Transepithelial Electrical Resistance Values Research Articles

P-Glycoprotein-Mediated Efflux Using a Rapidly Maturing Caco2 Clone (CLEFF4) in Only 5 Days without Requiring Modified Growth Medium.

In drug discovery it is essential that one of the parameters tested for any new chemical entity is its affinity for human efflux systems, most notably P-glycoprotein (P-gp). These efflux systems affect not only rates of oral absorption but also rates of excretion through the liver, blood-brain barrier, and accumulation in potential target cells that upregulate efflux systems. Current methods to determine drugs' P-gp transport potential include in vitro bidirectional transport studies, and the two most common cell lines used are Caco2 and MDR1-transfected MDCK models. Caco2 cells are human but slow growing and require more than 3 weeks to mature, while MDCK cells are canine, but when transfected with human P-gp become a rapid model of P-gp affinity. Our laboratory has generated a Caco2 subclone called CLEFF4 that is fully human, yet now approaches the rapid nature of the MDCK model. No special medium is required. We have shown, in as little as 5 days postseeding, high transepithelial electrical resistance values of more than 1000 Ω·cm2 plus P-gp expression more than threefold higher than that of 21-day-old cells. Currently tested drugs included rhodamine 123 (Rh123), vinblastine, and doxorubicin, and all drugs exhibited P-gp-mediated efflux that was inhibited by PSC833. By day 6, bidirectional transport of Rh123 was as potent as that of mature Caco2 cells, for use in comparative P-gp affinity studies. We now have a human P-gp model that is rapid and works without any need for special accelerating medium. We believe this could be a welcome addition to the testing regime of new chemical entities.

Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells.

PurposeTo investigate the role of protein misfolding in retinal pigment epithelial (RPE) cell dysfunction, the effects of R345W-Fibulin-3 expression on RPE cell phenotype were studied.MethodsPrimary RPE cells were cultured to confluence on Transwells and infected with lentivirus constructs to express wild-type (WT)- or R345W-Fibulin-3. Barrier function was assessed by evaluating zonula occludens-1 (ZO-1) distribution and trans-epithelial electrical resistance (TER). Polarized secretion of vascular endothelial growth factor (VEGF), was measured by Enzyme-linked immunosorbent assay (ELISA). Differentiation status was assessed by qPCR of genes known to be preferentially expressed in terminally differentiated RPE cells, and conversion to an epithelial–mesenchymal transition (EMT) phenotype was assessed by a migration assay.ResultsCompared to RPE cells expressing WT-Fibulin-3, ZO-1 distribution was disrupted and TER values were significantly lower in RPE cells expressing R345W-Fibulin-3. In cells expressing mutant Fibulin-3, VEGF secretion was attenuated basally but not in the apical direction, whereas Fibulin-3 secretion was reduced in both the apical and basal directions. Retinal pigment epithelial signature genes were downregulated and multiple genes associated with EMT were upregulated in the mutant group. Migration assays revealed a faster recovery rate in ARPE-19 cells overexpressing R345W-Fibulin-3 compared to WT.ConclusionsThe results suggest that expression of R345W-Fibulin-3 promotes EMT in RPE cells.

Cell barrier characterization in transwell inserts by electrical impedance spectroscopy

We describe an impedance-based method for cell barrier integrity testing. A four-electrode electrical impedance spectroscopy (EIS) setup can be realized by simply connecting a commercial chopstick-like electrode (STX-1) to a potentiostat allowing monitoring cell barriers cultivated in transwell inserts. Subsequent electric circuit modeling of the electrical impedance results the capacitive properties of the barrier next to the well-known transepithelial electrical resistance (TEER). The versatility of the new method was analyzed by the EIS analysis of a Caco-2 monolayer in response to (a) different membrane coating materials, (b) two different permeability enhancers ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) and saponin, and (c) sonoporation. For the different membrane coating materials, the TEERs of the standard and new protocol coincide and increase during cultivation, while the capacitance shows a distinct maximum for three different surface materials (no coating, Matrigel®, and collagen I). The permeability enhancers cause a decline in the TEER value, but only saponin alters the capacitance of the cell layer by two orders of magnitude. Hence, cell layer capacitance and TEER represent two independent properties characterizing the monolayer. The use of commercial chopstick-like electrodes to access the impedance of a barrier cultivated in transwell inserts enables remarkable insight into the behavior of the cellular barrier with no extra work for the researcher. This simple method could evolve into a standard protocol used in cell barrier research.

