full thickness samplesfrom Crohn's diseaseand in 0/8 of the corresponding isolated granulomas. Amplification of the smaller segment was positive in 2/8 full thickness samples and in 2/8 of the granulomas tested to date. Conclusions: (1) LCM of Crohn's granulomas can be used to search by for infectious agents by PCR, but formalin fixation requires that relatively short DNA segments be amplified; (2) With this technique, we cannot exclude a role M. paratuberculosis in a minority of cases of granulomatous Crohn's disease.
1678 Molecular Mechanism of CryptosporidiumParvum Invasion of Biliary Epithelia Xian-Ming Chen, 8ing Q. Huang, Patrick L Splinter, Mark A. McNiven, Nicholas F. Larusso, Mayo Medical Sch, Clin and Fdn, Rochester, MN Cryptosporidium parvum (CP) opportunistically infects intestinal and biliary epithelia causing considerable morbidity especially in patients with AIDS. To begin to explore the molecular mechanisms by which CP invadesepithelialcells, we previouslydemonstratedthat CP induces actin rearrangement at the epithelial cell-parasite interface using an in vitro model of CP infection of cultured human biliary epithelial cells (Hepstology28;906,1998 and Gastroenterology 118;368,2000). Since recent studies have demonstratedthat pp6Oc-src (a cytoskeletonassociated protein tyrosine kinase) and small GTP-binding proteins of the Rho family are involved in actin reorganization,we tested the HYPOTHESISthat CP stimulates pp6Oc-src and Rho GTP-bindingproteins causingactin rearrangementand epithelialcell invasion. METHODS: Bile duct epithelial cells (i.e., cholangiocytes) derived from normal human liver and immortalized by SM40transformation were exposedto freshly excysted CP sporozoites, and activation of pp6Oc-src, Rho GTP-bindingproteins and actin associatedproteins assessed by morphologicaland molecularapproaches.RESULTS:By immunoconfocal microscopy, pp6Ocsrc in cholangiocytes was found to be recruited to submembranous actin filaments at the parasite-cholangiocyteinterface during microbial invasion. Of the Rho GTP-bindingproteins, Rho and cdc42, hut not Rac, accumulated in cholangiocytesdirectly adjacentto the invading CP organism. Cortactin,an actin-binding protein previously identifiedas a substratefor ppBOcsrc, also showed strong staining within cholangiocytesat the parasite-cholangiocyteinterface by both confocal and immunoelectron microscopy. Moreover, inhibition of pp6Oc-src by a selective inhibitor (i.e., PP2) or by transfection of cholangiocyteswith a dominant negative mutant of pp6Oc-src, completely blocked CP invasion of cholangiocytes.CONCLUSIONS:CP invasion of cholangiocytes involves accumulation of cholangiocyte cortactin and Rho GTPbinding proteins and rearrangementof cholangiocyteactin at the parasite-cholangiocyteinterface, a process requiring recruitment and kinase activation of pp6Oc-src.
1676 Enterohaemorrhagic Escherichia coil (EHEC) Infection Induce IL-8 Productionvia MAP Kinases Activation in T84 Cells. Stephanie Dahan, Patrick Rampal, Dorota Czerucka, Univ Nice-SophiaAntipolis, Nice France BACKGROUND:TheShiga-liketoxin producing Escherichia coil (EHEC) infection is associated with bloody diarrhea and can lead to systemic complications including the haemolytic uremic syndrome (HUS). The high level of proinflammatory cytokine IL-8 was an indicator of risk for developing HUS (Cytokine, 2000, 12-6, pp822-827). Previously, we reported that enteropathogenic Escherichia co/i (EPEC) infection induced MAP kinases activation in intestinal cells. Thesesignaling pathwayswere implicated in invasion processand cytokinesexpression. AIM: The purpose of this study was to demonstrate the ability of EHECto activate host cell signal transduction pathways that lead to the production of proinflammatory cytokines such as IL-8. METHODS:T84 cells were infected for various times with the EDL931 EHECstrain (100 bacteria/cell). Prior infection, cells were serum deprived for 12 hours. The whole cell lysates (WCL) were analysed by western blots (WB) that were performed with antibodies toward the phosphorylatedforms of different members of MAPK family (ERK1/2, P3B and JNK). The IL-8 concentration of the culture supernatants was determinated by ELISA. When indicated, cells were treated with highly specific MEK1/2 MAPKK inhibitor, U0126 (IO/~M), or P38 MAPK inhibitor, SB203580 (IO~M), one hour before bacterial infection and during infection. RESULTS: Kinetic studies revealed that 3 hours after infection, EHEC induced activation of the three families of MAPK (ERK1/2, P38 and JNK) in T84 cells. EHECinfection of cultured intestinal cells results in time-dependant production of IL-8 that was dominant after 6 hours of infection (13.6 +-2.9 ng/mt vs 0.9 +-0.09 ng/ml in control cells). Pretreatment of cells with the highly specific MEK1/2 inhibitor U0126 or P38 inhibitor SB203580 lowered by about 70 percent the EHEC-induced IL-8 production (3.6 +-1.1 ng/ml and 4.5 +-1.1 ng/ ml respectivelyp-~O.O5vs EHECalone infected cells), CONCLUSIONS:EHECinduce activation of ERKI/2, P38 and JNK in infected T84 cells. We show that ERK1/2 and P38 MAPK are directly involved in IL-8 production. The implication of JNK in the physiopathologyand the nuclear responsesof EHECinfection are under current investigation.The researchwas funded by Laboratoires BIOCODEX,France.
