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tight junction [keywords]
- Bacillus subtilis Protects Porcine Intestinal Barrier from Deoxynivalenol via Improved Zonula Occludens-1 Expression. [Journal Article]
- Asian-Australas J Anim Sci 2014 Apr; 27(4):580-6.
Intestinal epithelial cells (IECs) forming the barrier for the first-line of protection are interconnected by tight junction (TJ) proteins. TJ alteration results in impaired barrier function, which causes potentially excessive inflammation leading to intestinal disorders. It has been suggested that toll-like receptor (TLR) 2 ligands and some bacteria enhance epithelial barrier function in humans and mice. However, no such study has yet to be claimed in swine. The aim of the present study was to examine whether Bacillus subtilis could improve barrier integrity and protection against deoxynivalenol (DON)-induced barrier disruption in porcine intestinal epithelial cell line (IPEC-J2). We found that B. subtilis decreased permeability of TJ and improved the expression of zonula occludens (ZO)-1 and occludin during the process of forming TJ. In addition, ZO-1 expression of IPEC-J2 cells treated with B. subtilis was up-regulated against DON-induced damage. In conclusion, B. subtilis may have potential to enhance epithelial barrier function and to prevent the cells from DON-induced barrier dysfunction.
- ZAP-70 genotype disrupts the relationship between microbiota and host leading to spondyloarthritis and ileitis. [JOURNAL ARTICLE]
- Arthritis Rheumatol 2014 Jul 21.
Objective: Spondyloarthropathies (SpA) including inflammatory arthritic, skin and bowel diseases (IBD) share genetic susceptibility, interleukin (IL)-23-dependence and involvement of the microbiota. It is unclear how host genetics influence gut microbiota, and the microbiota's relationship to organ inflammation in SpA. Methods: To model this, wild type SKG or TLR4-deficient SKG or BALB/c mice housed under specific pathogen-free (SPF) or germ-free (GF) conditions or GF conditions recolonized with altered Schaedler flora were injected intraperitoneally with microbial 1,3-D β-glucan (curdlan). Arthritis, spondylitis and ileitis were assessed histologically. Microbiome composition was analyzed in serial fecal samples of mice co-housed from weaning by 454 pyrosequencing. Infiltrating cells and cytokines in the peritoneal cavity were measured by flow cytometry and ELISA. Cytokine, endoplasmic reticulum (ER) stress marker and tight junction protein transcription were measured by qRT-PCR. Results: Microbiota content and response to curdlan varied in mice with normal or impaired T cell receptor signal strength due to the SKG ZAP70(W163C) mutation. Curdlan triggered acute inflammation regardless of the SKG allele or microbiota. However, no or limited microbiota content attenuated arthritis severity. In contrast, ileal IL-23 expression, ER stress, lymph node IL-17A production, goblet cell loss and ileitis development were microbiota-dependent. Ileitis but not arthritis was suppressed by microbiota transfer upon co-housing SKG with wild type BALB/c mice, as well as by TLR4 deficiency. Conclusion: The interaction of immunogenetic background and host microbiota leads to an IL-23-dependent loss of mucosal function triggering ileitis in response to curdlan. © 2014 American College of Rheumatology.
- Dietary tryptophan modulates intestinal immune response, barrier function, antioxidant status and gene expression of TOR and Nrf2 in young grass carp (Ctenopharyngodon idella). [JOURNAL ARTICLE]
- Fish Shellfish Immunol 2014 Jul 15.