Isolation and Characterization of a New Human Corneal Epithelial Cell Line: HCE-F

To isolate and characterize an epithelial cell (EC) line from a human donor cornea, which may serve as a reliable test cell line to address biomolecular issues and study the response of corneal epithelium to stressing events and therapeutic treatments. A corneal button from a donor patient was treated with enzymes to separate the epithelial sheet and to free EC, which were put in tissue culture. ECs were characterized by optic and electronic microscopies, cytokeratins and PAX6 were detected by SDS-PAGE and western immunoblotting, the barrier function was evaluated by transepithelial electric resistance and by the immune detection of membrane junction proteins, and the karyotype was characterized according to the classical methods. Morphological analyses returned the picture of classical homogeneous polygonal morphology as expecetd by EC that was maintained over time and several in vitro passages. Transepithelial electric resistance values were characteristic of a typical barrier-forming cell line. The cytokeratin expression pattern was the one expected for corneal EC with a predominance of CK3 and CK5 and different from a human keratocyte cell line. The male karyotype showed 2 trisomies, of chromosomes 8 and 11. All the data so far obtained with the HCE-F cells concur to certify this cell line as a stable, true primary human corneal EC line, which could then be used as a test cell line to study and address the questions concerning the biological response of human corneal epithelium to stressing and/or therapeutic treatments and as a term of comparison for EC derived from pathological corneas.

Nobiletin Attenuates DSS-Induced Intestinal Barrier Damage through the HNF4α-Claudin-7 Signaling Pathway.

The intestinal epithelium barrier functions to protect human bodies from damages such as harmful microorganisms, antigens, and toxins. In this study, we evaluated the protective effect and molecular mechanism of a dominant polymethoxyflavone nobiletin (NOB) from tangerine peels on intestinal epithelial integrity. The results from transepithelial electrical resistance (TEER) suggested that NOB pretreatment counteracts epithelial injury induced by inflammatory cytokines (TEER value in 48 h: vehicle, 135.6 ± 3.9 Ω/cm2; TNF-α + IL-1β, 90.7 ± 0.5 Ω/cm2; 10 μM NOB + TNF-α + IL-1β, 126.1 ± 0.8 Ω/cm2; 100 μM NOB + TNF-α + IL-1β, 125.3 ± 0.5 Ω/cm2. P < 0.001). Clinical and pathological test results suggested that administration of NOB effectively alleviates intestinal barrier injury induced by dextran sulfate sodium (DSS) as evidenced by the length of colon villi on day 7 (control, 253.7 ± 4.8 μm, DSS 131.6 ± 4.6 μm, NOB + DSS, 234.5 ± 5.1 μm. P < 0.001). Interestingly, when screening tight junction molecules for intestinal barrier integrity, we observed that independent treatment with NOB sharply increased claudin-7 levels (ratio of claudin-7 over GAPDH: control, 1.0 ± 0.06; DSS, 0.02 ± 0.001; NOB + DSS, 0.3 ± 0.07. P < 0.001), which was previously suppressed upon DSS stimulation. Furthermore, hepatocyte nuclear factor 4α (HNF-4α) transcriptional regulation of claudin-7 contributed to intestinal barrier homeostasis. Therefore, our study suggests potential intestinal protective strategies based on polymethoxyflavones of aged tangerine peels.

