Saccharomyces boulardii preserves the barrier function and modulates the signal transduction pathway induced in enteropathogenic Escherichia coli-infected T84 cells

Use of the nonpathogenic yeast Saccharomyces boulardii in the treatment of infectious diarrhea has attracted growing interest. The present study designed to investigate the effect of this yeast on enteropathogenic Escherichia coli (EPEC)-associated disease demonstrates that S. boulardii abrogated the alterations induced by an EPEC strain on transepithelial resistance, [(3)H]inulin flux, and ZO-1 distribution in T84 cells. Moreover, EPEC-mediated apoptosis of epithelial cells was delayed in the presence of S. boulardii. The yeast did not modify the number of adherent bacteria but lowered by 50% the number of intracellular bacteria. Infection by EPEC induced tyrosine phosphorylation of several proteins in T84 cells, including p46 and p52 SHC isoforms, that was attenuated in the presence of S. boulardii. Similarly, EPEC-induced activation of the ERK1/2 mitogen-activated protein (MAP) kinase pathway was diminished in the presence of the yeast. Interestingly, inhibition of the ERK1/2 pathway with the specific inhibitor PD 98059 decreased EPEC internalization, suggesting that modulation of the ERK1/2 MAP pathway might account for the lowering of the number of intracellular bacteria observed in the presence of S. boulardii. Altogether, this study demonstrated that S. boulardii exerts a protective effect on epithelial cells after EPEC adhesion by modulating the signaling pathway induced by bacterial infection.     

Enhanced uropathogenic Escherichia coli-induced infection in uroepithelial cells by sugar through TLR-4 and JAK/STAT1 signaling pathways

BackgroundPatients with diabetes mellitus (DM) have higher incidence and more severe urinary tract infections (UTIs) for longer duration than those of the patients without DM. It causes more complicated etiologies during uropathogenic Escherichia coli (UPEC) infection. However, studies regarding the molecular mechanism are scarce.MethodsThe present study (1) aimed to verify if sugar influences the process of UPEC-induced cystitis and invasion into the uroepithelial cells and (2) illustrated the mechanism of effects for sugar enhanced the UPEC infection into uroepithelial cells is related to TLR-4-mediated and JAK/STAT1-dependent pathway.ResultsThe results of the present study indicated that sugar can enhance UPEC infection in uroepithelial cells by up-regulating the transduced circuit between TLR-4-mediated UPEC interaction and JAK/STAT-1 signal pathways. The results of the inhibitor-co-incubating experiments demonstrated that the mechanism involved in the synergistic amplification of TLR-4-mediated UPEC interaction and JAK/STAT1 signaling pathways is responsible for the increased UPEC infection in uroepithelial cells.ConclusionThe results also proved that STAT-1 plays a critical role in the regulation of UPEC invasion and infection in the uroepithelial cells, especially those pretreated with glucose. The present study suggests a possible therapeutic approach to preferentially suppress UPEC infection during UTIs in the patients with diabetes.     

MAP-kinase signaling pathways in T cells.

The family of MAP-kinases include ERKs, p38 MAP-kinases and JNKs. Recently, the role of MAP-kinases in T lymphocytes has attracted particular interest. Genetically modified mouse models have brought insight into the specific function of each MAP-kinase pathway in T lymphocyte biology. Studies clearly show that these pathways are not redundant and that the role of each pathway depends on the T cell type and differentiation stage.     

Adherence of enterohemorrhagic Escherichia coli strains to a human colonic epithelial cell line (T84).

