Secreted bioactive factors from Bifidobacterium infantis enhance epithelial cell barrier function

Live probiotic bacteria are effective in reducing gut permeability and inflammation. We have previously shown that probiotics release peptide bioactive factors that modulate epithelial resistance in vitro. The objectives of this study were to determine the impact of factors released from Bifidobacteria infantis on intestinal epithelial cell permeability and tight junction proteins and to assess whether these factors retain their bioactivity when administered to IL-10-deficient mice. B. infantis conditioned medium (BiCM) was applied to T84 human epithelial cells in the presence and absence of TNF-alpha and IFN-gamma. Transepithelial resistance (TER), tight junction proteins [claudins 1, 2, 3, and 4, zonula occludens (ZO)-1, and occludin] and MAP kinase activity (p38 and ERK) were examined. Acute effects of BiCM on intestinal permeability were assessed in colons from IL-10-deficient mice in Ussing chambers. A separate group of IL-1-deficient mice was treated with BiCM for 4 wk and then assessed for intestinal histological injury, cytokine levels, epithelial permeability, and immune response to bacterial antigens. In T84 cells, BiCM increased TER, decreased claudin-2, and increased ZO-1 and occludin expression. This was associated with enhanced levels of phospho-ERK and decreased levels of phospho-p38. BiCM prevented TNF-alpha- and IFN-gamma-induced drops in TER and rearrangement of tight junction proteins. Inhibition of ERK prevented the BiCM-induced increase in TER and attenuated the protection from TNF-alpha and IFN-gamma. Oral BiCM administration acutely reduced colonic permeability in mice whereas long-term BiCM treatment in IL-10-deficient mice attenuated inflammation, normalized colonic permeability, and decreased colonic and splenic IFN-gamma secretion. In conclusion, peptide bioactive factors from B. infantis retain their biological activity in vivo and are effective in normalizing gut permeability and improving disease in an animal model of colitis. The effects of BiCM are mediated in part by changes in MAP kinases and tight junction proteins.     

Epithelial Barrier Modulation by a Channel Forming Peptide

NC-1059 is a synthetic channel-forming peptide that provides for ion transport across, and transiently reduces the barrier integrity of, cultured epithelial monolayers derived from canine kidney (MDCK cells). Experiments were conducted to determine whether epithelial cells derived from other sources were similarly affected. Epithelial cells derived from human intestine (T-84), airway (Calu-3), porcine intestine (IPEC-J2) and reproductive duct (PVD9902) were grown on permeable supports. Basal short circuit current (I _sc) was <3 μA cm⁻² for T-84, IPEC-J2 and PVD9902 cell monolayers and <8 μA cm⁻² for Calu-3 cells. Apical NC-1059 exposure caused, in all cell types, an increase in I _sc to >15 μA cm⁻², indicative of net anion secretion or cation absorption, which was followed by an increase in transepithelial conductance (in mS cm⁻²: T-84, 1.6 to 62; PVD9902, 0.2 to 51; IPEC-J2, 0.3 to 26; Calu-3, 2.3 to 13). These results are consistent with the peptide affecting transcellular ion movement, with a likely effect also on the paracellular route. NC-1059 exposure increased dextran permeation when compared to basal permeation, which documents an effect on the paracellular pathway. In order to evaluate membrane ion channels, experiments were conducted to study the dose dependence and stability of the NC-1059-induced membrane conductance in Xenopus laevis oocytes. NC-1059 induced a dose-dependent increase in oocyte membrane conductance that remained stable for greater than 2 h. The results demonstrate that NC-1059 increases transcellular conductance and paracellular permeation in a wide range of epithelia. These effects might be exploited to promote drug delivery across barrier epithelia.     