Corneal damage and its recovery after instillation of preservative-free versus preserved latanoprost eye drops

Purpose: Latanoprost ophthalmic solution is highly effective as a therapeutic agent for glaucoma and is applied worldwide. However, harmful effects on the corneal surface have been reported regarding the commercially available latanoprost ophthalmic solution. Corneal surface toxicity may be caused by the added preservative of the ophthalmic solution. In order to ascertain whether latanoprost itself can damage the cornea or if this is solely due to the added preservatives, this study attempted to determine the corneal changes that occur at different time periods following usage of preservative-free versus preserved latanoprost eye drops.Materials and methods: Preservative-free latanoprost eye drops (Monoprost®) or preserved latanoprost eye drops (Xalatan®) containing 0.02% benzalkonium chloride (BAC) were instilled in the corneas of rabbits. For each of the two different eye drop solutions, the rabbits used in this experiment were divided into three exposure groups: 1 minute, 24 hour, and 1 week groups. Corneal transepithelial electrical resistance (TER) and scanning electron microscopy (SEM) were examined immediately (1 minute) after instillation, at 24 hours after instillation, and at 24 hours after 1 week of daily instillations of latanoprost. Hank’s balanced salt solution was used in the control group.Results: The mean corneal TER of the control group was 933.8 ± 279.0 Ω cm2. In preservative-free latanoprost instilled corneas, there was no significant decrease in the TER or morphological changes at any of the time points, with the relative TER values of 117 ± 38%, 100 ± 34%, and 93 ± 21% for 1 minute, 1 day, and 1 week time points, respectively. In preserved latanoprost instilled corneas, SEM showed that only the immediate group exhibited superficial cell damage and a significant decrease in the corneal TER compared to the controls and other time points and to the immediate preservative-free latanoprost corneas. In the preserved latanoprost groups, the relative TER values were 18 ± 5%, 110 ± 28%, and 92 ± 10%, for the three respective observation time points.Conclusions: Preservative-free latanoprost can be safely instilled to the corneal epithelium. Latanoprost with 0.02% BAC has an immediate deleterious impact on the corneal epithelium; however, it disappears within 24 hours after instillation.

Dendrobium officinale Polysaccharide Protected CCl4-Induced Liver Fibrosis Through Intestinal Homeostasis and the LPS-TLR4-NF-κB Signaling Pathway.

We explored the therapeutic effects of Dendrobium officinale polysaccharide (DOP) on CCl4-induced liver fibrosis with respect to the intestinal hepatic axis using a rat model. Histopathological staining results showed that DOP alleviated extensive fibrous tissue proliferation in interstitium and lessened intestinal mucosal damage. Western blot and PCR results showed that DOP maintained intestinal balance by upregulating the expression of tight junction proteins such as occludin, claudin-1, ZO-1, and Bcl-2 proteins while downregulating the expression of Bax and caspase-3 proteins in the intestine. The transepithelial electrical resistance (TEER) value of the LPS-induced Caco-2 monolayer cell model was increased after DOP administration. These illustrated that DOP can protect the intestinal mucosal barrier function. DOP also inhibited activation of the LPS-TLR4-NF-κB signaling pathway to reduce the contents of inflammatory factors TGF-β and TNF-α, increased the expression of anti-inflammatory factor IL-10, and significantly decreased α-SMA and collagen I expression. These results indicated that DOP maintained intestinal homeostasis by enhancing tight junctions between intestinal cells and reducing apoptosis, thereby inhibiting activation of the LPS-TLR4-NF-κB signaling pathway to protect against liver fibrosis.

In vitro potential of human mesenchymal stem cells for corneal epithelial regeneration.

Aim: To determine the potential of mesenchymal stem cells (MSC) for corneal epithelial regeneration in vitro. Materials & methods: Bone marrow MSC (BM-MSC) and adipose tissue MSC were analyzed for corneal epithelial and mesenchymal markers, using limbal stem cells and corneal cells as controls. MSC with better potential were cultured with specific mediums for epithelial induction. Transepithelial electric resistance and wound healing assay with human corneal epithelial cells were performed. Results: BM-MSC showed better potential, increased corneal markers, and higher transepithelial electric resistance values when induced with limbal epithelial culture medium. Induced BM-MSC promoted better wound healing of human corneal epithelial cells by paracrine secretion. Conclusion: BM-MSC has potential for corneal epithelial induction in a protocol compatible with human application.

