A100 AGA ABSTRACTS

672

MOLECULAR STUDY OF H. PYLORI-EPITHELIAL CELL IN­TERACTION: A NOVEL IN VITRO SYSTEM BASED ON DIFFER­ENTIAL CULTURE CONDITIONS AND CELL POLARITY.Sandra Cottet, Irene E. Corthesy-Theulaz, Jean-Pierre Kraehenbuhl, BlaiseE. Corthesy, Immunology, Univ Hosp, Lausanne, Switzerland; Gastroen­terology, Univ Hosp, Lausanne, Switzerland; Swiss Institute for Experi­mental Research, Lausanne, Switzerland.

Animal models and human biopsy samples have provided a bulk ofinformation on the physiological consequences resulting from the infectionby H. pylori. However, in vitro dissection of the molecular and cellularmechanisms is complicated by the fact that H. pylori requires complexculture conditions and that no gastric polarized cell lines are available todate. Objectives: To design an in vitro system using polarized Caco-2 cells,maintaining an optimal environment for the epithelial cells and the bacte­rium in the basolateral and apical compartments, respectively. Methods:Polarized epithelial cells were grown on Transwell filters, and transepithe­Iial electrical resistance used as marker of monolayer integrity. Epithelialcells were exposed to H. pylori in a diffusion chamber with different apicaland basolateral media and gas conditions. Bacterial growth and adherencewere measured at different times, protein phosphorylation and activation oftranscription factor NF-kappaB were quantified in epithelial cell extractsand release of epithelial-specific cytokines measured by ELISA. Results:The Caco-2 transepithelial resistance is preserved for up to 48 hr when theapical medium is adapted to H. pylori growth, namely brain heart infusionmedium, pH 5.5, 5 mM urea, 5% 02-5% C02-90% N2. Under theseconditions, H. pylori keeps growing only in the presence of the epithelialcells, indicating that a careful definition of the bacterium needs is requiredto help dissecting the outcome of the interaction. Adherence of H. pylori toepithelial cells resulted in pedestal formation and brush border effacement,tyrosine phosphorylation, cytokines release (IL-8, MCP-l, Gro-alpha),decrease in urease A and B chain expression, and NF-kappaB nucleartranslocation. In contrast to other reports, these cellular markers weresustained for the time of the analysis (1-24 hrs). Conclusions: This novelsystem is adequate to examine various molecular aspects of H. pyloriinfection, including mechanisms of adherence, epithelial cell signal trans­duction, and the role of inflammatory cells by complementation of the sofar two partner system.

673

ENTEROPATHOGENIC ESCHERICHIA COU (EPEe) STIMU­LATE MITOGEN-ACTIVATED PROTEIN (MAP) KINASES IN T84CELLS.Dorota Czerucka, Stephanie Dahan, Baharia Mograbi, Bernard Rossi,Patrick Rampal, Univ Nice-Sophia Antipolis, Nice, France.

Recently, activation of MAP kinases have been reported in intestinal cellsinfected by Salmonella typhimurium (J.Immuno1.l997;159:5550-5559)andListeria monocytogenes (Inf. Immun.1998; 66:1106-1112). These signal­ing pathways were implicated either in the L. monocytogenes invasionprocess or cytokines expression induced by S. typhimurium. The purpose ofthis study was to investigate the ability of EPEC to activate MAP kinasesin intestinal cells and to correlate these signaling pathways with cellularresponses to EPEC infection. METHODS: T84 cells were infected forvarious times with the wild type (WT) EPEC strain E2348/69 or mutatedstrains: JPNl5 (~pMAR), CVD206(~eae A) and CVD452(~escN::aph

A-3). Prior infection, cells were serum deprived for 24 h. Western blotswere performed with anti-phosphotyrosine antibodies (aPtyr) and antibod­ies toward the phosphorylated forms of different members of MAP kinasefamily ( ERKI/2, p38/HOG and JNKlSAPK). Invasion was performed onfilter grown T84 cells by the gentamycin protection assay. Actin accumu­lation was followed by immunofluorescence using FITC-phalloidine. RE­SULTS: After 1 hour infection, E2348/69 induced tyrosine phosphoryla­tion of several proteins in T84 cells: the major substrats were localized atMW 52-46 kDa: we identified these proteins as p46 and p52 SHC isoforms.By contrast, SHC was not phosphorylated when the infection was per­formed with the JPNl5, CVD206 and CVD452 mutants. Kinetics studiesrevealed that all three MAP kinases (ERKII2, p38/hog and JNKlSAPK)were activated in the cells infected for 1 hour by the WT strain E2348/69but not by the mutants JPNI5, CVD206 and CVD452. Treatment of cellswith specific p38 MAP kinases inhibitor (SB203580 ), or MEKI inhibitor(PD98052) did not affect actin accumulation beneath the attached WTbacteria. But, PD98052 treatment reduced by 50 percent the number ofintracellular bacteria. CONCLUSIONS: EPEC induce activation ofERKI/2, p38 and JNK MAP kinases which is correlated with the fullyexpressed pathogenic phenotype of these bacteria (localized adherence,intimate attachment and type III protein secretion system). We show thatERKI/2 and p38 MAP kinases are not involved in the actin modificationinduced by EPEC, but ERK1I2 participate in bacterial internalization. Therespective implication of the three MAP kinases in cytokines expressionand control of epithelial cell apoptosis, are under investigation.

