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PubMed2004

Theta-isoform of PKC is required for alterations in cytoskeletal dynamics and barrier permeability in intestinal epithelium: a novel function for PKC-theta.

Using intestinal Caco-2 cells, we previously showed that assembly of cytoskeleton is required for monolayer barrier function, but the underlying mechanisms remain poorly understood. Because the theta-isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC-theta is crucial for changes in cytoskeletal and barrier dynamics. We have created the first multiple sets of gastrointestinal cell clones transfected with varying levels of cDNA to stably inhibit native PKC-theta (antisense, AS; dominant negative, DN) or to express its activity (sense). We studied transfected and WT Caco-2 cells. First, relative to WT cells, AS clones underexpressing PKC-theta showed monolayer injury as indicated by decreased native PKC-theta activity, reduced tubulin phosphorylation, increased tubulin disassembly (decreased polymerized and increased monomeric pools), reduced architectural integrity of microtubules, reduced stability of occludin, and increased barrier hyperpermeability. In these AS clones, PKC-theta was substantially reduced in the particulate fractions, indicating its inactivation. In WT cells, 82-kDa PKC-theta was constitutively active and coassociated with 50-kDa tubulin, forming an endogenous PKC-theta/tubulin complex. Second, DN transfection to inhibit the endogenous PKC-theta led to similar destabilizing effects on monolayers, including cytoskeletal hypophosphorylation, depolymerization, and instability as well as barrier disruption. Third, stable overexpression of PKC-theta led to a mostly cytosolic distribution of theta-isoform (<10% in particulate fractions), indicating its inactivation. In these sense clones, we also found disruption of occludin and microtubule assembly and increased barrier dysfunction. In conclusion, 1). PKC-theta isoform is required for changes in the cytoskeletal assembly and barrier permeability in intestinal monolayers, and 2). the molecular event underlying this novel biological effect of PKC-theta involves changes in phosphorylation and/or assembly of the subunit components of the cytoskeleton. The ability to alter the cytoskeletal and barrier dynamics is a unique function not previously attributed to PKC-theta.

Caco-2 Cells↗
PubMed2001

Hypoxia-inducible factor 1-dependent induction of intestinal trefoil factor protects barrier function during hypoxia.

Mucosal organs such as the intestine are supported by a rich and complex underlying vasculature. For this reason, the intestine, and particularly barrier-protective epithelial cells, are susceptible to damage related to diminished blood flow and concomitant tissue hypoxia. We sought to identify compensatory mechanisms that protect epithelial barrier during episodes of intestinal hypoxia. Initial studies examining T84 colonic epithelial cells revealed that barrier function is uniquely resistant to changes elicited by hypoxia. A search for intestinal-specific, barrier-protective factors revealed that the human intestinal trefoil factor (ITF) gene promoter bears a previously unappreciated binding site for hypoxia-inducible factor (HIF)-1. Hypoxia resulted in parallel induction of ITF mRNA and protein. Electrophoretic mobility shift assay analysis using ITF-specific, HIF-1 consensus motifs resulted in a hypoxia-inducible DNA binding activity, and loading cells with antisense oligonucleotides directed against the alpha chain of HIF-1 resulted in a loss of ITF hypoxia inducibility. Moreover, addition of anti-ITF antibody resulted in a loss of barrier function in epithelial cells exposed to hypoxia, and the addition of recombinant human ITF to vascular endothelial cells partially protected endothelial cells from hypoxia-elicited barrier disruption. Extensions of these studies in vivo revealed prominent hypoxia-elicited increases in intestinal permeability in ITF null mice. HIF-1-dependent induction of ITF may provide an adaptive link for maintenance of barrier function during hypoxia.

Animals↗
PubMed1999

Regulation of the intestinal epithelial paracellular barrier.

Paracellular transport of orally-administered drugs, the passage of molecules between adjacent intestinal epithelial cells, is impeded by a range of structural and functional features found in the intestine. An increased knowledge of the mechanisms that govern the paracellular barrier will enable the pharmaceutical scientist to design novel and rational formulations and delivery platforms that will improve the oral bioavailability of therapeutic molecules, particularly proteins and peptides, which would be taken-up by the paracellular pathway.

