Occludin

To describe the effects of epididymal and vasal ligation, in an experimental rat model, by using connexin 43 and occludin immunohistochemistry as well as transmission electron microscopy.Comparative and controlled experimental research study.University animal research and histology laboratories in Turkey.Wistar male rats in experimental and control groups.The control group underwent sham operation (n = 7). The first experimental group (n = 7) underwent unilateral epididymal ligation, whereas the second experimental group (n = 7) underwent unilateral vasal ligation to induce experimental epididymal and vasal obstruction models, respectively. All animals were then killed at 90 days.Immunohistochemical expression of connexin 43 and occludin for testicular tissues was determined after epididymal and vasal obstruction models. Ultrastructural morphological changes were examined by electron microscopy.Results of the semiquantitative analysis revealed that expressions of both occludin and connexin 43 in the rat testis were decreased in the experimental groups compared with in the sham-operated group. However, changes after vasal ligation were more prominent. Ultrastructural examination confirmed decreased intercellular communication as well as increased cellular degeneration among the ipsilateral and contralateral testicular tissues.Immunohistochemical expression of occludin and connexin 43 were decreased in the testis after vasal and epididymal ligation when compared with the sham-operated group. Ultrastructural changes indicating cell degeneration were more prominent after vasal ligation.

Epithelial permeability to ions and larger molecules in the gut is essential for fluid balance, and its dysregulation contributes to intestinal pathology. We investigated the effect of digestive serine proteases on epithelial paracellular permeability. Trypsin, chymotrypsin, and elastase elicited sustained increases in transepithelial resistance (RTE) in polarized monolayers of three intestinal epithelial cell lines. This effect was reflected by decreases in paracellular conductances of Na+ and Cl− and a concomitant decrease in permeability to 3,000 molecular weight dextran. The enzyme activities of the proteases were required, yet activators of known protease-activated receptors (PARs) did not reproduce the effect of these proteases on RTE. PKCζ isoform-specific inhibitor significantly reduced the trypsin-induced increase in RTE whereas PKCζ activity was increased in cells treated with trypsin and chymotrypsin compared with control cells; this activity was reduced to control levels...

Since ingestion is one of the main routes of entry of nanoparticles (NPs) in our organism, simple and fast in vitro models of the intestinal barrier can be helpful to evaluate NPs risk. The human colon adenocarcinoma Caco-2 cell line has been extensively used due to its ability to differentiate, forming a well-structured cell monolayer. In this study, we have used these differentiated cells as a model of intestinal barrier to evaluate a wide set of effects caused by exposure to silver nanoparticles (AgNPs) with an average size of 7.74 nm. Different parameters such as toxicity, monolayer integrity and permeability (assessed by changes in cells' morphology and gene expression pattern), internalization (uptake), translocation, and induction of DNA damage (DNA breaks and oxidative DNA damage) were evaluated. No significant effects were observed on the monolayer's integrity/permeability after exposure to silver nanoparticles, although cellular uptake was demonstrated by using con...

Deoxynivalenol (DON) and fumonisins (FB) are mycotoxins produced by Fusarium species, which naturally co-occur in animal diets. The gastrointestinal tract represents the first barrier met by exogenous food/feed compounds. The purpose of the present study was to investigate the effects of DON and FB, alone and in combination, on some intestinal parameters, including morphology, histology, expression of cytokines and junction proteins. A total of twenty-four 5-week-old piglets were randomly assigned to four different groups, receiving separate diets for 5 weeks: a control diet; a diet contaminated with either DON (3 mg/kg) or FB (6 mg/kg); or both toxins. Chronic ingestion of these contaminated diets induced morphological and histological changes, as shown by the atrophy and fusion of villi, the decreased villi height and cell proliferation in the jejunum, and by the reduced number of goblet cells and lymphocytes. At the end of the experiment, the expression levels of several cytokine...