Hydrogel co-networks of gelatine methacrylate and poly(ethylene glycol) diacrylate sustain 3D functional in vitro models of intestinal mucosa

Mounting evidence supports the importance of the intestinal epithelial barrier and its permeability both in physiological and pathological conditions. Conventional in vitro models to evaluate intestinal permeability rely on the formation of tightly packed epithelial monolayers grown on hard substrates. These two-dimensional models lack the cellular and mechanical components of the non-epithelial compartment of the intestinal barrier, the stroma, which are key contributors to the barrier permeability in vivo. Thus, advanced in vitro models approaching the in vivo tissue composition are fundamental to improve precision in drug absorption predictions, to provide a better understanding of the intestinal biology, and to faithfully represent related diseases. Here, we generate photo-crosslinked gelatine methacrylate (GelMA)—poly(ethylene glycol) diacrylate (PEGDA) hydrogel co-networks that provide the required mechanical and biochemical features to mimic both the epithelial and stromal compartments of the intestinal mucosa, i.e. they are soft, cell adhesive and cell-loading friendly, and suitable for long-term culturing. We show that fibroblasts can be embedded in the GelMA-PEGDA hydrogels while epithelial cells can grow on top to form a mature epithelial monolayer that exhibits barrier properties which closely mimic those of the intestinal barrier in vivo, as shown by the physiologically relevant transepithelial electrical resistance (TEER) and permeability values. The presence of fibroblasts in the artificial stroma compartment accelerates the formation of the epithelial monolayer and boosts the recovery of the epithelial integrity upon temporary barrier disruption, demonstrating that our system is capable of successfully reproducing the interaction between different cellular compartments. As such, our hydrogel co-networks offer a technologically simple yet sophisticated approach to produce functional three-dimensional (3D) in vitro models of epithelial barriers with epithelial and stromal cells arranged in a spatially relevant manner and near-physiological functionality.

Eye Irritation Potential of Microglycine and Microglycine-Containing Ointments: An in vitro Study on Reconstructed Human Corneal Epithelium.

ObjectiveTo assess the eye tolerability of a buffered ophthalmic solution containing microglycine (sodium hydroxymethylglycinate, mwaterTM) in an in vitro model.Materials and MethodsA multiple endpoint analysis (MEA) approach was applied to the reconstructed human corneal epithelium (HCE) model. Sodium hydroxymethylglycinate solution (0.04%) and two ophthalmic ointments containing microglycine (Protectorial, containing 0.02% of sodium hydroxymethylglycinate, and Edenight, containing 0.04% of sodium hydroxymethylglycinate) were investigated. The buffered solution and the ointments were tested on HCE after acute (one application in 24 hrs, followed or not by 16 hrs of recovery) or repeated (one application per day for three consecutive days) exposures; benzalkonium chloride (BAK) 0.01% and saline isotonic solution were used as positive and negative controls, respectively. Cellular viability, trans-epithelial electrical resistance (TEER), lactate dehydrogenase (LDH) release and histo-morphology were evaluated.ResultsBAK 0.01% toxicity in HCE was confirmed for the 24+16 hrs acute and repeated exposure protocols, while, after 24–hours acute treatment, only modifications of the superficial cell layer were visible compared with the negative control. Sodium hydroxymethylglycinate had a very good tolerability profile and a neutral impact on the corneal surface after acute or repeated exposure. The Protectorial and Edenight ointments preserved cell viability in the different exposure protocols, suggesting a good local tolerability profile. Modifications of the superficial layers were observed on histo-morphological analysis and confirmed by increased release of LDH after 24+16 hrs acute exposure (+65% and +76% for Protectorial and Edenight, respectively) and TEER values after 24+16 hrs and 72 hrs exposure protocols. These results were dependent on the ointments’ accumulation on the corneal epithelium due to their physical form (semi-solid) and lipophilic properties.ConclusionSodium hydroxymethylglycinate, alone or as part of eye ointments, was found to be non-toxic after acute or repeated exposure in the reconstructed HCE model.

Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro

Aeromonas sobria is a pathogen causing food-borne illness. In immunocompromised patients and the elderly, A. sobria can leave the intestinal tract, and this opportunistically leads to severe extraintestinal diseases including sepsis, peritonitis, and meningitis. To cause such extraintestinal diseases, A. sobria must pass through the intestinal epithelial barrier. The mechanism of such bacterial translocation has not been established. Herein we used intestinal (T84) cultured cells to investigate the effect of A. sobria serine protease (ASP) on junctional complexes that maintain the intercellular adhesion of the intestinal epithelium. When several A. sobria strains were inoculated into T84 monolayer grown on Transwell inserts, the strain with higher ASP production largely decreased the value of transepithelial electrical resistance exhibited by the T84 monolayer and markedly caused bacterial translocation from the apical surface into the basolateral side of T84 monolayer. Further experiments revealed that ASP acts on adherens junctions (AJs) and causes the destruction of both nectin-2 and afadin, which are protein components constituting AJs. Other studies have not revealed the bacterial pathogenic factors that cause the destruction of both nectin-2 and afadin, and our present results thus provide the first report that the bacterial extracellular protease ASP affects these molecules. We speculate that the destruction of nectin-2 and afadin by the action of ASP increases the ability of A. sobria to pass through intestinal epithelial tissue and contributes to the severity of pathological conditions.

Self-organized intestinal epithelial monolayers in crypt and villus-like domains show effective barrier function

Intestinal organoids have emerged as a powerful in vitro tool for studying intestinal biology due to their resemblance to in vivo tissue at the structural and functional levels. However, their sphere-like geometry prevents access to the apical side of the epithelium, making them unsuitable for standard functional assays designed for flat cell monolayers. Here, we describe a simple method for the formation of epithelial monolayers that recapitulates the in vivo-like cell type composition and organization and that is suitable for functional tissue barrier assays. In our approach, epithelial monolayer spreading is driven by the substrate stiffness, while tissue barrier function is achieved by the basolateral delivery of medium enriched with stem cell niche and myofibroblast-derived factors. These monolayers contain major intestinal epithelial cell types organized into proliferating crypt-like domains and differentiated villus-like regions, closely resembling the in vivo cell distribution. As a unique characteristic, these epithelial monolayers form functional epithelial barriers with an accessible apical surface and physiologically relevant transepithelial electrical resistance values. Our technology offers an up-to-date and novel culture method for intestinal epithelium, providing an in vivo-like cell composition and distribution in a tissue culture format compatible with high-throughput drug absorption or microbe-epithelium interaction studies.

Allium porrum Extract Decreases Effector Cell Degranulation and Modulates Airway Epithelial Cell Function.

Allium genus plants, such as leek (Allium porrum), are rich sources of anti-inflammatory and anti-oxidant secondary metabolites; this is of interest because it demonstrates their suitability as pharmacological alternatives for inflammatory processes, including allergy treatment. The composition of methanolic leek extract (LE) was analyzed by GC–MS and LC–IT/MS, and the total phenolic content and antioxidant capacity were quantified by colorimetric methods. Its pharmacological potential was analyzed in human bronchial epithelial Calu-3 cells, human mast cells LAD2, and humanized rat basophiles RBL-2H3. LE exhibited a cytotoxic effect on Calu-3 cells and HumRBL-2H3 cells only at high concentrations and in a dose-dependent manner. Moreover, LE decreased the degranulation of LAD2 and HumRBL-2H3 cells. LE treatment also significantly prevented alterations in transepithelial electrical resistance values and mRNA levels of glutathione-S-transferase (GST), c-Jun, and NFκB after treatment with H2O2 in ALI-cultured Calu-3 cells. Finally, ALI-cultured Calu-3 cells treated with LE showed lower permeability to Ole e 1 compared to untreated cells. A reduction in IL-6 secretion in ALI-cultured Calu-3 cells treated with LE was also observed. In summary, the results obtained in this work suggest that A. porrum extract may have potential anti-allergic effects due to its antioxidant and anti-inflammatory properties. This study provides several important insights into how LE can protect against allergy.

Cranberry Juice Phytochemicals Attenuated Ethanol-Induce Epithelial Barrier Dysfunction on Caco-2 Monolayers (P06-094-19)

