Drop In Transepithelial Electrical Resistance Research Articles

Melittin as a Permeability Enhancer II: In Vitro Investigations in Human Mucus Secreting Intestinal Monolayers and Rat Colonic Mucosae

Melittin has shown potential as a non-cytotoxic absorption enhancer in Caco-2 monolayers. Our objectives were to assess in vitro efficacy and cytotoxicity of melittin in two intestinal permeability models and investigate the potential mechanism by which melittin might enhance gastrointestinal absorption. The effects of melittin were examined in the mucus-secreting intestinal cell monolayers, HT29-MTX-E12 (E12), using transepithelial electrical resistance (TER), transmission electron microscopy (TEM) and the MTT viability assay. The effects of melittin on TER, permeability and short circuit current (Isc) were also investigated in rat colon mucosae mounted in Ussing chambers. Ion transporting capacity of tissue was measured in response to secretagogues as surrogate markers of cytotoxicity. Melittin stability was examined by a means of a hemolytic assay. The mechanism by which melittin decreases TER across the rat mucosa was examined with a range of enzymatic inhibitors. Apical addition of melittin resulted in a reversible non-cytotoxic concentration-dependent decrease in TER across E12 monolayers, which was independent of the presence of mucus. Apical addition of melittin reduced TER and increased the permeability of [(14)C]-mannitol across rat colonic mucosae. The melittin-induced drop in TER in rat colon was significantly attenuated by W7 suggesting partial mediation by calmodulin. The rapid and reversible nature of melittin's permeation enhancing properties and its limited cytotoxicity in polarized intestinal epithelia, suggests a potential drug delivery role for the peptide in oral formulations of poorly absorbed drugs.

Probiotics Reduce Enterohemorrhagic Escherichia coli O157:H7- and Enteropathogenic E. coli O127:H6-Induced Changes in Polarized T84 Epithelial Cell Monolayers by Reducing Bacterial Adhesion and Cytoskeletal Rearrangements

The aim of this study was to determine if probiotics reduce epithelial injury following exposure to Escherichia coli O157:H7 and E. coli O127:H6. The pretreatment of intestinal (T84) cells with lactic acid-producing bacteria reduced the pathogen-induced drop in transepithelial electrical resistance. These findings demonstrate that probiotics prevent epithelial injury induced by attaching-effacing bacteria.

Apical EGF receptors regulate epithelial barrier to gastric acid: endogenous TGF-alpha is an essential facilitator.

In previous studies, we found that apical and basolateral EGF receptors (EGFR) on primary canine gastric monolayers decreased paracellular permeability, evident by increased transepithelial electrical resistance (TER) and decreased flux of [(3)H]mannitol (MF). After studying monolayers in Ussing chambers, we now report that treatment with apical, but not basolateral, EGF enhanced tolerance to apical H(+), evident by a slower decay in TER and an attenuated rise in MF. Enhanced tolerance to apical acid was evident within 10 min of treatment with apical EGF. Immunoneutralization of endogenous transforming growth factor (TGF)-alpha accelerated the drop in TER and the rise in MF in response to apical acidification; apical EGF reversed these effects. Study of monolayers cultured in Transwell inserts showed that immunoblockade of basolateral, but not apical, EGFR also impaired the resistance to apical acidification and enhanced MF. We conclude that apical EGFR regulates the barrier to apical acidification via effects on paracellular resistance. Although exogenous basolateral EGF has a less apparent effect on the barrier to acid, endogenous ligand active at basolateral EGFR plays an important role in maintaining the barrier to apical acid. Our data implicate a role for an apical EGFR ligand, which may be EGF or another member of the EGF family.

