Membrane Of Intestinal Epithelial Cells Research Articles

GPR109A Is a G-protein–Coupled Receptor for the Bacterial Fermentation Product Butyrate and Functions as a Tumor Suppressor in Colon

Short-chain fatty acids, generated in colon by bacterial fermentation of dietary fiber, protect against colorectal cancer and inflammatory bowel disease. Among these bacterial metabolites, butyrate is biologically most relevant. GPR109A is a G-protein-coupled receptor for nicotinate but recognizes butyrate with low affinity. Millimolar concentrations of butyrate are needed to activate the receptor. Although concentrations of butyrate in colonic lumen are sufficient to activate the receptor maximally, there have been no reports on the expression/function of GPR109A in this tissue. Here we show that GPR109A is expressed in the lumen-facing apical membrane of colonic and intestinal epithelial cells and that the receptor recognizes butyrate as a ligand. The expression of GPR109A is silenced in colon cancer in humans, in a mouse model of intestinal/colon cancer, and in colon cancer cell lines. The tumor-associated silencing of GPR109A involves DNA methylation directly or indirectly. Reexpression of GPR109A in colon cancer cells induces apoptosis, but only in the presence of its ligands butyrate and nicotinate. Butyrate is an inhibitor of histone deacetylases, but apoptosis induced by activation of GPR109A with its ligands in colon cancer cells does not involve inhibition of histone deacetylation. The primary changes in this apoptotic process include down-regulation of Bcl-2, Bcl-xL, and cyclin D1 and up-regulation of death receptor pathway. In addition, GPR109A/butyrate suppresses nuclear factor-kappaB activation in normal and cancer colon cell lines as well as in normal mouse colon. These studies show that GPR109A mediates the tumor-suppressive effects of the bacterial fermentation product butyrate in colon.

A TRPM4‐LIKE NON‐SELECTIVE CATION CHANNEL IS EXPRESSED IN SMALL INTESTINAL EPITHELIAL CELLS

We used patch‐clamp techniques to characterize ion channels that are expressed in the apical membrane of small intestinal epithelial IEC6 cells. A nearly silent 23‐pS channel was recorded in cell‐attached patches, which was significantly activated when the intracellular Ca2+ was elevated by ionomycin. The channel was also strongly activated in excised inside‐out patches when the inner leaflet of the patch membrane exposed to 1 mM Ca2+, Change in Cl− gradient across the patch membrane did not affect the reversal potential. In contrast, the replacement of Na+ with NMDG+ in the patch pipette obviously shifted the reversal potential from 0 to ‐60 mV, which is very close to the equilibrium potential calculated according to the concentration gradient of Na+. However, the replacement of Na+ with K+ in the patch pipette did not affect the reversal potential. These biophysical properties are similar to those of native and expressed TRPM4 characterized in other types of cells. Recent studies suggest that the melastatin subfamily 4 of transient receptor potential channels (TRPM4) plays an important role in modulating the cell membrane potential. Therefore, we also performed confocal microscopy experiments and found that TRPM4 is expressed in IEC6 cells. These data suggest that a TRPM4‐like nonselective cation channel may regulate the transport function of small intestinal epithelial cells by affecting the membrane potential.

Probiotics restore ion transport in infected intestinal epithelial cells by modulating glycosphingolipids signaling.

Glycosphingolipids (GSL) are part of intestinal epithelial cell (IEC) membranes and are composed by a sugar moiety attached to ceramide. GSL contributes to many cellular functions, including protein sorting, cell adhesion, cell‐cell signaling and apoptosis.Probiotics are commensal bacteria with the ability to modify cellular processes in a beneficial way. We have shown that a probiotic preparation, S. thermophilus (ST) and L. acidophilus (LA), has the ability in vitro and in vivo to decrease chloride secretion and diarrhea by regulating kinase signaling and levels of ion transport proteins in IEC. Our ST strain expresses bacterial sphyngomyelinase , whereas LA does not. We hypothesized that ST/LA may regulate chloride secretion by altering GSL signaling in IEC. T84 and HT29/cl.19A cells were infected with enteroinvasive E.coli (EIEC) and S.dublin (SD), and were treated with wild type probiotics or mutated ST and/or LA. While wild type ST/LA, LA and ST alone were able to reverse the effects of EIEC and SD on chloride secretion in IEC, a ST strain carrying a mutation of the SMase gene and SMase‐negative commensal Streptococci had no effect on the dysregulated ion transport after pathogens infection. This effect was mediated by ERK1,2 and JNK, as shown by kinases inhibitors treatment. Moreover, we observed significantly altered CFTR and NKCC1 function and cellular distribution after treatment with ST/LA and LA and ST alone after EIEC and SD infection. Our findings provide insight on mechanisms of probiotics effects and on GSL cell signaling in the gut.

