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Background Epithelial barrier defects are well known in coeliac disease, but the mechanisms are only poorly defined. It is unclear, whether barrier disturbance reflects upregulated epithelial transcytosis or paracellular leakage. Objective To characterise the molecular structure and function of the epithelial tight junction (TJ) and mechanisms of its dysregulation. Methods Molecular analysis of proteins involved in TJ assembly and their regulation was performed by western blotting and confocal microscopy correlated to electrophysiology. Results A complex alteration of the composition of epithelial TJ proteins (with more pore-forming claudins like claudin-2 and a reduction in tightening claudins like claudin-3, -5 and -7) was found for protein expression and subcellular localisation, responsible for an increase in paracellular biotin-NHS uptake. In contrast, epithelial apoptosis was only moderately elevated (accounting for a minor portion of barrier defects) and epithelial gross lesions—for example, at cell extrusion zones, were absent. This TJ alteration was linked to an altered localisation/expression of proteins regulating TJ assembly, the polarity complex protein Par-3 and the serine-/threonine phosphatase PP-1. Conclusions Changes in cell polarity proteins Par-3 and PP-1 are associated with altered expression and assembly of TJ proteins claudin-2, -3, -5 and -7 and ZO-1, causing paracellular leakage in active coeliac disease.

The intestinal epithelium, the largest interface between the host and environment, regulates fluxes of ions and nutrients and limits host contact with the massive load of luminal antigens. Local protective and tolerogenic immune responses toward luminal content depend on antigen sampling by the gut epithelial layer. Whether, and how exaggerated, the entrance of antigenic macromolecules across the gut epithelium might initiate and/or perpetuate chronic inflammation as well as the respective contribution of paracellular and transcellular permeability remains a matter of debate. To this extent, experimental studies involving the in vivo assessment of intestinal permeability using small inert molecules do not necessarily correlate with the uptake of larger dietary antigens. This review analyzes both the structural and functional aspects of intestinal permeability with special emphasis on antigen handling in healthy and diseased states and consequences on local immune responses to food antigens.

Resume L’intestin est une zone d’absorption des nutriments et un lieu d’echantillonnage du contenu macromoleculaire (antigenes, bacteries) necessaire a l’information du systeme immunitaire et a sa reponse tolerogene ou inflammatoire. Deux voies de transport epithelial sont impliquees : les voies paracellulaire et transcellulaire. Les jonctions serrees intercellulaires determinent la permeabilite paracellulaire et en condition normale seules les petites molecules (type mannitol) sont permeables. Les antigenes alimentaires ne peuvent franchir les jonctions serrees, ils sont vehicules par transcytose a travers les enterocytes, partiellement degrades en peptides qui se lient aux molecules du CMH II enterocytaires et secretes sous forme de vesicules presentatrices d’antigenes appelees exosomes. Le contenu luminal peut aussi etre reconnu grâce au transport d’immun-complexes IgE dans l’allergie, d’immun-complexes IgA dans la maladie cœliaque ou parfois d’immun-complexes IgG. Ces complexes permettent une reponse inflammatoire qui est protectrice lorsqu’il s’agit d’eradiquer des bacteries pathogenes mais deletere lorsqu’il s’agit de complexes portant des antigenes ou allergenes alimentaires (allergies, maladie cœliaque).

Celiac disease is a multifactorial disorder and provides a privileged model to decipher how the interplay between environmental and genetic factors can alter mucosal tolerance to a food antigen, lead to chronic intestinal inflammation, and ultimately promote T-cell lymphomagenesis. Here we summarize how HLA-DQ2/8 molecules, the main genetic risk factor for this disease can orchestrate a CD4+ T-cell adaptive immune response against gluten, and discuss recent data which shed light on the innate and adaptive immune stimuli that collaborate to induce a proinflammatory TH1 response, a massive expansion of intraepithelial lymphocytes, and a cytolytic attack of the epithelium. The intestinal immune response driven in genetically predisposed patients by chronic exposure to gluten emerges as the pathological counterpart of normal acute intestinal responses to intracellular pathogens.

Celiac disease (CD) is an enteropathy resulting from an abnormal immune response to gluten-derived peptides in genetically susceptible individuals. This immune response is initiated by intestinal transport of intact peptide 31-49 (p31-49) and 33-mer gliadin peptides through an unknown mechanism. We show that the transferrin receptor CD71 is responsible for apical to basal retrotranscytosis of gliadin peptides, a process during which p31-49 and 33-mer peptides are protected from degradation. In patients with active CD, CD71 is overexpressed in the intestinal epithelium and colocalizes with immunoglobulin (Ig) A. Intestinal transport of intact p31-49 and 33-mer peptides was blocked by polymeric and secretory IgA (SIgA) and by soluble CD71 receptors, pointing to a role of SIgA–gliadin complexes in this abnormal intestinal transport. This retrotranscytosis of SIgA–gliadin complexes may promote the entry of harmful gliadin peptides into the intestinal mucosa, thereby triggering an immune response and perpetuating intestinal inflammation. Our findings strongly implicate CD71 in the pathogenesis of CD.

Background & Aims: 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. Methods: The capacity of exosomes derived from the HLA-DR4-expressing, intestinal epithelial cell line T84 to load the HLA-DR4-specific peptide 3H-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. Results: 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 (×10−3). Conclusions: 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.

Resume L’une des proprietes cles des micro-organismes probiotiques est la modulation du systeme immunitaire de l’hote. Selon que l’on s’adresse a l’individu sain ou presentant une pathologie liee a un dysfonctionnement immunitaire (allergies, maladies inflammatoires chroniques de l’intestin ou MICI), leurs effets peuvent etre tres differents : en effet, en situation normale, la plupart des micro-organismes probiotiques stimulent les defenses immunitaires innees (phagocytose, cytokines pro-inflammatoires) et agissent favorablement sur la duree des episodes infectieux ou sur la reponse vaccinale. Par ailleurs, un effet benefique anti-inflammatoire a aussi ete souligne dans certaines situations pathologiques (allergies, MICI), pour certaines souches de micro-organismes probiotiques. Cet effet « souche » doit imperativement rester en memoire, chaque souche d’une meme espece pouvant se conduire differemment. Les mecanismes impliques dans ces effets benefiques font actuellement l’objet de nombreuses etudes, l’effet immunostimulant chez l’homme sain engageant plus particulierement des composes bacteriens reconnaissant des recepteurs invariants des cellules immunitaires et l’effet anti-inflammatoire impliquant plutot des metabolites secretes (butyrate, NO) ou des constituants bacteriens (acides lipoteichoiques, sequence ADN riches en CpG).

