Model Of Intestinal Epithelial Barrier Research Articles

Attenuated replication and damaging effects of SARS-CoV-2 Omicron variants in an intestinal epithelial barrier model.

Many COVID-19 patients suffer from gastrointestinal symptoms and impaired intestinal barrier function is thought to play a key role in Long COVID. Despite its importance, the impact ofsevere acute respiratory syndrome coronavirus 2(SARS-CoV-2) on intestinal epithelia is poorly understood. To address this, we established an intestinal barrier model integrating epithelial Caco-2 cells, mucus-secreting HT29 cells and Raji cells. This gut epithelial model allows efficient differentiation of Caco-2 cells into microfold-like cells, faithfully mimics intestinal barrier function, and is highly permissive to SARS-CoV-2 infection. Early strains of SARS-CoV-2 and the Delta variant replicated with high efficiency, severely disrupted barrier function, and depleted tight junction proteins, such as claudin-1, occludin, and ZO-1. In comparison, Omicron subvariants also depleted ZO-1 from tight junctions but had fewer damaging effects on mucosal integrity and barrier function. Remdesivir, the fusion inhibitor EK1 and the transmembrane serine protease 2 inhibitor Camostat inhibited SARS-CoV-2 replication and thus epithelial barrier damage, while the Cathepsin inhibitor E64d was ineffective. Our results support that SARS-CoV-2 disrupts intestinal barrier function but further suggest that circulating Omicron variants are less damaging than earlier viral strains.

Availability and effects of n-TiO2 and PCB77 in fish in vitro models of the intestinal barrier and liver under single- and/or co-exposure scenarios

Titanium dioxide nanoparticles (n-TiO2) and polychlorinated biphenyls (PCBs) can be present in the food of fish, leading to intestinal exposure uptake, and accumulation in inner organs. This study examined combination effects of n-TiO2 and PCB77 in vitro models of the fish intestinal epithelium and liver, i.e., RTgut-GC cell cultures grown in ThinCerts™ and RTL-W1 cell cultures grown in standard tissue culture plates. Mass spectrometry and microscopy techniques were used to obtain information on nanoparticle translocation across the intestinal barrier model. In addition, the substances’ effect on intestinal barrier permeability, cell viability, expression of dioxin – and antioxidant response element -controlled genes, and induction of cytochrome P450 1a (Cyp1a)-dependent ethoxyresorufin-O-deethylase (EROD) activity were assessed. TiO2 nanoparticles were taken up by RTgut-GC cells and detected in the bottom compartment of the intestinal epithelial barrier model. It was not possible to conclude definitively if n-TiO2 translocation occurred via transcytosis or paracellular migration but observations of nanoparticles in the lateral space between adjacent epithelial cells were rare. PCB77 (1 and 10 µM, 24 h) did not affect barrier permeability, i.e., n-TiO2 translocation is probably not facilitated in case of co-exposure. Furthermore, previous and simultaneous exposure to n-TiO2 (1 and 10 mg/L, 24 h) did not have any influence on PCB77-induced Cyp1a mRNA and enzyme activity levels in RTL-W1 cells. Furthermore, there were no significant differences in expression of antioxidant response element-controlled genes comparing control, single substance, and mixture treatments, not even following long-term exposure (0.01–1 mg/L n-TiO2 + 1 nM PCB77, 4 weeks). While an underestimation of the effects of n-TiO2 and PCB77 cannot be fully excluded as concentration losses due to sorption to cell culture plastics were not measured, the results suggest that the test substances probably have a low potential to exhibit combination effects on the assessed endpoints when co-existing in fish tissues.

Invasiveness of Escherichia coli Is Associated with an IncFII Plasmid.