CD44v6 mediates neutrophil clearance from the apical surface of the intestinal epithelium

Neutrophil (PMN) infiltration and accumulation are pathognomonic of multiple inflammatory processes of the bowel, including ulcerative colitis, however, the signals governing PMN clearance remain to be defined. Our goal was to identify inflammation responsive epithelial ligands regulating PMN transepithelial migration and clearance. A panel of antibodies was generated against interferon‐gamma (IFNγ)‐stimulated T84 intestinal epithelial cell membranes. Once such antibody, GM35, recognizes an IFNγ‐responsive antigen and reduces PMN transepithelial migration in a time and dose dependent manner. Immunohistochemical staining and selective surface biotinylation localize the GM35 antigen to the apical surface of T84 cells, and further work indicates that the GM35 epitope is cleaved from T84 cells during PMN transepithelial migration. Antigen immunoprecipitation and confocal microscopic analysis of immunofluorescently labeled T84 cells indicate that GM35 binds a member of the CD44 family, while microsequencing more specifically identifies the GM35 ligand as the CD44 variant CD44v6. These studies identify CD44v6 as a candidate mediator of intestinal epithelial PMN clearance and a potential target for therapeutic intervention. This work is supported by R01 funding (Parkos), an NIH Career Development Award (Louis), and an Emory University Research Council seed grant.

Developmental changes in glycolipids and synchronized expression of nutrient transporters in the mouse small intestine

Small intestinal epithelial cells are rich in characteristic glycosphingolipids (GSLs) that are composed of phytosphingosine and α-hydroxy fatty acid, but the physiological roles of GSLs in the small intestine remain unclear. Here, we report the developmental changes in GSL composition in the mouse small intestine (duodenum through ileum) and their relationship with the temporal mRNA expression of nutrient transporters. Up to 2 weeks after birth, the major GSLs were hexosylceramide (HexCer), GM3, GM1 and GD1a. After 2 weeks of age, HexCer and asialo GM1 became the major GSLs. The ceramide moiety of both HexCer and asialo GM1 was composed mainly of phytosphingosine and α-hydroxy fatty acid, from birth through adulthood. Immunohistochemically, GM1 localized in the cytoplasm, and asialo GM1 localized exclusively in the apical microvillous membrane of small intestinal epithelial cells. The shift from sialylated GSLs to asialo GM1 was achieved by the combinational and tissue-specific transcriptional down-regulation of GM3 synthase and GM1-β-galactosidase at around 2 weeks of age. The temporal mRNA expression of various nutrient transporters also showed significant changes at around 2 weeks of age, including the up-regulation of the sodium/glucose cotransporter and the oligopeptide transporter, as well as the down-regulation of amino acid transporters. These synchronized changes in the mRNA expression of nutrient transporters with GSL composition during suckling-to-weanling transition suggest the contributions of GSLs to morphologic and functional development in the membrane of mouse small intestinal epithelial cells.

