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Expression of the thymus leukemia antigen in mouse intestinal epithelium.

The Qa and Tla regions of the mouse major histocompatibility complex contain a series of genes encoding proteins with structural similarity to the class I transplantation antigens of the same complex. In contrast to the genes encoding the transplantation antigens, the Qa and Tla genes show very little polymorphism. Function(s) of the proteins encoded by the Qa and Tla loci remain an enigma. Recently, the protein products of the Qa and Tla loci, often referred to as class Ib major histocompatibility complex molecules, have been proposed to present antigen to gamma delta T cells. In mice, gamma delta T cells have been found concentrated in several epithelial barriers and in the skin; yet, expression of serologically detectable Tla antigens is believed restricted to thymocytes, activated T lymphocytes, and some T-cell leukemias. Here we report that luminal epithelial cells of the mouse small intestine express the thymus leukemia antigen (TLA). We also find that, unlike T cells in Peyer's patches, a significant fraction of intestinal epithelial lymphocytes also express TLA. RNA prepared from intestinal cells contains transcripts of the T18d gene, which encodes TLA. These data extend the known expression profile of TLA molecules to mature lymphocytes and to nonhematopoietic cells. These data also demonstrate the specific expression of TLA on antigen-presenting cells in a site enriched for T cells that express gamma delta T-cell antigen receptor.

Animals↗

IV. Paneth cell antimicrobial peptides and the biology of the mucosal barrier.

The hypothesis that epithelial cells release preformed antibiotic peptides as components of mucosal innate immunity has gained experimental support in recent years. In the mammalian small intestine, Paneth cells secrete granules that are rich in alpha-defensins and additional antimicrobial peptides into the lumen of the crypt. The alpha-defensins are homologues of peptides that function as mediators of nonoxidative microbial cell killing in phagocytic leukocytes, and they are potent microbicidal agents in in vitro assays. Because certain mouse alpha-defensins stimulate cultured epithelial cells to secrete chloride ion, those peptides appear to be capable of interacting directly with the apical membranes of neighboring cells and perhaps influencing crypt physiology. In instances of crypt disruption or induced Paneth cell deficiency, crypt intermediate cells appear to compensate by accumulating and secreting Paneth cell antimicrobial peptides. Challenges for the future will be to understand the mechanisms of this epithelial plasticity and to show that Paneth cells contribute directly to innate immunity in the crypt microenvironment.

Animals↗

[The significance of lactic acid bacteria in treatment and prophylaxis of digestive tract disorders].

Probiotic bacteria are used to treat disturbed intestinal microflora and increased gut permeability which are characteristic to many intestinal disorders. Examples include children with acute rotavirus diarrhoea, subjects with food allergy, subjects with colonic disorders and patients undergoing pelvic radiotherapy and sometimes changes associated with colon cancer development. In all such disease states altered intestinal microflora, impaired gut barrier and different types of intestinal inflammation are present. Successful probiotic bacteria are able to survive gastric conditions and colonise the intestine, at least temporarily, by adhering to the intestinal epithelium. Already existing preparations containing viable lactic bacteria of human origin appear to have value in restoring normal microbial functions and alleviating symptoms in some patients with gastrointestinal infection and other conditions. Current scientifically based research efforts world-wide are now focusing on the development of high quality products containing microorganisms preselected for specific probiotic characteristics.

Child↗

System approach to the transepithelial transport across rat jejunal enterocytes: effect of cytochalasin B, colchicine, and ethylenediaminetetraacetic acid on butyric acid transport.