Enterohemorrhagic Escherichia coli (EHEC) produce Shiga-like toxins and attach to certain tissue culture cells. T84 cells are human colonic carcinoma cells. Unlike previously studied cell lines, T84 cells grown on collagen-coated surfaces polarize and produce tight junctions and desmosomes, forming a colonic epithelial cell layer in vitro. The purpose of this study was to examine the attachment of EHEC strains to the T84 cell line as a possibly more relevant in vitro model of EHEC adherence. Twelve EHEC strains were grown overnight in Penassay broth, suspended in minimal essential medium with and without 0.5% mannose, and incubated for 1 to 3 h with 5- to 7-day-old T84 cell monolayers grown on collagen-coated coverslips. The bacteria were removed, and attachment was quantitated microscopically. For both E. coli O157:H7 and other EHEC serotypes, there were marked differences in adherence between strains (range of 152 to 3 bacteria per oil immersion field). Mannose partially inhibited the adherence of some EHEC strains. Adherence to the T84 cells appeared to be related to the amount of pili present and not to the serotype. Electron micrographs showed that a highly adherent strain (strain 43-12) tended to form microcolonies in the area of tight junctions on the T84 cell monolayers. In addition, the attachment of these EHEC strains to T84 cells correlated with their ability to adhere to isolated rabbit colonocytes (r = 0.91, P = 0.00004; without mannose) (r = 0.60, P = 0.04; with mannose). These data show that there are EHEC strain-related differences in adherence which can be demonstrated in a human-derived colonic epithelial cell line (T84) and that these cells can be used to study EHEC adherence.     

Flagellin of enteropathogenic Escherichia coli stimulates interleukin-8 production in T84 cells

The type III secretion system (TTSS) of enteropathogenic Escherichia coli (EPEC) has been associated with the ability of these bacteria to induce secretion of proinflammatory cytokines, including interleukin-8 (IL-8), in cultured epithelial cells. However, the identity of the effector molecule directly involved in this event is unknown. In this study, we determined that the native flagellar filament and its flagellin monomer are activators of IL-8 release in T84 epithelial cells. Supernatants of wild-type EPEC strain E2348/69 and its isogenic mutants deficient in TTSS (escN) and in production of intimin (eae), grown in Luria-Bertani broth, elicited similar amounts of IL-8 secretion by T84 cells. In contrast, supernatants of EPEC fliC mutants and of B171, a nonflagellated EPEC strain, were defective in inducing IL-8 release, a phenotype that was largely restored by complementation of the fliC gene in the mutant lacking flagella. Purified flagella from E. coli K-12, EPEC serotypes H6 and H34, and enterohemorrhagic E. coli serotype H7 all induced IL-8 release in T84 cells. Induction of IL-8 by purified flagella or His-tagged FliC from EPEC strain E2348/69 was dose dependent and was blocked by a polyclonal anti-H6 antibody. Finally, the mitogen-activated protein kinases (Erk1 and -2 and Jnk) were phosphorylated in flagellin-treated T84 cells, and inhibition of the p38 and Erk pathways significantly decreased the IL-8 response induced by EPEC flagellin. Our data clearly indicate that FliC of EPEC is sufficient to induce IL-8 release in T84 cells and that activation of the Erk and p38 pathways is required for IL-8 induction.     

Saccharomyces boulardii inhibits lipopolysaccharide‐induced activation of human dendritic cells and T cell proliferation

Saccharomyces boulardii (Sb) is a probiotic yeast preparation that has demonstrated efficacy in inflammatory and infectious disorders of the gastrointestinal tract in controlled clinical trials. Although patients clearly benefit from treatment with Sb, little is known on how Sb unfolds its anti‐inflammatory properties in humans. Dendritic cells (DC) balance tolerance and immunity and are involved critically in the control of T cell activation. Thus, they are believed to have a pivotal role in the initiation and perpetuation of chronic inflammatory disorders, not only in the gut. We therefore decided to investigate if Sb modulates DC function. Culture of primary (native, non‐monocyte‐derived) human myeloid CD1c⁺CD11c⁺CD123^– DC (mDC) in the presence of Sb culture supernatant (active component molecular weight < 3 kDa, as evaluated by membrane partition chromatography) reduced significantly expression of the co‐stimulatory molecules CD40 and CD80 (P < 0·01) and the DC mobilization marker CC‐chemokine receptor CCR7 (CD197) (P < 0·001) induced by the prototypical microbial antigen lipopolysaccharide (LPS). Moreover, secretion of key proinflammatory cytokines such as tumour necrosis factor‐α and interleukin (IL)‐6 were notably reduced, while the secretion of anti‐inflammatory IL‐10 increased. Finally, Sb supernatant inhibited the proliferation of naive T cells in a mixed lymphocyte reaction with mDC. In summary, our data suggest that Sb may exhibit part of its anti‐inflammatory potential through modulation of DC phenotype, function and migration by inhibition of their immune response to bacterial microbial surrogate antigens such as LPS.     