Escherichia coli is one of the most prevalent pathogens, causing a variety of infections including bloodstream infections. At the same time, it can be found as a commensal, being part of the intestinal microflora. While it is widely accepted that pathogenic strains can evolve from colonizing E. coli strains, the evolutionary route facilitating the commensal-to-pathogen transition is complex and remains not fully understood. Identification of the underlying mechanisms and genetic changes remains challenging. To investigate the factors involved in the transition from intestinal commensal to invasive E. coli causing bloodstream infections, we compared E. coli isolated from blood culture to isolates from the rectal flora of the same individuals by whole genome sequencing to identify clonally related strains and potentially relevant virulence factors. in vitro invasion assays using a Caco- 2 cell intestinal epithelial barrier model and a gut organoid model were performed to compare clonally related E. coli. The experiments revealed a correlation between the presence of an IncFII plasmid carrying hha and the degree of invasiveness. In summary, we provide evidence for the role of an IncFII plasmid in the transition of colonization to invasion in clinical E. coli isolates.

Physico-Chemical Properties of Inorganic NPs Influence the Absorption Rate of Aquatic Mosses Reducing Cytotoxicity on Intestinal Epithelial Barrier Model.

Noble metals nanoparticles (NPs) and metal oxide NPs are widely used in different fields of application and commercial products, exposing living organisms to their potential adverse effects. Recent evidences suggest their presence in the aquifers water and consequently in drinking water. In this work, we have carefully synthesized four types of NPs, namely, silver and gold NPs (Ag NPs and Au NPs) and silica and titanium dioxide NPs (SiO2 NPs and TiO2 NPs) having a similar size and negatively charged surfaces. The synthesis of Ag NPs and Au NPs was carried out by colloidal route using silver nitrate (AgNO3) and tetrachloroauric (III) acid (HAuCl4) while SiO2 NPs and TiO2 NPs were achieved by ternary microemulsion and sol-gel routes, respectively. Once the characterization of NPs was carried out in order to assess their physico-chemical properties, their impact on living cells was studied. We used the human colorectal adenocarcinoma cells (Caco-2), known as the best representative intestinal epithelial barrier model to understand the effects triggered by NPs through ingestion. Then, we moved to explore how water contamination caused by NPs can be lowered by the ability of three species of aquatic moss, namely, Leptodictyum riparium, Vesicularia ferriei, and Taxiphyllum barbieri, to absorb them. The experiments were conducted using two concentrations of NPs (100 μM and 500 Μm as metal content) and two time points (24 h and 48 h), showing a capture rate dependent on the moss species and NPs type. Then, the selected moss species, able to actively capture NPs, appear as a powerful tool capable to purify water from nanostructured materials, and then, to reduce the toxicity associated to the ingestion of contaminated drinking water.

Effect of herb-partitioned moxibustion in improving tight junctions of intestinal epithelium in Crohn disease mediated by TNF-α-NF-κB-MLCK pathway

To explore the effect of herb-partitioned moxibustion (HPM) on tight junctions (TJs) of intestinal epithelial cells in Crohn disease (CD) mediated by tumor necrosis factor-α (TNF-α)-nuclear factor kappa B (NF-κB)-myosin-light-chain kinase (MLCK) pathway. Forty-eight male Sprague-Dawley rats were randomly divided into a normal control (NC) group, a model control (MC) group, an HPM group and a mesalazine (MESA) group, with 12 rats in each group. Trinitrobenzene sulfonic acid (TNBS) was administered to establish CD models. When the model was confirmed a success, the HPM group rats were treated with HPM at Tianshu (ST 25) and Qihai (CV 6), while the MESA group rats were given MESA solution by lavage. When the intervention finished, the colonic epithelial tissues were separated, purified and cultured in each group to establish the intestinal epithelial barrier model in vitro, and TNF-α was added (100 ng/mL) in the culture medium and maintained for 24 h to establish an increased epithelial permeability model. Transepithelial electrical resistance (TEER) was used to examine the permeability of the barrier; Western blot was used to observe the expressions of the proteins related to TJs of intestinal epithelial cells mediated by TNF-α-NF-κB-MLCK pathway; immunofluorescence staining was used to observe the expressions and distributions of tight junction proteins in the intestinal epithelium. After TNF-α induction, compared with the MC+TNF-α group, the TEER value increased significantly in the HPM+TNF-α and MESA+TNF-α groups (both P<0.001); the expressions of nuclear factor kappa B (NF-κB) p65, MLCK, myosin light chain (MLC), tumor necrosis factor receptor-associated factor 6 (TRAF6) and receptor interaction protein-1 (RIP1) decreased significantly (P<0.01 or P<0.05), and the expression of zinc finger protein A20 (A20) increased significantly (P<0.01); the expressions of occludin, claudin-1, zonula occludens protein 1 (ZO-1) and F-actin also increased significantly (all P<0.01). Compared with the MESA+TNF-α group, the expressions of MLC, occludin, claudin-1, ZO-1 and F-actin increased significantly in the HPM+TNF-α group (P<0.01 or P<0.05). HPM can protect or repair the damage of intestinal epithelial barrier in CD rats, which may be achieved through modulating the abnormal TJs in intestinal epithelium mediated by TNF-α-NF-κB-MLCK pathway.

