Listeria Adhesion Protein Induces Intestinal Epithelial Barrier Dysfunction for Bacterial Translocation

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Protective Effects of Carbon Monoxide-Releasing Molecule-2 on the Barrier Function of Intestinal Epithelial Cells

Objective

To investigate the protective effects and mechanisms of carbon monoxide-releasing molecule-2 (CORM-2) on barrier function of intestinal epithelial cells.

Materials and Methods

After pre-incubation with CORM-2 for 1 hour, cultured intestinal epithelial IEC-6 cells were stimulated with 50 µg/ml lipopolysaccharides (LPS). Cytokines levels in culture medium were detected using ELISA kits. Trans-epithelial electrical resistance (TER) of IEC-6 cell monolayers in Transwells were measured with a Millipore electric resistance system (ERS-2; Millipore) and calculated as Ω/cm² at different time points after LPS treatment. The permeability changes were also measured using FITC-dextran. The levels of tight junction (TJ) proteins (occludin and ZO-1) and myosin light chain (MLC) phosphorylation were detected using Western blotting with specific antibodies. The subsequent structural changes of TJ were visualized using transmission electron microscopy (TEM).

Results

CORM-2 significantly reduced LPS-induced secretion of TNF-α and IL-1β. The LPS-induced decrease of TER and increase of permeability to FITC-dextran were inhibited by CORM-2 in a concentration dependent manner (P<0.05). LPS-induced reduction of tight junction proteins and increase of MLC phosphorylation were also attenuated. In LPS-treated cells, TEM showed diminished electron-dense material and interruption of TJ and desmosomes between the apical lateral margins of adjoining cells, which were prevented by CORM-2 treatment.

Conclusions

The present study demonstrates that CORM-2, as a novel CO-releasing molecule, has ability to protect the barrier function of LPS-stimulated intestinal epithelial cells. Inhibition of inflammatory cytokines release, restoration of TJ proteins and suppression of MLC phosphorylation are among the protective effects of CORM-2.     

Effects of Kainic Acid on Brain Calcium Fluxes Studied In Vivo and In Vitro

The effect of in vivo administration of kainic acid into the rabbit hippocampus was studied with brain dialysis and subsequent determination of the Ca2+ concentration in the dialysate. When included in the perfusing medium, kainic acid as well as veratridine induced a decrease in extracellular Ca2+. The effect of kainic acid (but not of veratridine) was insensitive to tetrodotoxin. In vitro studies revealed no effect of kainic acid on 45Ca2+ uptake by isolated astrocytes, but showed an enhancement of synaptosomal 45Ca2+ accumulation. This was, however, only 25% of the stimulatory effect of high K+ depolarization. Glutamate activated synaptosomal Ca2+ uptake, whereas dihydrokainate had no effect. The uptake evoked by kainate and glutamate was independent of the K+ level in the medium which indicates the involvement of other than voltage-sensitive Ca2+ channels. The results confirm previous finding that kainic acid promotes the uptake of Ca2+ in brain cells. Kainate affects Ca2+ fluxes pre- and postsynaptically. Presynaptic Ca2+ influx may be mediated by chemically gated mechanisms.     

Effects of Sertraline and Fluoxetine on P-Glycoprotein at Barrier Sites: In Vivo and In Vitro Approaches

Background and Purpose

Retention of substances from systemic circulation in the brain and testes are limited due to high levels of P-glycoprotein (P-gp) in the luminal membranes of brain and testes capillary endothelial cells. From a clinical perspective, P-gp rapidly extrudes lipophilic therapeutic agents, which then fail to reach efficacious levels. Recent studies have demonstrated that acute administration of selective serotonin reuptake inhibitors (SSRI) can affect P-gp function, in vitro and in vivo. However, little is known concerning the time-course of these effects or the effects of different SSRI in vivo.

Experimental Approach

The P-gp substrate, tritiated digoxin ([³H] digoxin), was co-administered with fluoxetine or sertraline to determine if either compound increased drug accumulation within the brains and testes of mice due to inhibition of P-gp activity. We undertook parallel studies in endothelial cells derived from brain microvessels to determine the dose-response and time-course of effects.

