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PubMed2005

A strategy to identify stable membrane-permeant peptide inhibitors of myosin light chain kinase.

PURPOSE: A peptide inhibitor of myosin light chain kinase (MLCK), termed membrane permeant inhibitor of myosin light chain kinase (PIK), has previously been demonstrated to correct paracellular barrier defects associated with in vitro cell models of infectious and inflammatory intestinal disease. The current study describes a strategy to identify stable analogues of PIK required for future in vivo studies that has resulted in the identification of two promising candidates. METHODS: Because PIK functions at an intracellular site of epithelial cells and is envisaged to be administered orally, hydrolysis patterns were determined for PIK in both extracts of homogenized Caco-2 (a human intestinal epithelial cell line) and in luminal secretions isolated from rat intestine. Based on these hydrolysis patterns, four peptides Ac-RKKYKYRRK-NH(2) (acetylated PIK), rkkykyrrk-NH(2) (D PIK), krrykykkr-NH(2) (Dreverse PIK), and RKKykyRRK-NH(2) (Dpalindrome PIK) were synthesised. Studies were carried out to determine the stability, activity, and selectivity of these PIK analogues. RESULTS: D PIK and Dreverse PIK had much longer half-lives of 3.6 and 13.4 h, respectively, compared to PIK, acetylated (Ac)-PIK, or Dpalindrome PIK. All PIK analogues inhibited MLCK potently, although D PIK was a slightly better inhibitor than the other analogues. Similarly, all PIK analogues enhanced paracellular barrier function in Caco-2 monolayers studied in vitro. No appreciable inhibition of cAMP-dependent protein kinase (PKA) or calcium/calmodulin-dependent protein kinase II (CaMPKII) was detected with any of the analogues. CONCLUSIONS: PIK is quickly degraded within two enzyme-containing preparations that represent different aspects of the intestinal environment. The PIK analogues D PIK and Dreverse PIK demonstrated extended half-lives in these enzyme preparations while retaining the biological activity and specificity of the parent PIK peptide.

Animals↗
PubMed1998

Taxol resistance mediated by transfection of the liver-specific sister gene of P-glycoprotein.

The sister gene of P-glycoprotein (Spgp) is a liver-specific ATP-binding cassette protein highly related to the P-glycoprotein (Pgp) family (S. Childs et al, Cancer Res., 55: 2029-2034, 1995). Spgp appears to be related to the Pgp family by an ancient duplication occurring before the division of fish and mammals. P-Glycoproteins have diverse functions including broad specificity multidrug resistance in cell lines and tumors, detoxification of tissues such as the intestine and blood-brain barrier, and phosphatidylcholine transport in liver. Spgp is a Mr approximately 170,000 glycosylated plasma membrane protein localized to the canalicular surface of hepatocytes in the rat liver. The full-length cDNA of Spgp was isolated from rat, and its expression was characterized in situ and in transfected cells. The expression of Spgp correlates with the differentiation of hepatocytes and is seen only in late liver development. It is not observed in hepatoma cell lines. The physiological function of Spgp in liver is unknown, but it maps to 2q31 in humans, in the vicinity of liver transport disorders for bile acids and cholesterol. Spgp may therefore be involved in some aspect of bile acid or cholesterol metabolism. Spgp transfectants have a low level resistance to Taxol but not to other drugs that form part of the multidrug resistance phenotype. This resistance is reversible by the Pgp-reversing agents cyclosporin A, PSC833, and verapamil, suggesting a conservation in some functions of Pgps across large evolutionary distance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Ethanol impairs intestinal barrier defense by modulation of immunoglobulin A transport.

