Air-blood barrier in the respiratory intestine of the pond-loach, Misgurmus fossilis L. An ultrastructural study.
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Colitis and colonic mucosal barrier dysfunction.
Trauma, infection, neoplasia, and inflammation can all disrupt the intact intestinal mucosal barrier to intraluminal bacteria and bacterial antigens. This study investigated the relation between colonic inflammation and colonic mucosal barrier function in three experimental models of colitis. There were significantly increased systemic endotoxin concentrations in rats with acetic acid (7.5 (1.7-119.5) pg/ml), ethanol (13.7 (0-111.2) pg/ml), and hapten induced (14.4 (5-31.1) pg/ml) colitis compared with saline controls (3.3 (0-13.7) pg/ml). Data expressed as median (range). There were significant correlations between the systemic endotoxin concentration and both the severity of colitis and of illness in acetic acid induced colitis. A significant increase in colonic permeability to 14C-polyethylene glycol was shown in rats with acetic acid (3.42 (1.36-5.63)%) and hapten induced colitis (2.86 (1.03-8.10)%) compared with saline controls (1.20 (0.67-1.36)%). Data expressed as median (range) of percentage of the intracolonic bolus excreted in urine. There was a significant positive correlation between the severity of colitis and % colonic permeability to 14C-polyethylene glycol. This and other studies provide evidence that mucosal barrier dysfunction is a feature of colitis irrespective of aetiology or species. Such barrier dysfunction may be responsible for the systemic inflammatory response and complications seen in patients with inflammatory bowel disease.
Acetates↗
Increased intestinal permeability to differently sized polyethylene glycols in uremic rats: effects of low- and high-protein diets.
Intestinal mucosa forms an important barrier towards harmful agents in the intestinal lumen, besides being the site for absorption of nutrients. Little is known about the intestinal permeability properties in chronic uremia. The permeability toward differently sized polyethylene glycols (PEGs; range 326-1,162 Da) was studied in uremic groups compared to the control groups. The urinary recovery was also recovery of PEGs was increased in the uremic groups compared to the control groups. The urinary recovery was also increased in the groups on the high-protein diet compared to the corresponding group on the low-protein diet. This study suggests an increased permeability of PEG molecules in the range of 546-1,162 Da in uremic rats and a decreased intestinal permeability after a low-protein diet in both a uremic and nonuremic state. Thus, in chronic renal failure the intestinal barrier is impaired but returns towards normal with low-protein diets.
Animals↗
Validation of cell culture models for the intestine and the blood-brain barrier and comparison of drug permeation.
Cell culture models are useful tools to study the uptake of drugs across the barriers of the human body, like the intestine, the skin or the blood-brain barrier. Cell-based in vitro models not only help to reduce the number of animals used but are also much faster to perform, more cost effective and give more reproducible data than animal studies. Given the increasing number of new drugs and chemicals under development, there is an urgent need for the establishment of such in vitro models. However, the validity of such in vitro models is reflected by its ability to accurately predict the behaviour of a substance at the corresponding in vivo barrier. Here, we compare a well-established cell culture model for the intestine, based on Caco-2 colon carcinoma cells, with a primary cell culture model of the blood-brain barrier. We find that Caco-2 cells and cells of the blood-brain barrier have different barrier properties. Therefore, cells used for cell-based assays should be derived from the corresponding tissue to reflect the in vivo barrier characteristics.
Animal Testing Alternatives↗
Changes in barrier function of a model intestinal epithelium by intraepithelial lymphocytes require new protein synthesis by epithelial cells.
BACKGROUND: Elements of the mucosal immune system may play an important part in regulating epithelial barrier function in the intestinal tract. Intraepithelial lymphocytes (IELs) represent a subtype of immunocyte which is strategically placed to regulate epithelial function at most mucosal sites. AIMS AND METHODS: An IEL derived cell line (SC1) was used to examine its effects on the model epithelium T84--a tumour derived cell line which retains the phenotype of colonic crypt cells. Transepithelial electrical resistance (TER) was used as a marker of epithelial integrity. RESULTS: Coculture of T84 cells with SC1 produced a significant fall in TER as did exposure of T84 monolayers to IEL derived supernatant. Recombinant interferon-gamma (rIFN gamma) also reduced TER in T84 monolayers. Cycloheximide prevented the effects of IEL supernatant and of rIFN gamma on TER. The fall in TER in response to rIFN gamma was attenuated by blocking antibodies, which did not alter the fall in resistance induced by IEL supernatant. Fractions of IEL supernatant, separated on the basis of size, evoked temporally distinct changes in TER. Ultrastructural studies support the hypothesis that the slow onset but severe fall in TER indicates catastrophic effects on the monolayer. The more rapid onset fall in TER was not associated with gross changes in monolayer morphology. Reduction of TER by IEL supernatant was not influenced by inhibitors of tyrosine phosphatase or of protein kinase C. Although herbimycin did reduce the rapid onset change in TER, the tyrosine kinase inhibitor genistein did not alter responses to IEL supernatant. CONCLUSIONS: Mucosal T cells may influence barrier function by a process involving new protein synthesis by epithelial cells. This model may have relevance in some inflammatory conditions of the gastrointestinal tract.
