Localization of dysfunctional tight junctions in Salmonella enterica serovar Typhimurium-infected epithelial layers

Infection of polarized MDCK epithelial layers by Salmonella enterica serovar Typhimurium is accompanied by increased tight junction permeability and by contraction of perijunctional actinomyosin. We localized dysfunctional tight junctions in serovar Typhimurium-infected MDCK layers by imaging apical-basolateral intramembrane diffusion of fluorescent lipid and found that loss of the apical-basolateral diffusion barrier (tight junction fence function) was most marked in areas of prominent perijunctional contraction. The protein kinase inhibitor staurosporine prevented perijunctional contraction but did not reverse the effects of serovar Typhimurium on tight junction barrier function. Hence, perijunctional contraction is not required for Salmonella-induced tight junction dysfunction and this epithelial response to infection may be multifactorial.68127202720

E-cadherin and cell adhesion: a role in architecture and function in the pancreatic islet

Background/Aims: The efficient secretion of insulin from beta-cells requires extensive intra-islet communication. The cell surface adhesion protein epithelial (E)-cadherin (ECAD) establishes and maintains epithelial tissues such as the islets of Langerhans. In this study, the role of ECAD in regulating insulin secretion from pseudoislets was investigated. Methods: The effect of an immuno-neutralising ECAD on gross morphology, cytosolic calcium signalling, direct cell-to-cell communication and insulin secretion was assessed by fura-2 microfluorimetry, Lucifer Yellow dye injection and insulin ELISA in an insulin-secreting model system. Results: Antibody blockade of ECAD reduces glucose-evoked changes in [Ca2+](i) and insulin secretion. Neutralisation of ECAD causes a breakdown in the glucose-stimulated synchronicity of calcium oscillations between discrete regions within the pseudoislet, and the transfer of dye from an individual cell within a cell cluster is attenuated in the absence of ECAD ligation, demonstrating that gap junction communication is disrupted. The functional consequence of neutralising ECAD is a significant reduction in insulin secretion. Conclusion: Cell adhesion via ECAD has distinct roles in the regulation of intercellular communication between beta-cells within islets, with potential repercussions for insulin secretion. Copyright (C) 2007 S. Karger AG, Basel

Pig-to-Nonhuman Primates Pancreatic Islet Xenotransplantation: An Overview

The therapy of type 1 diabetes is an open challenging problem. The restoration of normoglycemia and insulin independence in immunosuppressed type 1 diabetic recipients of islet allotransplantation has shown the potential of a cell-based diabetes therapy. Even if successful, this approach poses a problem of scarce tissue supply. Xenotransplantation can be the answer to this limited donor availability and, among possible candidate tissues for xenotransplantation, porcine islets are the closest to a future clinical application. Xenotransplantation, with pigs as donors, offers the possibility of using healthy, living, and genetically modified islets from pathogen-free animals available in unlimited number of islets. Several studies in the pig-to-nonhuman primate model demonstrated the feasibility of successful preclinical islet xenotransplantation and have provided insights into the critical events and possible mechanisms of immune recognition and rejection of xenogeneic islet grafts. Particularly promising results in the achievement of prolonged insulin independence were obtained with newly developed, genetically modified pigs islets able to produce immunoregulatory products, using different implantation sites, and new immunotherapeutic strategies. Nonetheless, further efforts are needed to generate additional safety and efficacy data in nonhuman primate models to safely translate these findings into the clinic

TAT-Mediated Transduction of MafA Protein In Utero Results in Enhanced Pancreatic Insulin Expression and Changes in Islet Morphology

Alongside Pdx1 and Beta2/NeuroD, the transcription factor MafA has been shown to be instrumental in the maintenance of the beta cell phenotype. Indeed, a combination of MafA, Pdx1 and Ngn3 (an upstream regulator of Beta2/NeuroD) was recently reported to lead to the effective reprogramming of acinar cells into insulin-producing beta cells. These experiments set the stage for the development of new strategies to address the impairment of glycemic control in diabetic patients. However, the clinical applicability of reprogramming in this context is deemed to be poor due to the need to use viral vehicles for the delivery of the above factors. Here we describe a recombinant transducible version of the MafA protein (TAT-MafA) that penetrates across cell membranes with an efficiency of 100% and binds to the insulin promoter in vitro. When injected in utero into living mouse embryos, TAT-MafA significantly up-regulates target genes and induces enhanced insulin production as well as cytoarchitectural changes consistent with faster islet maturation. As the latest addition to our armamentarium of transducible proteins (which already includes Pdx1 and Ngn3), the purification and characterization of a functional TAT-MafA protein opens the door to prospective therapeutic uses that circumvent the use of viral delivery. To our knowledge, this is also the first report on the use of protein transduction in utero

Modulation of the epithelial barrier by dexamethasone and prolactin in cultured Madin-Darby canine kidney (MDCK) cells