BACKGROUND: Increased epithelial permeability may contribute to septic complications after alcohol intoxication. Prior ethanol (EtOH) exposure leads to structural and functional effects on cytoskeletal components important in maintaining barrier integrity. The cytoskeleton is also important in the transcellular transport of proteins including the polymeric immunoglobulin receptor (pIgR) mediated transport of immunoglobulin A (IgA) to the luminal surface of mucosal tissues. We (1) investigated the effects of acute EtOH exposure on cell monolayer barrier integrity and IgA transport and (2) assessed the importance of microtubule function on these functions in vitro. METHODS: Confluent Madin-Darby canine kidney (MDCK) epithelial cells transfected with pIgR cDNA were exposed to 1% or 5% EtOH, and transcytosis of dimeric IgA was measured. Paclitaxel was used to pretreat a subset of MDCK cells. Results. EtOH exposure resulted in a concentration and time dependent decrease in IgA transcytosis. This was associated with physical derangements in the integrity of the cell monolayer. Pretreatment with paclitaxel completely abrogated these effects. CONCLUSIONS: EtOH causes both physical and immune derangement of epithelial cell barrier function. Decreased secretory IgA at mucosal surfaces may initiate septic complications after EtOH intoxication.

Animals↗
PubMed2006

A defect in epithelial barrier integrity is not required for a systemic response to bacterial antigens or intestinal injury in T cell receptor-alpha gene-deficient mice.

BACKGROUND AND AIMS: Genetically induced disruption of the intestinal epithelial barrier leads to development of intestinal inflammation. In the interleukin-10 gene-deficient inflammatory bowel disease (IBD) mouse model, for instance, a primary defect in intestinal epithelial integrity occurs before the development of enterocolitis. In humans, a causal role for epithelial barrier disruption is still controversial. Although studies with first-degree relatives of IBD patients suggests an underlying role of impaired barrier function, a primary epithelial barrier defect in IBD patients has not been confirmed. The purpose of this article is to examine whether a primary epithelial barrier disruption is a prerequisite for the development of intestinal inflammation or whether intestinal inflammation can develop in the absence of epithelial disruption. We examined the intestinal epithelial integrity of the T cell receptor (TCR)-alpha gene-deficient mouse model of IBD. MATERIALS AND METHODS: In vivo colonic permeability, determined by mannitol transmural flux, was assessed in 6-week-, 12-week-, and 25-week-old TCR-alpha gene-deficient and wild-type control mice using a single-pass perfusion technique. Mice were scored for intestinal histological injury and intestinal cytokine levels measured in organ cultures. Systemic responses to bacterial antigens were determined through 48-h spleen cell cultures stimulated with sonicate derived from endogenous bacterial strains. RESULTS: In contrast with previous findings in the interleukin-10 gene-deficient IBD model, TCR-alpha gene-deficient mice did not demonstrate evidence of primary intestinal epithelial barrier disruption at any age, despite developing a moderate to severe colitis within 12 weeks. A rise in intestinal interferon (IFN)-gamma levels preceded the onset of mucosal inflammation and then correlated closely with the degree of intestinal inflammation and injury. Spleen cells from TCR-alpha gene-deficient mice released IFN-gamma in response to stimulation with endogenous luminal bacterial antigens, a finding that suggests that the systemic response to bacterial antigens occurred independently of epithelial barrier disruption. CONCLUSIONS: Intestinal inflammation and a systemic response to bacterial antigens can develop in the absence of a measurable disruption of intestinal permeability.

Age Factors↗
PubMed1996

The threshold for immune cell reactivity to milk antigens decreases in cow's milk allergy with intestinal symptoms.

BACKGROUND: In cow's milk allergy (CMA) with intestinal symptoms, peripheral blood mononuclear cells (PBMCs) secrete tumor necrosis factor-alpha (TNF-alpha), 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 Using chambers in HT29-19A intestinal cells. RESULTS: PBMCs from infants with active CMA secreted more TNF-alpha, when they were stimulated with intact rather than intestinally processed CMPs, and more TNF-alpha 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-alpha secretion capable of altering intestinal function was very small in infants with active CMA (approximately 2 micrograms/ml), but about 300 times higher in cured infants. CONCLUSION: Intact rather than intestinally processed proteins stimulate PBMCs to release TNF-alpha and alter intestinal barrier capacity. The threshold for PBMC reactivity to milk antigens drops considerably during active CMA with intestinal symptoms.