Journal Article↗
PubMed2005

theta Isoform of protein kinase C alters barrier function in intestinal epithelium through modulation of distinct claudin isotypes: a novel mechanism for regulation of permeability.

Using monolayers of intestinal Caco-2 cells, we discovered that the isoform of protein kinase C (PKC), a member of the "novel" subfamily of PKC isoforms, is required for monolayer barrier function. However, the mechanisms underlying this novel effect remain largely unknown. Here, we sought to determine whether the mechanism by which PKC- disrupts monolayer permeability and dynamics in intestinal epithelium involves PKC--induced alterations in claudin isotypes. We used cell clones that we recently developed, clones that were transfected with varying levels of plasmid to either stably suppress endogenous PKC- activity (antisense, dominant-negative constructs) or to ectopically express PKC- activity (sense constructs). We then determined barrier function, claudin isotype integrity, PKC- subcellular activity, claudin isotype subcellular pools, and claudin phosphorylation. Antisense transfection to underexpress the PKC- led to monolayer instability as shown by reduced 1) endogenous PKC- activity, 2) claudin isotypes in the membrane and cytoskeletal pools ( downward arrowclaud-1, downward arrowclaud-4 assembly), 3) claudin isotype phosphorylation ( downward arrow phospho-serine, downward arrow phospho-threonine), 4) architectural stability of the claudin-1 and claudin-4 rings, and 5) monolayer barrier function. In these antisense clones, PKC- activity was also substantially reduced in the membrane and cytoskeletal cell fractions. In wild-type (WT) cells, PKC- (82 kDa) was both constitutively active and coassociated with claudin-1 (22 kDa) and claudin-4 (25 kDa), forming endogenous PKC-/claudin complexes. In a second series of studies, dominant-negative inhibition of the endogenous PKC- caused similar destabilizing effects on monolayer barrier dynamics, including claudin-1 and -4 hypophosphorylation, disassembly, and architectural instability as well as monolayer disruption. In a third series of studies, sense overexpression of the PKC- caused not only a mostly cytosolic distribution of this isoform (i.e., <12% in the membrane + cytoskeletal fractions, indicating PKC- inactivity) but also led to disruption of claudin assembly and barrier function of the monolayer. The conclusions of this study are that PKC- activity is required for normal claudin assembly and the integrity of the intestinal epithelial barrier. These effects of PKC- are mediated at the molecular level by changes in phosphorylation, membrane assembly, and/or organization of the subunit components of two barrier function proteins: claudin-1 and claudin-4 isotypes. The ability of PKC- to alter the dynamics of permeability protein claudins is a new function not previously ascribed to the novel subfamily of PKC isoforms.

Caco-2 Cells↗
PubMed2004

Impaired intestinal immunity and barrier function: a cause for enhanced bacterial translocation in alcohol intoxication and burn injury.

Alcohol intoxication is being recognized increasingly as the major factor in pathogenesis after burn injury. Findings from multiple studies support the suggestion that, in comparison with burn-injured patients who sustained injury in the absence of alcohol intoxication, burn-injured patients who sustained injury under the influence of alcohol exhibit higher rates of infection and are more likely to die. Thus, infection becomes the primary cause of death in burn-injured patients. Because the intestine is considered to be a major source of bacteria, studies in experimental animals have been designed to examine whether alcohol intoxication before burn injury enhances bacterial translocation from the intestine. Results of these studies have shown a several-fold increase in bacterial translocation from the intestine in the group of animals receiving combined insult of alcohol intoxication and burn injury compared with findings for the groups receiving either insult alone. Alcohol intoxication and burn injury independent of each other have also been shown to cause an increase in bacterial translocation. The gastrointestinal tract normally maintains a physical mucosal and immunologic barrier that provides an effective defense in keeping bacteria within the intestinal lumen. However, in injury conditions these defense mechanisms are impaired. Intestinal bacteria consequently gain access to extraintestinal sites. Intestine-derived bacteria are implicated in causing systemic infection and in subsequent multiple organ dysfunction in both immunocompromised patients and patients with injury, such as burn and trauma. In this article, we discuss three potential mechanisms that are likely to contribute to the increase in bacterial translocation in alcohol intoxication and burn injury: (1) increase in bacterial growth in the intestine, (2) physical disruption of mucosal barrier of the intestine, and (3) suppression of the immune defense in the intestine.