Drug Permeability Assay Using Microhole-Trapped Cells in a Microfluidic Device

As orally administered drugs must be absorbed from the intestine into the blood circulation, permeability assays of drug candidates have been widely used in the early screening stages of drug discovery. In this study, a microfluidic device was developed for the drug permeability assay, considering the in vivo delivery path of drugs in humans. A microhole array for cell trapping was fabricated using the poly(dimethylsiloxane) (PDMS) molding technique by mimicking the intestinal epithelial cell membrane. On the basis of mathematical simulations, the configuration of the microfluidic device, including a microhole array and a mixing channel, was optimized to trap cells firmly in each microhole. At the flow rate under optimal conditions, cells were effectively trapped in a microhole array without cell damage. We measured the permeability of 10 drugs, including those with high and low permeability in microchannels, and compared the results with the reported values of permeability in the human and rat intestine. Most drugs had a high p value (p > 0.4), and only a few drugs had a low p value less than 0.05 by t test. Though their measured permeabilities are not the same as those in vivo human intestine, it shows that in vivo permeabilities in the human and rat intestine are highly correlated with those measured by the microfluidic device (R(2) = 0.9013 and R(2) = 0.8765, respectively). Also, the fraction of the dose absorbed in the human intestine (F(a)) indicated that the drug permeability measured using this device was significantly correlated (R(2) = 0.9641) with those in human subjects. As the microfluidic assay system is dependent on cells trapped inside a microhole array, it is a valuable tool in drug discovery as well as an alternative to animal testing.

Guanylate-binding protein-1 is expressed at tight junctions of intestinal epithelial cells in response to interferon-γ and regulates barrier function through effects on apoptosis

Guanylate-binding protein-1 (GBP-1) is an interferon inducible large GTPase involved in endothelial cell proliferation and invasion. In this report, expression and function of GBP-1 were investigated in vitro in intestinal epithelia after exposure to interferon-γ and in human colonic mucosa from individuals with inflammatory bowel disease (IBD). Interestingly, in contrast to other epithelia, GBP-1 distributed to the plasma membrane in intestinal epithelial cells where it colocalized with the tight junction protein coxsackie- and adenovirus receptor. In addition, expression of GBP-1 was upregulated in colonic epithelia of individuals with IBD. Downregulation of GBP-1 by siRNA resulted in enhanced permeability that correlated with increased apoptosis. Indeed, inhibition of caspase activity prevented the inhibition of barrier formation induced by the loss of GBP-1. These data suggest that GBP-1 is a novel marker of intestinal mucosal inflammation that may protect against epithelial apoptosis induced by inflammatory cytokines and subsequent loss of barrier function.

Direct Evidence for Efficient Transport and Minimal Metabolism of L-Cephalexin by Oligopeptide Transporter 1 in Budded Baculovirus Fraction

The oligopeptide transporter PEPT1 (SLC15A1) is responsible for absorption of peptidic nutrients in the small intestine. Although the L-diastereomer of the beta-lactam antibiotic cephalexin (L-cephalexin) is likely to be transported by PEPT1, there has been no direct demonstration of PEPT1-mediated L-cephalexin transport. Indeed, after the incubation with L-cephalexin, the intact form of L-cephalexin has not been identified inside vesicles/proteoliposomes prepared from brush border membrane of intestinal epithelial cells or cultured cell lines exogenously transfected with PEPT1 gene. Thus, it appears that L-cephalexin is rapidly metabolized by PEPT1 or PEPT1-associated proteins. Here, we attempted to verify whether L-cephalexin is transported by PEPT1 and whether it is hydrolyzed by PEPT1 itself, by using budded baculovirus expressing PEPT1 protein. Marked uptake of L-cephalexin in PEPT1-expressing budded baculovirus, compared with wild-type virus, indicated that L-cephalexin is a substrate for PEPT1. The uptake was found to be pH sensitive, and was strongly inhibited by the D-diastereomer of cephalexin and glycylsarcosine, but not by glycine. Thus, L-cephalexin is transported by PEPT1 itself. Upon the transport of both L- and D-cephalexin by PEPT1, dose-dependent membrane depolarization was observed; the EC(50) values of 0.18 and 2.9 mM, respectively, indicate that the affinity of L-cephalexin for PEPT1-mediated transport is much higher than that of the D-diastereomer. On the other hand, the L-cephalexin metabolite 7-aminodesacetoxycephalosporanic acid was not detected in PEPT1-expressing or wild-type virus at either pH 6.0 or 7.4. We conclude that L-cephalexin is transported by PEPT1 with high affinity, but is not metabolized by PEPT1 itself.