Glucagon-like peptide 2 attenuates intestinal mucosal barrier injury through the MLCK/pMLC signaling pathway in a piglet model.

Glucagon-like peptide-2 (GLP-2), an intestinotrophic hormone, has drawn considerable attention worldwide due to its potential to promote intestinal development. We investigated the effects and mechanisms of GLP-2 against lipopolysaccharide (LPS)-induced intestinal inflammation and injury both in vitro and in vivo. Forty healthy piglets weaned at the age of 28 days with similar body weight (BW) were assigned to four in vivo treatments with ten piglets each: (i) nonchallenged control; (ii) LPS-challenged control; (iii) LPS + low dose GLP-2; and (iv) LPS + high dose GLP-2. Piglets were subcutaneously injected with phosphate-buffered saline supplemented with GLP-2 at doses of 0, 0, 2, and 10 nmol/kg BW per day for seven consecutive days. The piglets were challenged with an intraperitoneal injection with 100 μg/kg LPS on day 14 to induce intestinal damage. After that, the gene and protein expression levels of representative tight junction proteins and myosin light-chain kinase (MLCK)/phosphorylated myosin light chain (pMLC), as well as proinflammatory cytokine levels were determined using quantitative reverse transcription polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay methods. A high dose of GLP-2 pretreatment increased intestinal permeability by downregulating and redistributing tight junction proteins (p < .05), for example, zona occluden-1 (ZO-1) and occludin. GLP-2 decreased the transcription of proinflammatory cytokines genes including interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor-α in small intestines (p < .05). GLP-2 prevented the LPS-induced increase in the expression of MLCK dose-dependently and the increase in pMLC levels in the duodenum, jejunum, and ileum. To assess further the protective effect of GLP-2 on LPS-induced intestinal barrier injury after weaning and its possible mechanism, an in vitro intestinal epithelial barrier model was established with IPEC-J2 monolayers and treated with 100 μg/ml LPS with or without 1 × 10-8 mol/L GLP-2 pretreatment. The in vitro analysis included control, LPS, and GLP-2 + LPS treatments. GLP-2 treatment alleviated the destructive effect of LPS on barrier permeability by restoring the expression and ultrastructure of ZO-1 and occludin (p < .05). In addition, GLP-2 reversed the LPS-induced MLCK hyperexpression and pMLC hyperphosphorylation (p < .05). Taken together, our findings revealed a mechanism by which GLP-2 alleviated LPS-challenged intestinal barrier injury and inflammation in weaned piglets and IPEC-J2 cells via the MLCK/pMLC signaling pathway.

Effect of uremic state in intestine through a co-culture in vitro intestinal epithelial model.