Alcoholic Intoxication↗
PubMed1979

[Permeability of histohematic barriers of the small intestine during perfusion with some preserving solutions].

The permeability changes in histohematic barriers of the isolated small intestine loops of laboratory white rats whose vessels were perfused with 0.85% sodium chloride, Ringer-Lock, Hanks and Collins-2 solutions as well as with hemodez and aminopeptide were studied. The amount of the fluid flowing off the vessels, perfusate penetration into the intestinal lumen and its transudation through the serous membrane were determined. It was concluded that the slightest disturbance in the small intestine histohematic barrier was observed during perfusion of the vessels with Collins-2 solution. The above method is recommended for testing the comparative characteristics of the preserving solution.

Animals↗
PubMed1999

A tungsten supplemented diet attenuates bacterial translocation in chronic portal hypertensive and cholestatic rats: role of xanthine dehydrogenase and xanthine oxidase.

BACKGROUND: Bacterial translocation (BT) plays a major role in the pathophysiological process of spontaneous infections in portal hypertension (PH) and cholestatic jaundice. The major mechanisms promoting BT in experimental animal models are the disruption of the intestinal ecological equilibrium and disruption of the intestinal mucosal barrier. The enzymes xanthine dehydrogenase (XD) and xanthine oxidase (XO) are often implicated as a significant source of oxidants which have a major impact on the impairment of intestinal barrier function. AIM: To investigate the incidence of BT in rats with PH and obstructive jaundice, and to evaluate the impact of XD and XO. METHODS: Animals were subjected to sham laparotomy (SL), PH by calibrated stenosis of the portal vein, and common bile duct ligation (CBDL). They were fed either a standard pellet diet or a tungsten supplemented molybdenum-free diet. Four weeks after the operative procedure, intestinal colonisation and BT to portal vein, vena cava, mesenteric lymph nodes, liver, and spleen were determined. Intestinal XD and XO activity were measured enzymatically and histochemically. RESULTS: Significant (p<0.01) intestinal bacterial overgrowth was present in all PH and CBDL groups compared with the SL group. In normally fed animals after SL, BT occurred in 12%. In PH and after CBDL, the rate of BT increased significantly (p<0.05) to 28% and 54% respectively. In the jejunum of normally fed animals subjected to PH or CBDL, a significant increase in XO was observed (p<0.01). Animals fed a tungsten supplemented diet showed a significant attenuation of BT to 14% in PH and 22% after CBDL (p<0. 05). Tungsten treatment completely suppressed jejunal XD and XO activities. CONCLUSIONS: Significant intestinal bacterial overgrowth, BT, and XD to XO conversion occurred in PH and after CBDL. XD and XO inactivation by a tungsten supplemented molybdenum-free diet significantly reduced the incidence of BT without affecting intestinal bacterial overgrowth. These data strongly support the hypothesis that increased XD to XO conversion may contribute to intestinal barrier failure in PH and after CBDL.

Animals↗
PubMed1985

Modelling of intestinal permeability in man to polyethylene glycols (PEG 400 and PEG 1000).