Key Results

In vitro, sertraline resulted in rapid and potent inhibition of P-gp function in brain endothelial cells, as determined by cellular calcein accumulation. In vivo, a biphasic effect was demonstrated. Brain accumulation of [³H] digoxin was increased 5 minutes after treatment with sertraline, but by 60 minutes after sertraline treatment, brain accumulation of digoxin was reduced compared to control. By 240 minutes after sertraline treatment brain digoxin accumulation was elevated compared to control. A similar pattern of results was obtained in the testes. There was no significant effect of fluoxetine on P-gp function, in vitro or in vivo.

Conclusions and Implications

Acute sertraline administration can modulate P-gp activity in the blood-brain barrier and blood-testes barrier. This clearly has implications for the ability of therapeutic agents that are P-gp substrates, to enter the brain when co-administered with SSRI.     

Neurochemical Changes Associated with Stress-Induced Sleep Disturbance in Rats: In Vivo and In Vitro Measurements

The goal of this study was to quantitatively assess the changes in the cerebral neurochemical profile and to identify those factors that contribute to the alteration of endogenous biomolecules when rats are subjected to stress-induced sleep disturbance. We exposed Sprague-Dawley rats (controls: n = 9; stress-induced sleep perturbation rats: n = 11) to a psychological stressor (cage exchange method) to achieve stress-induced sleep perturbation. In vivo magnetic resonance imaging assessments were carried out using a high-resolution 9.4 T system. For in vivo neurochemical analysis, a single voxel was localized in the right dorsal hippocampal region, and in vivo spectra were quantified for 17 cerebral neurochemical signals. Rats were sacrificed upon completion of the magnetic resonance spectroscopy protocol, and whole-brain tissue was harvested from twenty subjects. The dopamine and serotonin signals were obtained by performing in vitro liquid chromatography-tandem mass spectrometry on the harvested tissue. In the right dorsal hippocampal region, the gamma-aminobutyric-acid (GABA) and glutamine (Gln) concentrations were significantly higher in the sleep-perturbed rats than in the sham controls. The ratios of Gln/Glu (glutamate), Gln/tCr (total-creatine), and GABA/Glu were also significantly higher in the sleep-perturbed group, while serotonin concentrations were significantly lower in the sleep-perturbed rats. Pearson correlation results among individual rat data indicate that concentrations of dopamine (DA) and serotonin (5-HT) were significantly higher in SSP rats. A larger correlation coefficient was also observed for the SSP rats. Analysis of the correlation between the in vivo and in vitro signals indicated that the concentrations of Gln, 5-HT, and DA exhibited a significant negative correlation in the SSP rat data but not in that of control rats. The authors propose that the altered and correlated GABA, Gln, 5-HT, and DA concentrations/ratios could be considered key markers of neurological function in animal models of stress-induced sleep perturbation.     

Protective Effect of Borage Seed Oil and Gamma Linolenic Acid on DNA: In Vivo and In Vitro Studies

Borage (Borago officinalis L.) seed oil has been used as a treatment for various degenerative diseases. Many useful properties of this oil are attributed to its high gamma linolenic acid content (GLA, 18:3 ω-6). The purpose of this study was to demonstrate the safety and suitability of the use of borage seed oil, along with one of its active components, GLA, with respect to DNA integrity, and to establish possible in vivo toxic and in vitro cytotoxic effects. In order to measure these properties, five types of assays were carried out: toxicity, genotoxicity, antigenotoxicity, cytotoxicity (using the promyelocytic leukaemia HL60 cell line), and life span (in vivo analysis using the Drosophila model). Results showed that i) Borage seed oil is not toxic to D. melanogaster at physiological concentrations below 125 µl/ml and the studies on GLA indicated non-toxicity at the lowest concentration analyzed ii) Borage seed oil and GLA are DNA safe (non-genotoxic) and antimutagenic compared to hydrogen peroxide, thereby confirming its antioxidant capacity; iii) Borage seed oil and GLA exhibited cytotoxic activity in low doses (IC₅₀ of 1 µl/ml and 0.087 mM, respectively) iv) Low doses of borage seed oil (0.19%) increased the health span of D. melanogaster; and v) GLA significantly decreased the life span of D. melanogaster.Based on the antimutagenic and cytotoxic effects along with the ability to increase the health span, we propose supplementation with borage seed oil rather than GLA, because it protects DNA by modulating oxidative genetic damage in D. melanogaster, increases the health span and exerts cytotoxic activity towards promyelocytic HL60 cells.     