A three-compartment physical model is devised for transepithelial passive transport across intestinal cells. The mathematical equations derived from the model allow the simultaneous and quantitative measurements, in the form of permeability coefficients, of solute transport across both the luminal-serosal and serosal-blood barriers. The proposed model is used to study the involvement of the cytoskeleton in butyric acid absorption by the rat jejunum. Alterations in cytoskeletal functions are introduced by the administration of microfilamentous and microtubular altering agents such as cytochalasin, colchicine, or EDTA. An isolated jejunal segment perfused with a buffer containing labeled butyric acid was homogenized at the end of the experiment and assayed for its butyric acid content. During the perfusion, portal blood samples, as well as perfusate samples collected 10 cm distal to the perfusion site were drawn at 5-min intervals and assayed for their radioactivity. Cytochalasin was found to decrease the permeability of the mucosal membrane to butyric acid and to increase that of the serosal membrane. Colchicine did not have any effect either on the mucosal or on the serosal side. Cytochalasin and colchicine, when given in the same experiment, increased the permeability of the serosal membrane to butyric acid, but were without any effect on the mucosal barrier. Also, EDTA had no effect on the mucosal side, but decreased significantly the permeability of the serosal membrane to the fatty acid.

Animals↗

The importance of impermeant support in small bowel preservation: a morphologic, metabolic and functional study.

BACKGROUND: Ambiguity exists as to which preservation solution is the most effective for small bowel (SB); studies have shown equivalent results with normal saline and University of Wisconsin (UW) solution. This study was designed to investigate the requirement of SB for oncotic and osmotic support, thereby reassessing one of the key principles of static organ storage. METHODS: Rodent SB was vascularly flushed with the following solutions: group 1, 0.9% saline (154 mM NaCl); group 2, 154 mM NaCl +5% dextran; group 3, 104 mM NaCl +100 mM lactobionate + 5% dextran; and group 4, UW solution. Analysis of cellular energetics, permeability and histology (by electron microscopy) was performed over a 10-h time course of cold storage. RESULTS: The addition of dextran and lactobionate to a simple saline solution (group 3) resulted in superior maintenance of several key parameters of energy metabolism throughout prolonged storage. At all times, ATP/ADP and EC ratios in group 3 remained unchanged from those of freshly isolated tissue; storage in normal saline and UW solution resulted in a progressive decline between 1 and 10 h of storage. ATP was also notably greater in group 3 than in group 1 or UW after 10 h of storage. Functional parameters demonstrated significant improvements in maintaining barrier function and membrane ion/electrical activity in group 3. Of particular note, after 10 h of storage, permeability for groups 1, 3 and UW was 215, 76 and 400 nmol/cm2/h, respectively, compared with a fresh tissue value of 22 nmol/cm2/h. Scanning electron micrographs revealed complete epithelial denudation of bowel stored in simple saline and UW solutions at 10 h. The incorporation of 100 mM lactobionate plus 5% dextran in group 3 prevented extensive villus denudation; the presence of intact microvilli indicated normal epithelial cell morphology. CONCLUSION: The order of solution effectiveness was group 3 > group 2 > group 1 > group 4. Vascular supplied impermeants, when supplied in simple solution, provide markedly improved preservation of metabolism, barrier function, and morphology of SB compared with UW.

Adenosine↗

Heparan sulfate increases survival during gut-derived sepsis by decreasing bacterial translocation and enhancing host defense.

The effect of heparan sulfate (HS) on survival rate, bacterial translocation, and host defense was studied in a model of gut-derived sepsis that included transfusion-induced immunosuppression. Balb/c mice were treated pre- and postburn injury and bacterial challenge with HS, 5 mg/kg/day, or sterile phosphate-buffered saline. The HS pre- and postburn treated animals showed a significant improvement in survival compared to control animals (80 vs. 30%, p = .004, and 60 vs. 20%, p = .02, respectively). A lower amount of translocation was observed in the spleen (p < or = .001) of the HS group compared to control group. Quantitative colony counts and the calculated percentage of viable bacteria showed that the ability to kill translocated organisms was enhanced in all tissues of the animals receiving HS. These data suggest that treatment with HS positively affects the outcome in gut-derived sepsis. The beneficial effect was related both to an improved gut barrier function and to an enhanced host defense.

Analysis of Variance↗

Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms.

The phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibits Cl(-) secretion (short-circuit current, I(sc)) and decreases barrier function (transepithelial resistance, TER) in T84 epithelia. To elucidate the role of specific protein kinase C (PKC) isoenzymes in this response, we compared PMA with two non-phorbol activators of PKC (bryostatin-1 and carbachol) and utilized three PKC inhibitors (Gö-6850, Gö-6976, and rottlerin) with different isozyme selectivity profiles. PMA sequentially inhibited cAMP-stimulated I(sc) and decreased TER, as measured by voltage-current clamp. By subcellular fractionation and Western blot, PMA (100 nM) induced sequential membrane translocation of the novel PKC epsilon followed by the conventional PKC alpha and activated both isozymes by in vitro kinase assay. PKC delta was activated by PMA but did not translocate. By immunofluorescence, PKC epsilon redistributed to the basolateral domain in response to PMA, whereas PKC alpha moved apically. Inhibition of I(sc) by PMA was prevented by the conventional and novel PKC inhibitor Gö-6850 (5 microM) but not the conventional isoform inhibitor Gö-6976 (5 microM) or the PKC delta inhibitor rottlerin (10 microM), implicating PKC epsilon in inhibition of Cl(-) secretion. In contrast, both Gö-6976 and Gö-6850 prevented the decline of TER, suggesting involvement of PKC alpha. Bryostatin-1 (100 nM) translocated PKC epsilon and PKC alpha and inhibited cAMP-elicited I(sc). However, unlike PMA, bryostatin-1 downregulated PKC alpha protein, and the decrease in TER was only transient. Carbachol (100 microM) translocated only PKC epsilon and inhibited I(sc) with no effect on TER. Gö-6850 but not Gö-6976 or rottlerin blocked bryostatin-1 and carbachol inhibition of I(sc). We conclude that basolateral translocation of PKC epsilon inhibits Cl(-) secretion, while apical translocation of PKC alpha decreases TER. These data suggest that epithelial transport and barrier function can be modulated by distinct PKC isoforms.

Biological Transport↗

Changes in colonic mucosal permeability in mouse colitis induced with dextran sulfate sodium.

In this study we examined changes in colonic mucosal permeability induced by dextran sulfate sodium (DSS) during the acute phase of mouse colitis. To induce colitis, the mice were given drinking water containing 5% (w/v) DSS (MW = 40,000) ad libitum. Colonic mucosal permeability was evaluated by the permeation of Evans blue (EB) from the lumen into the wall of the colon on 1, 2, 3 and 7 days postadministration of DSS. Mucosal changes were also histologically examined daily for 7 days postadministration. The permeation of EB increased significantly by days 3 and 7 postadministration. Histological analysis showed that crypt loss was the initial change, with no inflammatory process and the surface mucosal epithelial cells remained morphologically intact. These histological changes developed on 2 to 3 days postadministration. Erosion was first recognized at 5 days postadministration. These findings indicated that the increase in colonic mucosal permeability may have occurred in 3 days postadministration, and the increase in mucosal permeability occurred before the appearance of the inflammatory process. This suggests that an increase in colonic mucosal permeability, leading to the destruction of mucosal barrier function, may play an important role in the induction of DSS-induced murine colitis.

Animals↗

Laminin stimulates rapid epithelial restitution of rabbit duodenal mucosa in vitro.

BACKGROUND: This study investigated the effect of the basal lamina constituents fibronectin, collagen IV, and laminin on epithelial restitution of rabbit duodenum in vitro. METHODS: Rabbit duodenal mucosal sheets were mounted in Ussing chambers, luminally exposed to 10 mM HCI for 10 min, and incubated with buffer or luminal buffer containing 25-100 micrograms/ml of collagen IV, fibronectin, laminin, or polyclonal antisera directed against these proteins (diluted 1:50-1:20) for 3 h. Resistance was calculated from potential difference and short-circuit current. Mucosal damage was assessed by morphometry on hematoxylin- and eosin-stained sections. RESULTS: Acid exposure caused a 40% drop in resistance (119 +/- 5 versus 71 +/- 5 Ohm.cm2 before versus after injury; P < 0.05, n = 6) and mucosal damage of 58 +/- 4% (n = 6). Three hours after injury resistance was 102 +/- 6, 117 +/- 4, and 48 +/- 5 Ohm.cm2 in the control, laminin, and anti-laminin groups, respectively. Furthermore, 36 +/- 2%, 16 +/- 2%, and 64 +/- 5% of the mucosa was damaged in the control, laminin, and anti-laminin groups, respectively, 3 h after injury (P < 0.05 versus controls). Laminin promoted epithelial wound closure by stimulation of enterocyte migration, which was inhibited by anti-laminin. Fibronectin, collagen IV, anti-fibronectin, and anti-collagen IV did not impair restitution. CONCLUSION: Our results show that laminin promotes electrophysiologic restoration and epithelial restitution of rabbit duodenum in vitro. We therefore suggest that laminin plays an important part in the orchestration of epithelial integrity and barrier function.