GASTROENTEROLOGY Vol. 118, No.4

674

SACCHAROMYCES BOULARDll EXERT AN EFFECT ON EPI­THELIAL CELL AND MODULATE SIGNAL TRANSDUCTIONPATHWAYS INDUCED IN EPEC-INFECTED T84 CELLS.Dorota Czerucka, Stephanie Dahan, Baharia Mograbi, Bernard Rossi,Patrick Rampal, Univ Nice-Sophia Antipolis, Nice, France.

The yeast Saccharomyces boulardii (S.b) is administrated in WesternEurope because of its antidiarrheal properties. In a previous study, we havedemonstrated that S.b maintain barrier function of T84 cells infected byEnteropathogenic Escherichia coli (EPEC). We have also demonstratedthat the yeast did not affect the number of cell-associated bacteria butdecreased the number of intracellular bacteria (Gastroenterology 1999;116:A560). AIM: Since EPEC pathogenesis was strickly correlated withthe induction of signal transduction pathways including phosphorylation ofhost cell proteins, the purpose of this study was to investigate the effect ofS.b on signal transduction pathways induced in EPEC-infected T84 cells.METHODS: T84 cells were infected for I hour with EPEC strain E2348/69alone (100 bacteria/cell) or in the presence of S.b (10 yeast/cell), and thewhole cell lysates were analyzed by western blotting using followingantibodies: anti-phosphotyrosine (aPtyr) or anti phosphorylated-ERKI/2.RESULTS: EPEC induced tyrosine phosphorylation of several proteins inT84 cells: the major phosphorylated substrates were proteins localized at52-46 kDa. We identified by immunoprecipitation that these proteinscorresponded to p46SHC and p52sHC: phosphorylation of these two SHCisoforms were significantly decreased in the presence of S.b. As SHC wasthe upstream regulatory protein of mitogen-activated-protein (MAP) ki­nases ERKI/2 pathway, ERKI/2 activation was assessed in cells infectedin the absence or presence of yeast. EPEC induced the activation ofERKI/2. When infection was performed in the presence of yeast, ERKII2were significantly less phosphorylated. CONCLUSION: EPEC infectioninduced tyrosine phosphorylation of several proteins in T84 cells: most ofthem were less phosphorylated when infection was performed in thepresence of yeast. Activation of ERK1I2 MAP kinase pathways waslowered in the presence of S.b. Recently, we have shown that activation ofERKI/2 MAP kinases was implicated in EPEC internalization, thus themodulation of this pathwaycould account for the decrease of intracellularbacteria observed in the presence of yeast. The consequence of inhibitionof tyrosine phosphorylation of other proteins observed in cells infected inthe presence of yeast and their implication in the protective effect of S.bareunder investigation. This research was funded by Laboratoires BIOCO­DEX, France.

675

ANALYSIS USING HIGH-DENSITY CDNA ARRAYS OF AL­TERED GENE EXPRESSION IN HUMAN INTESTINAL EPITHE­LIAL CELLS IN RESPONSE TO INFECTION WITH THE INVA­SIVE ENTERIC BACTERIA SALMONELLA.Lars Eckmann, Jennifer R. Smith, Michael P. Housley, Michael B.Dwinell, Martin F. Kagnoff, Univ of CA, San Diego, La Jolla, CA.

Many clinically important enteric pathogens initiate disease by invadingand passing through the intestinal epithelium, a process accompanied byincreased epithelial expression of proinfiammatory cytokines and probablyother, currently unidentified genes. New techniques allow the parallelanalysis of mRNA expession for a large number of genes. The aim of thesestudies was to apply these techniques to further define the repertoire ofintestinal epithelial cell responses to infection with invasive bacteria, usingSalmonella as a prototypic pathogen. Methods: Hybrid selection on twosets of high-density cDNA arrays (GF2l1 Gene Filter Release I fromResearch Genetics, Atlas CytokinelReceptor cDNA Expression Array fromClontech) was used to characterize the mRNA expression profile of-4,300 genes in human HT-29 intestinal epithelial cells after infectionwith Salmonella dublin. Selected findings were further evaluated by RT­PCR and protein assays. Results: cDNA array analysis revealed thatinfection of intestinal epithelial cells with Salmonella significantly upregu­lated mRNA expression of a relatively small fraction (5-20%) of all genestested. Of these, several cytokines (e.g. G-CSF, inhibin A, EBB, IL-8,MIP-2a), kinases (e.g. TKT, Eck, HEK), transcription factors (e.g. IRF-l)and HLA class I were the most prominent. RT-PCR analysis of mRNAexpression of a selected subset of genes shown to be both expressed andupregulated by cDNA array analysis confirmed expression, irrespective ofregulation, for 89% (16/18) of the tested genes, and both expression andupregulation for 63% (10/16) of the tested genes. Protein assays furtherconfirmed increased expression for 4/4 tested genes whose epithelialmRNA expression increased after Salmonella infection, as shown byeDNA array analysis and subsequent RT-PCR. Conclusions: These find­ings expand the repertoire of known epithelial cell responses to infectionwith an invasive enteric pathogen, and form a foundation for defining therole of the intestinal epithelium in the pathogenesis of enteric infections.The results also show that assessment of mRNA expression profiles bycDNA array analysis is a powerful approach to discovering genes notpreviously known to be regulated in response to microbial infection and forcharacterizing and understanding host-pathogen interactions. Supported byNIH DK35108 and the CCFA.