Caffeic Acid Derivatives: In Vitro and In Vivo Anti-inflammatory Properties

Objective: This study examined whether, daily fruit (blueberries) consumption (250 g) for three weeks or acute fruit ingestion (250 g) would attenuate angiotensin converting enzyme (ACE) activity and reduce oxidative stress in chronic cigarette smokers.

Methods: Twenty subjects were recruited and randomized into fruit or control groups. Blood samples and blood pressure were obtained at baseline and then pre and one hour post when subjects returned to the lab three weeks later. To examine acute effects, the fruit group immediately ingested 250 g of blueberries after returning and at least one hour prior to the post blood draw. Plasma samples were analyzed for ACE activity, F₂- isoprostanes and lipid hydroperoxides (LH) as measures of oxidative stress, and ferric reducing ability of plasma (FRAP) as a measure of antioxidant potential. A 2 (treatment) × 3 (time) repeated measures ANOVA was used for statistical analysis. If interaction was significant, then Student's t-tests were used to further examine this relationship. For these comparisons, a Bonferroni adjustment was made with statistical significance set at P < 0.025.

Results: The pattern of change between treatments was not significant for any variable except LH (P < 0.001).

Conclusion: This study indicates that LH are significantly reduced by daily fruit consumption, but not affected by acute ingestion. This finding could be one way in which fruit consumption contributes to prevention of cardiovascular disease.     

In Vitro and In Vivo Studies on Protective Action of N-Phenethyl Caffeamide against Photodamage of Skin

In our previous study, N-phenethyl caffeamide (K36) was proved to act as an antioxidant and an antiphotoaging agent by inhibiting type I procollagen degradation and stimulating collagen synthesis in human skin fibroblasts. In the present study, in vitro and in vivo experiments were conducted to investigate the mechanism of action and the antiinflammatory and antiphotoaging activity of K36. K36 reduced UVB-induced cyclooxygenase-2 (COX-2) and inducible nitric oxide synthases (iNOS) expression by regulating IκB and p-IκB expression. K36 also inhibited the nuclear translocation of NF-κB. Furthermore, the inhibition of mitogen-activated protein (MAP) kinases by K36 was attributed to the downregulation of COX-2. Topically applying K36 led to efficient antiwrinkle formation and reduced UVB-induced erythema and thickness of epidermis in hairless mice. In addition, K36 penetrated into the skin of hairless mice. Our findings show that K36 has significant beneficial effects on antioxidant, antiinflammatory, and antiphotoaging activity and suggest that K36 can be developed as an antiaging agent for cosmetic and skin care products.     

Mechanism of Reovirus Double-Stranded Ribonucleic Acid Synthesis In Vivo and In Vitro

The complementary strands of reovirus double-stranded ribonucleic acid (ds RNA) are synthesized sequentially in vivo and in vitro. In both cases, preformed plus strands serve as templates for the synthesis of the complementary minus strands. The in vitro synthesis of dsRNA is catalyzed by a large particulate fraction from reovirus-infected cells. Treatment of this fraction with chymotrypsin or with detergents which solubilize cellular membranes does not alter its capacity to synthesize dsRNA. The enzyme or enzymes responsible for dsRNA synthesis remain sedimentable at 10,000 x g after these enzyme or detergent treatments, indicating their particulate nature. Pretreatment of this fraction with ribonuclease, however, abolishes its ability to catalyze dsRNA synthesis, emphasizing the single-stranded nature of the template and its location in a structure permeable to ribonuclease. In contrast, the newly formed dsRNA is resistant to ribonuclease digestion at low salt concentrations and hence is thought to reside within a ribonuclease-impermeable structure.     

Ethanol Impairs Intestinal Barrier Function in Humans through Mitogen Activated Protein Kinase Signaling: A Combined In Vivo and In Vitro Approach

Background

Ethanol-induced gut barrier disruption is associated with several gastrointestinal and liver disorders.

Aim

Since human data on effects of moderate ethanol consumption on intestinal barrier integrity and involved mechanisms are limited, the objectives of this study were to investigate effects of a single moderate ethanol dose on small and large intestinal permeability and to explore the role of mitogen activated protein kinase (MAPK) pathway as a primary signaling mechanism.

Methods

Intestinal permeability was assessed in 12 healthy volunteers after intraduodenal administration of either placebo or 20 g ethanol in a randomised cross-over trial. Localization of the tight junction (TJ) and gene expression, phosphorylation of the MAPK isoforms p38, ERK and JNK as indicative of activation were analyzed in duodenal biopsies. The role of MAPK was further examined in vitro using Caco-2 monolayers.