The present research evaluated the effects of dietary tryptophan (Trp) on growth performance, intestinal mucosal immune, barrier function and antioxidant capacity and gene expression of young grass carp (Ctenopharyngodon idella). Fish were fed six different experimental diets containing graded levels of Trp at 0.7(control), 1.7, 3.1, 4.0, 5.2 and 6.1 g kg(-1) diet for 8 weeks. The results showed that Trp supplementation signiﬁcantly enhanced the percent weight gain (PWG), feed intake and feed efficiency (P < 0.05), and decreased the plasma ammonia content (PAC) (P < 0.05). After the 8-week feeding trail, an environmental copper exposure trail was conducted for 4 days. Results from the copper exposure trail showed that dietary Trp enhanced the lysozyme, acid phosphatase activities and complement 3 contents in the intestine of young grass carp (P < 0.05). In addition, Trp supplementation increased the copper/zinc superoxide dismutase (SOD1), glutathione peroxidase (GPx) activities and glutathione contents (P < 0.05), and decreased the protein carbonyl and malondialdehyde contents (P < 0.05). Furthermore, the relative gene expression levels of interleukin 10, transforming growth factor-β1, occludin, zonula occludens 1, claudin-b, -c, and -3, SOD1, GPx and NF-E2-related factor 2 in the intestine were signiﬁcantly up-regulated with increasing of dietary Trp up to a certain level (P < 0.05). Conversely, the mRNA levels of tumor necrosis factor α, interleukin 8, target of rapamycin, Kelch-like-ECH-associated protein 1, claudin-12 and -15a in the intestine were signiﬁcantly down-regulated by Trp (P < 0.05). Collectively, appropriate dietary Trp level improves ﬁsh growth, intestinal immune response, barrier function and antioxidant status, and regulated the mRNA levels of related signal molecules of young grass carp. Based on the quadratic regression analysis of the PWG and PAC, the dietary Trp requirement of young grass carp (287-699 g) was estimated to be 3.81 g kg(-1) diet (12.7 g kg(-1) protein) and 3.89 g kg(-1) diet (13.0 g kg(-1) protein), respectively.
- Dysregulation of nectin-2 in the testicular cells: An explanation of cadmium-induced male infertility. [JOURNAL ARTICLE]
- Biochim Biophys Acta 2014 Jul 18.
Nectin-2, a junction molecule, is found at the basal and apical ectoplasmic specializations (ES) for the formation of the blood-testis barrier (BTB) (constituted by tight junctions and basal ES) and Sertoli- spermatid adhesion. Loss of nectin-2 causes male infertility, suggesting nectin-2-based ES is crucial for spermatogenesis. Cadmium (Cd) has been known to induce severe testicular injury. Recent evidence has shown that the basal ES at the BTB and apical ES are the targets of Cd, suggesting that unique junction protein at the ES may explain why testis is more susceptible than other tissues. Since nectin-2 is expressed exclusively at the ES, it is highly possible that nectin-2 is the direct target of Cd. In this study, we investigate if nectin-2 is the target protein of Cd toxicity and the mechanism on how Cd down-regulates nectin-2 to achieve ES disruption. Our results revealed that Cd suppresses nectin-2 at transcriptional and post-translational levels. Inhibitor and shRNA knockdown have shown that Cd induces nectin-2 protein degradation via clathrin-dependent endocytosis. Immunofluorescence staining and endocytosis assays further confirmed that nectin-2 internalization is promoted upon Cd treatment. Besides, Cd directly represses nectin-2 transcription. EMSA and ChIP assays showed that Cd inhibits the binding of positive regulators to nectin-2 promoter. siRNA and overexpression analyses have demonstrated that Cd reduces the expression and binding affinity of positive regulators for transcription. Taken together, nectin-2 is the direct molecular target of Cd and its disruptive effects is mediated via direct repressing nectin-2 transcription and inducing endocytosis of nectin-2 for degradation.
- The Metastatic Microenvironment: Claudin-1 Suppresses the Malignant Phenotype of Melanoma Brain Metastasis. [JOURNAL ARTICLE]
- Int J Cancer 2014 Jul 21.
Brain metastases occur frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. Identification of molecular determinants regulating melanoma brain metastasis would advance the development of prevention and therapy strategies for this disease. Gene expression profiles of cutaneous and brain-metastasizing melanoma variants from three xenograft tumor models established in our lab revealed that expression of tight junction component CLDN1 was lower in the brain-metastasizing variants than in cutaneous variants from the same melanoma. The objective of this study was to determine the significance of CLDN1 down-regulation/loss in metastatic melanoma and its role in melanoma brain metastasis. An immunohistochemical analysis of human cells of the melanocyte lineage indicated a significant CLDN1 down-regulation in metastatic melanomas. Transduction of melanoma brain metastatic cells expressing low levels of CLDN1 with a CLDN1 retrovirus suppressed their metastatic phenotype. CLDN1 over-expressing melanoma cells expressed a lower ability to migrate and adhere to extracellular matrix, reduced tumor aggressiveness in nude mice, and most importantly, eliminated the formation of micro-metastases in the brain. In sharp contrast, the ability of the CLDN1 over-expressing cells to form lung micro-metastases was not impaired. CLDN1 mediated interactions between these cells and brain endothelial cells constitute the mechanism underlying these results. Taken together we demonstrated that down-regulation or loss of CLDN1 supports the formation of melanoma brain metastasis, and that CLDN1 expression could be a useful prognostic predictor for melanoma patients with a high risk of brain metastasis. © 2014 Wiley Periodicals, Inc.
