Subversion of human intestinal mucosa innate immunity by a Crohn's disease-associated E. coli.

Adherent-invasive Escherichia coli (AIEC), associated with Crohn's disease, are likely candidate contributory factors in the disease. However, signaling pathways involved in human intestinal mucosa innate host response to AIEC remain unknown. Here we use a 3D model of human intestinal mucosa explant culture to explore the effects of the AIEC strain LF82 on two innate immunity platforms, i.e., the inflammasome through evaluation of caspase-1 status, and NFκB signaling. We showed that LF82 bacteria enter and survive within a few intestinal epithelial cells and macrophages, without altering the mucosa overall architecture. Although 4-h infection with a Salmonella strain caused crypt disorganization, caspase-1 activation, and mature IL-18 production, LF82 bacteria were unable to activate caspase-1 and induce IL-18 production. In parallel, LF82 bacteria activated NFκB signaling in epithelial cells through IκBα phosphorylation, NFκBp65 nuclear translocation, and TNFα secretion. In addition, NFκB activation was crucial for the maintenance of epithelial homeostasis upon LF82 infection. In conclusion, here we decipher at the whole-mucosa level the mechanisms of the LF82-induced subversion of innate immunity that, by maintaining host cell integrity, ensure intracellular bacteria survival.

Galanin receptors in a hamster pancreatic beta-cell tumor: identification and molecular characterization.

High affinity binding sites for galanin are identified and characterized in membranes from a hamster pancreatic beta-cell tumor. Using the radioiodinated peptide [125I] galanin, interaction of the peptide with pancreatic membranes is shown to be saturable, reversible, and time, temperature, membrane protein concentration, pH, and ionic strength dependent. In optimized equilibrium conditions of binding (90 min at 10 C), native galanin competitively inhibits the binding of [125I]galanin in a dose-dependent manner (from 10(-11)-10(-8) M); half-maximal inhibition is induced by 1 nM peptide. Scatchard analysis indicates the existence of a single population of sites of high affinity (Kd = 1.5 nM) and low capacity (44 fmol/mg protein). The monophasic dissociation process confirms the homogeneity of galanin-binding sites. Galanin-binding sites are highly specific, since apart from native galanin, none of the numerous biologically active peptides tested competes with [125I] galanin for binding to pancreatic membranes. The cross-linking of [125I]galanin to beta-cell membranes is performed using the chemical bifunctional reagent ethylene glycol bis-(succinimidyl succinate). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in the presence or absence of dithiothreitol, one single band of 57,000 mol wt is observed, which may be corresponding to the [125I]galanin-receptor complex. Indeed, labeling of this 57,000 mol wt component is abolished only by native galanin but is unaffected by various other digestive peptides. Assuming one molecule of [125I]galanin is bound per molecule of protein, a 54,000 mol wt protein is identified as the pancreatic galanin receptor. In conclusion, our results indicate for the first time the identification of galanin receptors. Their presence in pancreatic beta-cells suggests a direct role of galanin in regulating endocrine beta-cell function.

Peptide-YY and neuropeptide-Y inhibit vasoactive intestinal peptide-stimulated adenosine 3',5'-monophosphate production in rat small intestine: structural requirements of peptides for interacting with peptide-YY-preferring receptors.

