Semaphorin 7A impairs barrier function in cultured human corneal epithelial cells in a manner dependent on nuclear factor-kappa B

●AIM:To evaluate the role of semaphorin 7A(Sema7A)and its associated regulatory mechanisms in modulating the barrier function of cultured human corneal epithelial cells(HCEs).●METHODS:Barrier models of HCEs were treated with recombinant human Sema7A at concentrations of 0,125,250,or 500 ng/mL for 24,48,or 72h in vitro.Transepithelial electrical resistance(TEER)as well as Dextran-fluorescein isothiocyanate(FITC)permeability assays were conducted to assess barrier function.To quantify tight junctions(TJs)such as occludin and zonula occludens-1(ZO-1)at the mRNA level,reverse transcriptionpolymerase chain reaction(RT-PCR)analysis was performed.Immunoblotting was used to examine the activity of the nuclear factor-kappa B(NF-κB)signaling pathway and the production of TJs proteins.Immunofluorescence analyses were employed to localize the TJs.Enzyme-linked immunosorbent assay(ELISA)and RT-PCR were utilized to observe changes in interleukin(IL)-1βlevels.To investigate the role of NF-κB signaling activation and IL^(-1)βin Sema7A’s anti-barrier mechanism,we employed 0.1μmol/L IκB kinase 2(IKK2)inhibitor IV or 500 ng/mL IL^(-1)receptor(IL-1R)antagonist.●RESULTS:Treatment with Sema7A resulted in decreased TEER and increased permeability of Dextran-FITC in HCEs through down-regulating mRNA and protein levels of TJs in a time-and dose-dependent manner,as well as altering the localization of TJs.Furthermore,Sema7A stimulated the activation of inhibitor of kappa B alpha(IκBα)and expression of IL-1β.The anti-barrier function of Sema7A was significantly suppressed by treatment with IKK2 inhibitor IV or IL-1R antagonists.●CONCLUSION:Sema7A disrupts barrier function through its influence on NF-κB-mediated expression of TJ proteins,as well as the expression of IL-1β.These findings suggest that Sema7A could be a potential therapeutic target for the diseases in corneal epithelium.

Increased paracellular permeability of tumor-adjacent areas in 1,2-dimethylhydrazine-induced colon carcinogenesis in rats

Objective: The morphology and functions of the proximal and distal large intestine are not the same. The incidence of colorectal cancer in these regions is also different, as tumors more often appear in the descending colon than in the ascending colon.Inflammatory bowel disease and colorectal cancer can increase transepithelial permeability, which is a sign of reduced intestinal barrier function. However, there is not enough evidence to establish a connection between the difference in colorectal cancer incidence in the proximal and distal colon and intestinal permeability or the effects of carcinogenesis on the barrier properties in various areas of the colon. The aim of the study was to assess the permeability of different segments of the large intestine according to a developed mapping methodology in healthy rats and rats with 1,2-dimethylhydrazine(DMH)-induced colon adenocarcinoma.Methods: The short circuit current, the transepithelial electrical resistance and the paracellular permeability to fluorescein of large intestine wall of male Wistar rats were examined in the Ussing chambers. The optical density of the solution from the serosa side to assess the concentration of the diffused fluorescein from mucosa to serosa was analyzed by spectrophotometry. The morphometric and histological studies were performed by optical microscopy.Results: Rats with DMH-induced colon adenocarcinomas showed elevated transepithelial electrical resistance in the areas of neoplasm development. In contrast, there was no change in the electrophysiological properties of tumor adjacent areas, however,the paracellular permeability of these areas to fluorescein was increased compared to the control rats and was characterized by sharply reduced barrier function.Conclusions: The barrier properties of the colon vary depending on tumor location. The tumors were less permeable than the intact intestinal wall and probably have a negative influence on tumor-adjacent tissues by disrupting their barrier function.