The intestinal permeability barrier was evaluated in four healthy individuals by the 6 h urinary recovery of a mixture of different-sized polyethylene glycols (PEG 400 and PEG 1000) after a liquid meal of probe molecules. The mixture of PEGs contained around 25 different PEG polymers, ranging from about 194 to 1294 dalton in size. Three models were employed to get a condensed formulation of the intestinal barrier, namely a filter function, describing exclusion of molecules due to the molecular weight, and two expressions for ultrafiltration through a pore due to the Stoke-Einstein radius. A factor reflecting unrestricted passage of probe molecules in the size-range used was also included in the mathematical expressions. It was found that a filter function showing half-maximum exclusion at 589-956 daltons (Da) PEG and a pore function yielding a pore-size of 8.0-8.5 A equally well described the intestinal barrier in the four subjects. The results are discussed in relation to structures of the intestinal epithelium that may restrict the passage of molecules.

Capillary Permeability↗
PubMed2026

Graded Mulberry Leaf Supplementation Shapes Gut Microbiota, Reprograms Intestinal Metabolism, and Maintains Intestinal Chemical-Immune Barrier Homeostasis in Amur Sturgeon: A Multi-Omics Study.

Mulberry leaf contains abundant phytochemicals with antioxidant and immunomodulatory activities. However, systematic insight into its dose-dependent regulatory effects on the intestinal health of Amur sturgeon remains limited. In the present study, multi-omics approaches, including 16S rRNA gene sequencing, untargeted metabolomics, transcriptomics, together with RT-qPCR, were applied to investigate graded dietary mulberry leaf supplementation in Acipenser schrenckii. Juvenile sturgeons were fed four experimental diets containing 0%, 2%, 4% and 6% mulberry leaf over a 10-week feeding trial. Dietary mulberry leaf caused no adverse impacts on growth performance or intestinal digestive capacity. Although the overall structure of the intestinal microbiota remained stable, beneficial bacterial taxa were enriched in a dose-dependent manner. Intestinal metabolism underwent hierarchical remodelling: low inclusion levels supported basal nutrient metabolism, medium inclusion strengthened antioxidant capacity, and high inclusion reprogrammed lipid metabolism and immune function. Mulberry leaf reinforced the intestinal chemical barrier by balancing redox homeostasis and reducing mucosal epithelial permeability. Moreover, intestinal immunity was modulated through three sequential phases: initial innate immune priming, B-cell homing, and the establishment of sustained immune tolerance. In conclusion, mulberry leaf maintains intestinal chemical-immune barrier homeostasis in a dosage-tunable manner, supporting its potential application as a functional aquafeed ingredient.

Amur sturgeon (Acipenser schrenckii)↗
PubMed1981

Development of gastrointestinal mucosal barrier. I. The effect of age on intestinal permeability to macromolecules.

Indirect evidence has suggested that increased quantities of antigen may penetrate the intestinal mucosa and enter the systemic circulation during the newborn period compared to adult life. However, no direct measurement of macromolecular transport has been reported as a function of perinatal age. To study this process, we administered 100 mg of tritiated bovine serum albumin ([3H]BSA) by gavage to rabbits at birth, one wk, 2 wk, 6 wk, and one year of age and measured plasma radioactivity 4 hr after gavage. Plasma concentration of trichloroacetic acid insoluble radioactivity and immunoreactive bovine serum albumin radioactivity decreased significantly after one wk of age. When adult animals were gavaged with the same amount of [3H]BSA per body weight as the one-wk-old animals, they failed to transport as much of the antigen as the younger animals. This study, therefore, provides objective evidence that the intestinal mucosal barrier of newborns may be incompletely developed at birth and allow increased intestinal transport of antigens into the circulation.

Age Factors↗
PubMed2000

[Shigella: from the rupture to the invasion and to the destruction of the colonic epithelium].

CROSSING THE EPITHELIAL BARRIER: Shigella acts on the immune system in order to cross the intestinal epithelial barrier which is normally impermeable to intestinal flora. Among the effectors of the immune system of the colonic mucosa are translocation sites corresponding to lymphatic follicles carrying a follicular epithelium in the presence of M cells. These cells capture molecules, particles and microorganisms from the intestinal barrier that they translocate to immune competent cells present in the follicles (antigen presenting cells including resident macrophages and then B and T lymphocytes). MACROPHAGE APTOTOSIS AND ITS CONSEQUENCES: The first cell infected by Shigella appears to be the macrophage once the translocation has been made via the M cells. This infection rapidly leads to apoptotic death of the macrophage allowing the Shigella to infect the laterobasal epithelial cells and the development of an inflammatory response. This response in turn facilitates the invasion, rupture and permeability of the intestinal epithelium and the initiation, by Shigella, of a cell-to-cell invasion pathway.

