Novel effects of the prototype translocating Escherichia coli, strain C25 on intestinal epithelial structure and barrier function

Intestinal bacteria play an etiologic role in triggering and perpetuating chronic inflammatory bowel disorders. However, the precise mechanisms whereby the gut microflora influences intestinal cell function remain undefined. Therefore, the effects of the non-pathogenic prototype translocating Escherichia coli, strain C25 on the barrier properties of human T84 and Madine-Darby canine kidney type 1 epithelial cells were examined. T-84 cells were also infected with commensal E. coil, strains F18 and HB101, and enterohaemorrhagic E. coli, serotype O157:H7. Strains F18 and HB101 had no effect on transepithelial electrical resistance (TER) of T84 monolayers. By contrast, epithelial cells infected with strain C25 displayed a time-dependent decrease in TER, preceded by an altered distribution of the cytoskeletal protein alpha-actinin, comparable to infection with E. coli O157:H7. E. coli C25 infection also led to activation of nuclear factor kappaB (NF-kappaB), interleukin-8 secretion and alterations in localization of claudin-1, but not zona occludens-1 or claudin-4, in T84 cells. There were adherent C25 bacteria on the intact apical surface of infected T84 cells, while mitochondria appeared swollen and vacuolated. These novel findings demonstrate the ability of a translocating commensal bacterium to adhere to and modulate intestinal epithelial barrier function and to induce morphological changes in a manner distinct from the known enteric pathogen, E. coli O157:H7.     

Transcellular translocation of Campylobacter jejuni across human polarised epithelial monolayers

The mechanisms whereby Campylobacter jejuni translocates across the host intestinal epithelium are not yet understood and the transepithelial route remains undefined. During C. jejuni translocation, the transmonolayer electrical resistance (TER) across polarised monolayers of Caco-2 cells is not affected and the penetration of [(14)C]inulin across the monolayers does not increase. Over 24 h, however, bacteria damage the monolayer integrity, causing a decrease in the TER. These results support C. jejuni translocation through the cytoplasm of invaded cells (transcellular) rather than via intercellular spaces (paracellular).     

Interferon-gamma signals via an ERK1/2-ARF6 pathway to promote bacterial internalization by gut epithelia

The barrier function of the epithelium lining the intestine is essential for health by preventing the free passage of colonic bacteria into the mucosa. Epithelia treated with interferon (IFN)-γ display increased bacteria transcytosis. Much is known of how IFNγ affects the tight junction and paracellular permeability, yet its role in modifying transcellular traffic of commensal bacteria remains poorly understood. Using immunoblotting, ELISA and immunolocalization, IFNγ was found to activate extracellular regulated kinase (ERK)1/2 in the human colon-like T84 epithelial cell line. Pharmacological inhibition of MEK/ERK1/2 signalling with U0126 significantly inhibited IFNγ-induced increases in the transcytosis of non-invasive Escherichia coli (strain HB101). IFNγ treatment enhanced epithelial internalization of E. coli, some of which subsequently escaped the enterocyte. Molecular analyses revealed that ERK1/2 inhibition prevented activation of the ADP-ribosylation factor (ARF)-6, a protein associated with endocytosis, and that siRNA knock-down of ARF6 expression reduced IFNγ-induced E. coli internalization into T84 cells. None of these interventions affected the drop in transepithelial resistance caused by IFNγ. Thus, increased transcellular passage may be a major component of IFNγ-induced increases in epithelial permeability, and ERK1/2 and ARF6 are presented as important molecules in IFNγ-evoked transcytosis of bacteria across gut epithelia.     