Probiotics prevent zonulin-mediated intestinal barrier dysfunction secondary to bacterial colonization

Background: In the mammalian host, the intestinal epithelium along with indigenous microflora serves as a powerful protective barrier. During pathologic conditions, this ecosystem gets disrupted leading to adverse consequences. We have previously shown that Gram (-) normal gut flora can adhere, invade, and trigger necrotizing enterocolitis-like changes in weanling rabbits. Recently, we described a novel eukaryotic protein, zonulin, and its regulatory role in gut permeability via tight junction disassembly. In this study, we evaluated the effect of a candidate probiotic strain, Lactobacillus plantarum strain PP-217 (Lp) on zonulin-mediated changes in the gut mucosa. Methods: Explant cultures from rabbit intestine were used to investigate the ability of a normal flora E. coli to induce zonulin release. Polarity of zonulin release, changes in transepithelial electrical resistance (TEER), transport of inulin, and transcytosis of E. coli were studied in the snapwell system using rabbit and rhesus monkey gut. Zonulin concentration was measured by sandwich ELISA. Results: E. coli induced significant release of zonulin from rabbit organ cultures (1.68 2.2 ng/mg protein.mrrr') compared to uninfected controls. Co-infection with Lp, resulted in a 3-8-fold reduction in zonulin release. Cultures of E. coli alone did not produce zonulin. In the snapwell systems, addition of 10 E. colilml to the apical side of the intestinal segments resulted in zonulin release from the luminal side, but not from the basolateral (BL) side, a drop in TEER (ll 20-57 n crrr'), an increased passage of inulin (11-34 ~mol.h/ crrr'), and transcytosis of E. coli to the BL side (3.3 XlO 1.8XlO in 1-6 hr). Lp reduced the release of zonulin (10-100 fold), prevented the drop in TEER (>50%), reduced inulin transcytosis (>75%), and caused a 4-100fold blockage of E. coli transcytosis. Addition of FIIO, a synthetic octapeptide analogue of zonulin provided similar protection in gut permeability. Conclusions: These results demonstrate that normal flora bacteria can induce zonulin release by the small intestine resulting in a reduction in barrier function and increased bacterial transcytosis. Candidate probiotic strains have the ability to prevent such changes. While the mechanism of action of probiotics in this system remains unidentified, the implications and its potential use can range from enteric bacterial infections to a myriad of conditions involving reduced intestinal permeability. Supported by NIH grants HD 29735 & 34974.

Penetration of clinical isolates of Pseudomonas aeruginosa through MDCK epithelial cell monolayers.

Pseudomonas aeruginosa causes both invasive (bacteremic) and chronic noninvasive infections. A simple in vitro system to screen P. aeruginosa clinical isolates for their capacity to penetrate MDCK cell monolayers has been developed. By means of this system, P. aeruginosa clinical isolates, including 32 blood and 45 respiratory isolates, were examined. When monolayers were infected with 3.5x107 cfu of bacteria, significantly more blood (93.7%) than respiratory (54.4%) isolates (P<.001) were detected in the basolateral medium after 3 h. Penetration ability was usually independent of cytotoxicity. Only 8 (4 blood and 4 respiratory) isolates were cytotoxic, possessed exoU, and passed through the monolayer after epithelial cell death, associated with a marked drop in transepithelial electrical resistance. Conversely, noncytotoxic isolates with high penetration ability but without severe epithelial damage were invasive. This system is well suited for screening clinical isolates and their mutants for specific genes conferring the invasiveness phenotype.

Evaluation of the Caco-2 monolayer as a model epithelium for iontophoretic transport.