Previous binding studies indicated that peptide-YY (PYY) and neuropeptide-Y (NPY) shared a common PYY-preferring receptor site in rat small intestinal epithelium. We showed here that PYY and NPY inhibited vasoactive intestinal peptide (VIP)-stimulated cAMP production in epithelial cells isolated from rat small intestine and examined their structure-activity relationship. Inhibition of VIP-stimulated cAMP by PYY or NPY is time and dose dependent; half-maximal effects were observed for 10 and 107 nM, respectively. In contrast, the structurally related peptide, pancreatic polypeptide, was only active at 1 microM. PYY or NPY reduced the efficacy of VIP by about 50% without altering its potency. Both peptides also suppressed prostaglandin E1-, prostaglandin E2-, and forskolin-stimulated cAMP production and reduced basal cAMP levels. Their inhibitory effects were observed throughout the small intestine, including duodenum, jejunum, and ileum, but not in large intestine. PYY or NPY and epinephrine (through alpha 2-adrenergic receptors) did not exert additive inhibitory effects on intestinal cAMP production. Several fragments of PYY and NPY were used to characterize their structural requirement for inhibiting VIP-stimulated cAMP production and competing with [125I]PYY for binding to intestinal membranes. A highly significant correlation was observed between IC50 values measured in the two assays. No partial sequence of PYY retained the full activity of intact PYY, but the C-terminal portion of PYY was shown to be much more important than the N-terminal portion. Deletion of 21 amino acids from the N-terminus [PYY-(22-36)] only resulted in a 4- to 5-fold decrease in potency compared to that of PYY-(1-36). In contrast, PYY-(27-36) exhibited a drastic loss of potency. The N-terminal fragments PYY-(1-22) and PYY-(1-28) also had very low potencies. Similar results were obtained with NPY fragments. These results provide the first insight on the negative coupling of PYY-preferring receptors with the cAMP production system in small intestine and evidence of the crucial role of the C-terminal portion of PYY in interaction with these receptors.

Immune modulation of blood leukocytes in humans by lactic acid bacteria: criteria for strain selection.

Lactic acid bacteria in food can transiently colonize the intestine and exert beneficial effects (probiotic). Survival during intestinal transit or adhesion to epithelium or both seem to be important for modifying the host's immune reactivity. Because Lactobacillus acidophilus strain La1 is adherent to enterocytes in vitro, we hypothesize that contact with immune cells may occur in vivo. However, Bifidobacterium bifidum strain Bb12, which shows high fecal colonization, is another potential immunomodulator. Twenty-eight volunteers were divided into two groups and given a fermented product containing one of the two strains. Lymphocyte subsets and leukocyte phagocytic activity were studied in blood. No modifications were detected in lymphocyte subsets. In contrast, phagocytosis of Escherichia coli ssp. was enhanced in both groups (P < 0.001 for both). Bacterial adhesion to enterocytes, fecal colonization, or both seem to be valuable selection criteria for immunomodulation. Antiinfective mechanisms of defense can be enhanced after ingestion of specific lactic acid bacteria strains.

Characterization of solubilized isoquinoline binding sites from rat intestine using 6,7-dimethoxy 4-(4'-amino, 3'(125I)iodobenzyl)isoquinoline.

In rat tissues, the specific binding of 6,7-dimethoxy 4-(4'-amino, 3'(125I)iodobenzyl)-isoquinoline is distributed as follows: aorta greater than pancreas greater than liver greater than intestine greater than stomach greater than lung greater than skeletal muscle greater than heart greater than brain. In aorta and intestine 125I-DMABI is specifically covalently incorporated after direct u.v. photolabeling, in a major polypeptide of Mr 36,000 daltons, and a minor polypeptide of Mr 52,000 daltons. In intestine another smaller minor polypeptide of Mr 26,000 is observed. In intestine a variety of isoquinolines are tested for their ability to inhibit the covalent photo-incorporation of 125I-DMABI. Inhibitory potency is influenced by 6,7-substitutions, e.g. 6,7-dimethoxy, and by the presence of benzyl ring in C-1 and C-4 positions. Isoquinoline is much more potent than tetrahydroisoquinoline. 125I-DMABI intestinal binding site is solubilized using Triton X-100. Layered on Sephadex G-25 column, a high specific peak of radioactivity is eluted in the void volume of the column. The 125I-DMABI binding protein loaded onto a Sephacryl S-300 column is eluted as a single peak corresponding to a species with a Stokes radius of 43.5 A. The sedimentation coefficient of the 125I-DMABI binding protein is measured by ultracentrifugation of 5.5 S, using 5-20% sucrose gradient. The calculated molecular weight of the intestinal 125I-DMABI binding protein is estimated at 110,000 daltons.

Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion.