PG-mediated closure of paracellular pathway and not restitution is the primary determinant of barrier recovery in acutely injured porcine ileum

Small bowel epithelium is at the frontline of intestinal barrier function. Restitution is considered to be the major determinant of epithelial repair, because function recovers in parallel with restitution after acute injury. As such, studies of intact mucosa have largely been replaced by migration assays of cultured epithelia. These latter studies fail to account for the simultaneous roles played by villous contraction and paracellular permeability in recovery of barrier function. NSAIDs result in increased intestinal permeability and disease exacerbation in patients with inflammatory bowel disease (IBD). Thus we examined the reparative attributes of endogenous PGs after injury of ileal mucosa by deoxycholate (6 mM) in Ussing chambers. Recovery of transepithelial electrical resistance (TER) from 20-40 Omega.cm2 was abolished by indomethacin (Indo), whereas restitution of 40-100% of the villous surface was unaffected despite concurrent arrest of villous contraction. In the presence of PG, resident crypt and migrating epithelial cells were tightly apposed. In tissues treated with Indo, crypt epithelial cells had dilated intercellular spaces that were accentuated in the migrating epithelium. TER was fully rescued from the effects of Indo by osmotic-driven collapse of the paracellular space, and PG-mediated recovery was significantly impaired by blockade of Cl- secretion. These studies are the first to clearly distinguish the relative contribution of paracellular resistance vs. restitution to acute recovery of epithelial barrier function. Restitution was ineffective in the absence of PG-mediated paracellular space closure. Failure of PG-mediated repair mechanisms may underlie barrier failure resulting from NSAID use in patients with underlying enteropathy.     

Molecular orbital theory applied to the study of nonsteroidal anti-inflammatory drug efficiency.

Using a simple quantum mechanical method, we calculated the energy of the highest-occupied molecular orbital (E(HOMO)) of three groups of anti-inflammatory compounds, and we have found correlations between E(HOMO) of these molecules and experimental data previously reported on (1) inhibition of sheep-vesicular-gland prostaglandin cyclooxygenase by phenolic compounds, (2) inhibition of prostaglandin cyclooxygenase in mouse macrophages by salicylates, benzoates and phenols, and (3) peroxyl-radical scavenging and radioprotection of a bacterial virus by NSAID drugs, including metiazinic acid, sulindac, D-penicillamine, piroxicam, indomethacin, benoxaprofen, and aspirin. Our correlations using a systematic evaluation of the HOMO energies can be of predictive value in the search for new anti-inflammatory drugs as well as for new radioprotectors.     

Decreased epithelial barrier function evoked by exposure to metabolic stress and nonpathogenic E. coli is enhanced by TNF-alpha

A defect in mitochondrial activity contributes to many diseases. We have shown that monolayers of the human colonic T84 epithelial cell line exposed to dinitrophenol (DNP, uncouples oxidative phosphorylation) and nonpathogenic Escherichia coli (E. coli) (strain HB101) display decreased barrier function. Here the impact of DNP on macrophage activity and the effect of TNF-alpha, DNP, and E. coli on epithelial permeability were assessed. DNP treatment of the human THP-1 macrophage cell line resulted in reduced ATP synthesis, and, although hyporesponsive to LPS, the metabolically stressed macrophages produced IL-1beta, IL-6, and TNF-alpha. Given the role of TNF-alpha in inflammatory bowel disease (IBD) and the association between increased permeability and IBD, recombinant TNF-alpha (10 ng/ml) was added to the DNP (0.1 mM) + E. coli (10(6) colony-forming units), and this resulted in a significantly greater loss of T84 epithelial barrier function than that elicited by DNP + E. coli. This increased epithelial permeability was not due to epithelial death, and the enhanced E. coli translocation was reduced by pharmacological inhibitors of NF-kappabeta signaling (pyrrolidine dithiocarbamate, NF-kappabeta essential modifier-binding peptide, BAY 11-7082, and the proteosome inhibitor, MG132). In contrast, the drop in transepithelial electrical resistance was unaffected by the inhibitors of NF-kappabeta. Thus, as an integrative model system, our findings support the induction of a positive feedback loop that can severely impair epithelial barrier function and, as such, could contribute to existing inflammation or trigger relapses in IBD. Thus metabolically stressed epithelia display increased permeability in the presence of viable nonpathogenic E. coli that is exaggerated by TNF-alpha released by activated immune cells, such as macrophages, that retain this ability even if they themselves are experiencing a degree of metabolic stress.     