The progressive loss of renal function in chronic kidney disease (CKD) leads to the accumulation of uremic toxins. Recent studies related uremic plasma as well dysbiotic gut microbiome to impaired intestinal barrier function, allowing the translocation of microorganisms or by-products from the intestinal lumen to systemic circulation, contributing to systemic inflammation, cardiovascular risk and progression of CKD. Our main goal was to evaluate the impact of different uremic conditions on an improved in vitro intestinal Caco-2/HT29-MTX/Raji B triple co-culture model. For that, the impact of CKD patients' plasma and elevated urea concentration and its by-products on the triple model was assessed. The results showed that uremic conditions did not potentiate the Escherichia coli (E. coli) translocation, although may interfere with the integrity and the permeability of the intestinal barrier. Also, results showed that E. coli translocation was higher in Caco-2 monoculture than in Caco-2/HT29-MTX/Raji B triple model, suggesting that the triple model creates a more effective intestinal barrier. This study allowed to conclude that the uremic state influences the integrity of the intestinal barrier, but this influence could not be directly translated in an increase on the E. coli translocation through the intestinal epithelium, at least in Caco-2/HT29-MTX/Raji B intestinal epithelial barrier model.

Antrodia cinnamomea Confers Obesity Resistance and Restores Intestinal Barrier Integrity in Leptin-deficient Obese Mice.

Obesity is associated with metabolic disorders. Thus, obesity prevention and treatment are essential for health. Antrodia cinnamomea (AC) is a multifunctional medicinal fungus used for the treatment of various diseases and for preventing diet-induced obesity. Leptin deficiency causes over-eating and spontaneous obesity. The concomitant metabolic symptoms are more severe than diet-induced obesity. Here, we used leptin-deficient (ob/ob) mice as an animal model for over-feeding to study the effect of AC on obesity. We fed C57BL/6 mice (WT, ob+/+) and ob/ob mice with AC for four weeks before performing qRT-PCR and immunoblot analysis to elaborate AC-modulated mechanisms. Further, we used Caco-2 cells as a human intestinal epithelial barrier model to examine the effect of AC on intestinal permeability. Our results suggested that AC reduces lipid deposits of the liver and epididymal white adipose tissue (EWAT) by promoting lipid metabolism and inhibiting lipogenesis-associated genes and proteins in ob/ob mice. Moreover, AC effectively repaired intestinal-barrier injury caused by leptin deficiency and enhanced intestinal barrier integrity in Caco-2 cells. Interestingly, AC significantly reduced body weight and EWAT with no compromise on food intake in ob/ob mice. Thus, AC effectively reduced obesity caused by leptin-deficiency and can potentially be used as a nutraceutical for treating obesity.

Effect of amyloid curli fibrils and curli CsgA monomers from Escherichia coli on in vitro model of intestinal epithelial barrier stimulated with cytokines

Amyloid curli fibrils produced by Escherichia coli are well-known virulence factor influencing E. coli adhesion and biofilm formation. However, the impact of curli on intestinal epithelial barrier stimulated with proinflammatory cytokines is unknown. In the study, we examined the effect of curli produced by nonpathogenic E. coli K-12 and wild-type E. coli EC32 strains, and purified CsgA proteins on differentiated Caco-2 cell monolayers stimulated with a mixture of IL-1β, TNF-α, and INFγ cytokines as a model of ‘inflamed intestinal epithelial barrier’ in vitro. The results of the study indicated that curliated E. coli adhered better to polarized Caco-2 cells than their curli-deficient mutants and the adherence was further augmented by stimulation of epithelial cells with proinflammatory cytokines. Interestingly, curli reduced internalization but enhanced intracellular survival of the wild-type E. coli strain EC32 within intestinal epithelial cells. Curli-expressing E. coli, as well as purified CsgA proteins, attenuated IL-8 secretion by unstimulated Caco-2 cells, although the effect was barely observed on cytokine-stimulated cells. The findings of the study revealed that curli fibrils are an important virulence factor enabling curliated E. coli to effectively colonize intestinal epithelium especially in individuals with inflammatory intestinal disorders.