Adenosine: An immune modulator of inflammatory bowel diseases

Inflammatory bowel disease (IBD) is a common and lifelong disabling gastrointestinal disease. Emerging treatments are being developed to target inflammatory cytokines which initiate and perpetuate the immune response. Adenosine is an important modulator of inflammation and its anti-inflammatory effects have been well established in humans as well as in animal models. High extracellular adenosine suppresses and resolves chronic inflammation in IBD models. High extracellular adenosine levels could be achieved by enhanced adenosine absorption and increased de novo synthesis. Increased adenosine concentration leads to activation of the A2a receptor on the cell surface of immune and epithelial cells that would be a potential therapeutic target for chronic intestinal inflammation. Adenosine is transported via concentrative nucleoside transporter and equilibrative nucleoside transporter transporters that are localized in apical and basolateral membranes of intestinal epithelial cells, respectively. Increased extracellular adenosine levels activate the A2a receptor, which would reduce cytokines responsible for chronic inflammation.

Ecto-Phosphorylation of CD98 Regulates Cell-Cell Interactions

Ecto-phosphorylation plays an important role in many cellular functions. The transmembrane glycoprotein CD98 contains potential phosphorylation sites in its extracellular C-terminal tail. We hypothesized that extracellular signaling through ecto-protein kinases (ePKs) might lead to ecto-phosphorylation of CD98 and influence its multiple functions, including its role in cell-cell interactions. Our results show that recombinant CD98 was phosphorylated in vitro by ePKs from Jurkat cells and by the commercial casein kinase 2 (CK2). Alanine substitutions at serines-305/307/309 or serines-426/430 attenuated CK2-mediated CD98 phosphorylation, suggesting that these residues are the dominant phosphorylation sites for CK2. Furthermore, CD98 expressed in the basolateral membranes of intestinal epithelial Caco2-BBE cells was ecto-phosphorylated by Jurkat cell-derived ePKs and ecto-CK2 was involved in this process. Importantly, cell attachment studies showed that the ecto-phosphorylation of CD98 enhanced heterotypic cell-cell interactions and that the extracellular domain of CD98, which possesses the serine phosphorylation sites, was crucial for this effect. In addition, phosphorylation of recombinant CD98 increased its interactions with Jurkat and Caco2-BBE cells, and promoted cell attachment and spreading. In conclusion, here we demonstrated the ecto-phosphorylation of CD98 by ePKs and its functional importance in cell-cell interactions. Our findings reveal a novel mechanism involved in regulating the multiple functions of CD98 and raise CD98 as a promising target for therapeutic modulations of cell-cell interactions.

Functional coupling of the downregulated in adenoma Cl−/base exchanger DRA and the apical Na+/H+exchangers NHE2 and NHE3

Non-nutrient-dependent salt absorption across the brush-border membrane of intestinal epithelial cells is primarily mediated by coupled apical Na(+)/H(+) (aNHE) and anion exchange transport, with the latter suspected of being mediated by DRA (downregulated in adenoma; SLC26A3) that is defective in congenital chloridorrhea. To investigate DRA in greater detail and determine whether DRA and NHE activities can be coupled, we measured (22)Na(+) and (36)Cl(-) uptake in Caco2BBE colon cells infected with the tet-off-inducible DRA transgene. Under basal conditions, DRA activity was low in normal and infected Caco2BBE cells in the presence of tetracycline, whereas NHE activities could be easily detected. When apical NHE activity was increased by transfection or serum-induced expression of the aNHE isoforms NHE2 and NHE3, increased (36)Cl(-) uptake was observed. Inhibition of DRA activity by niflumic acid was greater than that by DIDS as well as by the NHE inhibitor dimethylamiloride and the carbonic anhydrase inhibitor methazolamide. DRA activity was largely aNHE-dependent, whereas a component of DRA-independent aNHE uptake continued to be observed. Coupled aNHE and DRA activities were inhibited by increased cellular cAMP and calcium and were associated with synaptotagmin I-dependent, clathrin-mediated endocytosis. In summary, these data support the role of DRA in electroneutral NaCl absorption involving functional coupling of Cl(-)/base exchange and apical NHE.