Transcytosis of iota-toxin across polarized CaCo-2 cells

Iota-toxin from Clostridium perfringens type E is a binary toxin consisting of two independent proteins, an enzymatic Ia and binding Ib component. Ia catalyses ADP-ribosylation of actin monomers, thus disrupting the actin cytoskeleton. In this report, we show that Ia plus Ib applied apically or basolaterally induce a rapid decrease in the transepithelial resistance (TER) of CaCo-2 cell monolayers and disorganization of actin filaments as well as the tight and adherens junctions. Ib alone, on the apical or basolateral side, slowly decreased the TER without affecting the actin cytoskeleton, possibly via pore formation. Interestingly, the two iota-toxin components inoculated separately on each cell surface induced cytopathic effects and a TER decrease. Anti-Ib sera, raised against the whole molecule or the Ia docking domain and applied to the opposite cell side versus Ib, neutralized the TER decrease. In addition, radioactive Ib incubated in the basolateral compartment was detected on the apical side by selective cell surface biotinylation. This argues for a transcytotic routing of Ib to mediate internalization of Ia from the opposite cell surface. Bafilomycin A1 also prevented the cytopathic effects of Ia and Ib applied separately to each cell side, possibly by blocking translocation of Ia into the cytosol and/or the intracellular transport of Ib. Ib is either routed into the cell independently of Ia, trans-cytosed and permanently exposed on the opposite cell surface or continuously recycled between an endosomal compartment and the cell surface.     

Enterocyte TLR4 mediates phagocytosis and translocation of bacteria across the intestinal barrier

Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis, although the mechanisms by which bacterial translocation occurs remain largely unknown. We hypothesized that bacterial translocation across the intact barrier occurs after internalization of the bacteria by enterocytes in a process resembling phagocytosis and that TLR4 is required for this process. We now show that FcgammaRIIa-transfected enterocytes can internalize IgG-opsonized erythrocytes into actin-rich cups, confirming that these enterocytes have the molecular machinery required for phagocytosis. We further show that enterocytes can internalize Escherichia coli into phagosomes, that the bacteria remain viable intracellularly, and that TLR4 is required for this process to occur. TLR4 signaling was found to be necessary and sufficient for phagocytosis by epithelial cells, because IEC-6 intestinal epithelial cells were able to internalize LPS-coated, but not uncoated, latex particles and because MD2/TLR4-transfected human endothelial kidney (HEK)-293 cells acquired the capacity to internalize E. coli, whereas nontransfected HEK-293 cells and HEK-293 cells transfected with dominant-negative TLR4 bearing a P712H mutation did not. LPS did not induce membrane ruffling or macropinocytosis in enterocytes, excluding their role in bacterial internalization. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice having mutations in TLR4. These data suggest a novel mechanism by which bacterial translocation occurs and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.     

Mechanisms of extracellular vesicle uptake in stressed retinal pigment epithelial cell monolayers

PurposeExtracellular vesicles (EVs) can mediate long-distance communication in polarized RPE monolayers. Specifically, EVs from oxidatively stressed donor cells (stress EVs) rapidly reduced barrier function (transepithelial resistance, TER) in naïve recipient monolayers, when compared to control EVs. This effect on TER was dependent on dynamin-mediated EV uptake, which occurred rapidly with EVs from oxidatively stressed donor cells. Here, we further determined molecular mechanisms involved in uptake of EVs by naïve RPE cells.MethodsRPE cells were grown as monolayers in media supplemented with 1% FBS followed by transfer to FBS-free media. Cultures were used to collect control or stress EVs upon treatment with H₂O₂, others served as naïve recipient cells. In recipient monolayers, TER was used to monitor EV-uptake-based activity, live-cell imaging confirmed uptake. EV surface proteins were quantified by protein chemistry.ResultsClathrin-independent, lipid raft-mediated internalization was excluded as an uptake mechanism. Known ligand-receptor interactions involved in clathrin-dependent endocytosis include integrins and proteoglycans. Desialylated glycans and integrin-receptors on recipient cells were necessary for EV uptake and subsequent reduction of TER in recipient cells. Protein quantifications confirmed elevated levels of ligands and neuraminidase on stress EVs. However, control EVs could confer activity in the TER assay if exogenous neuraminidase or additional ligand was provided.ConclusionsIn summary, while EVs from both stressed cells and control contain cargo to communicate stress messages to naive RPE cells, stress EVs contain surface ligands that confer rapid uptake by recipient cells. We propose that EVs potentially contribute to RPE dysfunction in aging and disease.     