Novel polyurethane-based nanoparticles of infliximab to reduce inflammation in an in-vitro intestinal epithelial barrier model.

In this study we examined the potential of novel biodegradable polymers of polyesterurethane (PU), and its PEGylated (PU-PEG) form as nanocarriers of Infliximab (INF), to treat inflammation in an in-vitro epithelial model. Nanoparticles (NPs) formulated were of average size of 200-287 nm. INF loading of NPs (INF-NPs) resulted in an increase in size and zeta potential. No cytotoxicity was observed for any of the NPs. Cellular interaction and uptake of PU NPs were similar compared with polycaprolactone (PCL) NPs and significantly higher to Poly(lactic-co-glycolic) acid (PLGA) NPs. Cellular interaction was higher for corresponding PEG-NPs. INF-PU and INF-PU-PEG NPs showed a rapid rate and extent of recovery of the epithelial barrier function in inflamed Caco-2 cell monolayers and decreased cytokine levels in inflamed monocytes. Results obtained in this study are promising and the potential of PU and PU-PEG NPs for drug delivery and targeting to treat gastrointestinal inflammation warrants further investigation.

Inflammation induced ER stress affects absorptive intestinal epithelial cells function and integrity

Recent studies have linked impairment of intestinal epithelial function in inflammatory bowel disease to the disturbance of endoplasmic reticulum homeostasis (ER) in response to stress. Most studies are on goblet and Paneth cells, which are considered more susceptible to stress due to their role in the protection of intestinal epithelium against microbes and harmful substances. However, studies on the role of inflammation-induced ER stress in absorptive intestinal cells are scarce. In this study, we show, using Caco-2 cells as a model of intestinal epithelial barrier, that inducing ER stress using a cocktail mixture of pro-inflammatory mediators [TNFα (50ng/ml), MCP1 (50ng/ml), and IL-1β (25ng/ml)] as observed in IBD patients induces ER stress and leads to significant changes in key proteins of the apical (sucrase-isomaltase (SI), dipeptidyl-peptidase (DPPIV), and ezrin) and basolateral (E-cadherin, zonula occludens (ZO-1), and connexin-43) membranes. Aberrant trafficking of SI, DPPIV was observed as early as 8h post-inflammation-induced ER stress and even in the absence of loss of intestinal cell integrity. The observed effect was associated with a re-localization of ezrin, ZO-1, and connexin-43, key differentiation and junction proteins. Collectively, this study shows that disruption of the trafficking of key digestive enzymes of the intestinal epithelium occur in response to inflammation induced ER stress before the loss of monolayer integrity.

Proteomic analysis and bioluminescent reporter gene assays to investigate effects of simulated microgravity on Caco-2 cells.

Microgravity is one of the most important features in spaceflight. Previous evidence from in-vitro studies has shown that significant changes occur under simulated microgravity. For this reason, human colon adenocarcinoma Caco-2 cells were selected as cell model of intestinal epithelial barrier and their response to altered gravity conditions was investigated, especially on the protein level. In this study, we combined label-free shotgun proteomics and bioluminescent reporter gene assays to identify key proteins and pathways involved in the response of Caco-2 cells under reference and microgravity conditions. A two-dimensional clinostat was modified with 3D-printed adaptors to hold conventional T25 culture flasks. The comparative proteome analysis led to identify 38 and 26 proteins differently regulated by simulated microgravity after 48 and 72 h, respectively. Substantial fractions of these proteins are involved in regulation, cellular and metabolic processes and localization. Bioluminescent reporter gene assays were carried out to investigate microgavity-induced alterations on the transcriptional regulation of key targets, such as NF-kB pathway and CYP27A1. While no significant difference was found in the basal transcription, a lower NF-kB basal activation in simulated microgravity conditions was reported, corroborating the hypothesis of reduced immunity in microgravity conditions.