Interleukin 2 Modulates Intestinal Epithelial Cell Functionin Vitro

Although interleukin 2 (IL-2) has been presumed to have a highly circumscribed range of target cells limited largely to classic immune cell populations, the presence of functional IL-2 receptors in rat epithelial cell lines has recently been demonstrated. Limited information is available about the functional effects of IL-2 on intestinal epithelial cells. The effect of recombinant IL-2 on intestinal epithelial cell migration was assessed using a previously describedin vitromodel of epithelial restitution by quantitation of cells migrating into standard wounds established in confluent IEC-6 cell monolayers. Transforming growth factor β content was assessed by Northern blot and bioassay. Exogenous IL-2 enhanced epithelial cell restitutionin vitroon average 3.8-fold; this effect was independent of cell proliferation. Enhancement of restitution through IL-2 could be completely blocked through antibodies directed against TGFβ₁and interleukin-2 receptor, indicating that stimulation of epithelial cell restitution is specifically enhanced by interleukin-2 and mediated through a TGFβ-dependent pathway. In addition, increased expression of TGFβ₁mRNA and increased levels of bioactive TGFβ peptide in wounded monolayers treated with IL-2 compared to unwounded monolayers cultured in serum-deprived medium alone support the notion that enhancement of epithelial cell restitutionin vitrois mediated through a TGFβ-dependent pathway. These studies suggest that IL-2, a potent cytokine whose biological origin and targets have been presumed to be largely limited to lymphocyte and macrophage populations, may play a role in preserving the integrity of the intestinal epithelium following various forms of injuries.     

Calcitonin receptor-mediated CFTR activation in human intestinal epithelial cells

High levels of calcitonin (CT) observed in medullary thyroid carcinoma and other CT‐secreting tumours cause severe diarrhoea. Previous studies have suggested that CT induces active chloride secretion. However, the involvement of CT receptor (CTR) and the molecular mechanisms underlying the modulation of intestinal electrolyte secreting intestinal epithelial cells have not been investigated. Therefore, current studies were undertaken to investigate the direct effects of CT on ion transport in intestinal epithelial cells. Real time quantitative RT‐PCR and Western blot analysis demonstrated the expression of CTR in intestinal epithelial T84 cells. Exposure of T84 cells to CT from the basolateral but not from apical side significantly increased short circuit current (I_SC) in a dose‐dependent manner that was blocked by 1 μM of CTR antagonist, CT8–32. CT‐induced I_SC was blocked by replacing chloride in the bath solutions with equimolar gluconate and was significantly inhibited by the specific cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor, CFTR_127inh. Further, biotinylation studies showed that CT increased CFTR levels on the apical membrane. The presence of either the Ca²⁺ chelator, bis(2‐aminophenoxy)ethane tetraacetic acid‐acetoxymethyl (BAPTA‐AM) ester or the protein kinase A (PKA) inhibitor, H89, significantly inhibited I_SC induced by CT (～32–58% reduction). Response to CT was retained after permeabilization of the basolateral or the apical membranes of T84 cells with nystatin. In conclusion, the activation of CTR by CT induced chloride secretion across T84 monolayers via CFTR channel and the involvement of PKA‐ and Ca²⁺‐dependent signalling pathways. These data elucidate the molecular mechanisms underlying CT‐induced diarrhoea.     

Basolateral K channel activated by carbachol in the epithelial cell line T84

Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (I _SC), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T₈₄ cells. Carbachol had little effect on I _SC when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent I _SC response to calcium ionophores. Carbachol (100 m) transiently elevated intracellular free calcium ([Ca²⁺] _i ) by 3-fold in confluent cells cultured on glass coverslips with a time course resembling the I _sc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (–54 mV) with pipette solution containing 150 mm KCl (37°C). This rectification persisted when patches were bathed in symmetrical 150 mm KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mm barium. It is referred to as the K_BIC channel based on its most distinctive properties (Ba-insensitive, inwardly rectifying, Ca-activated). Like single K_BIC channels, the carbachol-stimulated I _SC was relatively insensitive to several blockers on the basolateral side and was unaffected by barium. These comparisons between the properties of the macroscopic current and single channels suggest that the K_BIC channel mediates basolateral membrane K conductance in T₈₄ cell monolayers during stimulation by cholinergic secretagogues.We thank Dr. Marcel Crest (Laboratoire de Neurobiologie, CNRS, Marseille) for providing a sample of kaliotoxin. This work was supported by the Canadian Cystic Fibrosis Foundation and the Respiratory Health Network of Centres of Excellence. J.W.H. is a Chercheur-Boursier of the Fonds de la recherche en santé du Québec.     