Benzoquinones↗
Effect of 'unstirred' water layer in the intestine on the rate and extent of absorption after oral administration.
The presence of an aqueous diffusion layer or 'unstirred' water layer adjacent to the intestinal membrane has long been regarded as a potential barrier for intestinal absorption of compounds. Theoretical analyses were performed in the present study to quantitatively determine the effect of this layer on the rate and extent of absorption of passively absorbed compounds with different membrane absorption half-lives (10 to 300 min) in humans, dogs, rabbits, rats and mice. Diffusion half-lives across this (40 microns thick) layer were estimated to be 5.8, 2.5, 1.1, 0.65 and 0.32 min, respectively, in the distended intestine of the above five species. These half-lives are reduced by about 5-fold when the intestine is about 80% 'flat' in fasting state. The results of extensive analysis indicate that the presence of such a layer is generally expected to have a relatively mild or insignificant effect on the rate of absorption and an insignificant effect on the extent of absorption. The study also indicates that an aqueous layer of 708 microns has practically no effect on the extent of absorption of progesterone, a highly lipophilic compound, in rats. For prediction of or correlation with the fraction of oral dose absorbed after oral administration, the present study indicates that use of apparent or effective permeability rather than unbiased or true wall (membrane) permeability, as advocated earlier by others, should generally suffice.
Administration, Oral↗
Epidermal growth factor prevents acetaldehyde-induced paracellular permeability in Caco-2 cell monolayer.
BACKGROUND: Intestinal permeability and endotoxemia play a crucial role in the pathogenesis of alcoholic liver disease. Previous studies showed that acetaldehyde disrupts intestinal epithelial barrier function and increases paracellular permeability by a tyrosine kinase-dependent mechanism. In the present study, the role of epidermal growth factor (EGF) in protection of epithelial barrier function from acetaldehyde was evaluated in Caco-2 intestinal epithelial cell monolayer. METHODS: Caco-2 cells on Transwell inserts were exposed to acetaldehyde in the absence or presence of EGF, and the paracellular permeability was evaluated by measuring transepithelial electrical resistance and unidirectional flux of inulin. Integrity of epithelial tight junctions and adherens junctions was analyzed by confocal immunofluorescence microscopy and immunoblot analysis of occludin, zonula occludens (ZO)-1, E-cadherin, and beta-catenin in the actin cytoskeleton. Reorganization of actin cytoskeletal architecture was examined by confocal microscopy. RESULTS: Acetaldehyde increased paracellular permeability to inulin and lipopolysaccharide, and EGF significantly reduced these effects of acetaldehyde in a time- and dose-dependent manner. EGF prevented acetaldehyde-induced reorganization of occludin, ZO-1, E-cadherin, and beta-catenin from the cellular junctions to the intracellular compartments. Acetaldehyde treatment induced a reorganization of actin cytoskeletal network and reduced the levels of occludin, ZO-1, E-cadherin, and beta-catenin associated with the actin cytoskeleton. EGF effectively prevented acetaldehyde-induced reorganization of actin cytoskeleton and the interaction of occludin, ZO-1, E-cadherin, and beta-catenin with the actin cytoskeleton. CONCLUSION: These results indicate that EGF attenuates acetaldehyde-induced disruption of tight junctions and adherens junctions and prevents acetaldehyde-induced reorganization of actin cytoskeleton and its interaction with occludin, ZO-1, E-cadherin, and beta-catenin.
Acetaldehyde↗
Intestinal nitric oxide output during reduced mucosal blood flow in healthy volunteers.