Salmonella typhimurium and enteropathogenic Escherichia coli (EPEC) were found to adhere to the brush border of differentiated human intestinal epithelial Caco-2 cells in culture, whereas Yersinia pseudotuberculosis and Listeria monocytogenes adhered to the periphery of undifferentiated Caco-2 cells. All these enterovirulent strains invaded the Caco-2 cells. Using a heat-killed human Lactobacillus acidophilus (strain LB) which strongly adheres both to undifferentiated and differentiated Caco-2 cells, we have studied inhibition of cell association with and invasion within Caco-2 cells by enterovirulent bacteria. Living and heat-killed Lactobacillus acidophilus strain LB inhibited both cell association and invasion of Caco-2 cells by enterovirulent bacteria in a concentration-dependent manner. The mechanism of inhibition of both adhesion and invasion appears to be due to steric hindrance of human enterocytic pathogen receptors by whole-cell lactobacilli rather than to a specific blockade of receptors.

Competitive exclusion of diarrheagenic Escherichia coli (ETEC) from human enterocyte-like Caco-2 cells by heat-killed Lactobacillus.

Diarrheagenic Escherichia coli (ETEC) bearing CFA/I or CFA/II adhesive factors specifically adhere onto the brush border of the polarized epithelial human intestinal Caco-2 cells in culture. Heat-killed Lactobacillus acidophilus strain LB, that adheres onto Caco-2 cells, inhibits diarrheagenic Escherichia coli adhesion in a concentration-dependent manner. Since the L. acidophilus does not express ETEC-CFA adhesive factors, it can be postulated that the heat-killed L. acidophilus LB cells inhibit diarrheagenic E. coli attachment by steric hindrance of the human enterocytic ETEC receptors.

Human cultured intestinal cells express attachment sites for uropathogenic Escherichia coli bearing adhesins of the Dr adhesin family.

We have recently demonstrated that cultured human intestinal HT-29 and Caco-2 cell lines express receptors for the F1845 fimbrial adhesin harbored by the diarrheagenic C1845 Escherichia coli (Kernéis et al., Infect. Immun. 59 (1991) 4013-4018). This adhesin belongs to a family of adhesins including the Dr hemagglutinin and the afimbrial adhesin AFA-I harbored by uropathogenic E. coli. Here we investigated the cell association of laboratory E. coli strains expressing the Dr hemagglutinin and the afimbrial adhesin AFA-I with human cultured enterocyte-like or mucosecreting cells. We observed that the E. coli strains bearing these adhesins adhere both to human intestinal undifferentiated and differentiated fluid-transporting cells, and to mucus-secreting cells. This result strongly suggests a high capacity of intestinal colonization for the uropathogenic E. coli harboring adhesive factors belonging to the Dr adhesin family. These results further corroborate the intestinal colonization by uropathogenic E. coli of the Dr family related to the fecal-perineal-urethral hypothesis of urinary tract infection pathogenesis.

Molecular properties of the papaverine binding sites identified in human colonic membranes with 6,7-dimethoxy-4-(4'-amino,3'-[125I]iodobenzyl) isoquinoline as probe.

The binding sites for papaverine in human intestine were studied with the radioiodinated papaverine derivative, 6,7-dimethoxy-4-(4'-amino,3'-[125I]iodobenzyl)isoquinoline [( 125I]DMABI) as a probe. This drug was shown to bind to two specific binding sites in human colon membranes; one with a low capacity (Kd = 0.02 +/- 0.01 microM, Bmax = 0.77 +/- 0.15 fmol/mg) and another with a high capacity (Kd = 12 +/- 1.5 microM, Bmax = 167 +/- 20 fmol/mg). The ability of various 4-benzyl isoquinolines derivatives, to inhibit radioligand binding was not influenced by 4'-substitutions, but was influenced by 6,7-substitutions, e.g. 6-hydroxy greater than 7-hydroxy greater than 6,7-dihydroxy; and by other substitutions, e.g. 1-CH3 greater than N-oxyde. The papaverine probe was further used to examine structural aspects of human colon papaverine binding sites. For this purpose, [125I]DMABI-labeled membranes were irradiated with U.V. for 15 min at 4 degrees C. Subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes revealed a major, specifically labeled protein of Mr = 36,000 Da. This photoaffinity labeling was protected by unlabeled DMABI in a dose-dependent manner (IC50: 0.5 microM).