Resume Les probiotiques sont des micro-organismes vivants, qui, ingeres en quantite adequate, sont susceptibles d'ameliorer certains aspects de la sante de l'hote, les effets sur le systeme immunitaire etant preponderants. Deux types d'effets paradoxaux sont decrits en fonction du statut immunologique initial de l'hote : d'une part, certains probiotiques peuvent stimuler une reponse immunitaire innee (phagocytose, cytokines) chez l'individu sain, raccourcissant ainsi l'evolution d'episodes infectieux (notamment les gastroenterites virales chez l'enfant) ou augmentant les anticorps protecteurs lors de vaccinations. D'autre part, une litterature importante demontre un effet anti-inflammatoire de souches probiotiques definies, dans certaines maladies inflammatoires chroniques de l'intestin, en particulier les rectocolites hemorragiques. Les mecanismes impliques commencent a etre mieux caracterises. L'effet immunostimulant engage des constituants de paroi bacterienne reconnaissant des recepteurs invariants (recepteurs toll-like) sur les cellules epitheliales et/ou immunitaires et l'effet anti-inflammatoire pourrait impliquer des composes secretes (butyrate, NO) ou des constituants bacteriens (acides lipoteichoiques, CpG-oligodeoxynucleotides). Les mecanismes de l'effet anti-inflammatoire sont moins bien caracterises.

The alteration of the intestinal epithelial barrier is often a consequence of various intestinal diseases but may also be the starting point of these diseases. Undigested food antigens are transported across the intestinal epithelium by a transcytotic mechanism, including a processing within the enterocytes, and leading to the passage of intact proteins, peptides, and amino acids to the underlying mucosa. Inflammation and infection lead to the upregulation of the transport and processing of food proteins; for example, IFN gamma increases the rate of transcytosis and alters, like TNF alpha, the tight junction permeability. Infection of gastric digestive epithelia with Helicobacter pylori also increases the antigenic load transmitted to the underlying immune system by inhibiting the enterocytic lysosomal degradation of proteins. In allergic diseases, such as cow's milk allergy, TNF alpha may be involved in the intestinal dysfunction and the associated enteropathy.

Intestinal permeability tests used in the diagnosis of allergic diseases in response to oral food challenge have led to the conclusion that constitutive defects of the intestinal barrier are not the primary cause of allergic diseases. However, perturbation of environmental factors (infection, stress), by increasing intestinal permeability and enhancing danger signals, may favour food allergy in susceptible individuals. The mechanisms of enhanced permeability to specific and bystander antigens have been delineated as well as the molecular events involved in the sequential phases of allergic reactions. Intestinal absorption of food antigens and immune responses are mutually dependent, and luminal (environmental) and serosal (intrinsic) factors synergize to maintain a self-perpetuating cycle in which antigens penetrate the mucosa and induce allergic inflammation.

Lactic acid bacteria or their secretion products can modulate immune responses differently in normal and inflammatory conditions. This comparative study analyzes the effect of oral administration of living lactic acid bacteria, or their conditioned media, on the epithelial and immune functions of colitis-prone C57BL/6 IL-10-deficient mice. Mice were untreated (control) or infected with Helicobacter hepaticus with or without oral treatment with living bacteria, Bifidobacterium breve C50 and Streptococcus thermophilus 065 (LB), or their culture-conditioned media (CM). Histology, cytokine mRNA, electrical resistance, and barrier capacity of colonic samples as well as cytokine secretion by mesenteric lymph node (MLN) cells were studied. Helicobacter hepaticus mice developed only mild colitis, which was not modified in LB or CM groups. In the CM (but not the LB) group, the colonic barrier was reinforced as compared to the other groups, as evidenced by decreased horseradish peroxidase (HRP) transcytosis and mannitol fluxes and increased electrical resistance. In MLN, the percentage of CD4+ and CD8+ T cells secreting IFNγ was significantly higher in CM (2.06% and 1.98%, respectively) mice than in H. hepaticus (1–1% and 0.47%, P < 0.05) or control mice. In addition, the nonspecific stimulation of IFNγ, TNFΑ, and IL-12 secretion by MLN cells was significantly higher in the CM group as compared to the other groups. In the absence of severe colitis, Bifidobacterium breve C50- and Streptococcus thermophilus 065-conditioned media can reinforce intestinal barrier capacity and stimulate Th1 immune response, highlighting the involvement of lactic acid bacteria–derived components in host defense.

Background & Aims: The resistance of prolamines to digestive enzymes is thought to be a key contributor to the pathogenesis of celiac disease by promoting the intestinal entrance of peptides able to trigger inflammation in at-risk individuals. Oral administration of a bacterial prolyl-endopeptidase (PEP) therefore was proposed as a treatment for celiac disease. To delineate the feasibility of this treatment, the effect of PEP on gliadin peptides was assessed in vitro, and ex vivo during their transport across intestinal biopsy specimens of active celiac disease patients. Methods: In vitro degradation by PEP of 3H-labeled gliadin peptides 56–88 (33-mer) and 31–49, was analyzed by radio–reverse-phase high-performance liquid chromatography and mass spectrometry. For ex vivo studies, PEP and 3H-peptides were added together onto the mucosal side of duodenal biopsy specimens mounted in Ussing chambers, and peptide transport and digestion were assessed by radio–reverse-phase high-performance liquid chromatography. Results: Gliadin peptides were degraded partly by 20 mU/mL PEP both in vitro and ex vivo. This concentration of PEP decreased the amount of intact peptides 31–49 and 56–88 crossing the intestinal biopsy specimens of celiac disease patients, but could not prevent the intestinal passage of toxic or immunostimulatory metabolites. PEP concentrations of at least 500 mU/mL for 3 hours were required to achieve full detoxification of peptides and to prevent intestinal transport of active fragments. Conclusions: After prolonged exposure to high concentrations of PEP, the amount of immunostimulatory gliadin peptides reaching the local immune system in celiac patients is decreased. These results provide a basis to establish whether such conditions are achievable in vivo.

The low affinity receptor for IgE (CD23) was recently implicated in the trans-epithelial transport of IgE-allergen complexes from the luminal side of enterocytes in animal models for intestinal allergy. Here, the respective functions of CD23 splice forms, b and bΔ5, in this apical to basolateral transport event have been investigated. First, the new bΔ5 splice form was further characterized, providing evidence that it binds IgE with high affinity, that its expression is induced by sensitization, and that bΔ5, unlike the classical b, undergoes constitutive internalization through clathrin-coated pits. These results suggested that the two CD23 splice forms were likely involved in different transcytotic events. MDCK cell lines expressing either b or bΔ5 were generated to directly test this hypothesis. In both cell lines, CD23 splice forms were localized at the apical membrane as in enterocytes from sensitized mice. Using mouse monoclonal IgE, we obtained evidence showing that bΔ5 mediates the apical to basolateral transport of free IgE, whereas classical b is much more efficient in mediating the transcytosis of IgE/allergen complexes. The present results shed new light on the role of CD23 species in IgE/allergen transepithelial transport and provide a new powerful physiological tool to study apical to basolateral transcytosis, a process which remains poorly characterized.