Critical roles for stx2, eae, and tir in enterohemorrhagic Escherichia coli-induced diarrhea and intestinal inflammation in infant rabbits

Enterohemorrhagic Escherichia coli (EHEC) is a group of food-borne pathogens that can cause diarrhea, colitis, and the hemolytic uremic syndrome (HUS). The importance of several of the proposed EHEC virulence factors lacks experimental verification in animal models. The limitations of current animal models led us to reexamine the infant rabbit model for the study of EHEC pathogenicity. Here, we report that intragastric inoculation of a Shiga toxin 2 (Stx2)-producing E. coli O157:H7 clinical isolate into infant rabbits led to severe diarrhea and intestinal inflammation but no signs of HUS. We constructed a set of isogenic derivatives of this isolate with deletions in several putative virulence genes, including stx₂, eae, tir, and ehxA, to investigate the contribution of individual virulence factors to EHEC pathogenicity. stx₂ increased the severity and duration of EHEC-induced diarrhea. Furthermore, although stx₂ had no role in EHEC intestinal colonization nor was it required for EHEC-induced inflammation, stx₂ altered how the host responded to EHEC infection by promoting heterophilic infiltration of the colonic epithelium and lamina propria. Intragastric inoculation of purified Stx2 also induced inflammation and diarrhea in this model. Diarrhea and intestinal inflammation were also dependent on EHEC colonization, as EHEC derivatives with deletions in eae and tir did not colonize, form attaching and effacing lesions, or develop clinical signs of disease. Our studies indicate that infant rabbits are a useful model for investigation of the intestinal stage of EHEC pathogenesis and suggest that Shiga toxin may play a critical role in causing diarrhea and inflammation in patients infected with EHEC.     

T84 cells in culture as a model for enteroaggregative Escherichia coli pathogenesis.

Enteroaggregative Escherichia coli (EAEC) is an important cause of persistent diarrhea in many developing parts of the world, yet the pathogenetic mechanisms of EAEC diarrhea are unknown. Experiments with animal models suggest that EAEC strains damage the intestinal mucosa, and a putative cytotoxin has been described. To characterize the mucosal effects of EAEC, we studied strain 042, which we have shown to cause diarrhea in adult volunteers. Strain 042 was incubated in an in vitro organ culture model with biopsy-derived normal intestinal mucosa from pediatric patients. Strain 042 adhered strongly to samples of jejunal, ileal, and colonic mucosa. In addition, scanning electron microscopic examination of in vitro-infected intestinal biopsies revealed cytotoxic effects marked by exfoliation of mucosal epithelial cells. To develop an in vitro model to study these effects, we incubated 042 with polarized monolayers of the human intestinal epithelial cell lines Caco-2 and T84. Strain 042 adhered strongly to T84 cells but not to Caco-2 cells. T84 cells infected with 042 displayed marked toxic effects, most prominently in areas where bacteria were adhering. The apical membrane of damaged cells exhibited vesiculation and shedding of microvilli. The cytoplasm of affected cells displayed subnuclear vacuolization, and in some cases, nuclei of affected cells became separated from the surrounding cytoplasm. Severely affected cells ruptured, releasing their nuclei. Vacuolated remnant cells were seen throughout the monolayer. Strain 042 was not internalized by T84 cells. We concluded that EAEC strain 042 alters intestinal cell morphology, ultimately leading to cell death. Although the factor(s) required for this effect remains to be elucidated, T84 cells may serve as a valuable model in EAEC pathogenesis studies.     