Primary human polarized small intestinal epithelial barriers respond differently to a hazardous and an innocuous protein

An experimental platform employing human derived intestinal epithelial cell (IEC) line monolayers grown on permeable Transwell® filters was previously investigated to differentiate between hazardous and innocuous proteins. This approach was effective at distinguishing these types of proteins and perturbation of monolayer integrity, particularly transepithelial electrical resistance (TEER), was the most sensitive indicator. In the current report, in vitro indicators of monolayer integrity, cytotoxicity, and inflammation were evaluated using primary (non-transformed) human polarized small intestinal epithelial barriers cultured on Transwell® filters to compare effects of a hazardous protein (Clostridium difficile Toxin A [ToxA]) and an innocuous protein (bovine serum albumin [BSA]). ToxA exerted a reproducible decrease on barrier integrity at doses comparable to those producing effects observed from cell line-derived IEC monolayers, with TEER being the most sensitive indicator. In contrast, BSA, tested at concentrations substantially higher than ToxA, did not cause changes in any of the tested variables. These results demonstrate a similarity in response to certain proteins between cell line-derived polarized IEC models and a primary human polarized small intestinal epithelial barrier model, thereby reinforcing the potential usefulness of cell line-derived polarized IECs as a valid experimental platform to differentiate between hazardous and non-hazardous proteins.

Establishment of Caco-2 cell monolayer model and barrier permeability

Objective To establish and evaluate intestinal epithelial barrier model using Caco-2 cell so as to play a foundation for next study of barrier permeability. Methods Caco-2 cells were cultured in vitro then seeded into Transwell cell culture inserts.The permeability of the intestinal epithelial barrier was detected by transepithelial electrical resistance(TEER)and lucifer yellow flux, and verified by transmission electron microscope.Different concentrations of PAF(0, 50, 100, and 200 nmol/L)were exposed for 24 hours to Caco-2 monolayer when cultured 21 days.The tight junction was observed under transmission electron microscope.Assessment of ZO-1 protein localization and expression were detected by immunofluorescence and Western blot analysis. Results Cultured Caco-2 cell confluencd as monolayer with time passed.From 5th day, TEER increased, then reached 600Ω·cm2 at 15th day and lasted to 21st day, there was little flux of lucifer yellow, transmission electron microscopy also found cells differentiated better, had well-arranged villi and polarity alined as monolayer, forming completed tight junction which was the marker of intestinal epithelial barrier model in vitro.TEER decreased and lucifer yellow flux increased in cells exposed to PAF.The permeability reached the peak when exposed to 100 nmol/L PAF(P< 0.01), tight junction disrupted, ZO-1 protein expression downregulated, abnormal localization and distribution was assessed by immunofluorescence staining. Conclusion Cultured Caco-2 cells for 2-3w can be used to study intestinal epithelial barrier as a model in vitro.PAF increased intestinal epithelial permeability, which would correlate to the decreased protein expression and abnormal distribution of ZO-1. Key words: Human colon carcinoma cell line(Caco-2); Permeability; Transepithelial electrical resistance; Platelet-activating factor(PAF); ZO-1

A TEM protocol for quality assurance of in vitro cellular barrier models and its application to the assessment of nanoparticle transport mechanisms across barriers.