Background: Probiotic bacteria have a beneficial effect on intestinal inflammation. In this study, we have examined the effect of lactic acid and commensal Gram positive (+) bacteria conditioned media (CM) on tumour necrosis factor α (TNF-α) release and the mechanisms involved. Methods: Lipopolysaccharide (LPS) induced TNF-α secretion by peripheral blood mononuclear cells or the THP-1 cell line was monitored in the presence or absence of bacteria CM obtained from two probiotic strains, Bifidobacterium breve (Bb) and Streptococcus thermophilus (St), and three commensal bacterial strains (Bifidobacterium bifidum, Ruminococcus gnavus, and unidentified Streptococcus). Bb and St bacteria CM were allowed to cross filter grown intestinal epithelial cell monolayers (HT29-19A) to assess intestinal transport of active bacterial products. These products were characterised and their effect on LPS binding to THP-1 cells and nuclear factor κB (NFκB) activation assessed. Results: Dose dependent inhibition of LPS induced TNF-α secretion was noted for both probiotic bacteria CM (64% and 71% inhibition for Bb and St, respectively) and to a lesser extent commensal bacteria CM (21–32% inhibition). Active products from Bb and St were resistant to digestive enzymes and had a molecular mass

We have previously shown that H. pylori increases the passage of intact protein across the digestive epithelium, in vitro and in vivo in mice. The aim of this study was to test whether H. pylori can alter gastric permeability to a food-type antigen in humans.Antral and corpus gastric biopsies obtained from H. pylori-positive and H. pylori-negative patients suffering from nonulcer dyspepsia with normal esogastroduodenal endoscopy were mounted in adapted Ussing chambers to measure gastric permeability. Electrical resistance (R, an index of epithelial integrity) and fluxes of 14C-mannitol (JMan, a marker of paracellular permeability) and 3H-horseradish peroxidase (HRP, a test protein of antigen transport and processing) under intact (JHRPi) and degraded (JD) form, across the biopsies, were measured.At the corpus level, biopsies obtained from H. pylori-positive patients, as compared to H. pylori-negative patients, presented significantly higher intact--but not degraded--HRP fluxes (JHRPi = 446 +/- 297 and 219 +/- 265 ng/h cm2, p0.05; JD = 4,247 +/- 3,884 and 3,575 +/- 2,594 ng/h x cm2, respectively), and an increase in paracellular permeability (Jman = 0.35 +/- 0.1 vs 0.24 +/- 0.08 micromol/h x cm2, p0.02, respectively). At the antrum level, a tendency to increased intact HRP fluxes in H. pylori-positive patients, as compared to H. pylori-negative patients, was observed (JHRPi = 284 +/- 250 vs 207 +/- 203 ng/h x cm2, p0.09), without modifications of paracellular permeability.H. pylori infection increases absorption of an intact food antigen across the corpus gastric mucosa. This phenomenon may contribute to the maintenance of gastric inflammation and could play a role in the development of allergic sensitization to dietary antigens in susceptible individuals.

Background: CD23 (FcΕRII) is expressed by intestinal epithelial cells (IEC) following allergic stimulation and increases the uptake of IgE/allergen complexes. The aim of this study was to further analyze the role of CD23 in the intraepithelial processing of food allergens during transepithelial transport. Methods: Balb-C mice were sensitized intraperitoneally with horseradish peroxidase (HRP) or β-lactoglobulin (β-LG) in the presence of pertussis toxin. In control and sensitized mice, 3H-HRP, intact HRP, or 14C-β-LG fluxes were measured across jejunal segments mounted in Ussing chambers, in the presence or absence of mucosal anti-CD23 antibodies. HPLC analysis of serosal buffer was performed to detect HRP- or β-LG-derived radiolabelled metabolites generated during transepithelial transport. Results: In HRP-sensitized mice, 3H-HRP fluxes and intact HRP fluxes (3,836 ± 476 and 290 ± 86 ng/h ·cm2, respectively) were significantly increased compared to control mice (1,677 ± 297 ng/h · cm2, p < 0.01, and 106 ± 23 ng/h · cm2, p < 0.02, respectively). HPLC analysis indicated the presence of intact HRP in the serosal compartment already 10 min after addition of HRP to the mucosal compartment, a result not observed in the control mice. In the presence of anti-CD23 antibodies, intact HRP fluxes were significantly decreased (131 ± 27 ng/h · cm2) compared to control values in sensitized mice (290 ± 86 ng/h · cm2, p < 0.02), suggesting that CD23 is involved is this ‘protected’ transport pathway. A similar protection during intestinal transport was observed for β-LG in β-LG sensitized mice. Conclusions: These results confirm that CD23 is involved in the rapid transepithelial transport of intact allergens in sensitized animals, and indicate that CD23 opens a ‘protected’ transport pathway in IECs.

Background: Intestinal epithelial cells secrete exosome-like vesicles. The aim of this study was to characterise murine intestinal epithelial exosomes and to analyse their capacity to inform the immune system in vivo in mice. Methods: Epithelial exosomes were obtained from the murine epithelial cell line MODE K incubated in the presence or absence of interferon γ (IFN-γ) together with pepsin/trypsin ovalbumin hydrolysate (hOVA) to mimic luminal digestion. Exosomes isolated from MODE K conditioned media (EXO-hOVA and EXO-hOVA-IFN) were characterised by western blot, peptide mapping, and mass spectrometry. They were injected intraperitoneally to C3H/HeN mice to test their immunocompetence. Results: MODE K epithelial exosomes displayed major histocompatibility complex (MHC) class I and class II (upregulated by IFN-γ) molecules and tetraspan proteins (CD9, CD81, CD82) potentially involved in the binding to target cells. A33 antigen, an Ig-like molecule highly specific for intestinal epithelial cells, was enriched in exosomes and was also found in mice mesenteric lymph nodes, suggesting exosome migration towards the gut associated lymphoid tissues. Intraperitoneal injection of EXO-hOVA or EXO-hOVA-IFN did not induce humoral or cellular tolerance to OVA in mice. In contrast, exosomes obtained after incubation with IFN-γ (EXO-hOVA-IFN), bearing abundant MHC class II/OVA complexes, induced a specific humoral immune response. Conclusions: Epithelial exosomes are antigen presenting vesicles bearing MHC class II/peptide complexes that prime for an immunogenic rather than tolerogenic response in the context of a systemic challenge. In the intestine, both the mucosal microenvironment and local effector cells are probably key players in determining the outcome of the immune response to exosome derived epitopes.

Rebamipide exerts a positive effect on the digestive epithelial barrier by reinforcing its integrity in normal and in inflammatory conditions, and by normalizing the macromolecular transport across this barrier, increased by Helicobacter infection. Moreover, in mice, rebamipide is capable of diminishing allergic sensitization and of counteracting the inhibitory effect of Helicobacter pylori on oral tolerance to dietary antigens. These properties of rebamipide could explain its anti-inflammatory activity with respect to the digestive mucosa and could provide protection against allergic sensitization to foreign antigens in susceptible individuals.