Palmitoylation of CD44 interferes with CD3-mediated signaling in human T lymphocytes

We studied the interactions between CD44 and four differentmonoclonal antl-CD44 antibodies. All four monoclonal anti-CD44antibodies studied (P3H9, Bu52, IM.7, and GKW.A3) act in synergywith human anti-CD2 antibodies in stimulating normal human peripheralblood lymphocytes to proliferate. GKW.A3 and IM.7 but not P3H9or Bu52 Inhibited the proliferation of normal human peripheralblood lymphocytes stimulated by antl-CD3. Interestingly, onlyGKW.A3 and IM.7 stimulated the incorporation of [3H]palmltlcacid and palmitoylation of CD44 molecules by normal human peripheralblood lymphocytes. The two monoclonal antl-CD44 antibodies (P3H9and Bu52) that failed to inhibit antl-CD3 induced proliferationalso failed to induce the incorporation of [³H]palmltlc acid.More Importantly, the inhibitory effects of GKW.A3 were reversedin the presence of cerulenln, an inhibitor of protein palmitoylation.Therefore, palmitoylation of CD44 may interfere with antl-CD3mediated signaling pathways. These data support the hypothesisthat palmitoylation of cell surface receptors may play an activerole in receptor and receptor interactions and signal transductlonin normal human T lymphocytes.     

Activation interferes with the APO-1 pathway in mature human T cells

One of the mechanisms to terminate a specific immune responsemay involve elimination of antigen activated T cells by programmedcell death, apoptosis. Apoptosis in activated T cells may beinduced via the TCR-CD3 complex or/and cell surface moleculeslike the APO-1 (Fas) antigen, a new member of the nerve growthfactor/tumor necrosis factor receptor superfamily. To investigateapoptosis in activated T cells we studied expression of APO-1and sensitivity to APO-1 mediated apoptosis in human peripheralT lymphocytes. APO-1 is not expressed on cord blood and themajority of resting T cells, but on activated T cells. One dayactivated T cells in culture showed activation induced resistanceto apoptosis (ARA). However, after prolonged in vitro culture,6 day activated T cells acquired sensitivity to activation inducedsensitivity to apoptosis (ASA). Restimulation of the ASA⁺ activatedT cells by triggering TCR-CD3 or CD2 induced prollferation andapoptosis in a fraction of the cells. In the surviving fractionof ASA⁺ activated T cells, however, this treatment reinduceda transient ARA⁺ phenotype. Thus, activation of resting matureT cells or restimulation of activated T cells may induce a translentresistance to apoptotic signals. Activation signals may interferewith the APO-1 pathway and may prevent elimination of activatedT cells in the periphery (peripheral selection).     

Possible Role of Catheters in Saccharomyces boulardii Fungemia

 Four cases of Saccharomyces boulardii fungemia, a very rare side effect of Saccharomyces boulardii therapy, are reported. The clinical impact of Saccharomyces boulardii infection appeared to be moderate. However, even though organ involvement was never demonstrated, septic shock with no other etiology was observed in one of our patients. All patients had an indwelling vascular catheter. Contamination of the air, environmental surfaces, and hands following the opening of a packet suggests that catheter contamination may have been a source of infection. To prevent catheter contamination it is recommended that packets or capsules of Saccharomyces boulardii be opened with gloves, outside the patient's room.     