Animals↗
PubMed2001

The subpopulation of CF-1 mice deficient in P-glycoprotein contains a murine retroviral insertion in the mdr1a gene.

A subpopulation of the CF-1 mouse strain is sensitive to neurotoxicity following exposure to avermectins, a family of structurally related antiparasitic agents. This unusual sensitivity is the result of a deficiency in the mdr1a P-glycoprotein that normally contributes to a functional blood-brain barrier. Previous studies demonstrated a correlation between P-glycoprotein levels in the brain, intestine, testis, and placenta with an restriction fragment length polymorphism (RFLP) pattern from DNA isolated from the animals. We have demonstrated that only P-glycoprotein derived from the mdr1a gene is deficient in these mice. In this article, we describe the genetic defect in the subpopulation of CF-1 mice resulting in an absence of P-glycoprotein. The data presented describes a reverse transcription--polymerase chain reaction (RT-PCR) protocol that specifically amplifies mdr1a mRNA from tissue and confirms that the P-glycoprotein defect results from a truncated mRNA with a deleted exon 23. Genomic amplification and sequencing of the intron between exon 22 and 23 in Pgp-deficient animals reveals an insertion of approximately 8.35 kb of DNA at the exon 23 intron--exon junction corresponding to a murine leukemia virus. This insertion results in the aberrant splicing of the mRNA and the loss of exon 23 during RNA processing.

Animals↗
PubMed2002

Piperine modulates permeability characteristics of intestine by inducing alterations in membrane dynamics: influence on brush border membrane fluidity, ultrastructure and enzyme kinetics.

Piperine (1-Piperoyl piperidine) is a major alkaloid of Piper nigrum Linn. and Piper longum Linn. It is shown to possess bioavailability-enhancing activity with various structurally and therapeutically diverse drugs. The mechanism of enhancing the bioavailability, is, however, not understood. We hypothesize that piperine's bioavailability-enhancing property may be attributed to increased absorption, which may be due to alteration in membrane lipid dynamics and change in the conformation of enzymes in the intestine. Results of membrane fluidity studies using an apolar fluorescent probe, pyrene (which measures the fluid properties of hydrocarbon core), showed an increase in intestinal brush border membrane (BBM) fluidity. Piperine also stimulated Leucine amino peptidase and Glycyl-glycine dipeptidase activity, due to the alteration in enzyme kinetics. This suggests that piperine could modulate the membrane dynamics due to its apolar nature by interacting with surrounding lipids and hydrophobic portions in the protein vicinity, which may decrease the tendency of membrane lipids to act as stearic constrains to enzyme proteins and thus modify enzyme conformation. Ultra structural studies with piperine showed an increase in microvilli length with a prominent increase in free ribosomes and ribosomes on the endoplasmic reticulum in enterocytes, suggesting that synthesis or turnover of cytoskeletal components or membrane proteins may be involved in the observed effect. In conclusion, it is suggested that piperine may be inducing alterations in membrane dynamics and permeation characteristics, along with induction in the synthesis of proteins associated with cytoskeletal function, resulting in an increase in the small intestine absorptive surface, thus assisting efficient permeation through the epithelial barrier.

Alkaloids↗
PubMed1998

Is early enteral nutrition in acute pancreatitis dangerous? About 20 patients fed by an endoscopically placed nasogastrojejunal tube.

Bowel rest during treatment of acute pancreatitis deprives the gut of nutrients and affects its structure and function. Enteral feeding is usually performed late in the course of acute pancreatitis and therefore cannot prevent intestinal barrier dysfunction and possible bacterial translocation. To assess the effect of early enteral nutrition we performed a prospective study on 21 patients (11 males/10 females) presenting with severe acute pancreatitis (13 biliary, 6 alcoholic, and 2 miscellaneous). Severity was established by a mean Ranson score of 3.57. All but one patient could be fed through a double-lumen nasogastrojejunal tube within 60 h after admission. No significant complication of the technique was observed. We conclude that early jejunal feeding can be used safely in severe acute pancreatitis. Further comparative studies are necessary to demonstrate any superiority to total parenteral nutrition.