Actin depolymerization disrupts tight junctions via caveolae-mediated endocytosis

The tight junction (TJ) determines epithelial barrier function. Actin depolymerization disrupts TJ structure and barrier function, but the mechanisms of this effect remain poorly understood. The goal of this study was to define these mechanisms. Madin-Darby canine kidney (MDCK) cells expressing enhanced green fluorescent protein-, enhanced yellow fluorescent protein-, or monomeric red fluorescent protein 1-fusion proteins of beta-actin, occludin, claudin-1, ZO-1, clathrin light chain A1, and caveolin-1 were imaged by time-lapse multidimensional fluorescence microscopy with simultaneous measurement of transepithelial electrical resistance (TER). Actin depolymerization was induced with latrunculin A (LatA). Within minutes of LatA addition TER began to fall. This coincided with occludin redistribution and internalization. In contrast, ZO-1 and claudin-1 redistribution occurred well after maximal TER loss. Occludin internalization and TER loss, but not actin depolymerization, were blocked at 14 degrees C, suggesting that membrane traffic is required for both events. Inhibition of membrane traffic with 0.4 M sucrose also blocked occludin internalization and TER loss. Internalized occludin colocalized with caveolin-1 and dynamin II, but not with clathrin, and internalization was blocked by dominant negative dynamin II (K44A), but not by Eps15Delta95-295 expression. Inhibition of caveolae-mediated endocytosis by cholesterol extraction prevented both LatA-induced TER loss and occludin internalization. Thus, LatA-induced actin depolymerization causes TJ structural and functional disruption by mechanisms that include caveolae-mediated endocytosis of TJ components.     

Escherichia coli lipopolysaccharides produce serotype-specific hypothermic response in biotelemetered rats

We investigated whether LPS-induced hypothermia develops in a serotype-specific manner in biotelemetered conscious rats. Two different Escherichia coli serotypes of LPSs were injected at a dose of 250 mug/kg ip. E. coli O55:B5 LPS elicited an initial hypothermia and subsequent fever, but E. coli O111:B4 LPS caused more potent monophasic hypothermia. Serum tumor necrosis factor (TNF)-alpha levels were dramatically elevated at the initial phase of the hypothermia induced by both LPSs. This elevation tended to subside at the nadir of E. coli O55:B5 LPS-induced response but progressively increased at the nadir of E. coli O111:B4 LPS hypothermia. Serum IL-10 levels were moderately elevated at the initial phase of the hypothermia and persisted at the same level at the nadir of each LPS-induced response. No change was observed at the serum IL-18 levels. A selective cyclooxygenase (COX)-1 enzyme inhibitor, valeryl salicylate (20 mg/kg sc), abolished the hypothermia without any effect on the elevated cytokine levels. Another COX-1-selective inhibitor, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560; 1 mg/kg sc) inhibited hypothermic responses as well. Meanwhile, cytokine levels were also reduced by SC-560 treatment. These findings suggest that LPS-induced hypothermia may have serotype-specific characteristics in rats. E. coli O111:B4 LPS has more potent hypothermic activity than E. coli O55:B5 LPS; that may presumably be related to its higher or sustained capability to release antipyretic cytokines, such as TNF-alpha. COX-1 enzyme may be involved in the generation of the hypothermia, regardless of the type of LPS administered.     