- Structure and function of the ependymal barrier and diseases associated with ependyma disruption. [Journal Article, Review]
- Tissue Barriers 2014.:e28426.
The neuroepithelium is a germinal epithelium containing progenitor cells that produce almost all of the central nervous system cells, including the ependyma. The neuroepithelium and ependyma constitute barriers containing polarized cells covering the embryonic or mature brain ventricles, respectively; therefore, they separate the cerebrospinal fluid that fills cavities from the developing or mature brain parenchyma. As barriers, the neuroepithelium and ependyma play key roles in the central nervous system development processes and physiology. These roles depend on mechanisms related to cell polarity, sensory primary cilia, motile cilia, tight junctions, adherens junctions and gap junctions, machinery for endocytosis and molecule secretion, and water channels. Here, the role of both barriers related to the development of diseases, such as neural tube defects, ciliary dyskinesia, and hydrocephalus, is reviewed.
- Safeguarding Stem Cell-Based Regenerative Therapy against Iatrogenic Cancerogenesis: Transgenic Expression of DNASE1, DNASE1L3, DNASE2, DFFB Controlled By POLA1 Promoter in Proliferating and Directed Differentiation Resisting Human Autologous Pluripotent Induced Stem Cells Leads to their Death. [JOURNAL ARTICLE]
- J Stem Cell Res Ther 2013 Jul 22; Suppl 9(5)
The worst possible complication of using stem cells for regenerative therapy is iatrogenic cancerogenesis. The ultimate goal of our work is to develop a self-triggering feedback mechanism aimed at causing death of all stem cells, which resist directed differentiation, keep proliferating, and can grow into tumors.The specific aim was threefold: (1) to genetically engineer the DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter; (2) to bioengineer anti-SSEA-4 antibody guided vectors delivering transgenes to human undifferentiated and proliferating pluripotent stem cells; (3) to cause death of proliferating and directed differentiation resisting stem cells by transgenic expression of the human recombinant the DNases (hrDNases).The DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter were genetically engineered. The vectors targeting specifically SSEA-4 expressing stem cells were bioengineered. The healthy volunteers' bone marrow mononuclear cells (BMMCs) were induced into human, autologous, pluripotent stem cells with non-integrating plasmids. Directed differentiation of the induced stem cells into endothelial cells was accomplished with EGF and BMP. The anti-SSEA 4 antibodies' guided DNA vectors delivered the transgenes for the human recombinant DNases' into proliferating stem cells.Differentiation of the pluripotent induced stem cells into the endothelial cells was verified by highlighting formation of tight and adherens junctions through transgenic expression of recombinant fluorescent fusion proteins: VE cadherin, claudin, zona occludens 1, and catenin. Proliferation of the stem cells was determined through highlighting transgenic expression of recombinant fluorescent proteins controlled by POLA promoter, while also reporting expression of the transgenes for the hrDNases. Expression of the transgenes for the DNases resulted in complete collapse of the chromatin architecture and degradation of the proliferating cells' genomic DNA. The proliferating stem cells, but not the differentiating ones, were effectively induced to die.Herein, we describe attaining the proof-of-concept for the strategy, whereby transgenic expression of the genetically engineered human recombinant DNases in proliferating and directed differentiation resisting stem cells leads to their death. This novel strategy reduces the risk of iatrogenic neoplasms in stem cell therapy.
- Recruitment and retention of human autologous CD34+ CD117+ CD133+ bone marrow stem cells to infarcted myocardium followed by directed vasculogenesis: Novel strategy for cardiac regeneration. [JOURNAL ARTICLE]
- Mol Cell Ther 2013 Dec 13.