Results

Ethanol increased small and large intestinal permeability, paralleled by redistribution of ZO-1 and occludin, down-regulation of ZO-1 and up-regulation of myosin light chain kinase (MLCK) mRNA expression, and increased MAPK isoforms phosphorylation. In Caco-2 monolayers, ethanol increased permeability, induced redistribution of the junctional proteins and F-actin, and MAPK and MLCK activation, as indicated by phosphorylation of MAPK isoforms and myosin light chain (MLC), respectively, which could be reversed by pretreatment with either MAPK inhibitors or the anti-oxidant L-cysteine.

Conclusions

Administration of moderate ethanol dosage can increase both small and colon permeability. Furthermore, the data indicate a pivotal role for MAPK and its crosstalk with MLCK in ethanol-induced intestinal barrier disruption.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00928733","term_id":"NCT00928733"}}NCT00928733     

Fatty Acid Ethyl Esters Induce Intestinal Epithelial Barrier Dysfunction via a Reactive Oxygen Species-Dependent Mechanism in a Three-Dimensional Cell Culture Model

Background & Aims

Evidence is accumulating that ethanol and its oxidative metabolite, acetaldehyde, can disrupt intestinal epithelial integrity, an important factor contributing to ethanol-induced liver injury. However, ethanol can also be metabolized non-oxidatively generating phosphatidylethanol and fatty acid ethyl esters (FAEEs). This study aims to investigate the effects of FAEEs on barrier function, and to explore the role of oxidative stress as possible mechanism.

Methods

Epithelial permeability was assessed by paracellular flux of fluorescein isothiocyanate-conjugated dextran using live cell imaging. Cell integrity was evaluated by lactate dehydrogenase release. Localization and protein levels of ZO-1 and occludin were analyzed by immunofluorescence and cell-based ELISA, respectively. Intracellular oxidative stress and cellular ATP levels were measured by dichlorofluorescein and luciferase driven bioluminescence, respectively.

Results

In vitro, ethyl oleate and ethyl palmitate dose dependently increased permeability associated with disruption and decreased ZO-1 and occludin protein levels, respectively, and increased intracellular oxidative stress without compromising cell viability. These effects could partially be attenuated by pretreatment with the antioxidant, resveratrol, pointing to the role of oxidative stress in the FAEEs-induced intestinal barrier dysfunction.

Conclusions

These findings show that FAEEs can induce intestinal barrier dysfunction by disrupting the tight junctions, most likely via reactive oxygen species-dependent mechanism.     

Effects of Lead In Vivo and In Vitro on GABAergic Neurochemistry

Abstract: Alterations in aspects of neurotransmission utilizing -γ-aminobutyric acid (GABA) are associated with in vivo exposure of rats to lead at doses that do not produce convulsions, but sensitize animals to convulsant agents. These effects are observed regionally and include: decreased GABA levels in cerebellum; increased activity of glutamate decarboxylase (GAD) in caudate; and decreased GABA release (both resting and K⁺-stimulated) in cortex, caudate, cerebellum and substantia nigra. Sodium-dependent uptake of GABA by synaptosomes of cerebellum, substantia nigra and caudate was also affected: in these regions, affinity (K_m) was increased and maximal velocity (V_max) was reduced. Sodium-independent binding of GABA to synaptic membranes was increased in cerebellum, but was observed only when tissue was Tritonized and prepared without freezing and washing. No effects on GAD or on GABA uptake, release, or binding were observed when lead was added to brain tissue in vitro in concentrations as high as 100 μM. The results suggest that lead may produce chronic inhibition of presynaptic GABAergic function, notably in the cerebellum, which is associated with supersensitivity of postsynaptic GABA receptors. Failure of lead to affect GABAergic function in vitro may indicate that these effects are secondary to another neurotoxic action of lead in the CNS or are consequent to a nonneuronal metabolic action of lead.     

In Vivo and In Vitro Synthesis of Human Rhinovirus Type 2 Ribonucleic Acid

HeLa cells infected with human rhinovirus type 2 synthesize a mixture of single-and double-stranded ribonucleic acid (RNA). The RNA synthesized by the membrane-bound RNA polymerase complex in vitro is also a mixture of single- and double-stranded RNA, whereas the deoxycholate-treated RNA polymerase complex synthesized only double-stranded RNA. Although twice as much cell-associated viral RNA is synthesized in vivo at 34 C than at 37 C, there is no difference in the rate of RNA synthesized in vitro at 34 C and 37 C by the polymerase complex. The RNA polymerase complex, after treatment with deoxycholate, sediments as a broad peak with an average sedimentation value of 120S.     