The Lactobacillus plantarum strain ACA-DC287 isolated from a Greek cheese demonstrates antagonistic activity in vitro and in vivo against Salmonella enterica serovar Typhimurium.

AIMS: The purpose of this study was to investigate the antibacterial activity of the Xynotyri cheese isolate Lactobacillus plantarum ACA-DC287 using a set of in vitro and in vivo assays. METHODS AND RESULTS: The co-culture of L. plantarum strain ACA-DC287 and Salmonella enterica serovar Typhimurium strain SL1344 results in the killing of the pathogen. The killing activity was produced mainly by non-lactic acid molecule(s) that were present in the cell-free culture supernatant of the L. plantarum strain ACA-DC287. The culture of the L. plantarum strain ACA-DC287 inhibited the penetration of S. typhimurium SL1344 into cultured human enterocyte-like Caco-2/TC7 cells. In conventional mice infected with S. typhimurium SL1344, the intake of L. plantarum strain ACA-DC287 results in a decrease in the levels of Salmonella associated with intestinal tissues or those present in the intestinal contents. In germ-free mice, the L. plantarum strain ACA-DC287 colonized the gastrointestinal tract. CONCLUSIONS: The L. plantarum strain ACA-DC287 strain exerts anti-Salmonella activity similar that of the established probiotic strains Lactobacillus rhamnosus GG, Lactobacillus casei Shirota YIT9029 and Lactobacillus johnsonii La1. SIGNIFICANCE AND IMPACT OF THE STUDY: The observation that a selected cheese Lactobacillus strain exerted antibacterial activity that was similar to those of probiotic Lactobacillus strains, is of interest for the use of this strain as an adjunct strain for the production of health-giving cheeses.

In vitro antibacterial activity of Lactobacillus helveticus strain KS300 against diarrhoeagenic, uropathogenic and vaginosis-associated bacteria.

AIMS: The purpose of this study was to investigate in vitro the antibacterial activity of the Lactobacillus helveticus strain KS300 against vaginosis-associated bacteria including Gardnerella vaginalis and Prevotella bivia, uropathogenic Escherichia coli, and diarrhoeagenic Salmonella enterica serovar Typhimurium. METHODS AND RESULTS: The KS300 strain inhibited the growth of G. vaginalis, P. bivia, S. typhimurium, and pathogenic E. coli. After direct co-culture, data show that the Lactobacillus strain decreased the viability of G. vaginalis, P. bivia, S. typhimurium, and pathogenic E. coli. The adhering KS300 strain inhibited the adhesion of G. vaginalis DSM 4944 and uropathogenic Dr-positive E. coli IH11128 onto HeLa cells. Moreover, the KS300 strain inhibited the internalization of uropathogenic Dr-positive E. coli IH11128 within HeLa cells and S. typhimurium SL1344 within Caco-2/TC7 cells. CONCLUSIONS: The findings demonstrate that L. helveticus strain KS300 is adhesive onto cultured human cells and has antagonistic activities against vaginosis-associated, uropathogenic and diarrhoeagenic pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Adhering L. helveticus strain KS300 is a potential probiotic strain displaying a strain-specific array of in vitro antibacterial activities.

Rotavirus-induced structural and functional alterations in tight junctions of polarized intestinal Caco-2 cell monolayers.

We provide here new insights into rotavirus (RRV) pathogenicity by showing that RRV infection promotes structural and functional injuries localized at the tight junctions (TJ) in the cell-cell junctional complex of cultured polarized human intestinal Caco-2 cells forming monolayers. RRV infection resulted in a progressive increase in the paracellular permeability to [(3)H]mannitol as a function of the time postinfection. We observed a disorganization of the TJ-associated protein occludin as a function of the time postinfection, whereas distribution of the zonula adherens associated E-cadherin was not affected. These structural and functional RRV-induced TJ injuries were not accompanied by alteration in cell and monolayer integrity, as assessed by the lack of change in transepithelial membrane resistance and lactate dehydrogenase release. Finally, using the stabilizer of actin filaments Jasplakinolide, we demonstrated that the RRV-induced structural and functional alterations in TJ are independent of the RRV-induced apical F-actin rearrangements.