Up-regulation and Cytoprotective Role of Epithelial Multidrug Resistance-associated Protein 1 in Inflammatory Bowel Disease

MRP1 (multidrug resistance-associated protein 1) is well known for its role in providing multidrug resistance to cancer cells. In addition, MRP1 has been associated with both pro- and anti-inflammatory functions in nonmalignant cells. The pro-inflammatory function is evident from the fact that MRP1 is a high affinity transporter for cysteinyl-leukotriene C4 (LTC4), a lipid mediator of inflammation. It remains unexplained, however, why the absence of Mrp1 leads to increased intestinal epithelial damage in mice treated with dextran-sodium sulfate, a model for inflammatory bowel disease (IBD). We found that MRP1 expression is induced in the inflamed intestine of IBD patients, e.g. Crohn disease and ulcerative colitis. Increased MRP1 expression was detected at the basolateral membrane of intestinal epithelial cells. To study a putative role for MRP1 in protecting epithelial cells against inflammatory cues, we manipulated MRP1 levels in human epithelial DLD-1 cells and exposed these cells to cytokines and anti-Fas. Inhibition of MRP1 (by MK571 or RNA interference) resulted in increased cytokine- and anti-Fas-induced apoptosis of DLD-1 cells. Opposite effects, e.g. protection of DLD-1 cells against cytokine- and anti-Fas-induced apoptosis, were observed after recombinant MRP1 overexpression. Inhibition of LTC4 synthesis reduced anti-Fas-induced apoptosis when MRP1 function was blocked, suggesting that LTC4 is the pro-apoptotic compound exported by epithelial MRP1 during inflammation. These data show that MRP1 protects intestinal epithelial cells against inflammation-induced apoptotic cell death and provides a functional role for MRP1 in the inflamed intestinal epithelium of IBD patients.

Heterologous expression of cholera toxin B subunit in Saccharomyces cerevisiae

The cholera toxin B subunit (CTB), which consists of five identical polypeptides and adopts a pentameric structure, has been shown to bind to the GM1-gangliosides at the cellular surface. Recombinant CTB has attracted much attention due to its non-toxicity and potential as a strong immunogenic antigen and immuno adjuvant for both system and mucosal immune responses. In this study, CTB was expressed in Saccharomyces cerevisiae and the resulting recombinant CTB was extensively characterized. PCR and back-transformation into E. coli confirmed the presence of the CTB gene-containing plasmid in the transformants and northern analysis showed the presence of the CTB-specific transcript. Western blot analysis of the yeast-derived protein extract showed the presence of CTB with mobility similar to purified CTB from Vibrio cholerae suggesting that the expressed CTB assembled into the desired pentameric form. Quantitative ELISA revealed that the recombinant CTB comprised approximately 0.5∼1.3% of the total cell-free extract. In addition, 0.5∼2 mg of CTB protein per liter of cultured media was detected 1 day, at the earliest after cultivation. The GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA) confirmed that the yeast-derived CTB bound specifically to the GM1-ganglioside receptor, indicating that it retained its native function and pentameric form, which is required for binding to intestinal epithelial cell membrane glycolipid receptors. In addition to the development of a yeast-derived edible vaccine against cholera, this study regarding the expression and assembly of recombinant CTB into biologically active oligomers in recombinant S. cerevisiae enables the efficient production of a GRAS microorganism-based adjuvant, as well as the development of carriers for foreign vaccine molecules.

Expression of Chlamydophila psittaci MOMP heat-labile toxin B subunit fusion gene in transgenic rice

A DNA fragment encoding the MOMP gene of Chlamydophila psittaci was fused to the heat-labile toxin B subunit gene (LTB-MOMP) and transferred into rice callus by Agrobacterium tumefaciens-mediated transformation. The LTB-MOMP fusion gene was detected in genomic DNA from transformed rice leaves by Southern blot and RT-PCR amplification. Synthesis and assembly of the LTB-MOMP fusion protein into pentamers was detected in transformed leaf extracts by immunoblot analysis. Binding of the pentamers to intestinal epithelial cell membrane glycolipid receptors was quantified by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). The ELISA results indicated that LTB-MOMP fusion protein made up 0.0033–0.0054% of the total soluble leaf protein. Meanwhile, this suggested that the fusion protein retained both its native antigenicity and the ability to form pentamers.