Drug Transfer Across the Blood-Brain Barrier: Correlation Between In Vitro and In Vivo Models

To assess the drug transport across the blood-brain barrier (BBB), we compared the maximal brain extraction values at time 0 [E(0) values] obtained using either in vitro or in vivo methods. The in vitro BBB model consisted of a coculture of brain capillary endothelial cells growing on one side of a filter and astrocytes on the other. The in vivo model used intracarotid injection in anesthetized rats. Eleven compounds were tested. They were selected because they exhibit quantitatively different brain extraction rates: very low for inulin and sucrose, low for oxicam-related nonsteroidal antiinflammatory drugs and diclofenac, and high for propranolol and diazepam. As these compounds are apparently transferred by a passive diffusion mechanism, two others, glucose and leucine, were added that cross the BBB by a known carrier-mediated process. The in vivo and in vitro E(0) values showed a strong correlation as indicated by the Spearman's correlation coefficient (r = 0.88, p less than 0.01). The relative ease with which such cocultures can be produced in large quantities could facilitate the screening of new centrally acting drugs.     

A Deafness-Associated Mutant Human Connexin 26 Improves the Epithelial Barrier In Vitro

A large proportion of recessive nonsyndromic hearing loss is due to mutations in the GJB2 gene encoding connexin 26 (Cx26), a component of a gap junction. Within different ethnic groups there are specific common recessive mutations, each with a relatively high carrier frequency, suggesting the possibility of heterozygous advantage. Carriers of the R143W GJB2 allele, the most prevalent in the African population, present with a thicker epidermis than noncarriers. In this study, we show that (R143W)Cx26-expressing keratinocytes form a significantly thicker epidermis in an organotypic coculture skin model. In addition, we show increased migration of cells expressing (R143W)Cx26 compared to (WT)Cx26-overexpressing cells. We also demonstrate that cells expressing (R143W)Cx26 are significantly less susceptible to cellular invasion by the enteric pathogen Shigella flexneri than (WT)Cx26-expressing cells. These in vitro studies suggest an advantageous effect of (R143W)Cx26 in epithelial cells. The first two authors contributed equally to this work.     

High Uric Acid Induces Insulin Resistance in Cardiomyocytes In Vitro and In Vivo

Clinical studies have shown hyperuricemia strongly associated with insulin resistance as well as cardiovascular disease. Direct evidence of how high uric acid (HUA) affects insulin resistance in cardiomyocytes, but the pathological mechanism of HUA associated with cardiovascular disease remains to be clarified. We aimed to examine the effect of HUA on insulin sensitivity in cardiomyocytes and on insulin resistance in hyperuricemic mouse model. We exposed primary cardiomyocytes and a rat cardiomyocyte cell line, H9c2 cardiomyocytes, to HUA, then quantified glucose uptake with a fluorescent glucose analog, 2-NBDG, after insulin challenge and detected reactive oxygen species (ROS) production. Western blot analysis was used to examine the levels of insulin receptor (IR), phosphorylated insulin receptor substrate 1 (IRS1, Ser307) and phospho-Akt (Ser473). We monitored the impact of HUA on insulin resistance, insulin signaling and IR, phospho-IRS1 (Ser307) and phospho-Akt levels in myocardial tissue of an acute hyperuricemia mouse model established by potassium oxonate treatment. HUA inhibited insulin-induced glucose uptake in H9c2 and primary cardiomyocytes. It increased ROS production; pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger, reversed HUA-inhibited glucose uptake induced by insulin. HUA exposure directly increased the phospho-IRS1 (Ser307) response to insulin and inhibited that of phospho-Akt in H9C2 cardiomyocytes, which was blocked by NAC. Furthermore, the acute hyperuricemic mice model showed impaired glucose tolerance and insulin tolerance accompanied by increased phospho-IRS1 (Ser307) and inhibited phospho-Akt response to insulin in myocardial tissues. HUA inhibited insulin signaling and induced insulin resistance in cardiomyocytes in vitro and in vivo, which is a novel potential mechanism of hyperuricemic-related cardiovascular disease.