Cyclooxygenase 2 (COX-2) inhibition increases the inflammatory response in the brain during systemic immune stimuli

Non-steroidal anti-inflammatory drugs (NSAIDs) and inhibitors of the cyclooxygenase (COX) pathways are currently recommended for the prevention and treatment of several inflammatory diseases, including neurodegenerative disorders. However non-selective blockade of COX was found to have pro-inflammatory properties, because they have the ability to alter the plasma glucocorticoid levels that play a critical role in the control of the innate immune response. The present study investigated the role of non-selective (ketorolac or indomethacin) or specific inhibitors of COX-1 (SC-560) and COX-2 (NS-398) in these effects. Mice challenged systemically with the endotoxin lipopolysaccharide (LPS) exhibited a robust hybridization signal for numerous inflammatory genes in vascular-associated cells of the brain and microglia across the cerebral tissue. Ketorolac, indomethacin and NS-398 significantly increased the ability of LPS to trigger such an innate immune response at time 3 h post challenge, whereas SC-560 failed to change gene expression in the brain of animals treated with the endotoxin. These data together with the crucial role of COX-2-derived prostaglandin E2 (PGE2) in the increase of glucocorticoids during systemic immune stimuli provide evidence that inhibition of this pathway results in an exacerbated early innate immune reaction. This may have a major impact on the use of these drugs in diseases where inflammation is believed to be a contributing and detrimental factor.     

Identification of specific miRNAs targeting proteins of the apical junctional complex that simulate the probiotic effect of E. coli Nissle 1917 on T84 epithelial cells

In the intestine, dysregulation of miRNA is associated with inflammation, disruption of the gastrointestinal barrier, and the onset of gastrointestinal disorders. This study identifies miRNAs involved in the maintenance of intercellular junctions and barrier integrity. For the functional identification of barrier affecting miRNAs, we took advantage of the barrier-enforcing effects of the probiotic bacterium Escherichia coli Nissle 1917 (EcN) which can be monitored by enhanced transepithelial resistance (TER). miRNA-profiling of T84 monolayers prior and after co-incubation with EcN revealed for the first time differentially regulated miRNAs (miR-203, miR-483-3p, miR-595) targeting tight junction (TJ) proteins. Using real-time PCR, Western blotting and specific miRNA mimics, we showed that these miRNAs are involved in the regulation of barrier function by modulating the expression of regulatory and structural components of tight junctional complexes. Furthermore, specific inhibitors directed at these miRNA abrogated the disturbance of tight junctions induced by enteropathogenic E. coli (EPEC). The half-maximal inhibitory concentration (IC(50)) was determined to 340 nM by monitoring inhibitor kinetics. In summary, we conclude that specific miRNAs effect regulatory as well as structural proteins of the junctional complex which in turn are involved in the barrier enhancing effect of EcN. Hence, we suggest that the application of miRNAs might be refined and further developed as a novel supportive strategy for the treatment of gastrointestinal disorders.     

Inhibition of adenine nucleotide synthesis: effect on tight junction structure and function of clone 4 MDCK cells.

The formation and maintenance of tight junctions as a barrier to the diffusion of ions and other water-soluble across epithelia is an energy-dependent process. The administration of N-formyl-hydroxyaminoacetic acid (Hadacidin), an analog of aspartate and a competitive inhibitor of adenylosuccinate synthetase, has been shown to inhibit the multiplication of clone 4 MDCK cells and concomitantly reduce the levels of ATP and cAMP (J. Cell. Physiol. 140, 186-194 (1989)). When added to mitotically quiescent confluent cultures of clone 4 MDCK cells, millimolar concentrations of Hadacidin inhibited the generation of transepithelial electrical resistance (TER). In such cultures passive Na+ permeability was similar to controls indicating that the effect of Hadacidin was not on the transcellular pathway. That these cells were viable was demonstrated by their ability to exclude Trypan Blue, and the fact that they remained competent to develop steady state TER upon removal of the inhibitor. Suppression of TER was completely reversed within 48 h of replacing the Hadacidin-supplemented medium with one containing aspartate. Adenosine, but not aspartate, when added simultaneously with the drug, obviated the latter's effect on TER. A mixture of dibutyryl cAMP (db-cAMP) and theophylline was only partially effective in overcoming the effects of Hadacidin on the development of TER and, in fact, markedly delayed its development in control cultures not treated with the drug. When monolayers with established steady state TER were exposed to Hadacidin, no change was noted during the first 24 h. By 48 h, however, TER had decreased to very low values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apolipoprotein AI could be a significant determinant of epithelial integrity in rainbow trout gill cell cultures: a study in functional proteomics