Identification and molecular characterization of the isoquinoline rat intestinal binding site using 6,7-dimethoxy-4-(4'-amino-3'-[125I]iodobenzyl) isoquinoline.

The mechanism of the relaxant action of isoquinolines on smooth muscle is conjectural. In order to gain further insight into the intestinal action of isoquinolines, we have synthesized an isoquinoline derivative which can be radioiodinated, resulting in the obtention of a ligand with a high specific activity. 6,7-Dimethoxy-4-(4'-aminobenzyl) isoquinoline (DMABI) is an arylamine analogue of the most relaxating isoquinoline derivative, i.e., 6,7-dimethoxy-4-(4'-chlorobenzyl) isoquinoline. Its iodinated derivative, 6,7-dimethoxy-4-(4'-amino-3'-[125I]iodobenzyl) isoquinoline (125I-DMABI) binds reversibly to rat intestinal membranes. Binding is rapid, saturable, and temperature dependent. The binding of 125I-DMABI to intestinal membranes is competitively inhibited by identical concentrations of unlabeled DMABI or iodo-DMABI in the range between 10(-8) and 10(-5)M. Scatchard analysis indicates the existence of two classes of binding sites: a class with a low capacity (14 +/- 2 pmol/mg of protein) and a Kd = 0.10 +/- 0.02 microM, and a class with a high capacity (240 +/- 31 pmol/mg of protein) and a Kd = 8.0 +/- 1.1 microM. Specific binding of the radioiodinated ligand is inhibited by a variety of 4-benzyl isoquinolines and 1-benzyl isoquinolines. Structure-activity relationship demonstrates the primordial role of C-6 and C-7 methoxy groups and the important role of 4-benzyl on configuration related to the isoquinoline nucleus. A high significant correlation between competitive binding (Ki) and relaxant effect in rat intestine (IC50) is observed and strongly suggests that the isoquinoline-binding site mediates the pharmacologic response. Upon photolysis, this ligand incorporates irreversibly into rat intestinal membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography reveal a major 125I-DMABI-labeled protein with molecular weight of 36,000 and two minor proteins with molecular weights of 52,000 and 26,000. The labeling of these proteins is specific since it is completely abolished by 100 microM DMABI. Scanning of autoradiographs and integration of peaks show that the probe binds with the same apparent affinity to the three proteins. These findings indicate the utility of this novel high affinity radioiodinated probe as a tool for elucidating the mechanism of action of isoquinoline.

Piracy of decay-accelerating factor (CD55) signal transduction by the diffusely adhering strain Escherichia coli C1845 promotes cytoskeletal F-actin rearrangements in cultured human intestinal INT407 cells.

Diffusely adhering Escherichia coli (DAEC) C1845 (clinical isolate) harboring the fimbrial adhesin F1845 can infect cultured human differentiated intestinal epithelial cells; this process is followed by the disassembly of the actin network in the apical domain. The aim of this study was to examine the mechanism by which DAEC C1845 promotes F-actin rearrangements. For this purpose, we used a human embryonic intestinal cell line (INT407) expressing the membrane-associated glycosylphosphatidylinositol (GPI) protein-anchored decay-accelerating factor (DAF), the receptor of the F1845 adhesin. We show here that infection of INT407 cells by DAEC C1845 can provoke dramatic F-actin rearrangements without cell entry. Clustering of phosphotyrosines was observed, revealing that the DAEC C1845-DAF interaction involves the recruitment of signal transduction molecules. A pharmacological approach with a subset of inhibitors of signal transduction molecules was used to identify the cascade of signal transduction molecules that are coupled to the DAF, that are activated upon infection, and that promote the F-actin rearrangements. DAEC C1845-induced F-actin rearrangements can be blocked dose dependently by protein tyrosine kinase, phospholipase Cgamma, phosphatidylinositol 3-kinase, protein kinase C, and Ca2+ inhibitors. F-actin rearrangements and blocking by inhibitors were observed after infection of the cells with two E. coli recombinants carrying the plasmids containing the fimbrial adhesin F1845 or the fimbrial hemagglutinin Dr, belonging to the same family of adhesins. These findings show that the DAEC Dr family of pathogens promotes alterations in the intestinal cell cytoskeleton by piracy of the DAF-GPI signal cascade without bacterial cell entry.