1679
Bacteroides Fragilis Entedoxin Induces c-Myc Expression And Cellular Proliferation Shaoguang Wu, Johns Hopkins Univ, Baltimore, MD; Pat J. Morin, National Institute on Aging, Baltimore, MD; Djik Maouyo, Cynthia L Sears, Johns Hopkins Univ, Baltimore, MD BACKGROUND:EnterotoxigenicBacteroides fragi/is (ETBF) are associatedwith diarrheal diseases in children, adults, and livestock. The pathogenicity of ETBFis ascribed to a heat-labile -20 kD metafloproteasetoxin (B. fragflistoxin,BFT). 8FT stimulates secretion in ligated lamb intestinal segments,alters cell morphologyand increasesthe permeabilityof intestinalepithelial cell monolayers and human colonic mucosa in vitro. We have previously demonstrated that BFT specifically cleaves E-cadherin resulting in a complete loss of cellular E-cadherin. Oownregulation of E-cadherin on epithelial tumor cells has been associated with enhanced metastatic potential of these cells. ~catenin, a portion of which is bound to E-cadherin, is known to trigger the Wnt signal transduction pathway,an important pathwayin tumorigenesis. The objective of this study was to evaluate if the cleavageand temporary downregulation of E-cadherin due to BFT treatment triggers ,8-catenin signaling in a cloned intestinal cell line, HT29/C1. METHODSAND RESULTS: Using confocal immunofluorascent microscopy, ,8catecin staining localizedmainly at the cell membrane in control HT2g/C1 cells. After 3 hours of BFT treatment, ~satenin staining was detected diffusely in the cytoplasm and nuclei. Accumulation of ~catenin in HT29/C1 cell nuclei persistedfor > 24 hours after BFTtreatment. To evaluatethe functional consequencesof ~-catenin nucleartranslocation, c-myctranscription and translation was evaluatedby ribonucleaseprotection assay (RPA) and Western Blot. RPA analyses indicated that c-myc mRNA increased as early as 6 hours after BFT treatment of HT29/C1 cells. Western blot analyses revealed a significant increase in c-Myc protein 7 to 24 hours after DR treatment when compared to control cells (P
1677 EPEC Infection Allows Migration of .81 Integrin to Apical Membrane Which Contributes to Functional Perturbations Gall Hecht, Athanasia Koutsouris, Michelle M. Muza, Univ of Illinois at Chicago, Chicago, IL Enteropathogenic=Ecoil (EPEC) disrupts host intestinal epithelial tight junctions (TJs) which serve as paracellular barriers and maintain the polar distribution of membrane proteins. In polarizedcells, ,81 integrin and Na+/K* ATPaseare restrictedto the basolateral(BL) membrane. The outer membrane EPEC protein intimin can bind to both the EPECtranslocated intimin receptor(Tir) and ,81 integrin. Restrictionof,81 integrin to the BL domain precludesopportunity for interaction. We have reported that EPEC perturbs the TJ barrier but have not examined the impact on cell polarity. We hypothesizethat EPEC-induceddisruption of TJs allows BL proteins, like ,81 integrin, to migrate to the apical (AP) membrane of host cells thus allowing interaction with intimin. The aim of this study was to determine if EPECperturbs the polar distribution of BL proteins and if such redistribution contributes to pathogenesis.Cultured human intestinal epithelial cells, T84, and EPECE2348/69 were used. The effect of EPECon the polar distribution of select BL proteins was determined by labeling AP or BL surfaces of T84 monolayerswith biotin, immunoprecipitating Na+/K÷ ATPaseor/TI from the AP and BL fractions, and quantifying by streptavidin immunoblot. After 6h of infection, the AP:BL ratio
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