Trichomonas vaginalis perturbs the junctional complex in epithelial cells

Trichomonas vaginalis,a protist parasite of the urogenital tract in humans,is the causative agent of trichomonosis,which in recent years have been associated with the cervical cancer development.In the present study we analyzed the modifications at the junctional complex level of Caco-2 cells after interaction with two isolates of T.vaginalis and the influence of the iron concentration present in the parasite’s culture medium on the interaction effects.Our results show that T.vaginalis adheres to the epithelial cell causing alterations in the junctional complex,such as:(a)a decrease in transepithelial electrical resistance;(b)alteration in the pattern of junctional complex proteins distribution as observed for E-cadherin,occludin and ZO-1;and(c)enlargement of the spaces between epithelial cells.These effects were dependent on(a)the degree of the parasite virulence isolate,(b)the iron concentration in the culture medium,and(c)the expression of adhesin proteins on the parasite surface.

FBXW7 regulates epithelial barrier impairment in human bronchial epithelial cells in vitro by targeting apoptosis signal-regulating kinase1 via the p38 pathway

Bronchial asthma is a common chronic inflammatory disease characterized by airway hyperresponsiveness(AHR),inflammatory cell infiltration,and airway remodeling.F-box/WD repeat-containing protein 7(FBXW7),an E3 ubiquitin ligase,is required for various endothelial functions,such as cell migration,inflammation,and endothelial integrity.This study aimed to investigate the role of FBXW7 in lipopolysaccharide(LPS)-induced epithelial barrier impairment in bronchial epithelial cells in vitro.By using lentivirus-based technology,FBXW7 was overexpressed or silenced(24 h)in human bronchial epithelial(16HBE)cells,which were treated with LPS or not(24 h).Immunoprecipitation(IP)detection and Western blot analysis were used to evaluate the interaction of target proteins.Cell permeability was measured using transepithelial electrical resistance and FITC dextran flux(48 h).IL-1β,IL-18 and TNF-αin cell supernatants were measured using ELISA(48 h).The results showed that LPS stimulation suppressed FBXW7 expression in a time-and dose-dependent manner.LPS exposure decreased cell proliferation,elevated IL-1β,IL-18 and TNF-α,increased epithelial permeability,and p38 phosphorylation.These LPS-induced changes were partly compromised by FBXW7 overexpression.Similar to LPS stimulation,FBXW7 knockdown increased epithelial permeability and levels of inflammatory cytokines and p38 phosphorylation,which were,in part,blocked by apoptosis signal-regulating kinase(ASK)1 knockdown or p38 pathway inhibition.IP and Western blot analysis showed that FBXW7 interacted with ASK1.ASK1 expression was inversely associated with FBXW7 expression.FBXW7 overexpression markedly enhanced ASK1 ubiquitination.These data revealed that FBXW7 counter against inflammation and protects epithelial barrier integrity in bronchial epithelial cells by promoting ubiquitination-mediated degradation of ASK1 via the p38 pathway.

High-risk HPVs, microbiota and epithelial carcinogenesis: state of the art and research contribution of in vitro 3D models

Persistent high-risk human papillomavirus (HR-HPV) infection is associated with anogenital and head & neck squamous epithelial (HNSCC) tumors, which altogether cause about 550,000 new cases every year. Several evidences suggest that the microbiota could have a role on the inflammatory, epithelial mesenchymal transition and tumorigenesis processes promoted by HR-HPV infection, yet the mechanisms involved remain to be clarified. In this review we report the state of the art on this topic and on the most promising in vitro developed models for studying the host-pathogen interactions. Using MEDLINE, several terms were searched and combined to select the most pertinent papers. The investigation was limited to the international indexed articles published in PubMed in the last 10 years. This review reports the latest knowledge in the field of the microbial-associated anogenital tumors and HNSCC. In addition, we also discuss the in vitro epithelial culture systems that reproduce the pathophysiological features of the tumoral microenvironment and the in vivo response to microbial agents, thus representing a useful tool for analyzing at cellular and molecular levels the role played by infective agents in tumorigenesis.