Acute Disease↗
PubMed1998

Helicobacter pylori alters exogenous antigen absorption and processing in a digestive tract epithelial cell line model.

To study the influence of Helicobacter pylori on epithelial barrier function, bacteria, bacterial sonicates, or broth culture supernatants were incubated for 24 h with HT29-19A intestinal cells grown as monolayers. Subsequently, the monolayers were mounted in Ussing chambers, and electrical resistance (R), fluxes of 22Na (JNa) and 14C-mannitol (JMan) (markers of the paracellular pathway), and fluxes of horseradish peroxidase (HRP) in total (J3H-HRP), intact (JHRPi), and degraded forms were measured. H. pylori did not induce any modification of the paracellular pathway (R = 148 +/- 10 versus 174 +/- 16 Omega. cm2; JNa = 4.16 +/- 0.44 versus 3.51 +/- 0.41 microEq/h. cm2; JMan = 0.081 +/- 0.01 versus 0.058 +/- 0.009 micromol/h. cm2), nor did it modify J3H-HRP (2,201 +/- 255 versus 2, 110 +/- 210 ng/h. cm2 for H. pylori-infected and control cells, respectively). However, in the presence of H. pylori, we observed a significant increase in JHRPi (520 +/- 146 versus 171 +/- 88 ng/h. cm2). This effect was not dependent of the cag status of the strain and was not reproduced by the sonicates or the culture supernatants. It was related to the presence of urease, since a urease-negative mutant of H. pylori did not induce this effect. Ammonia and bafilomycin A1, two agents known to increase the endolysosomal pH, reproduced the increase in JHRPi. In conclusion, H. pylori does not affect directly the integrity of intercellular junctions of epithelial cells in vitro, but it increases the passage of intact HRP, probably by inhibition of the intralysosomal degradation due to the release of ammonia. The increased transport of intact macromolecules may contribute to the induction and maintenance of gastric inflammation by H. pylori.

Ammonium Chloride↗
PubMed2006

Osteopontin and mucosal protection.

Protection of mucosal tissues of the oral cavity, intestines, respiratory tract, and urogenital tract from the constant challenge of pathogens is achieved by the combined barrier function of the lining epithelia and specialized immune cells. Recent studies have indicated that osteopontin (OPN) has a pivotal role in the development of immune responses and in the tissue destruction and the subsequent repair processes associated with inflammatory diseases. While expression of OPN is increased in immune cells--including neutrophils, macrophages, T- and B-lymphocytes--and in epithelial, endothelial, and fibroblastic cells of inflamed tissues, deciphering the specific functions of OPN has been difficult. In part, this is due to the broad range of biological activities of OPN that are mediated by multiple receptors which recognize several signaling motifs whose activities are influenced by post-translational modifications and proteolytic processing of OPN. Understanding the role of OPN in mucosal inflammation is further complicated by its contributions to the barrier function of the lining epithelia and the complexity of the specialized mucosal immune system. In an attempt to provide some insights into the involvement of OPN in mucosal diseases, this review summarizes current knowledge of the biological activities of OPN involved in the development of inflammatory responses and in wound healing, and indicates how these activities may affect the protection of mucosal tissues.

Animals↗
PubMed2005

The influence of whole wheat feeding on Salmonella infection and gut flora composition in broilers.

In an experiment with broilers infected with a rifampicin-resistant Salmonella Typhimurium strain at 15 days of age, it was demonstrated that whole wheat feeding influenced the course of infection. The pH in the contents of the gizzard decreased as the amount of whole wheat in the diet increased over time. Following infection, lower numbers of Salmonella Typhimurium were found in the gizzard and ileum of birds receiving whole wheat compared to pellet-fed birds. However, there was no difference with respect to the numbers of Salmonella Typhimurium in the ceca and rectum between the two experimental groups. Quantitative cultivation of different groups of bacteria belonging to the natural intestinal microflora showed that whole wheat feeding significantly reduced the numbers of Clostridium perfringens in the intestinal tract of the birds. The present results indicate that the gizzard has an important function as a barrier organ, one that prevents pathogenic bacteria from entering the distal digestive tract.