Escherichia coli strains colonising the gastrointestinal tract protect germfree mice against Salmonella typhimurium infection.

BACKGROUND: Escherichia coli is part of the normal gastrointestinal microflora which exerts a barrier effect against enteropathogens. Several E coli strains develop a protective effect against other Enterobacteriaceae. AIMS: Two E coli strains, EM0, a human faecal strain, and JM105 K-12 were tested for their ability to prevent in vivo and in vitro infection by Salmonella typhimurium C5. METHODS: Inhibition of C5 cell invasion by E coli was investigated in vitro using Caco-2/TC7 cells. The protective effect of E coli was examined in vivo in germfree or conventional C3H/He/Oujco mice orally infected by the lethal strain C5. RESULTS: EMO expresses haemolysin and cytotoxic necrotising factor in vitro. In vitro, the two strains did not prevent the growth of C5 by secreted microcins or modified cell invasion of C5. In vivo, establishment of EM0 or JM105 in the gut of germfree mice resulted in a significant increase in the number of surviving mice: 11/12 and 9/12, respectively, at 58 days after infection (2x10(6)/mouse) versus 0/12 in control germfree group at 13 days after infection. Colonisation level and translocation rate of C5 were significantly reduced during the three days after infection. In contrast, no reduction in faecal C5 excretion was observed in C5 infected conventional mice (1x10(8)/mouse) receiving the EM0 or JM105 cultures daily. CONCLUSIONS: Establishment of E coli strains, which do not display antimicrobial activity, protects germfree mice against infection and delays the establishment of C5 in the gut. Possible mechanisms of defence are discussed.

Pharmacokinetics of cysteine ethyl ester in rat.

1. The pharmacokinetics of the mucolytic compound 35S-cysteine ethyl ester in rat show that it is completely absorbed after oral administration, with a bioavailability of 0.59. 2. By autoradiography, tissue distribution of 35S-cysteine ethyl ester showed a high affinity for the lung, different to that observed with 35S-cysteine. 3. Unchanged cysteine ethyl ester, cysteine and inorganic sulphates were present in the lung. 4. After oral or i.v. administration of 35S-cysteine ethyl ester the total radioactivity excreted in the urine amounted to 16% dose and 40% was excreted in the faeces, following biliary excretion. It was metabolized to inorganic sulphate, cysteine and taurine, which were excreted in the urine.

Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions.

Thirteen human bifidobacterial strains were tested for their abilities to adhere to human enterocyte-like Caco-2 cells in culture. The adhering strains were also tested for binding to the mucus produced by the human mucus-secreting HT29-MTX cell line in culture. A high level of calcium-independent adherence was observed for Bifidobacterium breve 4, for Bifidobacterium infantis 1, and for three fresh human isolates from adults. As observed by scanning electron microscopy, adhesion occurs to the apical brush border of the enterocytic Caco-2 cells and to the mucus secreted by the HT29-MTX mucus-secreting cells. The bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage. The adhesion to Caco-2 cells of bifidobacteria did not require calcium and was mediated by a proteinaceous adhesion-promoting factor which was present both in the bacterial whole cells and in the spent supernatant of bifidobacterium culture. This adhesion-promoting factor appeared species specific, as are the adhesion-promoting factors of lactobacilli. We investigated the inhibitory effect of adhering human bifidobacterial strains against intestinal cell monolayer colonization by a variety of diarrheagenic bacteria. B. breve 4, B. infantis 1, and fresh human isolates were shown to inhibit cell association of enterotoxigenic, enteropathogenic, diffusely adhering Escherichia coli and Salmonella typhimurium strains to enterocytic Caco-2 cells in a concentration-dependent manner. Moreover, B. breve 4 and B. infantis 1 strains inhibited, dose dependently, Caco-2 cell invasion by enteropathogenic E. coli, Yersinia pseudotuberculosis, and S. typhimurium strains.