Nitazoxanide is a thiazolide compound that exhibits antimicrobial properties against helminths, protozoa, anaerobic bacteria and also Helicobacter pylori. The mucosal diffusion of this new drug has not been studied. The aim of this study was to examine the transport of radiolabelled nitazoxanide across the epithelial barrier according to the mode (mucosal or serosal) of drug administration. HT29-19A intestinal epithelial cells, grown as monolayers on microporous filters, were used as an epithelial model. In a short-term (100 min) transport study, the apical to basal and the basal to apical transport of nitazoxanide across the monolayers was studied in an Ussing chamber. In a long-term (24 h) study, the transport of the drug and its intracellular accumulation were studied in filter-grown epithelial monolayers kept in culture plates. In the short-term transport study, both the apical and basal fluxes achieved a steady state after 70 min and there was no significant difference between the apical to basal (3991 ± 1001 ng h−1 cm−2) and the basal to apical (4246 ± 856 ng h−1 cm−2) nitazoxanide fluxes. In the long-term transport study, after apical or basal drug application, a gradual increase in the drug concentration in the opposite compartment was noted, which reached similar values for apical and basal fluxes (2497 ± 125 and 2309 ± 81 ng mL−1, respectively) after 24 h. Moreover, a rapid, although transitory, intracellular accumulation of nitazoxanide was observed at 10 min after apical (299 ± 25 ng/106 cells) and basal (124 ± 10 ng/106 cells) drug application, but decreased thereafter. There is an important transepithelial transport of nitazoxanide across the digestive epithelial monolayer with a rapid intracellular accumulation of the drug. No difference between the apical to basal and basal to apical fluxes of the drug was observed, suggesting that both topical and systemic modes of action of this antibiotic are successful.

Intestinal epithelial cells (IEC) are located at the strategic interface between the external environment and the most extensive lymphoid compartment in the body. Besides their central role in the absorption of nutrients, they also provide sample information to the immune system on soluble or particulate antigens present in the intestinal lumen. Like professional antigen-presenting cells, IEC have recently been shown to secrete 30- to 90-nm diameter vesicles named exosomes from their apical and basolateral surfaces. These vesicles carry molecules that are implicated in adhesion and antigen presentation, such as major histocompatibility complex (MHC) class I molecules, MHC class II molecules, CD63, CD26/dipeptidyl-peptidase IV, tetraspan proteins, and A33 antigen. IEC exosomes therefore, constitute a link by which IEC may influence antigen presentation in the mucosal or systemic immune system independent of direct cellular contact with effector cells.

Infection with Helicobacter increases the transcellular passage of macromolecules across the epithelium, and this effect can be prevented by a gastroprotective agent rebamipide. The aim was to gain insight into the mechanisms involved. The HT29-19A intestinal epithelial cells grown on microporous filters as monolayers were incubated in the presence or absence of rebamipide (1 or 2 mM) with: (1) suspension of a wild H. pylori strain, (2) IL-1β (0.5 ng/ml) + IFN-γ (2 units/ml). After incubation, the monolayers were submitted to evaluation of apoptosis by using the apoptotic cell death detection ELISA kit and to assessment of epithelial permeability in Ussing chamber where the ionic conductance (G), fluxes of mannitol (JMan) and of horseradish peroxidase in both intact (JHRPi)- and degraded (JD) form, were measured. H. pylori increased the intact HRP fluxes across the barrier (JHRPi = 17 ± 20 vs 97 ± 70 ng/hr/cm2, P < 0.007), an effect prevented by rebamipide (JHRPi = 33 ± 34 ng/hr/cm2, P < 0.006). IL-1β increased the ionic conductance (G = 5.5 ± 1.0 and 21.0 ± 7.0 mS/cm2, P < 0.006), the intact HRP fluxes (JHRPi = 18 ± 15 and 476 ± 344 ng/hr/cm2, P < 0.006), and the apoptotic index of the cells (AI = 1 ± 0 vs 3.7 ± 0.8), all effects prevented by rebamipide (G = 12 ± 4.9 mS/cm2, JHRPi = 79 ± 38, AI = 1.6 ± 0.6, P < 0.03 as compared to IL-β-treated cells). In basal conditions, rebamipide increased the integrity of the barrier (G = 7.5 ± 2.3 vs 6.0 ± 1.8 mS/cm2 for controls, P < 0.007). In conclusion, H. pylori as well as IL-1β, may alter epithelial permeability and rebamipide may exert its protective effect on gastric mucosa by reinforcing the epithelial barrier in normal conditions and by counteracting the deleterious effect of Helicobacter pylori and IL-1β on macromolecular permeability.

Background: Despite Helicobacter pylori eradication, gastric inflammation persists for months or years. Preliminary results have indicated that an increase in gastric permeability could be one reason. Our aim was to evaluate the effect of a mucosal protective drug, rebamipide, on gastric permeability in a model of H. felis-infected mice. Methods: Thirty-three C57/BL6 mice were inoculated with H. felis, and seven controls were kept non-inoculated. After 2 months 20 infected mice were treated with omeprazole, amoxicillin, and clarithromycin for 1 week and then for 4 weeks with either rebamipide (n = 9) or placebo (n = 11). The 13 remaining mice were kept untreated. After cessation of treatment, a fragment of antrum obtained from each mouse was mounted in a small Ussing chamber to study the electric resistance of the tissue (R) and antral permeability. Results: No modification of the paracellular permeability (R, JNa, JMan) was observed in either group. However, the transcellular permeability to horseradish peroxidase (HRP) was significantly increased in H. felis-infected non-treated mice (1131 463, 948 339, and 182 312 ng/hcm 2 ) as compared with non-infected controls (469 262, 458 261, and 10 6 ng/ hcm 2 , for J 3 H-HRP, JD, and JHRPi, respectively) (P< 0.003). Eradication of the bacteria by antibiotics without subsequent treatment with rebamipide led to a non-significant decrease in the HRP fluxes. However, when rebamipide was used after the antibiotic treatment, a significant (P< 0.01) decrease in HRP fluxes as compared with non-treated mice was observed. Conclusions: These results confirm that gastric permeability to macromolecules remains increased despite H. felis eradication and show that rebamipide can facilitate the normalization of gastric permeability to macromolecules after bacterial eradication.