Animal Husbandry↗
PubMed1992

Total parenteral nutrition-induced changes in gut mucosal function: atrophy alone is not the issue.

BACKGROUND: Total parenteral nutrition (TPN) has been implicated in gut atrophy and breakdown of barrier function leading to bacterial translocation (BT) in animals. BT during TPN, however, is not found consistently, and it has therefore been suggested that macromolecular permeability may occur independently of BT during TPN. METHODS: Male Sprague-Dawley rats were administered isocaloric standard TPN enterally, parenterally, or split equally between the two routes or allowed food ad lib. A second group of rats was administered isocaloric TPN with and without 4% lipids, and changes in gut barrier function were assessed by measuring lactulose permeability. RESULTS: Rats receiving TPN both enterally and parenterally maintained histologic intestinal structure to the same degree as rats fed enterally and those allowed food. Although parenteral feeding led to significant gut atrophy and cecal bacterial overgrowth, BT was not increased. Gut permeability to lactulose, however, was increased significantly in the TPN groups. Lipid content did not affect outcome. CONCLUSIONS: These results suggest that gut atrophy, BT, and permeability to macromolecules are not necessarily related. Gut-origin septic states during TPN or trauma may be caused by an increased escape of macromolecules from the gut, and BT may be an end result rather than a primary cause of such septic episodes.

Animals↗
PubMed2003

Oral and intestinal microflora in 5-fluorouracil treated rats, translocation to cervical and mesenteric lymph nodes and effects of probiotic bacteria.

Serious systemic infections may occur during cancer chemotherapy due to disturbances in the oropharyngeal and gastrointestinal microflora, impaired mucosal barrier functions and immunosuppression. Bacteria may spread from the gastrointestinal tract to the regional lymph nodes. The routes for bacterial spread from the oral cavity are less well known. In the present study we investigated changes in the oral and intestinal microfloras in rats given 50 mg/kg 5-fluorouracil (5-FU) i.v. for 6 days. Bacterial dissemination to the lymph nodes draining the oral cavity and the lymph nodes draining the gastrointestinal tract was examined. Effects of adding the probiotic strain Lactobacillus plantarum 299v in the drinking water to the rats were measured. 5-FU treatment caused an increase in the number of facultative and strictly anaerobic bacteria in biopsies from the oral cavity and an increase in the number of facultative anaerobes in the large intestine. The proportion of facultative gram-negative rods increased in both the oral cavity and intestine. Bacteria translocated to both the cervical and mesenteric lymph nodes in untreated animals and increased in numbers after 5-FU treatment due to an increase in the number of facultative gram-negative rods. Treatment with L. plantarum 299v improved food intake and body weight in 5-FU-treated rats. It also reduced the 5-FU-induced raise in the total numbers of facultative anaerobes in the intestine, but did not reduce translocation and did not prevent diarrhea. This study reinforces the oral cavity, along with the gastrointestinal tract, as a source for bacterial dissemination. The use of probiotic bacteria may reduce some side effects of 5-FU treatment.

Animals↗
PubMed2005

Intestinal flora of animal models of human diseases as an environmental factor.

Genetically-engineered animals are known to be useful in clarifying the functions of many genes and as animal models for human diseases. However, it has been widely reported that pathophysiology is not expressed in these animals when they become germfree or SPF animals, i.e., the pathophysiology is not the result of genes alone and a combination of gene function and intestinal flora as an environmental factor are necessary. It is important to determine the roles of each of these two factors by pathophysiological analysis. Gnotobiotic mice were produced by establishment of specified bacterial species in germfree animals to form the intestinal flora of SPF animals and they were placed in barrier facilities. Measures have been taken against infections by bacteria such as Pseudomonas aeruginosa and Enterobacter cloacae. In addition, gnotobiotic mice with a highly normal physiology are required. Analysis of the effects of each bacterial species and combinations of bacteria on in vivo functions, i.e., the cross-talk between the host and intestinal flora, is essential in the creation of better laboratory animals. Monitoring of the intestinal flora, a key factor in the colonies produced, is a topic for future research.