The freshwater fish gill forms a barrier against an external hypotonic environment. By culturing rainbow trout gill cells on permeable supports, as intact epithelia, this study investigates barrier property mechanisms. Under symmetrical conditions the apical and basolateral epithelial surfaces contact cell culture media. Replacing apical media with water, to generate asymmetrical conditions (i.e. the situation encountered by the freshwater gill), rapidly increases transepithelial resistance (TER). Proteomic analysis revealed that this is associated with enhanced expression of pre-apolipoprotein AI (pre-apoAI). To test the physiological relevance, gill cells were treated with a dose of 50 microg ml(-1) human apolipoprotein (apoAI). This was found to elevate TER in those epithelia which displayed a lower TER prior to apoAI treatment. These results demonstrate the action of apoAI and provide evidence that the rainbow trout gill may be a site of apoAI synthesis. TER does not differentiate between the trans-cellular (via the cell membrane) and para-cellular (via intercellular tight junctions) pathways. However, despite the apoAI-induced changes in TER, para-cellular permeability (measured by polyethylene glycol efflux) remained unaltered suggesting apoAI specifically reduces trans-cellular permeability. This investigation combines proteomics with functional measurements to show how a proteome change may be associated with freshwater gill function.     

Surface-layer protein extracts from Lactobacillus helveticus inhibit enterohaemorrhagic Escherichia coli O157:H7 adhesion to epithelial cells

Adherence of intestinal pathogens, including Escherichia coli O157:H7, to human intestinal epithelial cells is a key step in pathogenesis. Probiotic bacteria, including Lactobacillus helveticus R0052 inhibit the adhesion of E. coli O157:H7 to epithelial cells, a process which may be related to specific components of the bacterial surface. Surface-layer proteins (Slps) are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. However, the ability of S-layer protein extract derived from probiotic bacteria to block adherence of enteric pathogens has not been investigated. Human epithelial (HEp-2 and T84) cells were treated with S-layer protein extract alone, infected with E. coli O157:H7, or pretreated with S-layer protein extract prior to infection to determine their importance in the inhibition of pathogen adherence. The effects of S-layer protein extracts were characterized by phase-contrast and immunofluorescence microscopy and measurement of the transepithelial electrical resistance of polarized monolayers. Pre-treatment of host epithelial cells with S-layer protein extracts prior to E. coli O157:H7 infection decreased pathogen adherence and attaching-effacing lesions in addition to preserving the barrier function of monolayers. These in vitro studies indicate that a non-viable constituent derived from a probiotic strain may prove effective in interrupting the infectious process of an intestinal pathogen.     

Characterisation of Escherichia coli strains involved in transcytosis across gut epithelial cells exposed to metabolic and inflammatory stress