OBJECTIVE: Nitric oxide regulates epithelial permeability and other properties of the intestinal mucosal barrier. It previously has been shown in animals that intestinal mucosal nitric oxide production is impaired during gut hypoperfusion. The study was performed to confirm the presence of intestinal mucosal nitric oxide production in humans and to investigate the effect of gut hypoperfusion due to moderate arterial hypotension on intestinal nitric oxide concentrations. DESIGN: Open study where each subject served as his own control. SETTING: Clinical research laboratory. SUBJECTS: Nine healthy volunteers were intubated with a nasogastrointestinal tube for recordings in the distal duodenum. Intestinal nitric oxide output and motility were assessed by tonometry and manometry, respectively. Laser Doppler flowmetry and plasma angiotensin II concentration were used to investigate mucosal perfusion and a vasoregulatory response. INTERVENTIONS: Moderate hypotension was induced with lower body negative pressure over 1 hr. MEASUREMENTS AND MAIN RESULTS: Intestinal nitric oxide production varied in parallel with the migrating motor complex. Low values were obtained during phase I and peak values during phase III. Lower body negative pressure was initiated at a well-defined point in the migrating motor complex cycle. It was followed by a 40 +/- 6% reduction of laser Doppler flow signal, a 778 +/- 138% increase in angiotensin II, and a reduction in intestinal mucosal nitric oxide production by 48 +/- 8%. After lower body negative pressure, laser Doppler signal and angiotensin II concentrations returned to baseline levels within 1 hr, whereas intestinal nitric oxide output remained decreased. CONCLUSIONS: Intestinal tonometry in humans exhibits a considerable mucosal nitric oxide formation that varies in relation to intestinal motility. Intestinal nitric oxide production is depressed during conditions with lowered mucosal blood perfusion.
Adult↗
Expression of intestinal trefoil factor in developing rat intestine.
Intestinal trefoil factor (ITF or TFF3), a small peptide secreted at the mucosal surface by goblet cells throughout the mature intestine, appears to play important roles in the maintenance and repair of the intestinal mucosal barrier. To study the expression of TFF3 during development, intestinal tissues were collected from rats at different development stages and examined by Northern blot analysis, Western blot analysis and immunohistochemical staining for TFF3 mRNA and protein expression. The results demonstrate that rat TFF3 mRNA is not detected until the 17th gestational day (term = 22 days), the expression is greater on gestational day 20 and increased further postnatally. TFF3 protein is first detected by Western blotting and immunohistochemical staining on gestational day 20. Further increases in TFF3 protein expression are demonstrated at around the weaning period. In conclusion, significant expression of rat TFF3 commences late in gestation and its expression is relatively deficient in immature rats. Expression of TFF3 may be deficient in premature infants and, therefore, may have a role in the development of necrotizing enterocolitis.
Animals↗
Enhanced expression of iNOS in inflamed colons of IL-2-deficient mice does not impair colonic epithelial barrier function.
On the basis of recently observed high levels of iNOS expression that correlated with intestinal inflammation in interleukin-2-deficient [IL-2(-/-)] mice, it was postulated that nitric oxide may damage colonic epithelial cells or impair intestinal epithelial barrier function. This damage may result in an increased permeability of the colonic epithelium leading to high antigenic exposure of the intestinal immune system, which may perpetuate chronic inflammation. Our data demonstrate that high expression of iNOS in IL-2(-/-) mice is correlated with the length/weight ratio (L/W ratio), a widely accepted marker for intestinal inflammation. However, no reduction of epithelial resistance was observed, as would be expected in case of a damaged, leaky epithelium. Our results suggest that enhanced formation of NO in IL-2(-/-) mice does not cause impairment of epithelial barrier function.
Animals↗
Role of the mucosal barrier in toxin/microbial attachment to the gastrointestinal tract.
An important component of bacterial infection of the gastrointestinal tract, particularly when enterotoxin disruption of gut function occurs, relates to the interaction of the bacterium and toxin with the intestinal surface. Adherence or attachment of bacteria lead to colonization and toxic/invasive diarrhoeal states. The purpose of this review is to consider the intestinal mucosal barrier as a deterrent to microbial/toxin attachment, particularly emphasizing the mucosal surface itself, which includes the mucus coat and microvillous membrane. Consequences of mucosal barrier deficiency, particularly the incomplete development of the mucosal barrier, result in bacterially induced diarrhoeal states such as toxigenic diarrhoea and necrotizing enterocolitis. To illustrate the importance of the mucosal barrier as a factor in controlling the external environment containing bacteria and bacterial toxins, recent research in our laboratory on the development of the mucosal barrier is presented. This compares mucosal surface functional control of antigen/toxin attachment and penetration with mucosal surface compositional changes. Finally, evidence of a preliminary nature will be provided to suggest that modifications of the underdeveloped mucosal barrier of the immature intestine by the ingestion of breast milk may act to prevent pathological interactions between the gut and microbes/toxins.