Animals↗
PubMed1995

[Evaluation of the long-term results of the surgical treatment in patient with vaginal aplasia in the presence of functioning uterus].

Results of surgical treatment of 28 adolescent girls with vaginal aplasia and functioning uterus are analyzed. The uterus had to be removed in all the patients 1.5 to 6 months after abdominal colpopoiesis with preservation of functioning uterus; after sigmoidal colpopoiesis the uterus was preserved in 85% of patients. Analysis of the contents of the uterine cavity showed endometritis, which was evidently caused by the absence of the cervix uteri, a natural barrier preventing the penetration of intestinal flora of neovaginal mucosa into the uterine cavity. The studies permitted a new interpretation of the results of abdominal and sigmoidal colpopoiesis with preservation of functioning uterus in adolescent girls with vaginal aplasia.

Adolescent↗
PubMed1996

Clinical uses of probiotics for stabilizing the gut mucosal barrier: successful strains and future challenges.

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 colonize the intestine, at least temporarily, by adhering to the intestinal epithelium. Such probiotic microorganisms appear to be promising candidates for the treatment of clinical conditions with abnormal gut microflora and altered gut mucosal barrier functions. They are also promising ingredients to future functional foods and clinical foods for specific disease states provided that basic requirements for strains and clinical studies are carefully followed.

Animals↗
PubMed1993

Effect of cytokines on the epithelial function of the human colon carcinoma cell line HT29 cl 19A.

In various intestinal diseases, lymphoid cell infiltration of the lamina propria might be an important factor causing intestinal dysfunction through cytokine release. The effects of such cytokines were therefore measured on chloride secretion and macromolecular transport in intestinal HT29 cl 19A cells. Cells were grown on transwell filters and serosally exposed for two to 48 hours to 1% or 5% concentrations of phytohaemagglutinin (PHA) activated or non-activated healthy human mononuclear cell culture supernatants. These supernatants were tested for their interferon gamma (IFN gamma), tumour necrosis factor alpha (TNF alpha), and interleukin 6 (IL6) concentration. The mean (SE) were 4.72 (1.63), 3.17 (0.14), and 4.47 (1.12) ng/ml in activated supernatants. Intestinal function was studied in Ussing chambers: chloride secretion was assessed from the variations in short circuit current, and epithelial barrier capacity was monitored by the electrical resistance of the HT29 cl 19A cell monolayers and by measuring intact or degraded fluxes of the macromolecular tracer horseradish peroxidase. Release of lactate dehydrogenase (LDH) in the presence of various concentrations of cytokines was assessed as a test for cellular injury. The results indicate that serosal cytokines do not directly stimulate short circuit current. Long term (48 hours) exposure, however, significantly reduced both short circuit current (6.79 (0.65) v 2.90 (1.16) microA/cm2 in control v treated cells) and barrier capacity as shown by the decrease in resistance of the HT29 cl 19A cell monolayers from 158 (10) to 36 (9) ohms.cm2, and the increase in intact fluxes from 167 (33) in controls to 874 (206) ng/h.cm2, in cytokine rich supernatant treated cells and in degraded fluxes from 1113 (114) to 2327 (234) ng/h.cm2. This decreased barrier capacity was associated with an increase in release of LDH F(21.7 (1.2) and 43.6 (2.0) UI/h.cm2 in 1% and 5% cytokine treated monolayers v 10.8 (2.1) UI/h.cm2 in control monolayers). The results indicate that cytokines do not directly stimulate intestinal chloride secretion but that prolonged exposure to these agents reduced both electrolyte transport and barrier capacity to macromolecules through cellular and paracellular pathways.

Cell Membrane Permeability↗