Adhering heat-killed human Lactobacillus acidophilus, strain LB, inhibits the process of pathogenicity of diarrhoeagenic bacteria in cultured human intestinal cells.

Heat-killed L. acidophilus, strain LB, was tested for its ability to adhere in vitro onto human enterocyte-like Caco-2 and muco-secreting HT29-MTX cells in culture. The heat-killed LB bacteria exhibited a high adhesive property. A diffuse pattern of adhesion was observed to the undifferentiated cells, the apical brush border of the enterocytic cells, and to the mucus layer that covered the surface of the mucus-secreting cells. The inhibitory effect of heat-killed LB organisms against the human intestinal Caco-2 cell-adhesion and cell-invasion by a large variety of diarrhoeagenic bacteria was investigated. The following dose-dependent inhibitions were obtained: (i) against the cell-association of enterotoxigenic, diffusely-adhering and enteropathogenic Escherichia coli, Listeria monocytogenes, Yersinia pseudotuberculosis, and Salmonella typhimurium; (ii) against the cell-invasion by enteropathogenic Escherichia coli, Yersinia pseudotuberculosis, Listeria monocytogenes and Salmonella typhimurium.

Adhesion of three Lactobacillus strains to human urinary and intestinal epithelial cells.

Three strains of urogenital lactobacilli were found to adhere in phosphate buffered saline to human uroepithelial cells in vitro according to thermodynamic principles, and to adhere in culture medium to intestinal cells with no such correlation. The most hydrophilic strain (water contact angle 54 degrees) L. casei RC-17 was the most adherent to uroepithelial (118 bacteria per cell) and intestinal cells (165 bacteria per cell). A direct correlation was found between bacterial hydrophilicity and adhesion to uroepithelial cells for the three strains tested. An extracellular adhesin, which appeared to be proteinaceous, and a trypsin-insensitive cell wall adhesin were identified. It was evident that high levels of bacterial retention could be reproduced in vitro, perhaps indicating the potential colonizing capacity of these strains in vivo.

How intestinal epithelial cell differentiation inhibits the cell-entry of Yersinia pseudotuberculosis in colon carcinoma Caco-2 cell line in culture.

In the human intestine, target cells of enteropathogens differentiate during cell migration along the crypt-villus axis. We have recently provided evidence that intestinal cell differentiation up-regulates intestinal cell infection by the noninvasive enterotoxigenic Escherichia coli [5, 23]. Several enterovirulent bacteria can penetrate intestinal epithelial cells, which are normally nonphagocytic. To document the role of intestinal epithelial cell differentiation in the pathogenesis of enteroinvasive bacteria, we examined here the intestinal cell-association and cell-entry of Yersinia pseudotuberculosis as a function of cell differentiation. For this purpose we used the colon carcinoma Caco-2 cell line in culture, which provides the most useful tool for the study of intestinal epithelial cell differentiation, because of its unique ability to spontaneously differentiate upon reaching confluence in normal culture condition. We report here that the thermoregulated inv and ail loci of Y. pseudotuberculosis have distinct roles in infection of Caco-2 cells. The ail locus initiates the cell-association and the inv locus initiates both the cell-association and the cell-entry processes. Moreover, we observed that: (i) both the bacterial cell-association (ail) and the bacterial cell-invasion (inv) occur at subconfluence when the Caco-2 cells are undifferentiated, and (ii) these processes are arrested when the differentiation commences. Since the integrin-beta 1 heterodimers are involved in cell-entry of Y. pseudotuberculosis in several mammalian cells, we further examined which beta 1 integrin promotes bacterial cell-entry in Caco-2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)