Glucocorticoids and prolactin (PRL) have a direct effect on the formation and maintenance of tight junctions (TJs) in cultured endothelial and mammary gland epithelial cells. In this work, we investigated the effect of a synthetic glucocorticoid dexamethasone (DEX) and PRL on the paracellular barrier function in MDCK renal epithelial cells. DEX (4 mu M) + PRL (2 mu g/ml) and DEX alone increased significantly the transepithelial electrical resistance after chronic treatment (4 days) of confluent MDCK monolayers or after 24 h treatment of subconfluent monolayers. Immunoblotting and immunocytochemistry revealed no changes in the expression and distribution of TJ-associated proteins occludin, ZO-1 and claudin-1 in confluent monolayers after hormone addition. However, a marked increase in junctional content for occludin and ZO-1 with no changes in their total expression was observed in subconfluent MDCK monolayers 24 h exposed to DEX or DEX + PRL. No change in cell proliferation/growth was detected at subconfluent conditions following hormone treatment. An increase in the total number of viable cells was observed only in confluent MDCK monolayers after exposure to DEX + PRL suggesting that the main effect of these hormones on already established barrier may be associated with the inhibition of cell death. In Conclusion, our data Suggest that these hormones (specially dexamethasone) have an effect on TJ structure and function only during the formation of MDCK epithelial barrier by probably modulating the localization, stability or assembly of TJ proteins to membrane sites of intercellular contact. (c) 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.30210111

Modulation of gap and adherens junctional proteins in cultured neonatal pancreatic islets

Fetal and neonatal pancreatic islets have lower insulin secretory responses compared with adult islets. In culture conditions and after treatment with mammosomatotropic hormones, neonatal islets undergo maturation of the secretory machinery that might involve regulation of cell-cell contacts within the islet. This study is an investigation of the effect of prolonged culturing and in vitro treatment with prolactin on the expression of the gap junction-associated connexin 43 and the adherens junction-associated beta -catenin in cultured neonatal rat islets. Pancreatic islets from neonatal Wistar rats were cultured for 24 hours or 7 days, and the treated group was exposed to 2 mug/mL prolnctin daily for 7 days. Connexin 43 and beta -catenin were barely detected at the cell-cell contacts in 24-hour-cultured islets, as revealed by immunocytochemical analysis. Nevertheless, both junctional proteins were well expressed at the junctional region in islet cells cultured for 7 days and showed even greater staining in islets after long-term prolactin treatment. In accordance with the morphologic data, neonatal islets cultured for 24 hours displayed a relatively low level of connexin 43, as determined by Western blot analysis. Culturing for 7 days or combined prolactin treatment induced a significant increase in connexin 43 expression; this was 40% greater in the prolactin-treated group than in the control group. Furthermore, an enhancement of the expression of beta -catenin and translocation of this protein to the cell-cell contact site was also observed in neonatal islets cultured for 7 days compared with those cultured for 24 hours. In vitro prolactin treatment induced even greater expression of beta -catenin in islet cells. A correlation was observed between the increased expression of these junctional proteins and an increase in insulin secretion in cultured neonatal islets. In conclusion, prolonged culturing and in vitro treatment with prolactin induce the modulation of gap and adherens junctional proteins in pancreatic islets, which may be an important event in the in vitro maturation process of neonatal islet cells.23217718

Upregulation of the expression of tight and adherens junction-associated proteins during, maturation of neonatal pancreatic islets in vitro

Cell-cell contacts mediated by intercellular junctions are crucial for proper insulin secretion in (he endocrine pancreas. The biochemical composition of the intercellular junctions in this organ and the role of junctional proteins in endocrine pancreatic dysfunctions are still unclear. In this study. we investigated the expression and cellular location of junctional and cytoskeletal proteins in cultured neonatal rat pancreatic islets. Neonatal B-cells had an impaired insulin secretion compared to adult cells. Cultured neonatal islets showed a time-dependent increase in the glucose-induced secretory response. The maturation of B-cells in vitro was accompanied by upregulation of the expression of some junctional proteins in islet cells. Neonatal islets cultured for only 24 h showed a low expression and a diffuse cytoplasmic location of the tight junctional proteins occludin and ZO-1 and of the adherens junctional proteins alpha- and beta-catenins, as demonstrated by immunoblotting and immunocytochemistry. Culturing islets for up to 8 days significantly increased the cell expression of these junctional proteins but not of the cytoskeletal proteins vinculin and a-actinin. A translocation of ZO-1 and catenins to the cell-cell contact region. as well as a higher association of F-actin with the intercellular junction. were also observed in neonatal islets following prolonged culturing. ZO-1 and beta-catenin were immunolocated in the endocrine pancreas of adult rats indicating that these junctional proteins are also expressed in this organ in situ. In conclusion. endocrine pancreatic cells express several junctional proteins that are upregulated following differentiation of the endocrine pancreas in vitro.354189081182