PDZK1 Regulates Two Intestinal Solute Carriers (Slc15a1 and Slc22a5) in Mice

Gastrointestinal (GI) absorption of certain therapeutic agents is thought to be mediated by solute carrier (SLC) transporters, although minimal in vivo evidence has been reported. Here, we show key roles of postsynaptic density 95/disk-large/ZO-1 (PDZ) domain-containing protein, PDZK1, as a regulatory mechanism of two solute carriers, Slc15a1 (oligopeptide transporter PEPT1) and Slc22a5 (carnitine/organic cation transporter OCTN2) in mouse small intestine by using pdzk1 gene knockout (pdzk1(-/-)) mice. GI absorption of cephalexin, a substrate of PEPT1, after p.o. administration was delayed in pdzk1(-/-) mice compared with wild-type mice. Absorption of carnitine, a substrate of OCTN2, was also decreased in pdzk1(-/-) mice. Immunohistochemical analysis revealed the localization of both PEPT1 and OCTN2 at apical membrane of small intestinal epithelial cells in wild-type mice, whereas such apical localization was reduced in pdzk1(-/-) mice, with a concomitant decrease in their protein levels assessed by Western blotting in intestinal brush-border membranes. Electron microscopy revealed localization of PEPT1 in intracellular vesicular structures in pdzk1(-/-) mice. In addition, we first identified interaction between PEPT1 and PDZK1 in mouse small intestine and found that PDZK1 stimulates transport activity of PEPT1 by increasing its expression level in human embryonic kidney 293 cells. Taken together, the present findings provide direct evidence that PDZK1 regulates two intestinal SLC transporters in vivo as an adaptor protein for these transporters and affects oral absorption of their substrates. These findings also raise the possibility that intestinal absorption of the substrate drugs for PEPT1 and OCTN2 is governed by the protein network of these transporters and their adaptor PDZK1.

Osmotic water permeability of rat intestinal brush border membrane vesicles: involvement of aquaporin-7 and aquaporin-8 and effect of metal ions

Water channels AQP7 and AQP8 may be involved in transcellular water movement in the small intestine. We show that both AQP7 and AQP8 mRNA are expressed in rat small intestine. Immunoblot and immunohistochemistry experiments demonstrate that AQP7 and AQP8 proteins are present in the apical brush border membrane of intestinal epithelial cells. We investigated the effect of several metals and pH on the osmotic water permeability (Pf) of brush border membrane vesicles (BBMVs) and of AQP7 and AQP8 expressed in a cell line. Hg2+, Cu2+, and Zn2+ caused a significant decrease in the BBMV Pf, whereas Ni2+ and Li+ had no effect. AQP8-transfected cells showed a reduction in Pf in the presence of Hg2+ and Cu2+, whereas AQP7-transfected cells were insensitive to all tested metals. The Pf of both BBMVs and cells transfected with AQP7 and AQP8 was not affected by pH changes within the physiological range, and the Pf of BBMVs alone was not affected by phlorizin or amiloride. Our results indicate that AQP7 and AQP8 may play a role in water movement via the apical domain of small intestine epithelial cells. AQP8 may contribute to the water-imbalance-related clinical symptoms apparent after ingestion of high doses of Hg2+ and Cu2+.