Background: TNF-α secretion by blood mononuclear cells stimulated with cow’s milk proteins is significantly higher in infants with active cow’s milk allergy (CMA) manifested by digestive symptoms than in children who have recovered from CMA. Objective: The current study was undertaken to analyze the kinetics of TNF-α secretion and to evaluate the usefulness of the measurement of TNF-α release in whole blood cultures in the prediction of clinical outcome after milk challenge. Methods: Blood samples were obtained from 83 children maintained on a cow’s milk–free diet and examined just before a cow’s milk provocation. Children were divided into 4 groups according to clinical outcome: group I (active CMA with cutaneous symptoms), group II (active CMA with predominantly digestive symptoms), group III (children recovered from CMA), and group IV (control). The kinetics of TNF-α secretion was measured in blood cultured for 1 to 5 days at different cow’s milk protein concentrations. Results: On day 1 TNF-α secretion was significantly higher in group I (485 [453] pg/mL, mean [SD], P < .005) and in group II (269 [102] pg/mL, P < .005) than that observed in groups III and IV (149 [95] and 87 [71] pg/mL, respectively). Then TNF-α was rapidly degraded and a second peak of secretion was observed on day 5 but only in group II (278 [221] pg/mL), whereas in groups I, III, and IV a low secretion was observed (70 [61], 45 [40], and 11 [12] pg/mL, respectively, P < .02). Conclusion: These results show that the pattern of TNF-α secretion in response to cow’s milk proteins is different in CMA infants with cutaneous or digestive symptoms and suggest that TNF-α release might predict clinical relapse on challenge. (J Allergy Clin Immunol 1999;104:863-9.)

The respective effect of milk and fermented milks on intestinal barrier capacity and on sensitization to beta-lactoglobulin was studied using a guinea pig model of cow's milk allergy.Guinea pigs were fed a control diet or the same diet supplemented with milk, fermented milk (Streptococcus thermophilus and Bifidobacterium breve), or dehydrated fermented milk. Intestinal barrier capacity to macromolecules was assessed in an Ussing chamber, and sensitization to cow's milk proteins was measured by systemic anti-beta-lactoglobulin immunoglobulin G1 titers and by intestinal anaphylaxis, the latter assessed by the beta-lactoglobulin-induced increase in short-circuit current of jejunal fragments (deltaIsc(beta-LG)).The electrical resistance of jejunum was similar in the four groups (approximately 80 omega/cm2) suggesting the same paracellular permeability. The transport of 14C-beta-lactoglobulin from mucosa to serosa was significantly decreased in the animals fed dehydrated fermented milk (403+/-131 ng / hr x cm2) compared with that in control animals or animals fed milk (767+/-250 ng / hr x cm2 and 749+/-475 ng / hr x cm2, respectively; p0.05). Milk fermentation did not modify native beta-lactoglobulin concentration but anti-beta-lactoglobulin immunoglobulin G1 titers were higher in fermented milk and dehydrated fermented milk (log10 titer = 2.86 and 2.79, respectively) than in guinea pigs fed milk (log10 titer = 2.5; p0.007). However, beta-lactoglobulin-induced intestinal anaphylaxis remained the same in the three groups (deltaIsc(beta-LG), 9.6+/-4.1 microA/cm2, 8.5+/-4.3 microA/cm2, and 8.5+/-3.4 microA/cm2 in milk-fed, fermented milk-fed, and dehydrated fermented milk-fed guinea pigs, respectively).The intestinal barrier capacity to milk proteins seems to be reinforced by dehydrated fermented milk, but milk and fermented milks are equally efficient in inducing cow's milk allergy in guinea pigs.

International audience; We have previously shown that milk sensitization aggravates intestinal dysfunction in the malnourished guinea pigs, suggesting that it may also impair the recovery from malnutrition. To test this hypothesis, the growing guinea pigs were malnourished by feeding only maize for 7 d and then were refed for 21 d with a balanced diet containing either intact or hydrolyzed cow's milk proteins. The control animals received the hydrolyzed milk protein diet for 28 d. After an initial period of total inhibition of growth owing to maize, guinea pigs gained weight regularly, with both balanced diets, and there was no evidence of mucosal damage at the end of the refeeding period. However, refeeding with intact milk proteins induced milk sensitization, which was demonstrated on the systemic level by the presence of anti-beta-lactoglobulin IgG1 antibodies, and on the local level by the intestinal anaphylaxis measured by the increase in short circuit current induced by beta-lactoglobulin (16.4 +/- 2.6 mu A/cm(2)) in jejunal segments mounted in Ussing chambers. Such an immune sensitization was associated with impaired intestinal permeability, as both the ionic conductance (21.0 +/- 1.6 versus 14.6 +/- 0.7 mS/cm(2)) and the transepithelial fluxes of horseradish peroxidase (537 +/- 203 versus 152 +/- 28 ng/h.cm(2)) were significantly increased in guinea pigs refed with the intact milk proteins compared with controls. In contrast, there was no difference in intestinal permeability between controls and guinea pigs refed with the hydrolyzed milk protein diet. These data show that sensitization to cow's milk proteins can develop in guinea pigs recovering from severe malnutrition and may impair full intestinal repair.

Non-live probiotic bacteria and their fermentation products can be used in milk-based formula intended for healthy infants. The effects of a milk formula fermented with Bifidobacterium breve and Streptococcus thermophilus and heated/dehydrated to inactivate the micro-organisms have been reported over the last few years to decrease the intestinal permeability to macromolecules in experimental animals in vivo and more recently to down-regulate inflammatory condition in vitro. Feeding guinea-pigs with such dehydrated fermented milk reinforced the intestinal barrier resistance to food proteins (HRP, β-lactoglobulin). In addition, the products secreted by bacteria were capable of inhibiting the lipopolysaccharide (LPS)-induced TNF-a secretion by human peripheral mononuclear blood cells. The active secretion products were resistant to digestive enzymes and their anti-inflammatory properties were preserved after transepithelial transport across the filter-grown intestinal epithelial cell line, especially in inflammatory conditions. The binding of LPS to monocytes as well as NFκB nuclear translocation leading to pro-inflammatory cytokine transcription were inhibited by bacteria-culture supernatants. Conclusion: B. breve and S. thermophilus used as non-live micro-organisms in fermented infant formula seem to induce a reduction in macromolecular absorption and release metabolites exerting an anti-TNF-a effect, which persists after intestinal transport. Thus, specific lactic acid bacteria and their metabolites seem to affect positively the intestinal function.