Apoptosis↗
PubMed2006

Immune privilege in the gut: the establishment and maintenance of non-responsiveness to dietary antigens and commensal flora.

Immune privilege in the gut is the result of a complex interplay between the gut microbiome, gut luminal antigens, and the intestinal epithelial barrier. Composed of both physical and immunochemical components, the intestinal barrier secretes immunoregulatory mediators that promote the generation of tolerogenic antigen-presenting cells, phagocytic innate immune cells characterized by 'inflammatory anergy', and regulatory cells of the adaptive immune system. Innate immune cells mediate controlled transepithelial transport of luminal antigens as far as the mesenteric lymph nodes, where the intestinal and peripheral immune systems intersect. This promotes the generation of adaptive regulatory lymphocytes that actively suppress effector cell responses against gut luminal antigens and flora. The net result is the generation of tolerance to dietary antigens and the maintenance of gut homeostasis. Dysregulation of this complex immunoregulatory network leads to diseases such as food allergy and inflammatory bowel disease. Future therapies for these diseases will likely involve the functional restoration of the barrier and regulatory cell functions at the epithelial/luminal interface.

Animals↗
PubMed1998

[Portal and systemic bacteremia as a manifestation of the functional failure of the enteral barrier in acute intestinal obstruction].

A complex investigation of 72 patients, with acute bowel obstruction (ABO) having clinico-laboratory signs and symptoms of endotoxicosis (ET), was carried out. It was proven that ABO was accompanied by profound decrease of the immuno-secretory function of the small bowel and its extensive bacterial contamination as a result of significant increase in the concentration of gram-negative symbiotic microflora, which leads to increased permeability of bowel barrier. Consequently there is massive translocation of the internal medium of the organism by enteral microflora and most of all in the portal zone and in the presence of inadequate hepatic barrier function--the systemic blood supply which corresponds to the clinical picture of endotoxic shock.

Acute Disease↗
PubMed1985

Macromolecular transport in the fetal rat intestine.

Macromolecular barrier function of the fetal rat small intestine and colon was analyzed from 16 to 22 days gestation (birth). During this period the epithelium is converted from stratified to simple columnar. To assess permeability, horseradish peroxidase (HRP) was introduced by microinjection into the lumen or into the umbilical circulation. Proximal small intestine, distal small intestine, and colon were examined after 10-20 min. Paracellular passage of HRP through occluding junctions was not observed after either intraluminal or intravascular injection. After intraluminal injection, transepithelial transport of HRP from lumen to blood occurred in all regions at all ages studied. Horseradish peroxidase was present in cytoplasmic vesicles of most cells in the primitive stratified epithelia, during epithelial conversion, and in simple columnar epithelia. After intravascular injection, HRP was present in the lamina propria and in intercellular spaces of the epithelium, but HRP did not enter tight junctions. Tracer was taken up into cytoplasmic vesicles of both stratified and simple columnar epithelial cells, but was only rarely seen in the lumen. We conclude that there is rapid transcellular, vesicle-mediated transport from lumen to blood across both stratified and simple columnar epithelia of fetal rat small intestine and colon; after intravascular injection, macromolecules may be taken up into vesicles at basolateral epithelial cell surfaces but are not rapidly transported into the lumen; paracellular passage does not occur in the fetal ages studied.

Animals↗
PubMed2025

Recent evolution of the developing human intestine affects metabolic and barrier functions.