Bacterial Infections↗
The Caco-2 cell monolayer system as an in vitro model for studying bacterial-enterocyte interactions and bacterial translocation.
Partly because of inherent limitations of in vivo models, the cellular mechanisms underlying the process of bacterial translocation across the intestinal epithelial barrier are incompletely understood. We therefore used the Caco-2 intestinal cell line as an in vitro model to examine the bacterial translocation process under controlled conditions. Caco-2 cells were grown on porous membranes in the upper compartment of a two-compartment system. Caco-2 cells were cultured for 7, 14, 21, or 28 days. Cellular confluence and tight junction integrity were verified by measurements of dextran permeability and transepithelial electrical resistance. Bacterial translocation was measured by culturing the bacteria (E. coli C25) that were able to cross the Caco-2 cell monolayer. The passage of E. coli C-25 and dextran across the Caco-2 monolayer was higher and the transepithelial electrical resistance lower after 7 days of culture than after 14 or 21 days of culture. The Caco-2 cells became impermeable to dextran blue after 14 days of culture with an average transepithelial electrical resistance of 173.1 +/- 9.24 ohms.cm2. When increasing doses of (10(2)-10(9) colony-forming units) of E. coli were tested in 14-day-old Caco-2 monolayers, bacterial translocation occurred in a time- and dose-dependent fashion. Once cellular confluence and tight junction integrity have been established, bacterial translocation across Caco-2 cells appears to be a time- and dose-dependent process.
Cell Movement↗
[Pancreatitis and translocation--approaches for nutritional strategies].
Translocation of intestinal bacteria from the gut into pancreatic necrosis is an important factor in the development of septic complications in acute pancreatitis. Bacterial translocation is promoted by an impaired intestinal mucosal barrier, which can be attributed to the reduced oxygen and substrate supply of the intestine. A rat model of severe acute pancreatitis has been used to confirm the hypothesis that an impaired mucosal barrier can be stabilized by supplying certain nutrients, vitamins, and trace elements. A reduction in secondary pancreas infections with intestinal bacteria and improved survival was achieved under intravenous glutamine substitution, an essential amino acids in stress situations for enterocytes, colonocytes and immunocompetent cells. The positive experimental results are currently being investigated in a controlled randomized multicenter trial. Comparable studies need to be performed for verifying the effect of other nutritive factors on bacterial translocation and the disease course in acute pancreatitis.
Animals↗
Passage of vasoactive intestinal peptide across the blood-brain barrier.
We investigated the ability of vasoactive intestinal peptide (VIP) to cross the blood-brain barrier (BBB), the interface between the peripheral circulation and central nervous system (CNS). VIP labeled with 131I (I-VIP) and injected intravenously into mice was taken up by brain as determined by multiple-time regression analysis. Excess unlabeled VIP was unable to impede the entry of I-VIP, indicating that passage is by nonsaturable transmembrane diffusion. High pressure liquid chromatography (HPLC) showed the radioactivity entering the brain to be intact I-VIP. After intracerebroventricular (i.c.v.) injection, I-VIP was sequestered by brain, slowing its efflux from the CNS. In summary, VIP crosses the BBB unidirectionally from blood to brain by transmembrane diffusion.
Animals↗
Interplay between CYP3A-mediated metabolism and polarized efflux of terfenadine and its metabolites in intestinal epithelial Caco-2 (TC7) cell monolayers.
PURPOSE: To further characterize cytochrome P450 (CYP) and P-glycoprotein (Pgp) expression in monolayers of the Caco-2 cell clone TC7, a cell culture model of the human intestinal epithelium. To study the interplay between CYP3A and Pgp as barriers to intestinal drug absorption in TC7 cells using terfenadine and its metabolites as substrates. METHODS: mRNA expression of eight CYPs and Pgp was investigated in TC7 and parental Caco-2 (Caco-2p) cell monolayers using RT-PCR. The CYP3A kinetics was determined in microsomes from both cell lines. The transport, metabolism and efflux of terfenadine and its metabolites were investigated in TC7 monolayers. RESULTS: Both TC7 and Caco-2p cells expressed mRNA for Pgp and several important CYPs. However, mRNA for CYP3A4 was detectable anly from TC7 cells. The relative affinity of CYP3A for terfenadine metabolism in the two cell lines was comparable, but the maximum reaction rate in the TC7 cells was 8-fold higher. The rate of transport of terfenadine and its metabolites hydroxy-terfenadine (HO-T) and azacyclonol across TC7 monolayers was 7.1-, 3.5- and 2.1-fold higher, respectively, in the basolateral to apical direction than it was in the apical to basolateral (AP-BL) direction. Inhibition studies indicated that the efflux was mediated by Pgp. Ketoconazole increased the AP-BL transport terfenadine dramatically by inhibiting both terfenadine metabolism and Pgp efflux. CONCLUSIONS: Cell culture models such as TC7 provide qualitative information on drug interactions involving intestinal CYP3A and Pgp.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
[The state of the gastrointestinal tract in reactive arthritis].