Zinc supplementation could favor recovery from diarrhea in malnourished children. As the recent experimental evidence suggests that oxidative stress and intestinal anaphylaxis may contribute to the intestinal dysfunction associated with malnutrition, we postulated that zinc could act through antioxidant or antianaphylactic properties. Control (C), malnourished (M), and malnourished zinc-treated (MZ) guinea pigs were, respectively, fed a normal 30% protein diet, a low 4% protein diet, and a low 4% protein diet plus 1800 ppm of zinc. Milk proteins were included in the diets to trigger intestinal anaphylaxis. Milk sensitization was assessed by passive cutaneous anaphylaxis (PCA) against beta-lactoglobulin and by intestinal anaphylaxis measured in Ussing chambers by the increase in short circuit-current after addition of beta-lactoglobulin (deltaIsc(betaLg)). Oxidative stress was assessed by intestinal lipid peroxidation. The intestinal secretion was assessed by deltaIsc induced by inflammatory mediators. Malnutrition increased the level of anti-betaLg reaginic antibodies [PCA = 1.19 +/- 0.79 and 0.69 +/- 0.67 log(l/titer) in M versus C guinea pigs, p = 0.07] and enhanced intestinal anaphylaxis (deltaIsc(betaLg)) = 16.4 +/- 9.9 and 9.1 +/- 5.8 microA/cm2 in M versus C guinea pigs, p = 0.07), without inducing intestinal lipid peroxidation. Moreover, malnutrition enhanced significantly the intestinal secretory response to histamine and 5-hydroxytryptamine. Administration of pharmacologic doses of zinc during malnutrition inhibited the increase in milk sensitization induced by malnutrition, both at the systemic [PCA = 0.35 +/- 0.55 log(l/titer) in MZ guinea pigs, p = 0.03 versus M] and intestinal (deltaIsc(betaLg)) = 2.8 +/- 2.5 microA/cm2 in MZ guinea pigs; p = 0.001 versus M) level, and prevented the hypersecretion in response to histamine and 5-hydroxytryptamine. These data suggest that zinc has antianaphylactic and antisecretory properties that may contribute to its capacity to prevent intestinal dysfunction during malnutrition.

Summary Background In infants with cow's milk allergy and intestinal symptotns, peripheral blood mononuclear cells stimulated in vitro with cow's milk proteins, secrete large amounts of the proinflammatory cytokine TNFα thus altering intestinal barrier capacity. Terfenadine, an antihistaminic drug, inhibits the release of several inflammatory mediators, including histamine, prostaglandins and leukotrienes. Objectives To test the potential ability of terfenadine to inhibit TNFα secretion by mononuclear cells from infants with cow's milk allergy. Methods Mononuclear cells from infants allergic to cow's milk proteins were stimulated in vitro for 6 days by a mixture of milk proteins (β-lactoglobulin, α-lactalbumin and casein) with or without terfenadine (0.1 –1 μM) and culture supernatants were assayed for TNFα by enzyme immunoassay. The effect of culture supernatants on intestinal barrier capacity was evaluated by measuring the electrical resistance (index of integrity) of filter-grown HT29–19 A intestinal cells in Ussing chambers. Results During active cow's milk allergy, mononuclear cells stimulated with cow's milk proteins secreted large amounts of TNFα which significantly reduced the electrical resistance of HT29–19 A intestinal cells. There was a dose-dependent decrease in TNFα secretion in the presence of terfenadine, with a maximal inhibition of 62% of this secretion at 1 μM. Accordingly, terfenadine-treated mononuclear cells supernatants did not alter the electrical resistance of intestinal HT29. 19 A cells. Conclusion These results indicate that in infants with intestinal dysfunction due to cow's milk allergy, terfenadine is a potent inhibitor of the TNFα secretion induced by sensitizing milk protein antigens. This inhibition prevents the degradation of intestinal function as measured in an intestinal cell line, in vitro.

Non-live probiotic bacteria and their fermentation products can be used in milk-based formula intended for healthy infants. The effects of a milk formula fermented with Bifidobacterium breve and Streptococcus thermophilus and heated/dehydrated to inactivate the micro-organisms have been reported over the last few years to decrease the intestinal permeability to macromolecules in experimental animals in vivo and more recently to down-regulate inflammatory condition in vitro. Feeding guinea-pigs with such dehydrated fermented milk reinforced the intestinal barrier resistance to food proteins (HRP, beta-lactoglobulin). In addition, the products secreted by bacteria were capable of inhibiting the lipopolysaccharide (LPS)-induced TNF-alpha secretion by human peripheral mononuclear blood cells. The active secretion products were resistant to digestive enzymes and their anti-inflammatory properties were preserved after transepithelial transport across the filter-grown intestinal epithelial cell line, especially in inflammatory conditions. The binding of LPS to monocytes as well as NFkappaB nuclear translocation leading to pro-inflammatory cytokine transcription were inhibited by bacteria-culture supernatants.B. breve and S. thermophilus used as non-live micro-organisms in fermented infant formula seem to induce a reduction in macromolecular absorption and release metabolites exerting an anti-TNF-alpha effect, which persists after intestinal transport. Thus, specific lactic acid bacteria and their metabolites seem to affect positively the intestinal function.

International audience; : In celiac disease, enhanced permeability to gliadin peptides can result from their apico-basal transport by secretory immunoglobulin A1 (SIgA1) binding to the CD71 receptor ectopically expressed at the gut epithelial surface. Herein, we have established a mouse model in which there is apico-basal transport of the model antigen ovalbumin (OVA) by specific SIgA1 and have analyzed local T-cell activation. Transgenic DO11.10 mice were grafted with a hybridoma-secreting OVA-specific humanized IgA1, which could bind mouse CD71 and which were released in the intestinal lumen as SIgA. CD71 expression was induced at the gut apical surface by treating the mice with tyrphostin A8. Following gavage of the mice with OVA, OVA-specific CD4(+) T cells isolated from the mesenteric lymph nodes displayed higher expression of the activation marker CD69 and produced more interferon gamma in mice bearing the hybridoma-secreting OVA-specific IgA1, than in ungrafted mice or in mice grafted with an irrelevant hybridoma. These results indicate that the protective role of SIgA1 might be jeopardized in human pathological conditions associated with ectopic expression of CD71 at the gut surface.Mucosal Immunology advance online publication 10 July 2013; doi:10.1038/mi.2013.49.

Background and aimsPresence of serum antibodies against Mycobacterium avium paratuberculosis (MAP) in Crohn's Disease (CD) as a disease characteristic remains controversial. In the present work, we assessed antibody reactivity of serum and intestinal fluid against four distinct MAP-antigens, including the recently identified MAP-specific lipopentapeptide (L5P).MethodsImmunoglobulin concentrations and specificity against 3 non MAP-specific antigens: glycosyl-transferase-d (GSD), purified protein derivative from MAP (Johnin-PPD), heparin binding haemagglutinin (MAP-HBHA) and one MAP-specific antigen: synthetic L5P were determined by ELISA in gut lavage fluids from adult controls or patients with CD, and in sera of children or adult controls or patients with CD, ulcerative colitis or celiac disease.ResultsTotal IgA and IgG concentrations were increased in sera of children with CD but were decreased in sera of adults with CD, thereof specificity against MAP antigens was assessed by normalizing immunoglobulin concentrations between samples. In CD patients, IgG reactivity was increased against the four MAP antigens, including L5P in gut lavage fluids but it was only increased against L5P in sera. By contrast, anti-L5P IgG were not increased in patients with ulcerative colitis or celiac disease.ConclusionsA significant increase in anti-L5P IgG is observed in sera of children and adults with CD but not in patients with other intestinal inflammatory diseases. Anti-L5P antibodies may serve as serological marker for CD.