We report here a protocol to characterise and monitor the quality of in vitro human cellular barrier models using Transmission Electron Microscopy (TEM), which can be applied for transport assays, mechanistic studies and screening of drug/compound (including nanoparticle) penetration across such biological barriers. Data from two examples of biological barriers are given, namely the hCMEC/D3 endothelial blood-brain barrier model, and the Caco-2 intestinal epithelial barrier model, to show the general applicability of the method. Several aspects of this method are applicable to the quality assurance of in vitro barrier models, e.g., assessment of the multi or mono-layer structure of the endothelial cells; identification of any potential "holes" in the barrier that could confound transport assay results; validation of tight junction expression; and determination of the types and amounts of key cellular organelles present in the barrier to account for any significant changes in phenotype that may occur compared to the in vivo situation. The method described here provides a key advantage in that it prevents loss of the filter membrane during monolayer sectioning, thereby preserving critical details associated with the basal cell membrane. Applicability of the protocol for other in vitro biological barriers, such as the blood-foetus, blood-testes, blood-cerebrospinal fluid (CSF) and lung alveolar-capillary barriers is also discussed. Additionally, we demonstrate the use of the method for assessment of nanoparticle transport across cellular barriers and elucidation of transcytosis mechanisms. Sequential events of cellular endocytosis, localisation and transcytosis can be described in detail by TEM imaging, revealing useful sub-cellular details that provide evidence for the mechanism of nanoparticle transport in the hCMEC/D3 blood-brain barrier model and the Caco-2 intestinal epithelial cell model. Potential artefacts resulting from the nanoparticles interacting with the Transwell membranes can also be assessed.

FRET-based dual-emission and pH-responsive nanocarriers for enhanced delivery of protein across intestinal epithelial cell barrier.

The oral route is a convenient and commonly employed way for drug delivery. However, therapeutic proteins have poor bioavailability upon oral administration due to the impermeable barrier from intestinal epithelial tight junction (TJ). Moreover, the pH of the small intestine varies among different regions of the intestinal tract where digestion and absorption occur at different levels. In this study, a tunable dual-emitting and pH-responsive nanocarrier that can alter the fluorescent color and emission intensity in response to pH changes and can trigger the opening of intestinal epithelial TJ at different levels were developed from chitosan-N-arginine and poly(γ-glutamic acid)-taurine conjugates. As pH increased from 6.0 to 8.0, the binding affinity of the oppositely charged polyions decreased, whereas the ratio of the intensity of the donor-to-acceptor emission intensity (ID/IA) increased by 27-fold. The fluorescent and pH-responsive nanocarrier was able to monitor the pH change of intestinal environment and to control the release of an anti-angiogenic protein in response to the pH gradient. The nanocarrier triggered the opening of intestinal epithelial TJ and consequently enhanced the permeation of the released protein through the intestinal epithelial barrier model (Caco-2 cell monolayer) to inhibit tube formation of human umbilical vein endothelial cells.

Effects of Ethanol and Oxidized Metabolites of Linoleic Acid on Caco‐2 cell Model of Intestinal Epithelial Barrier

IntroductionThe intestinal epithelial barrier plays a critical role in host defense against luminal pathogens. Ethanol (EtOH) and dietary components may alter barrier integrity resulting in increased intestinal permeability, endotoxemia and multi‐organ pathology. The aim of the present study was to evaluate the effects of EtOH, linoleic acid (LA, a major unsaturated fatty acid in the Western diet), and its bioactive oxidized metabolites (OXLAMs) on Caco‐2 cells, an in vitro model of intestinal epithelial barrier.Materials and MethodsCaco‐2 cell monolayers were exposed to LA, OXLAMs (9‐and 13‐hydroxyoctadecadienoic acids, HODEs) and/or EtOH. Transepithelial resistance (TEER), expression of intestinal tight junction (TJ) proteins, and pro‐inflammatory response were evaluated.ResultsDecreased TEER in response to EtOH, LA, and multiple HODE isoforms was observed after 24 hours of exposure. Combined effects of EtOH and 13(S)HODE or +13HODE on TEER were greater compared to EtOH and 9(S)HODE or +9HODE. The combination of EtOH and 13(S)HODE or +13HODE resulted in greater down‐regulation of TJ proteins and increased expression of TNF‐α, IL‐1β, and IL‐6 mRNA compared to EtOH alone.ConclusionCombined exposure of EtOH and OXLAMs exacerbates disruption of intestinal barrier integrity and pro‐inflammatory response in Caco‐2 cells.