ObjectivesThe objective of this study was to determine whether cranberry juice phytochemicals protect intestinal barrier using Caco-2 monolayer models, and to elucidate the underlying mechanisms of cranberry juice phytochemicals on ethanol-induced barrier dysfunction. MethodsCranberry juice phytochemicals were extracted using Amberlite FPX66 resin and phytochemicals composition were analyzed on HPLC. Caco-2 monolayers were incubated with cranberry juice phytochemicals at different concentrations for 2 hours before treatment with 4.5% ethanol. The transepithelial electrical resistance (TEER) and fluorescein isothiocyanate-labeled dextran (FITC) was used to evaluate paracellular permeability. Epithelial integrity and distribution of tight junction proteins (Occludin and ZO-1) in Caco-2 monolayers were determined by immunofluorescence labeling method. Protein levels of ZO-1, Occludin and Claudin-1 were determined by immunoblotting. ResultsCranberry juice phytochemicals consisted of malvidin 3,5-diglucoside, petunidin 3-glucoside, chlorogenic acid and caffeic acid. Ethanol induced decrease of TEER while pretreatment of cranberry juice phytochemicals protected Caco-2 monolayer integrity in a dose-dependent manner. Monolayers treated with 7.5, 15, and 30 μg/mL of cranberry juice phytochemicals had TEER value 11.6%, 22.7% and 30.5% higher than the ethanol-treatment monolayers, respectively. This was accompanied by 40%, 55%, and 50% decreases of the paracellular permeability compared to ethanol-treated monolayers. Immunofluorescent analysis showed that occludin was localized tightly around cell membrane and colocalize with ZO-1 in the normal Caco-2 monolayers. Exposure to 4.5% ethanol disrupted both occludin and ZO-1 networks and resulted in gap formation among the cells. Cranberry juice phytochemicals attenuated these changes induced by ethanol exposure. Ethanol exposure significantly reduced the protein levels of occludin and ZO-1. Pretreatment with cranberry juice phytochemicals increased the protein levels of occludin dose dependently but did not affect ZO-1 level. ConclusionsCurrent study suggested that cranberry juices phytochemicals attenuate ethanol-induced intestinal barrier dysfunction in part by enhancing the tight junction assemblies. Funding SourcesNA.

Creating an Intestinal Epithelial Cell Culture Model to Screen Novel Phytochemical Therapies for Postmenopausal Women (FS09-05-19)

ObjectivesThe intestinal epithelium serves as barrier maintained by tight junction (TJ) proteins. TJ integrity is compromised by release of matrix-metalloproteases (MMPs). Degradation of TJ proteins may lead to increased permeability of the small intestine. Previous research has shown that MMPs are up-regulated with estrogen deficiency. In postmenopausal women, there may be a causal relationship between intestinal permeability and inflammation-related disease. The purpose of this project is to create an in vitro cell culture model, representative of the estrogen-deficient small intestine in vivo, to screen phytochemicals that may attenuate increased intestinal permeability observed with a decline in estrogen levels. MethodsHuman colorectal adenocarcinoma (CaCo-2) cells seeded on 12 mm Transwell® inserts were co-cultured with 3T3 Swiss fibroblasts, a major source of MMPs in the intestines, in 12-well plates. Cells were pre-treated with 10 μM universal estrogen receptor inhibitor (ICI 181,780; ABCAM, Boston, MA) for 1 hour, then treated with 10 nM estrogen (17 β-Estradiol; Sigma-Aldrich, St. Louis, MO) with or without MMP-9 Inhibitor (MMP-9 Inhibitor I; Millipore Sigma, Burlington, MA). Transepithelial electrical resistance (TER) was measured at 24, 48, and 72 hours following treatment. Confocal immunocytochemistry was used to determine the expression and distribution of TJ proteins. Differences between treatment groups were analyzed via ANOVA using SAS 9.3. ResultsInhibition of the estrogen receptors resulted in downregulation of TJ proteins in this model, suggesting a decline in intestine epithelial barrier integrity. MMP 9 inhibition concomitant with suppression of estrogen signaling resulted in increased TER values. ConclusionsMMP 9 inhibition when estrogen signaling is blocked enhances the intestinal epithelial barrier integrity. Future work will use this model to identify food components, especially those known to inhibit MMP 9, that may attenuate the decline in intestine epithelial barrier integrity observed with estrogen deficiency. Our long-term goal is to identify nutrition-related therapeutic targets to decrease the various inflammation-related comorbidities associated with estrogen deficiency, thereby improving the health of postmenopausal women. Funding SourcesCollege of Allied Health Student Research and Creativity Grant.