Regulation of the eicosanoid pathway by tumour necrosis factor alpha and leukotriene D4 in intestinal epithelial cells

In this study the mRNA and protein levels of the key enzymes involved in eicosanoid biosynthesis and the cysteinyl leukotriene receptors (CysLT₁R and CysLT₂R) have been analysed in non-transformed intestinal epithelial and colon cancer cell lines. Our results revealed that tumour necrosis factor alpha (TNF-α), and leukotriene D₄ (LTD₄), which are inflammatory mediators implicated in carcinogenesis, stimulated an increase of cyclooxygenase-2 (COX-2), in non-transformed epithelial cells, and 5-lipoxygenase (5-LO) in both non-transformed and cancer cell lines. Furthermore, these mediators also stimulated an up-regulation of LTC₄ synthase in cancer cells as well as non-transformed cells. We also observed an endogenous production of CysLTs in these cells. TNF-α and LTD₄, to a lesser extent, up-regulate the CysLT₁R levels. Interestingly, TNF-α also reduced CysLT₂R expression in cancer cells. Our results demonstrate that inflammatory mediators can cause intestinal epithelial cells to up-regulate the expression of enzymes needed for the biosynthesis of eicosanoids, including the cysteinyl leukotrienes, as well as the signal transducing proteins, the CysLT receptors, thus providing important mechanisms for both maintaining inflammation and for tumour progression.     

Methylated N-(4-N,N-Dimethylaminobenzyl) Chitosan, a Novel Chitosan Derivative, Enhances Paracellular Permeability Across Intestinal Epithelial Cells (Caco-2)

The aim of this study was to investigate the effect of methylated N-(4-N,N-dimethylaminobenzyl) chitosan, TM-Bz-CS, on the paracellular permeability of Caco-2 cell monolayers and its toxicity towards the cell lines. The factors affecting epithelial permeability, e.g., degree of quaternization (DQ) and extent of dimethylaminobenzyl substitution (ES), were evaluated in intestinal cell monolayers of Caco-2 cells using the transepithelial electrical resistance and permeability of Caco-2 cell monolayers, with fluorescein isothiocyanate dextran 4,400 (FD-4) as a model compound for paracellular tight-junction transport. Cytotoxicity was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide viability assay. The results revealed that, at pH 7.4, TM-Bz-CS appeared to increase cell permeability in a concentration-dependent manner, and this effect was relatively reversible at lower doses of 0.05–0.5 mM. Higher DQ and the ES caused the permeability of FD-4 to be higher. The cytotoxicity of TM-Bz-CS depended on concentration, %DQ, and %ES. These studies demonstrated that this novel modified chitosan has potential as an absorption enhancer.     

Transepithelial transport of fluorescein in Caco-2 cell monolayers and use of such transport in in vitro evaluation of phenolic acid availability

Fluorescein is a marker-dye customary applied to the evaluation of tight-junctional permeability of epithelial cell monolayers. However, the true mechanism for the permeation has not been elucidated. Transepithelial transport of fluorescein in Caco-2 cell monolayers was therefore examined. Fluorescein transport was dependent on pH, and in a vectorical way in the apical-basolateral direction, but it was independent of the tight-junctional permeability of monolayers of these human intestinal cells. The permeation of fluorescein was concentration-dependent and saturable; the Michaelis constant was 7.7 mM and the maximum velocity was 40.3 nmol min(-1) (mg protein)(-1). Benzoic acid competitively inhibited fluorescein transport, suggesting that fluorescein is transported by a monocarboxylic acid transporter (MCT). Antioxidative polyphenolic compounds such as ferulic acid from dietary sources, competitively inhibited the permeation of fluorescein. These compounds probably share a transport carrier with fluorescein. Measurement of the effects of phenolic acids on fluorescein transport across Caco-2 monolayers would be a useful way to evaluate the intestinal absorption or bioavailability of dietary phenolic acids.     

Neutrophil transepithelial migration: evidence for sequential,contact-dependent signaling events and enhanced paracellular permeability independent of transjunctional migration