Animals↗

Intestinal intraepithelial lymphocytes are primed for gamma interferon and MIP-1beta expression and display antiviral cytotoxic activity despite severe CD4(+) T-cell depletion in primary simian immunodeficiency virus infection.

Intraepithelial lymphocytes (IEL) are a critical effector component of the gut-associated lymphoid tissue (GALT) and play an important role in mucosal immunity as well as in the maintenance of the epithelial cell integrity and barrier function. The objective of this study was to determine whether simian immunodeficiency virus (SIV) infection of rhesus macaques would cause alterations in the immunophenotypic profiles of IEL and their mitogen-specific cytokine (gamma interferon [IFN-gamma] and MIP-1beta) responses (by flow cytometry) and virus-specific cytotoxic T-cell (CTL) activity (by the chromium release assay). Virally infected IEL were detected through the entire course of SIV infection by in situ hybridization. Severe depletion of CD4(+) single-positive and CD4(+)CD8(+) double-positive T cells occurred early in primary SIV infection, which was coincident with an increased prevalence of CD8(+) T cells. This was in contrast to a gradual depletion of CD4(+) T cells in peripheral blood. The CD8(+) IEL were the primary producers of IFN-gamma and MIP-1beta and were found to retain their potential to produce both IFN-gamma and MIP-1beta through the entire course of SIV infection. SIV-specific CTL activity was detected in primary IEL at 1, 2, and 4 weeks post-SIV infection. These results demonstrated that IEL may be involved in generating antiviral immune responses early in SIV infection and in suppressing viral infection thereafter. Alterations in homeostasis in epithelia due to severe CD4(+) T-cell depletion accompanied by changes in the cytokine and chemokine production by IEL may play a role in the enteropathogenesis of SIV infection.

Animals↗

Cytoskeleton of intestinal goblet cells: role of actin filaments in baseline secretion.

Although microtubules appear necessary to maintain mucin granule transport in intestinal goblet cells, the role of microfilaments in mucus secretion is unknown. To determine the functional significance of microfilaments in goblet cell secretion, fluorescent cytochemistry of microfilaments and autoradiographic studies on granule movement were performed on rabbit intestinal goblet cells, with and without the actin depolymerizing agents, cytochalasin D (cyto D), and dihydro-cytochalasin B (dihydro B). In normal goblet cells, cytochemical localization of F-actin with NBD-phallacidin demonstrated their restriction to the apical surface of the goblet cell. Visualization of the goblet cell apical surface by electron microscopy revealed the presence of a thin layer of cytoplasm overlying the granule mass. Treatment with cyto D and dihydro B eliminated NBD-phallacidin staining of the apical cell surface. Quantitative analysis of baseline granule translocation demonstrated that treatment with cyto D and dihydro B resulted in dramatic acceleration of granule movement through goblet cells. This cellular response results from an increase in baseline secretion and facilitation of secretion of newly synthesized mucins, not stimulation of an accelerated secretory event. These data imply that actin filaments fulfill a barrier function in baseline secretion by hindering granule access to the plasma membrane; once the granule contacts the plasma membrane, exocytosis occurs. Secretion is balanced by the translocation of subjacent granules. In contrast, an accelerated secretory event is not triggered by plasma membrane access alone; this event requires a regulatory signal. We hypothesize that, unlike accelerated secretion, baseline secretion is constitutive, with exocytosis limited solely by the physical constraint of secretory granule access to the apical plasma membrane.