The gastrointestinal tract status (GIT) was evaluated in 23 reactive arthritis (RA) patients: in 17 after intestinal infection, in 2 after urogenital infection, and in 4 after mixed infection. All the examined were found to have signs of diffuse variously pronounced chronic inflammation of the small and large intestine, impaired barrier function of the stomach, liver disorders, and moderate-severe intestinal dysbacteriosis with developing transitory bacteraemia in most severe cases. The GIT changes were correlated with the severity of the RA course. In 20 control-group patients not afflicted with joint disease and operated on for cicatricial stricture of the oesophagus, no signs of chronic inflammation of the mucosa were revealed in the biopsy samples of the large and small intestine. The obtained results may be indicative of the role played by the GIT in the development of pathogenic processes in RA.
Adult↗
Bacterial translocation across enterocytes: results of a study of bacterial-enterocyte interactions utilizing Caco-2 cells.
Due to the inherent limitations of in vivo studies, the cellular mechanisms underlying the process of bacterial translocation (BT) across the intestinal epithelial barrier are poorly understood. Thus, we have utilized the Caco-2 intestinal cell line to study this process. Caco-2 cells were grown to confluence on semipermeable membranes contained in the upper compartment of a 2 compartment system. Cellular confluence and tight junction integrity was verified by measurements of the transepithelial electrical resistance in ohms cm2.BT was measured by culturing the bacteria (nonpathogenic Escherichia coli) that were able to cross the Caco-2 monolayer and were present in the bottom compartment, as well as by monitoring the passage of 1-micron fluorescent beads. Caco-2 cells were pretreated with several metabolic inhibitors: 1.0 mM sodium azide (oxidative phosphorylation), 10 micrograms/ml nocodazole (microtubule), 10 microM phalloidine (microfilament), and 5.0 micrograms/ml cytochalasin D (microfilament). To investigate the mechanisms of BT. Both bacteria and fluorescent beads crossed the Caco-2 monolayer. Azide had no effect on BT, while both nocodazole (n = 17) and phalloidine (n = 14) significantly decreased translocation of E. coli versus control monolayers (p < .05). Cytochalasin D increased BT versus control membranes, however this was associated with loss of tight junction integrity (transepithelial electrical resistance decreased from 201 +/- 79 to 87 +/- 6.4). BT across Caco-2 cells appears to be a polar process which is to some extent microtubule- and microfilament-dependent.
Azides↗
Short chain fatty acids but not lactate or succinate stimulate mucus release in the rat colon.
BACKGROUND: Short chain fatty acids (SCFAs) affect various intestinal functions. Mucus is an important physiological component of the intestinal mucosal barrier. However, the effect of SCFAs or other organic acids on the intestinal mucus release is poorly understood. The aim of this study was to investigate whether lumen SCFA stimulates mucus release into the rat colon. METHODS: A solution of SCFA, lactate or succinate was infused into the colon of anesthetized rats, and we then measured the hexose content of the effluent. We also examined the influence of cholinergic antagonists on the effects of SCFA. RESULTS: A SCFA mixture (75 mM acetate, 35 mM propionate and 20 mM butyrate) or individual SCFAs (130 mM) increased the mucus release into the colon in a similar manner. The individual SCFAs, but not lactate or succinate, stimulated colonic mucus secretion in similar concentration-dependent manners. Butyrate stimulated colonic mucus secretion at 20 mM, but acetate, propionate, lactate and succinate at this concentration did not. Pretreatment with an anti-cholinergic agent diminished the stimulatory effects of SCFAs on mucus secretion. CONCLUSIONS: Lumen SCFAs, but not lactate or succinate, stimulate mucus release from the rat colon via a cholinergic nerve mechanism.
Animals↗