Clinical Saccharomyces cerevisiae isolates cannot cross the epithelial barrier in vitro

Saccharomyces cerevisiae is generally considered to be a safe organism and is essential to produce many different kinds of foods as well as being widely used as a dietary supplement. However, several isolates, which are genetically related to brewing and baking yeasts, have shown virulent traits, being able to produce human infections in immunodeficient patients. Previously it has been shown that the administration of S. cerevisiae clinical isolates can lead to systemic infections, reaching several organs in murine systems. In this work, we studied S. cerevisiae clinical isolates in an in vitro intestinal epithelial barrier model, comparing their behaviour with that of several strains of the related pathogens Candida glabrata and Candida albicans. The results showed that, in contrast to C. glabrata and C. albicans, S. cerevisiae was not able to cross the intestinal barrier. We concluded that S. cerevisiae can only perform opportunistic or passive crossings when epithelial barrier integrity is previously compromised.

Tumor necrosis factor-α increases the paracellular permeability of Caco-2 cell monolayersin vitro

AIM: To investigate the effects of tumor necrosis factor-α (TNF-α) on the paracellular permeability of Caco-2 cell monolayers and to explore potential mechanisms involved. METHODS: Caco-2 cells were cultured in vitro to establish a model of intestinal epithelial barrier and divided randomly into two groups: control group and TNF-α treatment group. The TNF-α treatment group was further divided into four subgroups for testing at 3, 6, 12 and 24 h after TNF-α treatment. The changes in transepithelial electrical resistance (TEER) of Caco-2 cells were measured using an electrical resistance system. The changes in cytoskeleton were observed by direct staining with rhodamine-phalloidin. The activation of nuclear factor-κB (NF-κB) was determined by luciferase reporter gene assay. RESULTS: The TEER significantly decreased in all four treatment subgroups compared to the control group (all P＜0.05). The TEER was significantly lower in the 24-h treatment subgroup than in other subgroups (all P＜0.05). The activation level of NF-κB was significantly raised in 3-, 6- and 12-h treatment subgroups compared to the control group (all P＜0.05). The activation level of NF-κB in 12-h treatment subgroup was significantly higher than those in other subgroups (all P＜0.05). In the control group, rhodamine-phalloidin staining showed that F-actin was visualized around the cell membrane and exhibited a honeycomb pattern of fluorescent staining. After treatment with TNF-α, the fluorescence signals were weakened and distributed in a serrated pattern. CONCLUSION: TNF-α increases the paracellular permeability of Caco-2 cell monolayers perhaps by inducing NF-κB activation and actin reorganization.

Metabolism and transfer of the mycotoxin zearalenone in human intestinal Caco-2 cells

The mycotoxin zearalenone (ZEA) is found worldwide as contaminant in cereals and grains. It is implicated in reproductive disorders and hyperestrogenic syndromes in animals and humans exposed by food. We investigated metabolism and transfer of ZEA using the human Caco-2 cell line as a model of intestinal epithelial barrier. Cells exposed to 10–200 μM ZEA showed efficacious metabolism of the toxin. α-zearalenol and β-zearalenol were the measured preponderant metabolites (respectively 40.7 ± 3.1% and 31.9 ± 4.9% of total metabolites, after a 3 h exposure to 10 μM ZEA), whereas ZEA-glucuronide and α-zearalenol glucuronide were less produced (respectively 8.2 ± 0.9% and 19.1 ± 1.3% of total metabolites, after a 3 h exposure to 10 μM ZEA). Cell production of reduced metabolites was strongly inhibited by α-and β-hydroxysteroid dehydrogenase inhibitors, and Caco-2 cells exhibited α-hydroxysteroid dehydrogenase type II and β-hydroxysteroid dehydrogenase type I mRNA. After cell apical exposure to ZEA, α-zearalenol was preponderantly found at the basal side, whereas β-zearalenol and both glucuronides were preferentially excreted at the apical side. As α-zearalenol shows the strongest estrogenic activity, the preferential production and basal transfer of this metabolite suggests that intestinal cells may contribute to the manifestation of zearalenone adverse effects.