Characterization of the binding surface of the translocated intimin receptor, an essential protein for EPEC and EHEC cell adhesion

The translocated intimin receptor (TIR) of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) is required for EPEC and EHEC infections, which cause widespread illness across the globe. TIR is translocated via a type-III secretion system into the intestinal epithelial cell membrane, where it serves as an anchor for E. coli attachment via its binding partner intimin. While many aspects of EPEC and EHEC infection are now well understood, the importance of the intermolecular contacts made between intimin and TIR have not been thoroughly investigated. Herein we report site-directed mutagenesis studies on the intimin-binding domain of EPEC TIR, and how these mutations affect TIR-intimin association, as analyzed by isothermal titration calorimetry and circular dichroism. These results show how two factors govern TIR's binding to intimin: A three-residue TIR hot spot is identified that largely mediates the interaction, and mutants that alter the beta-hairpin structure of TIR severely diminish binding affinity. In addition, peptides incorporating key TIR residues identified by mutagenesis are incapable of binding intimin. These results indicate that hot spot residues and structural orientation/preorganization are required for EPEC, and likely EHEC, TIR-intimin binding.

In vitro assessment of gastrointestinal viability of two photosynthetic bacteria, Rhodopseudomonas palustris and Rhodobacter sphaeroides

The objectives of this study were to assess the potential of two photosynthetic bacteria (PSB), Rhodopseudomonas palustris HZ0301 and Rhodobacter sphaeroides HZ0302, as probiotics in aquaculture. The viability of HZ0301 and HZ0302 in simulated gastric transit conditions (pH 2.0, pH 3.0 and pH 4.0 gastric juices) and in simulated small intestinal transit conditions (pH 8.0, with or without 0.3% bile salts) was tested. The effects of HZ0301 and HZ0302 on the viability and permeability of intestinal epithelial cell in primary culture of tilapias, Oreochromis nilotica, were also detected. All the treatments were determined with three replicates. The simulated gastric transit tolerance of HZ0301 and HZ0302 strains was pH-dependent and correspondingly showed lower viability at pH 2.0 after 180 min compared with pH 3.0 and pH 4.0. Both HZ0301 and HZ0302 were tolerant to simulated small intestine transit with or without bile salts in our research. Moreover, there was no significant difference (P>0.05) among three treatments including the control and the groups treated with HZ0301 or HZ0302 both in intestinal epithelial cell viability and membrane permeability, showing no cell damage. In summary, this study demonstrated that HZ0301 and HZ0302 had high capacity of upper gastrointestinal transit tolerance and were relatively safe for intestinal epithelial cells of tilapias.

Caught in the traffic

In mice, deletion of the Rab8 protein disrupts organized molecular distribution to membranes of intestinal epithelial cells. Death by starvation follows, exactly as it does in humans with microvillus inclusion disease.

T84-Intestinal Epithelial Exosomes Bear MHC Class II/Peptide Complexes Potentiating Antigen Presentation by Dendritic Cells

Intestinal epithelial cells release antigen-presenting vesicles (exosomes) bearing major histocompatibility complex class II/peptide complexes stimulating specific immune responses in vivo. To characterize further the role of human epithelial exosomes in antigen presentation, their capacity to load antigenic peptides, bind immune target cells, and induce T-cell activation was analyzed in vitro. The capacity of exosomes derived from the HLA-DR4-expressing, intestinal epithelial cell line T84 to load the HLA-DR4-specific peptide (3)H-HSA 64-76 and to activate a HLA-DR4-restricted T-cell hybridoma was tested in the presence or absence of human monocyte-derived dendritic cells (DCs). Interaction of fluorescein isothiocyanate-labeled exosomes with T cells and DCs was analyzed by flow cytometry and confocal microscopy. T84-derived exosomes, enriched in CD9, CD81, CD82, and A33 antigen, were capable of binding specifically human serum albumin (HSA) 64-76 peptide on HLA-DR4 molecules and of interacting preferentially with DCs. HSA-loaded exosomes were unable to activate the T-cell hybridoma directly but induced a productive T-cell activation through DCs. When HSA peptide was bound to exosomal HLA-DR4 molecules instead of in a soluble form, the threshold of peptide presentation by DCs was markedly decreased (x10(-3)). Exosomes released by intestinal epithelial cells bear exogenous peptides complexed to major histocompatibility complex class II molecules and interact preferentially with DCs, strongly potentiating peptide presentation to T cells. Epithelial exosomes constitute a powerful link between luminal antigens and local immune cells by mediating the transfer of tiny amounts of luminal antigenic information and facilitating immune surveillance at mucosal surfaces.