Escherichia coli K12: An evolving opportunistic commensal gut microbe distorts barrier integrity in human intestinal cells

Commensal enteric microbes under specific conditions viz. immunocompromised system, altered microbiota or uncompetitive niche induce their otherwise dormant pathogenic phenotype to distort host cellular functioning. Here we investigate how under in vitro environment established by using Caco-2 cells, commensal gut microbe E. coli K12 (ATCC 14849) disrupt intestinal epithelial barrier function. Caco-2 cells exposed to E. coli showed the time dependent significant (P < 0.01) decrease in transepithelial electrical resistance (TEER) and concomitantly increased phenol red flux across cell monolayer in contrast to non infected control cells. E. coli infected intestinal cells were observed with suppressed (p < 0.05) mRNA levels of ZO-1, Claudin-1, Occludin and Cingulin-1 in contrast to significantly (p < 0.05) higher PIgR and hbd-2 mRNA fold changes. Immunofluorescent and electron micrographs revealed the disrupted distribution and localisation of specific tight junction proteins (Zo-1 and Claudin-1) and actin filament in E. coli infected Caco-2 cells that ultimately resulted in deformed cellular morphology. Taken together, E. coli K12 under compromised in vitro milieu disrupted the intestinal barrier functions by decreasing the expression of important tight junction genes along with the altered distribution of associated proteins that increased the intestinal permeability as reflected by phenol red flux and TEER values.

Construction of Vascularized Oral Mucosa Equivalents Using a Layer-by-Layer Cell Coating Technology.

There have been many advances in tissue engineering with respect to in vitro and in vivo models of oral mucosa equivalents (OMEs). To apply in vitro reconstructed oral mucosa models to regenerative medicine and alternatives to animal testing, it is necessary to develop the technology of reconstructing different types of oral tissues, such as control of epithelial differentiation and introduction of appendages. We previously reported that functional three-dimensional (3D) tissue models could be quickly constructed by using a layer-by-layer (LbL) cell coating technique that assembles extracellular matrix (ECM) nanofilms to a cell surface. In this study, 3D human OMEs composed of lamina-propria, keratinized or non-keratinized epithelium, and blood capillaries were constructed by using the LbL cell coating technology. Human oral mucosal fibroblasts (HOMFs) were coated with ECM nanofilms and accumulated for the construction of oral mucosal lamina-propria. To construct OMEs with keratinized or non-keratinized epithelium, human oral keratinocytes isolated from gingiva (human oral gingival keratinocytes: HOGKs) or human oral keratinocytes isolated from oral mucosa (human oral mucosal keratinocytes: HOMKs) were used in this study. We further studied the construction of epithelialized OMEs with density- and size-controlled blood capillary networks by using human umbilical vein endothelial cells (HUVECs). It was revealed that these constructions had barrier functions in accordance with their histological characterization. The OMEs with keratinization (K-OMEs) showed higher transepithelial electrical resistance (TEER) values compared with OMEs with non-keratinization (N-OMEs). The constructed epithelialized OMEs with blood capillaries are useful for in vitro/ex vivo research models and regenerative medicine as in oral tissue regeneration. The results suggest that OMEs with oral tissue appendages are more promising alternatives to animal testing and can be applied to the design of in vitro oral models that mimic human tissue organs.

The effects and mechanisms of myeloid differentiation protein 2 on intestinal mucosal permeability in mice with chronic colitis.

The present study was designed to investigate the mechanism of myeloid differentiation protein 2 (MD2) on intestinal mucosa destruction in mice with chronic colitis. Briefly, a chronic colitis mouse model was established by the administration of dextran sulfate sodium (DSS) in transgenic mice of MD2 overexpression (Transgenic, MD2-Tg) and C57BL/6 wild-type mice (MD2-WT). In addition, Caco-2 cells were cultured to form a monolayer cell model in vitro. The small interfering RNA was utilized to silence the MD2 gene in Caco-2 cells, and tumor necrosis factor-α (TNF-α) was used to establish the model of intestinal mucosal inflammation. After DSS induction, the intestinal mucosal tissue inflammation was more severe in MD2-Tg mice than MD2-WT. In addition, the intestinal mucosa was severely damaged, the intestinal mucosal permeability was increased, bacterial translocation was obvious, and the expression levels of MD2, MyD88, Toll-like receptor 4 (TLR4), and HMGB1 in mucosal tissues were significantly increased, while the expression levels of tight junction proteins, occludin, and claudin-1 were significantly lower in MD2-Tg mice compared with those in MD2-WT mice. TNF-α could induce inflammatory apoptosis in Caco-2 cell models. After MD2 silencing, the apoptotic level was decreased, the value of transepithelial electrical resistance was increased, the permeability of intestinal mucosa was decreased, the cellular expression levels of MD2, MyD88, TLR4, and HMGB1 were decreased, while the expression levels of tight junction proteins, occludin and claudin-1 were increased. MD2 could aggravate the destruction of intestinal mucosa in chronic colitis through the HMGB1-TLR4-MyD88 pathway.