Actins↗

Markedly high eosinophilia and an elevated serum IL-5 level in an infant with cow milk allergy.

BACKGROUND: Interleukin-5 (IL-5) promotes the production and function of eosinophils, and an increase in the serum soluble CD23 (sCD23) level is suggestive of enhanced type-2 helper T-cell activity. The secretion of a large amount of the proinflammatory cytokine, tumor necrosis factor alpha (TNF-a), has been reported to alter the intestinal barrier capacity. OBJECTIVE: To determine whether or not distinct profiles of cytokine production were involved in the marked peripheral eosinophilia of as high as 20,000/mm3 and the gastrointestinal symptoms seen in an infant with cow milk allergy. METHODS: The levels of IL-5, sCD23, and TNF-alpha in serum and the culture supernatants of mononuclear cells were compared with those in infants with anaphylaxis to cow milk and nonallergic infants. RESULTS: Interleukin-5 was detected in the serum (19 pg/mL) but became undetectable after 2 weeks on a milk-free diet together with clinical remission. A kinetic decrease in the serum sCD23 level was also observed during the administration of a milk-free diet with improvement of the eosinophilia in 2 months. The TNF-alpha produced in vitro after stimulation with cow milk protein was not different from in controls. CONCLUSION: It seems likely that the allergic inflammation due to cow milk can induce marked eosinophilia with an associated increase in IL-5 production.

Animals↗

Cutting-edge technology: IV. Genomic engineering for studies of the gastrointestinal tract in mice.

Advances in our understanding of the complex, dynamic interactions that exist among the gastrointestinal microflora, the epithelium of the gastrointestinal mucosae, and the immune system have been facilitated by powerful new genetic tools. Recent understanding that the gastrointestinal epithelium performs not only a barrier function but is also an active sensor of the microflora and an important intermediary in regulating and integrating cross-talk between it and cells of the innate and adaptive immune systems provides one of the most fertile and challenging areas for application of these technologies. The intestinal epithelium also represents an important model system for study of programs of cell lineage commitment and differentiation, given its continual and rapid regeneration throughout life and the regional differences in these programs that exist along the gastrocolonic and crypt-villous axes. This review will highlight current and emerging technologies that are available in the mouse model for identification and manipulation of genetic elements that regulate the normal and pathological physiology of the intestinal tissues in the post-genomic era.

Animals↗

TLR signaling in the gut in health and disease.

The human intestine has evolved in the presence of diverse enteric microflora. TLRs convert the recognition of pathogen-associated molecules in the gut into signals for anti-microbial peptide expression, barrier fortification, and proliferation of epithelial cells. Healing of injured intestinal epithelium and clearance of intramucosal bacteria require the presence of intact TLR signaling. Nucleotide oligomerization domain (Nod)1 and Nod2 are additional pattern recognition receptors that are required for defense against invasive enteric pathogens. Through spatial and functional localization of TLR and Nod molecules, the normal gut maintains a state of controlled inflammation. By contrast, patients with inflammatory bowel disease demonstrate inflammation in response to the normal flora. A subset of these patients carry polymorphisms in TLR and CARD15/NOD2 genes. A better understanding of the delicate regulation of TLR and Nod molecules in the gut may lead to improved treatment for enteric infections and idiopathic inflammatory bowel diseases.

Adaptor Proteins, Signal Transducing↗

Conservative treatment in acute pancreatitis.

In the absence of causative therapy of acute pancreatitis the management of the disease focus on treatment and prevention of complications and symptoms. In mild acute pancreatitis intravenous fluid administration, analgetics and avoidance of oral fluid or food intake is sufficient in most cases. The treatment of severe pancreatitis involves identification of pancreatic and peripancreatic necroses, best demonstrated and evaluated by contrast-enhanced computerized tomography (CT). Infection of such necroses is the most common cause of death from acute pancreatitis. Recent data suggest that antibiotic prophylaxis is worthwhile and it should therefore always be instituted in the severe form of the disease. Careful monitoring of vital functions is mandatory and early treatment with assisted ventilation and renal dialysis is advised. Adequate volume replacement and nutritional support is important. The role of the gut in the development of infected necroses is becoming increasingly obvious. Also, the absence of food in the intestine may increase the intestinal barrier damage. Therefore, enteral nutrition is discussed as a logical step in pancreatitis treatment. However, the food should be delivered below the ligament of Treitz (below the area of the cholecystokinin (CCK) cells) as CCK stimulation probably worsen the course of the disease.