To assess the Caco-2 monolayer as a model for iontophoresis of drugs across a model epithelium. The apparent permeability co-efficient (Papp) of mannitol, thyrotrophin releasing hormone (TRH), dexamethasone and a range of sizes of fluorescein isothiocyanate (FITC) dextrans across Caco-2 monolayers was measured under passive and electrically stimulated conditions. Trans-epithelial electrical resistance (TEER) was determined throughout. Transmission electron micrographs (TEM) of the monolayers were taken. Confocal laser scanning microscopy (CLSM) was used to visualize the iontophoretic transport route of FITC-Dextran (MW = 20 kDa) across a Caco-2 monolayer. Application of 14.3 micro-Eq x cm(-2) across the monolayer evoked a transient drop in TEER. The drop in TEER was accompanied by statistically significant increases in fluxes of all the agents in the mucosal to serosal direction except for FD-70. TEM of test samples exhibited tight junction dilatation, in addition to intracellular vacuolisation. The iontophoresis of FD-20 was visualised with confocal laser scanning microscopy and was localised in paracellular spaces of the monolayer. The fluxes of mannitol, TRH, dexamethasone, FD-4, FD-10 and FD-20 across the Caco-2 monolayer were significantly enhanced when electric field was applied. The iontophoretic effect appeared to be directly upon tight junctions

Structure—activity relationships for enhancement of paracellular permeability by 2-alkoxy-3-alkylamidopropylphosphocholines across Caco-2 cell monolayers

The oral route is the preferred route of delivery for a large number of drug molecules. However, the intestinal epithelium presents a formidable barrier for delivery of drugs into systemic circulation. Phospholipids are among compounds that enhance the absorption of drugs across the intestinal epithelium. In this paper, we describe structure–activity relationships for phospholipid derivatives as enhancers of paracellular permeability across Caco-2 cell monolayers. In a series of 2-alkoxy-3-alkylamidopropylphosphocholine derivatives, compounds with a long chain at C-3 (R3) and short chain at C-2 (R2) were potent in causing a decrease in transepithelial electrical resistance (TEER) and an increase in mannitol transport, but also showed significant cytotoxicity. Compounds with 9–11 carbons at C-3 and 6–10 carbons at C-2 provided good separation (up to 2.7-fold) between activity and cytotoxicity. Notably, a good correlation (r2 ) 0.93) was observed between EC50 (TEER) [concentration that caused a drop in TEER to 50% of its control (untreated) value] and EC10x(mannitol) [concentration that caused 10-fold increase in mannitol transport over the control (untreated) value], confirming that a decrease in TEER is associated with enhanced permeability of the hydrophilic compounds across Caco-2 cell monolayers. Compounds with medium to long carbon chains at C-2 and C-3, and the total carbons in the alkyl chains > 20, showed poor activity and no cytotoxicity.

Different invasion phenotypes of Campylobacter isolates in Caco-2 cell monolayers.

The pathogenesis of campylobacter enteritis is not well understood, but invasion into and translocation across intestinal epithelial cells may be involved in the disease process, as demonstrated for a number of other enteric pathogens. However, the mechanisms involved in these processes are not clearly defined for campylobacters. In this study, isolates were compared quantitatively in established assays with the enterocyte-like cell line, Caco-2, to determine the extent to which intracellular invasion contributes to translocation across epithelial cell monolayers, and whether isolates vary in this respect. Ten fresh Campylobacter isolates were compared and shown to differ in invasiveness by a factor of 10-fold by following their recovery from gentamicin-treated Caco-2 cells grown on nonpermeable tissue-culture wells. Four of these isolates with contrasting invasive ability were also shown to vary in their ability to translocate across Caco-2 cells grown on semipermeable Transwell inserts by a factor >10. However, translocation did not quantitatively correlate with the intracellular invasiveness of these isolates. Isolate no. 9752 was poorly invasive but had modest translocation ability, isolate no. 10392 was very invasive but did not translocate significantly and remained within the monolayer, isolate no. 9519 both translocated and invaded well, whereas, isolate no. 235 translocated very efficiently but was poorly invasive. Isolate no. 9519 also uniquely caused a transitory flattening of the Caco-2 cells and a possible drop in trans-epithelial electrical resistance (TEER) of the Transwell monolayers, whereas isolate no. 235 did not show these effects. Together these data demonstrate that there are significantly different 'invasion' phenotypes among Campylobacter strains involving different degrees of intracellular invasion, and either different rates of transcellular trafficking or, alternatively, paracellular trafficking.