Background: In cow’s milk allergy (CMA) with intestinal symptoms, peripheral blood mononuclear cells (PBMCs) secrete tumor necrosis factor-α (TNF-α), altering intestinal function. However, the type of cow’s milk protein (CMP) that triggers symptoms (intact or intestinally processed) is not known, and neither is the minimal amount required. Methods: PBMCs were isolated from infants with active CMA or cured infants just before a new challenge and stimulated with intact or intestinally processed CMP. Supernatants were tested for cytokine content and for their ability to perturb intestinal barrier capacity, measured in Ussing chambers in HT29-19A intestinal cells. Results: PBMCs from infants with active CMA secreted more TNF-α, when they were stimulated with intact rather than intestinally processed CMPs, and more TNF-α than PBMCs from cured infants. Accordingly, supernatants from PBMCs stimulated with intact but not intestinally processed CMPs significantly increased intestinal permeability. The CMP concentration required to trigger TNF-α secretion capable of altering intestinal function was very small in infants with active CMA (≅2 μg/ml), but about 300 times higher in cured infants. Conclusion: Intact rather than intestinally processed proteins stimulate PBMCs to release TNF-α and alter intestinal barrier capacity. The threshold for PBMC reactivity to milk antigens drops considerably during active CMA with intestinal symptoms. (J A LLERGY C LIN I MMUNOL 1996;98:781-9.)

This paper discusses the relationship between food antigens, lymphocytes and the epithelial properties of the jejunum in children with cow's milk allergy. Experimental results indicate that increased protein permeability is not the primary cause of cow's milk allergy. Rather, results are interpreted as a secondary effect of an abnormal immunological response leading to mucosal inflammation and impairment of the endocytic process by the intestinal epithelial cells. Stimulation by cow's milk proteins caused the lymphocytes from infants with cow's milk allergy to release more tumor necrosis factor-alpha TNF alpha than those from control infants. After appropriate antigenic stimulation, the cytokines released by the activated lymphocytes from these infants perturbed epithelial function, in particular its barrier capacity. Tumor necrosis factor alpha, together with gamma interferon are involved in these adverse effects. It is thought that bovine beta-lactoglobulin present in the intestinal lumen may be responsible for the secretory diarrhea observed in children with cow's milk allergy, as a consequence of stimulation of electrogenic chloride secretion. In addition, luminal foreign protein may stimulate the submucosal cells. As a consequence, the submucosal release of mediators, including lymphokines, might alter the intestinal epithelial barrier. In conclusion, in physiological conditions, the subepithelial tissue that comprises the immune system and many other systemic systems receive information on the antigenic content within the intestinal lumen via the intestinal epithelium.

Coeliac disease is a gut disease driven by an abnormal immune response towards dietary gluten in genetically susceptible individuals. Whether and, if so, how abnormal transport of gluten across the gut epithelium may participate in the pathogenesis of coeliac disease remains debatable. This paper summarises the interactions of gluten-derived peptides with the intestinal epithelium and discusses the mechanisms that control their transport across the epithelium. It shows how recent data point to a key role for the transcellular pathway and highlights the ‘Trojan horse’ role of secretory IgA which can hijack the transferrin receptor and allow the rapid translocation of intact gluten peptides into the mucosa. These recent findings might be useful for the design of new treatments.

Tumor promoter phorbol esters directly activate protein kinase C (PKC). Similarly tumor promoter bile salts activate PKC in the human colon epithelial cell line HT-29 cl.19A, as well as in platelets. Bile salts were also able to directly activate the purified brain enzyme. We designed experiments to investigate the effects of bile salts on Cl- secretion in HT-29cl.19A cell monolayers and the involvement of PKC in this process. Sodium deoxycholate (DOG) gradually evoked a slow increase of the short-circuit current (I-sc) in the Ussing chamber-mounted cell monolayers. By contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited a large increase in I-sc which rapidly plateaued. The I-sc response was representative of enhanced Cl- secretion, since it was abrogated in Cl--free medium, and was prevented by Cl- channel blocker diphenylamine-2-carbonylate as well as by bumetanide, a Na+/K+/Cl- transporter inhibitor. Sequential applications of TPA and DOC at suboptimal doses elicited additive effects. Neither DOC nor TPA increased the levels of cAMP in these cells. However DOG, at variance with TPA, increased the intracellular content of inositol trisphosphate, presumably due to triggering of the phosphoinositide pathway. From our studies and others, it is suggested that DOG-induced unspecific activation of some membrane-associated proteins, elicits a rise in Ca2+ from extracellular and internal sources which combines its effects with those due to PKC activation.

The intestinal permeability of undegraded α9-gliadin peptide 31-49 (p31-49) and 33-mer gliadin peptides is increased in active celiac disease. Two distinct transport pathways have been proposed: paracellular leakage through epithelial tight junctions and protected transcellular transport. To analyze the relative contribution of these pathways, we compared mucosa-to-serosa permeability of small and large permeability markers [ionic conductance (G), mannitol, 182 Da; horseradish peroxidase, 40 kDa] and gliadin peptides [33-mer (p56-88, 3900 Da), 19-mer (p31-49, 2245 Da; and p202-220, 2127 Da), and 12-mer (p57-68, 1453 Da)] in duodenal biopsy specimens mounted in Ussing chambers. The permeability of intact peptides was much higher for p31-49 or 33-mer than for horseradish peroxidase, p202-220, and p57-68. A positive correlation was observed between G, an index of paracellular diffusion of ions, and mannitol permeability. The absence of correlation between G and permeability to intact 33-mer or p31-49 did not favor paracellular diffusion of the peptides. Immunofluorescence studies indicated that 33-mer enters the early endosome antigen 1–positive compartment but escapes the lysosomal-associated protein 2–positive compartment. The results underline that mannitol and ionic conductance G cannot be considered markers of permeability to gliadin peptides. In active celiac disease, increases in transcellular permeability to intact gliadin peptides might be considered in treatment strategies aimed at controlling epithelial permeability to gluten.