Active migration of polymorphonuclear leukocytes (PMN) through the intestinal crypt epithelium is a hallmark of inflammatory bowel disease and correlates with patient symptoms. Previous in vitro studies have shown that PMN transepithelial migration results in increased epithelial permeability. In this study, we modeled PMN transepithelial migration across T84 monolayers and demonstrated that enhanced paracellular permeability to small solutes occurred in the absence of transepithelial migration but required both PMN contact with the epithelial cell basolateral membrane and a transepithelial chemotactic gradient. Early events that occurred before PMN entering the paracellular space included increased permeability to small solutes (<500 Da), enhanced phosphorylation of regulatory myosin L chain, and other as yet undefined proteins at the level of the tight junction. No redistribution or loss of tight junction proteins was detected in these monolayers. Late events, occurring during actual PMN transepithelial migration, included redistribution of epithelial serine-phosphorylated proteins from the cytoplasm to the nucleus in cells adjacent to migrating PMN. Changes in phosphorylation of multiple proteins were observed in whole cell lysates prepared from PMN-stimulated epithelial cells. We propose that regulation of PMN transepithelial migration is mediated, in part, by sequential signaling events between migrating PMN and the epithelium.     

Immune cell activation and subsequent epithelial dysfunction by Staphylococcus enterotoxin B is attenuated by the green tea polyphenol (-)-epigallocatechin gallate

Bacterial superantigens (SAg) are potent T cell activators and when delivered systemically elicit a self-limiting enteropathy in mice. Also, SAg-stimulated human peripheral blood mononuclear cells (PBMC) increase enteric epithelial cell monolayer permeability in vitro. Epigallocatechin gallate (EGCG), the major polyphenol component of green tea (Camilla sinesis) leaf, has been presented as an anti-inflammatory agent. We tested the hypothesis that EGCG (10-100 microM) would block PBMC activation by the SAg, Staphylococcus aureus enterotoxin B (SEB, 1 microg/ml), thus preventing disruption of the epithelial barrier. Pretreatment or co-treatment of human PBMC or murine lymphnode cells with EGCG significantly reduced SEB-induced proliferation and IL-2, IFNgamma, and TNFalpha production. ConA-induced proliferation was also inhibited by EGCG (50 microM) co-treatment. These effects of EGCG were not due to induction of immune cell apoptosis, and were independent of EGCGs anti-oxidant activity, and inhibition of NF-kappaB or AP-1 activation. Moreover, addition of exogenous IL-2 (20 ng/ml) to the cultures could not overcome the immunosuppressive effect of EGCG. Culture supernatant from PBMC stimulated in the presence of EGCG failed to increase the permeability of T84 epithelial cell monolayers: a finding consistent with the reduced IFNgamma and TNFalpha production by SAg+EGCG treated PBMC. These data promote EGCG as a suppressor of T cell activation, and given the prominent role that bacteria and T cells play in inflammatory disease we suggest that EGCG could be a useful addition to current treatments for enteric immune disorders and T cell driven immunopathologies.     

Stress-Derived Corticotropin Releasing Factor Breaches Epithelial Endotoxin Tolerance

Background and aims

Loss of the endotoxin tolerance of intestinal epithelium contributes to a number of intestinal diseases. The etiology is not clear. Psychological stress is proposed to compromise the intestinal barrier function. The present study aims to elucidate the role of the stress-derived corticotropin releasing factor (CRF) in breaching the established intestinal epithelial endotoxin tolerance.

Methods

Epithelial cells of HT-29, T84 and MDCK were exposed to lipopolysaccharide to induce the endotoxin tolerance; the cells were then stimulated with CRF. The epithelial barrier function was determined using as indicators of the endotoxin tolerant status. A water-avoid stress mouse model was employed to test the role of CRF in breaching the established endotoxin tolerance in the intestine.

Results

The established endotoxin tolerance in the epithelial cell monolayers was broken down by a sequent exposure to CRF and LPS manifesting a marked drop of the transepithelial resistance (TER) and an increase in the permeability to a macromolecular tracer, horseradish peroxidase (HRP). The exposure to CRF also increased the expression of Cldn2 in the epithelial cells, which could be mimicked by over expression of TLR4 in epithelial cells. Over expression of Cldn2 resulted in low TER in epithelial monolayers and high permeability to HRP. After treating mice with the 10-day chronic stress, the intestinal epithelial barrier function was markedly compromised, which could be prevented by blocking either CRF, or TLR4, or Cldn2.

Conclusions

Psychological stress-derived CRF can breach the established endotoxin tolerance in the intestinal mucosa.     