Cryptotanshinone Attenuates Oxygen-Glucose Deprivation/ Recovery-Induced Injury in an in vitro Model of Neurovascular Unit.

Cryptotanshinone (CTs), an active component isolated from the root of Salvia miltiorrhiza (SM), has been shown to exert potent neuroprotective property. We here established an oxygen-glucose deprivation/recovery (OGD/R)-injured Neurovascular Unit (NVU) model in vitro to observe the neuroprotective effects of CTs on cerebral ischemia/reperfusion injury (CIRI), and explore the underlying mechanisms. CTs was observed to significantly inhibit the OGD/R-induced neuronal apoptosis, and decease the activation of Caspase-3 and the degradation of poly-ADP-ribose polymerase (PARP), as well as the increase of Bax/Bcl-2 ratio in neurons under OGD/R condition. The inhibitory effects of CTs on neuron apoptosis were associated with the blocking of mitogen-activated protein kinase (MAPK) signaling pathway. CTs also remarkably ameliorated OGD/R-induced reduction of transepithelial electrical resistance (TEER) values and the increase of transendothelial permeability coefficient (Pe) of sodium fluorescein (SF) by upregulating the expression of ZO-1, Claudin-5, and Occludin in brain microvascular endothelial cells (BMECs), which might be related to the down-regulation of matrix metalloproteinase (MMP)-9 expression. Based on these findings, CTs may play a neuroprotective role in OGD/R injure in NVU models in vitro by inhibiting cell apoptosis and alleviating the damage of blood-brain barrier (BBB).

Eleutheroside B increase tight junction proteins and anti-inflammatory cytokines expression in intestinal porcine jejunum epithelial cells (IPEC-J2).

Eleutheroside B (EB) is a phenylpropanoid glycoside with anti-inflammatory properties, neuroprotective abilities, immunomodulatory effects, antinociceptive effects, and regulation of blood glucose. The aim of this study was to investigate the effects of EB on the barrier function in the intestinal porcine epithelial cells J2 (IPEC-J2). The IPEC-J2 cells were inoculated into 96-well plates at a density of 5×103 cells per well for 100% confluence. The cells were cultured in the presence of EB at concentrations of 0, 0.05, 0.10, and 0.20mg/ml for 48hr. Then, 0.10mg/ml was selected as the suitable concentration for the estimation of transepithelial electric resistance (TEER) value, alkaline phosphatase activity, proinflammatory cytokines mRNA expression, tight junction mRNA and protein expression. The results of this study indicated that the supplementation of EB in IPEC-J2 cells decreased cellular membrane permeability and mRNA expression of proinflammatory cytokines, including interleukin-6 (IL-6), interferon-γ (INF-γ), and tumour necrosis factor-α (TNF-α). The supplementation of EB in IPEC-J2 cells increased tight junction protein expression and anti-inflammatory cytokines, interleukin 10 (IL-10) and transforming growth factor beta (TGF-β). In addition, the western blotting and real-time quantitative polymerase chain reaction (RT-qPCR) results indicated that EB significantly (p<0.05) increased the mRNA and protein expression of intestinal tight junction proteins, Claudin-3, Occludin, and Zonula Occludins protein-1 (ZO-1). Therefore, dietary supplementation of EB may increase intestinal barrier function, tight junction protein expression, anti-inflammatory cytokines, and decrease proinflammatory cytokines synthesis in IPEC-J2 cells.