In vivo and in vitro studies recently showed that intestinal permeability to cow's milk proteins such as beta-lactoglobulin increased transiently in infant rabbits infected by the enteroadherent Escherichia coli strain RDEC-1 at weaning. The consequences of this enhanced permeability for local anaphylactic and/or systemic immune responses were studied in infected and age-matched control rabbits at weaning, given either water or cow's milk diluted in water in addition to their solid diet. The systemic immune response was determined by measuring IgG, IgA, IgM, and IgE antibodies to cow's milk proteins. A rise in IgG and IgM, but not IgA or IgE, antibody titres to milk protein was observed. Further, infected milk-drinking rabbits had significantly higher beta-lactoglobulin and casein IgG titres than milk-drinking controls. The anaphylactic response was tested by mounting ileal segments in Ussing chambers, adding the milk proteins to the serosal side of the tissue and recording the variation in short-circuit current in order to detect any electrogenic chloride secretion. No change in short-circuit current was observed in the presence of milk proteins in infected or age-matched control milk-drinking rabbits, indicating the absence of any immediate hypersensitivity reaction. These results indicate that at weaning, the rabbit is not prone to developing anaphylactic responses to milk proteins. The rise in milk protein IgG antibodies that followed infection with RDEC-1 might favour the elimination of milk protein-IgG immune complexes from the systemic circulation. The present results emphasize the evidence that genetic background and/or animal species greatly influence the immune response to an antigenic load.

Celiac disease (CD) is an inflammatory enteropathy induced by gluten/gliadins in genetically susceptible individuals. In patients with active CD, an abnormal retro-transport of IgA/gliadin immune complexes is observed. This retro-transport is mediated by the expression of CD71 on the apical pole of enterocytes and promotes the entry of harmful gliadin peptides in the intestinal mucosa and the triggering of abnormal immune responses to gliadin peptides. Our results indicate a CD71-mediated transcytosis of gliadin peptides that may participate in the pathogenesis of CD in genetically predisposed individuals.

The transepithelial absorption of food-type proteins has been shown to proceed by endocytosis along two functional pathways: a minor direct pathway allowing transport of intact protein and a major lysosomal degradative pathway. The human colon carcinoma cell line CaCo-2 grown on Millipore filters was used here further to characterize these pathways by measuring HRP transport across the cell monolayer in Ussing chambers. In the apical-basal direction, this transport mainly occurred along the degradative pathway and was inhibited at 4 degrees C (7.41 +/- 1.26 pmoles/h.cm2 vs. 27.40 +/- 8.91 at 37 degrees C). The amount conveyed via the direct pathway was very small (0.89 +/- 0.35 pmoles/h.cm2) and did not diminish at 4 degrees C (1.43 +/- 0.59 pmoles/h.cm2). In the basal-apical direction, HRP transport along the degradative pathway at 37 degrees C was similar to the apical-basal value and was inhibited at 4 degrees C (16.40 +/- 4.05 vs. 2.72 +/- 2.52 pmoles/h.cm2), but along the direct pathway, it was eight times the apical-basal value (8.36 +/- 3.11 pmoles/h.cm2) and was inhibited at 4 degrees C (2.43 +/- 0.78 pmoles/h.cm2). Intact HRP fluxes were not correlated with the electrical resistance of the filters, indicating transport via a transcellular route. Monensin at 10(-5) M did not affect direct or degradative transport in the apical-to-basal direction. These results suggest that in CaCo-2 cells HRP undergoes bidirectional transcytosis by a fluid-phase mechanism, but the extent of degradation during that transport varies according to the membrane (apical or basal) where it is presented.

The systemic and local immune responses and the intestinal barrier function were examined in orally or parenterally milk-sensitized guinea pigs. Both types of sensitization led to positive passive cutaneous anaphylactic responses and high IgG titers against β-lactoglobulin (β-LG), especially in parenterally immunized animals. In Ussing chambers, sensitized jejunum had higher short-circuit current (Isc) than control jejunum, with and without β-LG challenge. The further increase in Isc induced by serosal β-LG treatment was higher in parenterally than orally sensitized animals. Barrier function was tested as the intestinal transport and degradation of horseradish peroxidase (HRP) in the presence and absence of β-LG. There was a 5-fold increase in degraded HRP transport in sensitized versus control animals, with and without β-LG challenge. Serosally applied β-LG enhanced the transport of intact HRP in sensitized but not in control animals. These results indicate that sensitization of guinea pigs to cow’s milk permanently increases endocytic and electrogenic activities. The challenge with β-LG induced a further transient rise in Isc and increased intact HRP transport.

BackgroundCARD15/NOD2 mutations are associated with susceptibility to Crohn's Disease (CD) and Graft Versus Host Disease (GVHD). CD and GVHD are suspected to be related with the dysfunction of Peyer's patches (PP) and isolated lymphoid follicles (LFs). Using a new mouse model invalidated for Card15/Nod2 (KO), we thus analysed the impact of the gene in these lymphoid formations together with the development of experimental colitis.Methodology/principal findingsAt weeks 4, 12 and 52, the numbers of PPs and LFs were higher in KO mice while no difference was observed at birth. At weeks 4 and 12, the size and cellular composition of PPs were analysed by flow cytometry and immunohistochemistry. PPs of KO mice were larger with an increased proportion of M cells and CD4(+) T-cells. KO mice were also characterised by higher concentrations of TNFalpha, IFNgamma, IL12 and IL4 measured by ELISA. In contrast, little differences were found in the PP-free ileum and the spleen of KO mice. By using chamber experiments, we found that this PP phenotype is associated with an increased of both paracellular permeability and yeast/bacterial translocation. Finally, KO mice were more susceptible to the colitis induced by TNBS.ConclusionsCard15/Nod2 deficiency induces an abnormal development and function of the PPs characterised by an exaggerated immune response and an increased permeability. These observations provide a comprehensive link between the molecular defect and the Human CARD15/NOD2 associated disorders: CD and GVHD.

A life-long but constraining gluten-free diet is the only treatment currently available for coeliac disease. The human gastrointestinal tract does not possess the enzymatic equipment to efficiently cleave the gluten-derived proline-rich peptides driving the abnormal immune intestinal response in patients with coeliac disease. Oral therapy by exogenous prolylendopeptidases able to digest ingested gluten was therefore propounded as an alternative treatment to the diet. The feasibility of this approach is discussed by reviewing recent data on the intestinal transport of gliadin peptides, properties of available enzymes and preliminary clinical assays. Development of new enzymes or enzymatic cocktails offers potentially more potent therapeutic tools that, however, need meticulous evaluation based on clinical, biological and histological criteria.

International audience; Our aim was to study the effect of a mucosal protective agent, rebamipide, on the colonic barrier and the immune response in colitis-prone interleukin-10 deficient (IL-10-/-) C57BL/6 mice infected with Helicobacter hepaticus. After sacrifice, in all mice, control, or previously infected with H. hepaticus, or previously infected and treated with rebamipide enema, a histological examination of colonic samples was performed, intestinal permeability was studied in Ussing chamber, and mesenteric lymph node proliferation and cytokine secretion were measured. Mice treated with rebamipide, presented a reinforcement of the distal colonic epithelial barrier, an increase of mesenteric lymph node cells proliferation and of IFNΓ and IL-12 secretion. These results indicate that in IL-10-/- mice with mild colitis, rectally administered rebamipide reinforces the distal colonic barrier and has a slight Th1 immuno-stimulatory effect on mesenteric lymph node cells. These properties could be helpful in the management of some inflammatory bowel diseases.