Hydroxyxanthone as an inhibitor of cAMP-activated apical chloride channel in human intestinal epithelial cell

AimsPrevious investigation showed that polyphenols abundantly found in many plants could inhibit Cl^? secretion. The present study was aimed to investigate the effect of phenol containing xanthone derivatives on cAMP-activated intestinal Cl^? secretion and evaluate potential benefits of these compounds in the treatment of cholera.Main methodsFour hydroxy xanthones were synthesized via oxidative coupling reaction of the corresponding ortho-hydroxybenzoic acids and hydroxyphenols. Short-circuit current and apical Cl^? current measurements across monolayers of human intestinal epithelial (T84) cell and Fisher rat thyroid cells transfected with human CFTR (FRT-hCFTR cell) were performed to determine the effect of hydroxyxanthones on cAMP-activated Cl^? secretion. Intracellular cAMP was measured by immunoassay methods. Anti-diarrheal efficacy was evaluated using closed loop model of cholera.Key findingsAmong the tested xanthones, 1,3,6-trihydroxyxanthone (THX-001) was found to be the most potent derivative in the inhibition of cAMP-activated Cl^? secretion across T84 cell monolayers (IC₅₀ ~ 100 μM). Electrophysiological analysis of T84 cells and FRT-hCFTR cells revealed that THX-001 targeted two distinct cAMP-activated Cl^? channels in the apical membrane of T84 cells, namely, CFTR and inward rectifying Cl^? channel (IRC). In contrast, THX-001 had no effect on intracellular cAMP levels in these cells. Importantly, THX-001 completely abolished cholera toxin-induced Cl^? secretion across T84 cell monolayers and significantly inhibited cholera toxin-induced intestinal fluid secretion in mouse closed loop models.SignificanceThis study revealed that hydroxyxanthone represents another chemical class of polyphenolic compounds that may hold promise as anti-secretory therapy for cholera.     

Role for IL-4 in macromolecular transport across human intestinal epithelium

Increased epithelial permeability is associated with intestinalinflammation, but there is little information on factors that regulatebarrier function in the absence of or before inflammation. We examinedif interleukin (IL)-4, or serum from atopic individuals, could alterthe barrier function of human colonic epithelial (T84) monolayers toantigenic-sized macromolecules. IL-4 and atopic serum significantlydecreased T84 monolayer resistance and increased transepithelialhorseradish peroxidase (HRP) transport. Bidirectional transport studiesdemonstrated that IL-4 selectively enhanced apical-to-basal movement ofHRP. HRP transport induced by IL-4 was inhibited by cold (4°C) andthe tyrosine kinase inhibitor genistein, but not the protein kinase Cinhibitor staurosporine. Electron microscopic analysis demonstratedthat both transcellular and paracellular pathways were affected.Anti-IL-4 antibodies abolished the increase in HRP transport inresponse to both IL-4 and serum. We speculate that enhanced productionof IL-4 in allergic conditions may be a predisposing factor toinflammation by allowing uptake of luminal antigens that gain access tothe mucosal immune system.     

Nippostrongylus-Induced Intestinal Hypercontractility Requires IL-4 Receptor Alpha-Responsiveness by T Cells in Mice

Gut-dwelling helminthes induce potent IL-4 and IL-13 dominated type 2 T helper cell (T_H2) immune responses, with IL-13 production being essential for Nippostrongylus brasiliensis expulsion. This T_H2 response results in intestinal inflammation associated with local infiltration by T cells and macrophages. The resulting increased IL-4/IL-13 intestinal milieu drives goblet cell hyperplasia, alternative macrophage activation and smooth muscle cell hypercontraction. In this study we investigated how IL-4-promoted T cells contributed to the parasite induced effects in the intestine. This was achieved using pan T cell-specific IL-4 receptor alpha-deficient mice (iLck^creIL-4Rα^−/lox) and IL-4Rα-responsive control mice. Global IL-4Rα^−/− mice showed, as expected, impaired type 2 immunity to N. brasiliensis. Infected T cell-specific IL-4Rα-deficient mice showed comparable worm expulsion, goblet cell hyperplasia and IgE responses to control mice. However, impaired IL-4-promoted T_H2 cells in T cell-specific IL-4Rα deficient mice led to strikingly reduced IL-4 production by mesenteric lymph node CD4⁺ T cells and reduced intestinal IL-4 and IL-13 levels, compared to control mice. This reduced IL-4/IL-13 response was associated with an impaired IL-4/IL-13-mediated smooth muscle cell hypercontractility, similar to that seen in global IL-4Rα^−/− mice. These results demonstrate that IL-4-promoted T cell responses are not required for the resolution of a primary N. brasiliensis infection. However, they do contribute significantly to an important physiological manifestation of helminth infection; namely intestinal smooth muscle cell-driven hypercontractility.     