Recruitment of cytoskeletal and signaling proteins to enteropathogenic and enterohemorrhagic Escherichia coli pedestals

Enteropathogenic Escherichia coli (EPEC) is a human pathogen that attaches to intestinal epithelial cells and causes chronic watery diarrhea. A close relative, enterohemorrhagic E. coli (EHEC), causes severe bloody diarrhea and hemolytic-uremic syndrome. Both pathogens insert a protein, Tir, into the host cell plasma membrane where it binds intimin, the outer membrane ligand of EPEC and EHEC. This interaction triggers a cascade of signaling events within the host cell and ultimately leads to the formation of an actin-rich pedestal upon which the pathogen resides. Pedestal formation is critical in mediating EPEC- and EHEC-induced diarrhea, yet very little is known about its composition and organization. In EPEC, pedestal formation requires Tir tyrosine 474 phosphorylation. In EHEC Tir is not tyrosine phosphorylated, yet the pedestals appear similar. The composition of the EPEC and EHEC pedestals was analyzed by examining numerous cytoskeletal, signaling, and adapter proteins. Of the 25 proteins examined, only two, calpactin and CD44, were recruited to the site of bacterial attachment independently of Tir. Several others, including ezrin, talin, gelsolin, and tropomyosin, were recruited to the site of EPEC attachment independently of Tir tyrosine 474 phosphorylation but required Tir in the host membrane. The remaining proteins were recruited to the pedestal in a manner dependent on Tir tyrosine phosphorylation or were not recruited at all. Differences were also found between the EPEC and EHEC pedestals: the adapter proteins Grb2 and CrkII were recruited to the EPEC pedestal but were absent in the EHEC pedestal. These results demonstrate that although EPEC and EHEC recruit similar cytoskeletal proteins, there are also significant differences in pedestal composition.     

Altered T and B lymphocyte signaling pathways in lupus

Systemic lupus erythematosus (SLE) has long been recognized to be characterized by dysregulated signaling pathways in T and B lymphocytes, beginning with observations of cellular hyperactivity and hyperresponsiveness, and evolving to recent studies focused upon the genetic and molecular bases of such phenomena. This review focuses on recently elucidated signaling abnormalities currently thought to be intrinsic to T and/or B cells in human SLE.     

Triggering of toll-like receptor signaling pathways in T cells contributes to the anti-tumor efficacy of T cell responses

Traditionally, expression of toll-like receptors (TLRs) has been associated with innate immune cells in particular professional antigen presenting cells and natural killer cells. This led to the concept that the adjuvant effects of ligation of TLR in a host occur mainly in innate immune cells. However, this concept has been challenged by recent studies including ours demonstrating that T cells express appreciated levels of different TLRs, which can serve as costimulatory co-receptors during polyclonal and antigen-specific stimulation of T cells. Because T cells express low levels of TLRs as compared to innate immune cells, increasing the expression levels of TLRs in T cells can significantly maximize their responses to the costimulatory effects of TLR ligation. This review article focuses on the potential role of TLR expression in T cells in their responses to vaccination regimen containing TLR agonists and how it can be modulated to optimize anti-tumor immunity.     

Immunopharmacological effects of Saccharomyces boulardii in healthy human volunteers

Investigation of oral administration of Saccharomyces boulardii in healthy volunteers demonstrates several cellular and humoral changes in peripheral blood. Among its effects are the increase of erythrocytes, leucocytes, polymorphs, neutrophils, complement components C3, C5, C3d, serum anticomplementary activity and leucocyte chemokinesis, specially when autologous serum and antigen have been added to the culture medium and decrease of complement haemolytic activity (CH50, classic and alternative pathways). We have also demonstrated that in vitro S. boulardii was able to activate complement directly, to fix C3b to its surface and that its phagocytosis by mononuclear cells was complement-dependent. The overall changes in serum proteins suggested changes of acute phase proteins typical of an inflammatory process. Furthermore S. boulardii had no mitogenic response of lymphocyte populations. Our results demonstrated that S. boulardii activates the reticuloendothelial system and complement system and suggest that S. boulardii merits therapeutic trial in a variety of clinical situations.