Diet, microbiota, and other exposures make the intestinal epithelium a nexus for evolutionary change; however, little is known about genomic changes associated with adaptation to a distinctly human environment. In this work, we interrogate the evolution of cell types in the developing human intestine by comparing tissue and organoids from humans, chimpanzees, and mice. We find that recent changes in primates are associated with immune barrier function and lipid and xenobiotic metabolism and that human-specific genetic features affect these functions. Enhancer assays, genetic deletion, and in silico mutagenesis resolve evolutionarily important enhancers of lactase (LCT) and insulin-like growth factor binding protein 2 (IGFBP2). Altogether, we identify the developing human intestinal epithelium as a rapidly evolving system and show that great ape organoids provide insight into human biology.

Animals↗
PubMed1997

Review article: Intestinal epithelia and barrier functions.

The mucosal epithelia of the digestive tract acts as a selective barrier, permeable to ions, small molecules and macromolecules. These epithelial cells aid the digestion of food and absorption of nutrients. They contribute to the protection against pathogens and undergo continuous cell renewal which facilitates the elimination of damaged cells. Both innate and adaptive defence mechanisms protect the gastrointestinal-mucosal surfaces against pathogens. Interaction of microorganisms with epithelial cells triggers a host response by activating specific transcription factors which control the expression of chemokines and cytokines. This host response is characterized by the recruitment of macrophages and neutrophils at the site of infection. Disruption of epithelial signalling pathways that recruit migratory immune cells results in a chronic inflammatory response. The adaptive defence mechanism relies on the collaboration of epithelial cells (resident sampling system) with antigen-presenting and lymphoid cells (migratory sampling system); in order to obtain samples of foreign antigen, these samples must be transported across the barriers without affecting the integrity of the barrier. These sampling systems are regulated by both environmental and host factors. Fates of the antigen may differ depending on the way in which they cross the epithelial barrier, i.e. via interaction with motile dendritic cells or epithelial M cells in the follicle-associated epithelium.

Adaptation, Physiological↗
PubMed1999

Ethanol modulation of intestinal epithelial tight junction barrier.

Previous studies have shown that high concentrations of ethanol (>/=40%) cause functional damage of the gastrointestinal epithelial barrier by direct cytotoxic effect on the epithelial cells. The effects of lower noncytotoxic doses of ethanol on epithelial barrier function are unknown. A major function of gastrointestinal epithelial cells is to provide a barrier against the hostile substances in the gastrointestinal lumen. The apicolaterally located tight junctions (TJs) form a paracellular seal between the lateral membranes of adjacent cells and act as a paracellular barrier. In this study, we investigated the effects of lower doses of ethanol on intestinal epithelial TJ barrier function using filter-grown Caco-2 intestinal epithelial monolayers. The Caco-2 TJ barrier function was assessed by measuring epithelial resistance or paracellular permeability of the filter-grown monolayers. Ethanol (0, 1, 2.5, 5, 7.5, and 10%) produced a dose-related drop in Caco-2 epithelial resistance and increase in paracellular permeability. Ethanol also produced a progressive disruption of TJ protein (ZO-1) with separation of ZO-1 proteins from the cellular junctions and formation of large gaps between the adjacent cells. Ethanol, at the doses used (</=10%), did not cause cytotoxicity (lactate dehydrogenase release) to the Caco-2 cells. Ethanol produced a disassembly and displacement of perijunctional actin and myosin filaments from the perijunctional areas. On ethanol removal, actin and myosin filaments rapidly reassembled at the cellular borders. Ethanol stimulated the Caco-2 myosin light chain kinase (MLCK) activity but did not affect the MLCK protein levels. Specific MLCK inhibitor ML-7 inhibited both ethanol increases in MLCK activity and TJ permeability without affecting the MLCK protein levels. Consistent with these findings, metabolic inhibitors sodium azide and 2,4-dinitrophenol significantly prevented ethanol-induced increase in Caco-2 TJ permeability, whereas cycloheximide or actinomycin D had no effect. The results of this study indicate that ethanol at low noncytotoxic doses causes a functional and structural opening of the Caco-2 intestinal epithelial TJ barrier by activating MLCK.

Azepines↗