Ongoing clinical trials, in regenerative therapy of patients suffering from myocardial infarctions, rely primarily upon administration of bone marrow stem cells to the infarcted zones. Unfortunately, low retention of these cells, to the therapeutic delivery sites, reduces effectiveness of this strategy; thus it has been identified as the most critical problem for advancement of cardiac regenerative medicine.The specific aim of this work was three-fold: (1) to isolate highly viable populations of human, autologous CD34+, CD117+, and CD133+ bone marrow stem cells; (2) to bioengineer heterospecific, tetravalent antibodies and to use them for recruiting of the stem cells to regenerated zones of infarcted myocardium; (3) to direct vasculogenesis of the retained stem cells with the defined factors.Cardiac tissue was biopsied from the hearts of the patients, who were receiving orthotopic heart transplants after multiple cardiac infarctions. This tissue was used to engineer fully human in vitro models of infarcted myocardium. Bone marrow was acquired from these patients. The marrow cells were sorted into populations of cells displaying CD34, CD117, and CD133. Heterospecific, tetravalent antibodies were bioengineered to bridge CD34, CD117, CD133 displayed on the stem cells with cardiac myosin of the infarcted myocardium. The sorted stem cells were administered to the infarcted myocardium in the in vitro models.Administration of the bioengineered, heterospecific antibodies preceding administration of the stem cells greatly improved the stem cells' recruitment and retention to the infarcted myocardium. Treatment of the retained stem cells with vascular endothelial growth factor and angiopoietin efficiently directed their differentiation into endothelial cells, which expressed vascular endothelial cadherin, platelet / endothelial cell adhesion molecule, claudin, and occludin, while forming tight and adherens junctions.This novel strategy improved retention of the patients' autologous bone marrow stem cells to the infarcted myocardium followed by directed vasculogenesis. Therefore, it is worth pursuing it in support of the ongoing clinical trials of cardiac regenerative therapy.
- The effect of kiwifruit (Actinidia deliciosa) cysteine protease actinidin on the occludin tight junction network in T84 intestinal epithelial cells. [JOURNAL ARTICLE]
- Food Chem Toxicol 2014 Jul 17.
Actinidin, a kiwifruit cysteine protease, is a marker allergen for genuine sensitization to this food allergen source. Inhalatory cysteine proteases have the capacity for disruption of tight junctions (TJs) enhancing the permeability of the bronchial epithelium. No such properties have been reported for allergenic food proteases so far. The aim was to determine the effect of actinidin on the integrity of T84 monolayers by evaluating its action on the TJ protein occludin. Immunoblot and immunofluorescence were employed for the detection of occludin protein alterations. Gene expression was evaluated by RT-PCR. Breach of occludin network was assessed by measuring transepithelial resistance, blue dextran leakage and passage of allergens from the apical to basolateral compartment. Actinidin exerted direct proteolytic cleavage of occludin; no alteration of occludin gene expression was detected. There was a reduction of occludin staining upon actinidin treatment as a consequence of its degradation and dispersion within the membrane. There was an increase in permeability of the T84 monolayer resulting in reduced transepithelial resistance, blue dextran leakage and passage of allergens actinidin and thaumatin-like protein from the apical to basolateral compartment. Opening of TJs by actinidin may increase intestinal permeability and contribute to the process of sensitization in kiwifruit allergy.
- Sucrose Esters Increase Drug Penetration, But Do Not Inhibit P-Glycoprotein in Caco-2 Intestinal Epithelial Cells. [JOURNAL ARTICLE]
- J Pharm Sci 2014 Jul 16.
Sucrose fatty acid esters are increasingly used as excipients in pharmaceutical products, but few data are available on their toxicity profile, mode of action, and efficacy on intestinal epithelial models. Three water-soluble sucrose esters, palmitate (P-1695), myristate (M-1695), laurate (D-1216), and two reference absorption enhancers, Tween 80 and Cremophor RH40, were tested on Caco-2 cells. Caco-2 monolayers formed a good barrier as reflected by high transepithelial resistance and positive immunostaining for junctional proteins claudin-1, ZO-1, and β-catenin. Sucrose esters in nontoxic concentrations significantly reduced resistance and impedance, and increased permeability for atenolol, fluorescein, vinblastine, and rhodamine 123 in Caco-2 monolayers. No visible opening of the tight junctions was induced by sucrose esters assessed by immunohistochemistry and electron microscopy, but some alterations were seen in the structure of filamentous actin microfilaments. Sucrose esters fluidized the plasma membrane and enhanced the accumulation of efflux transporter ligands rhodamine 123 and calcein AM in epithelial cells, but did not inhibit the P-glycoprotein (P-gp)-mediated calcein AM accumulation in MES-SA/Dx5 cell line. These data indicate that in addition to their dissolution-increasing properties sucrose esters can enhance drug permeability through both the transcellular and paracellular routes without inhibiting P-gp. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.