Translocation of normally non-pathogenic bacteria across the gut may drive inflammatory responses associated with sepsis and inflammatory bowel disease. Recent evidence suggests translocation may not be purely passive, but occurs via novel transcellular pathways activated in enterocytes by inflammatory and metabolic stress. The specificity of this pathway with respect to different E. coli strains and other bacterial species, and possible molecular determinants of the "translocating" phenotype have been investigated. Translocation of E. coli strains and other bacteria was studied across Caco-2 monolayers exposed to different forms of cellular stress. All bacteria, apart from the pathogen Shigella sonnei, exhibited low levels of translocation in untreated monolayers. However, following enterocyte stress, translocation of E. coli strains C25 and HBTEC-1 was markedly stimulated, accompanied by increased internalisation into enterocytes. C25 and HBTEC-1 were typed to ECOR group A and group D respectively. Pathoarray analysis showed both strains had profiles quite different to those predicted for typical ExPEC isolates, lacking many of the genes associated with pathogenicity, although they contained several ORFs in common with ExPEC isolates. These data suggest translocating E. coli strains associated with infections are not opportunistic ExPEC strains but may comprise a separate group of E. coli strains.     

Differential effects of COX inhibitors against beta-amyloid-induced neurotoxicity in human neuroblastoma cells

Retrospective epidemiological studies have suggested that chronic treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) provides some degree of protection from Alzheimer's disease (AD). Although most NSAIDs inhibit the activity of cyclooxygenase (COX), the rate-limiting enzyme in the production of prostanoids from arachidonic acid (AA), the precise mechanism through which NSAIDs act upon AD pathology remains to be elucidated. Classical NSAIDs like indomethacin inhibit both the constitutive COX-1 and the inducible COX-2 enzymes. In the present work, we characterize the protective effect of the indomethacin on the neurotoxicity elicited by amyloid-β protein (Aβ, fragments 25–35 and 1–42) alone or in combination with AA added exogenously as well as its effects on COX-2 expression. We also compared the neuroprotective effects of indomethacin with the selective COX-1, COX-2 and 5-LOX inhibitors, SC-560, NS-398 and NDGA, respectively. Our results show that indomethacin protected from Aβ and AA toxicity in naive and differentiated human neuroblastoma cells with more potency than SC-560 while, NS-398 only protected neurons from AA-mediated toxicity. Present results suggest that Aβ toxicity can be reversed more efficiently by the non-selective COX inhibitor indomethacin suggesting its role in modulating the signal transduction pathway involved in the mechanism of Aβ neurotoxicity.     

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.     

Anti-inflammatory effect of two isoforms of COX in H. pylori-induced gastritis in mice: possible involvement of PGE2

Neutrophil infiltration mediated by TNF-alpha is associated with various types of gastric injury, whereas PGs play a crucial role in gastric defense. We examined roles of two isoforms of cyclooxygenase (COX) and PGE2 in Helicobacter pylori-induced gastritis in mice. Mice infected with H. pylori were given selective COX-1 inhibitor SC-560 (10 mg/kg), selective COX-2 inhibitor NS-398 (10 mg/kg), or nonselective COX inhibitor indomethacin (2 mg/kg) with or without 16,16-dimethyl PGE2 for 1 wk. H. pylori infection increased levels of mRNA for COX-1 and -2 in gastric tissue by 1.2-fold and 3.3-fold, respectively, accompanied by a significant increase in PGE2 production by gastric tissue. H. pylori infection significantly elevated MPO activity, a marker of neutrophil infiltration, and epithelial cell apoptosis in the stomach. SC-560 augmented MPO activity and epithelial cell apoptosis with associated reduction in PGE2 production, whereas NS-398 had the same effects without affecting PGE2 production. Inhibition of both COX-1 and -2 by indomethacin or concurrent treatment with SC-560 and NS-398 resulted in a stronger increase in MPO activity and apoptosis than inhibition of either COX-1 or -2 alone. H. pylori infection elevated TNF-alpha mRNA expression in the stomach, which was further increased by indomethacin. Effects of COX inhibitors on neutrophil infiltration, apoptosis, and TNF-alpha expression in H. pylori-infected mice were abolished by exogenous 16,16-dimethyl PGE2. In conclusion, PGE2 derived from either COX-1 or -2 is involved in regulation of gastric mucosal inflammation and contributes to maintenance of mucosal integrity during H. pylori infection via inhibition of TNF-alpha expression.