Acute Disease↗

IL-4 directly modulates function of a model human intestinal epithelium.

Intestinal epithelia are in intimate contact with subepithelial and intraepithelial lymphocytes. When stimulated, mucosal lymphocytes generate inflammatory cytokines such as IL-4 and IFN-gamma. We have shown that IFN-gamma directly regulates epithelial function. It is unknown whether IL-4 might influence epithelial function and, if so, whether such influences are similar to or differ from those exerted by IFN-gamma. In this study, we examine the effect of human IL-4 on barrier function, ion transport, and immune accessory ligand expression on T84 cells, a crypt-like epithelial cell line. Basolateral exposure of epithelial monolayers to IL-4 attenuated epithelial barrier function by greater than 65% in a dose (50% of effective dose = 1 U/ml)- and time (t1/2 = 24 h)-dependent fashion, and was inhibitable by neutralizing anti-IL-4 and anti-IL-4R Ab. Stimulated Cl- secretion, as measured by epithelial short circuit current, was diminished by as much as 70% by IL-4. Epithelial preexposure to IL-4 brought about a greater than twofold increase in beta 2 integrin-dependent neutrophil adhesion to epithelial, but retarded neutrophil migration into and across epithelial monolayers. ELISAs revealed that epithelial exposure to IL-4 had no effect on cell surface expression of MHC class I, MHC class II, or ICAM-1. These results indicate that IL-4, like IFN-gamma, may serve to regulate intestinal epithelial function, but that resulting phenotypes may be cytokine specific. We speculate from these data that activation of the basolateral receptor for IL-4 potentially provides a new strategy for damping the cellular component of active inflammation in the intestine.

Cell Adhesion Molecules↗

Intestinal function in glycogen storage disease type I.

Glycogen storage disease type I (GSD I) (McKusick 232200) is caused by inherited defects of the glucose-6-phosphatase complex. Patients with GSD Ia as well as patients with GSD lb may suffer from intermittent diarrhoea, which seems to worsen with age. The cause of this diarrhoea is unknown. This study describes the results of investigations of intestinal functions and morphology in patients with GSD Ia and GSD lb, which were performed to detect a common cause for chronic diarrhoea in GSD I. The following were investigated: faecal fat excretion, faecal alpha1-antitrypsin and faecal chymotrypsin, expiratory H2 concentrations, persorption of cornstarch in urine and colonic biopsies. With the investigations presented in this study, no common cause for diarrhoea in GSD I was found. In GSD lb loss of mucosal barrier function due to inflammation, documented by increased faecal alpha1-antitrypsin excretion (3.5-9.6 mg/g dry faeces) and inflammation in the colonic biopsies, seems to be the main cause. The inflammation is most likely related to disturbed neutrophil function, which is often found in GSD lb. Whether another cause is involved in GSD Ia and in GSD Ib, related to the disturbed function of glucose-6-phosphatase in the enterocyte, remains to be investigated.

Adolescent↗

Glutamine.

Glutamine is the most abundant free amino acid in the circulation. It is a primary fuel for rapidly dividing cells and plays a key role in the transport of nitrogen between organs. Although glutamine is absent from conventional regimens aimed at nutritional support, glutamine deficiency can occur during periods of metabolic stress; this has led to the reclassification of glutamine as a conditionally essential amino acid. Experiments with various animal models have demonstrated that the provision of glutamine can result in better nitrogen homoeostasis, with conservation of skeletal muscle. There is also considerable evidence that glutamine can enhance the barrier function of the gut. This review concludes by discussing the clinical evidence that supports the inclusion of stable forms of glutamine in solutions of nutrients.

Enteral Nutrition↗