Estrogen receptor-β signaling modulates epithelial barrier function

Impaired epithelial barrier function and estrogens are recognized as factors influencing inflammatory bowel disease (IBD) pathology and disease course. Estrogen receptor-β (ERβ) is the most abundant estrogen receptor in the colon and a complete absence of ERβ expression is associated with disrupted tight-junction formation and abnormal colonic architecture. The aim of this study was to determine whether ERβ signaling has a role in the maintenance of epithelial permeability in the colon. ERβ mRNA levels and colonic permeability were assessed in IL-10-deficient mice and HLA-B27 rats by RT-PCR and Ussing chambers. ERβ expression and monolayer resistance were measured in HT-29 and T84 colonic epithelial monolayers by RT-PCR and electric cell-substrate impedance sensing. The effect of 17β-estradiol and an estrogen agonist [diarylpropionitrile (DPN)] and antagonist (ICI 182780) on epithelial resistance in T84 cells was measured. Expression of ERβ and proinflammatory cytokines was investigated in colonic biopsies from IBD patients. Levels of ERβ mRNA were decreased, whereas colonic permeability was increased, in IL-10-deficient mice and HLA-B27 transgenic rats prior to the onset of colitis. T84 cells demonstrated higher resistance and increased levels of ERβ mRNA compared with HT-29 cells. 17β-estradiol and DPN induced increased epithelial resistance in T84 cells, whereas an ERβ blocker prevented the increased resistance. Decreased ERβ mRNA levels were observed in colonic biopsies from IBD patients. This study suggests a potential role for ERβ signaling in the modulation of epithelial permeability and demonstrates reduced ERβ mRNA in animal models of colitis and colon of patients with inflammatory bowel disease.     

Trolox and Ascorbic Acid Reduce Direct and Indirect Oxidative Stress in the IPEC-J2 Cells,an In Vitro Model for the Porcine Gastrointestinal Tract

Oxidative stress in the small intestinal epithelium is a major cause of barrier malfunction and failure to regenerate. This study presents a functional in vitro model using the porcine small intestinal epithelial cell line IPEC-J2 to examine the effects of oxidative stress and to estimate the antioxidant and regenerative potential of Trolox, ascorbic acid and glutathione monoethyl ester. Hydrogen peroxide and diethyl maleate affected the tight junction (zona occludens-1) distribution, significantly increased intracellular oxidative stress (CM-H₂DCFDA) and decreased the monolayer integrity (transepithelial electrical resistance and FD-4 permeability), viability (neutral red) and wound healing capacity (scratch assay). Trolox (2 mM) and 1 mM ascorbic acid pre-treatment significantly reduced intracellular oxidative stress, increased wound healing capacity and reduced FD-4 permeability in oxidatively stressed IPEC-J2 cell monolayers. All antioxidant pre-treatments increased transepithelial electrical resistance and viability only in diethyl maleate-treated cells. Glutathione monoethyl ester (10 mM) pre-treatment significantly decreased intracellular oxidative stress and monolayer permeability only in diethyl maleate-treated cells. These data demonstrate that the IPEC-J2 oxidative stress model is a valuable tool to screen antioxidants before validation in piglets.     

The NCI-N87 cell line as a gastric epithelial barrier model for drug permeability assay

The objective of this study was to evaluate the human NCI-N87 cell line as a model for gastric permeability drug studies under pH conditions of the stomach. The optimal conditions that led NCI-N87 cells to form a typical differentiated gastric epithelial barrier were a seeding density of 2.5 × 10⁵ cells/cm² on porous inserts and growth in serum-complemented RPMI-1640 medium until 18–27 days post-confluency. The resulting cell monolayers showed moderately high transepithelial electrical resistance (TEER) values of about 500 Ω cm², cells of polygonal morphology expressing E-cadherin and ZO-1 proteins at their contact surfaces, and production of mucus clusters. The monolayers withstood apical pH of 7.4 down to 3.0 with the basal pH fixed at 7.4. The apparent permeability coefficients (P_app) of model compounds were evaluated in the apical-to-basolateral and basolateral-to-apical directions under different pH gradients. The monolayers were impermeable to the integrity marker Lucifer Yellow (low P_app of 0.3–1.1 × 10⁻⁶ cm/s). The furosemide P_app (0.4–1.5 × 10⁻⁵ cm/s) were slightly dependent on pH but remained moderate. The caffeine P_app (4.2–5.0 × 10⁻⁵ cm/s) were higher and insensitive to pH changes. The NCI-N87 cell line provides a useful in vitro tool to assess gastric drug permeability and absorption under physiologic conditions prevailing in the human stomach.     

Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils

In human intestinal disease induced by Salmonella typhimurium, transepithelial migration of neutrophils (PMN) rapidly follows attachment of the bacteria to the epithelial apical membrane. In this report, we model those interactions in vitro, using polarized monolayers of the human intestinal epithelial cell, T84, isolated human PMN, and S. typhimurium. We show that Salmonella attachment to T84 cell apical membranes did not alter monolayer integrity as assessed by transepithelial resistance and measurements of ion transport. However, when human neutrophils were subsequently placed on the basolateral surface of monolayers apically colonized by Salmonella, physiologically directed transepithelial PMN migration ensued. In contrast, attachment of a non-pathogenic Escherichia coli strain to the apical membrane of epithelial cells at comparable densities failed to stimulate a directed PMN transepithelial migration. Use of the n-formyl-peptide receptor antagonist N-t-BOC-1-methionyl-1-leucyl-1- phenylalanine (tBOC-MLP) indicated that the Salmonella-induced PMN transepithelial migration response was not attributable to the classical pathway by which bacteria induce directed migration of PMN. Moreover, the PMN transmigration response required Salmonella adhesion to the epithelial apical membrane and subsequent reciprocal protein synthesis in both bacteria and epithelial cells. Among the events stimulated by this interaction was the epithelial synthesis and polarized release of the potent PMN chemotactic peptide interleukin-8 (IL-8). However, IL-8 neutralization, transfer, and induction experiments indicated that this cytokine was not responsible for the elicited PMN transmigration. These data indicate that a novel transcellular pathway exists in which subepithelial PMN respond to lumenal pathogens across a functionally intact epithelium. Based on the known unique characteristics of the intestinal mucosa, we speculate that IL-8 may act in concert with an as yet unidentified transcellular chemotactic factor(s) (TCF) which directs PMN migration across the intestinal epithelium.     

Prior stimulation of antigen-presenting cells with Lactobacillus regulates excessive antigen-specific cytokine responses in vitro when compared with Bacteroides

The development of allergy is related to differences in the intestinal microbiota. Therefore, it is suggested that the immune responses induced by different genera of bacteria might be regulated through adaptive as well as innate immunity. In this study, we examined whether antigen-specific immune responses were affected by stimulation with the different genera of intestinal bacteria in vitro. Mesenteric lymph node (MLN) cells isolated from germ-free ovalbumin (OVA)-specific T cell receptor transgenic (OVA-Tg) mice were stimulated with OVA and intestinal bacteria. Cecal contents from conventional mice but not germ-free mice could induce OVA-specific cytokine production. Among the murine intestinal bacteria, Bacteroides acidofaciens (BA) enhanced OVA-specific IFN-γ and IL-10 production while Lactobacillus johnsonii (LA) increased OVA-specific IL-10 production only. The expression of cell surface molecules and cytokine production by antigen-presenting cells (APCs) from germ-free Balb/c mice were analyzed. BA increased the expression of MHC II and co-stimulatory molecules on APCs compared with LA. BA increased IL-6 and IL-10 production but induced less IL-12p40 than LA. To examine the effects of prior stimulation of APCs by intestinal bacteria on the induction of antigen-specific immune responses, cytokine production was determined following co-culture with OVA, CD4⁺ T cells from OVA-Tg mice, and APCs which were pre-stimulated with the bacteria or not. APCs pre-stimulated with LA did not enhance OVA-specific cytokine production while BA stimulated OVA-specific IL-10 production. These results suggest that the prior stimulation of intestinal immunocytes by Lactobacillus might regulate excessive antigen-specific cytokine responses via APCs when compared with prior stimulation by Bacteroides.