Homeostasis of the central nervous system (CNS) microenvironment is maintained by the blood-brain barrier (BBB) which regulates the transport of molecules from blood into brain and back. Many disorders change the functionality and integrity of the BBB. Glucocorticoids are being used sucessfully in the treatment of some disorders while their effects on others are questionable. In addition, conflicting results between clinical and experimental experience using animal models has arisen, so that the results of molecular studies in animal models need to be revisited in an appropriate in vitro model of the human BBB for more effective treatment strategies. Using the human brain microvascular endothelial cell line hCMEC/D3, the influence of glucocorticoids on the expression of barrier constituting adherens junction and tight junction transmembrane proteins (VE-cadherin, occludin, claudins) was investigated and compared to other established BBB models. In hCMEC/D3 cells the administration o...

African trypanosomiasis, sleeping sickness in humans, is caused by the systemic infection of the host by the extracellular parasite, the African trypanosome. The pathogenetic mechanisms of the severe symptoms of central nervous system involvement are still not well understood. The present study examined the routes of haematogenous spread of Trypanosoma brucei brucei (Tbb) to the brain, in particular on the question whether parasites can cross the blood-brain barrier, as well as their effect on tight junction proteins. Rats were infected with Tbb and at various times post-infection, the location of the parasite in the central nervous system was examined in relation to the brain vascular endothelium, visualized with an anti-glucose transporter-1 antibody. The tight junction-specific proteins occludin and zonula occludens 1, and the possible activation of the endothelial cell adhesion molecules ICAM-1 and VCAM-1 were also studied. At 12 and 22 days post-infection, the large majority of parasites were confined within blood vessels. At this stage, however, some parasites were also clearly observed in the brain parenchyma. This was accompanied by an upregulation of ICAM-1/VCAM-1. At later stages, 42, 45 and 55 days post-infection, parasites could still be detected within or in association with blood vessels. In addition, the parasite was now frequently found in the brain parenchyma and the extravasation of parasites was more prominent in the white matter than the cerebral cortex. A marked penetration of parasites was seen in the septal nuclei. In spite of this, occludin and zonula occludens 1 staining of the vessels was preserved. The results indicate that the Tbb parasite is able to cross the blood-brain barrier in vivo, without a generalized loss of tight junction proteins.

Tight junction integral membrane proteins such as claudins and occludin are tethered to the actin cytoskeleton by adaptor proteins, notably the closely related zonula occludens (ZO) proteins ZO-1, ZO-2, and ZO-3. All three ZO proteins have recently been inactivated in mice. Although ZO-3 knockout mice lack an obvious phenotype, animals deficient in ZO-1 or ZO-2 show early embryonic lethality. Here, we rescue the embryonic lethality of ZO-2 knockout mice by injecting ZO-2(-/-) embryonic stem (ES) cells into wild-type blastocysts to generate viable ZO-2 chimera. ZO-2(-/-) ES cells contribute extensively to different tissues of the chimera, consistent with an extraembryonic requirement for ZO-2 rather than a critical role in epiblast development. Adult chimera present a set of phenotypes in different organs. In particular, male ZO-2 chimeras show reduced fertility and pathological changes in the testis. Lanthanum tracer experiments show a compromised blood-testis barrier. Expression le...

The initiating etiologic factor in Crohn's disease (CD) remains unclear. SAMP1/YitFc (SAMP) mice develop chronic ileitis similar to human CD. We used bone marrow chimeras to determine if SAMP ileitis results from a primary immunological defect or from dysregulated mucosal immunity secondary to intrinsic, nonhematopoietic (e.g., epithelial) dysfunction. SAMP mice receiving wild-type (AKR) BM developed severe ileitis, whereas SAMP BM did not confer ileitis to WT recipients. WT lymphocytes from reconstituted SAMP mice resembled native SAMP populations in regard to surface phenotype and cytokine production. Ilea from native SAMP mice and SAMP recipients of wild-type BM displayed decreased epithelial barrier resistance ex vivo and increased epithelial permeability in vivo compared to native WT mice and AKR recipients of SAMP BM. This permeability defect preceded the development of ileal inflammation, was present in the absence of commensal bacteria, and was accompanied by altered ile...

The apical-most epithelial intercellular junction, referred to as the tight junction (TJ), regulates paracellular solute flux in diverse physiological and pathological states. TJ affiliations with the apical filamentous actin (F-actin) cytoskeleton are crucial in regulating TJ function. F-actin organization is influenced by the Rho GTPase family, which also controls TJ function. To explore the role of Rho GTPases in regulating TJ structure and function, we utilized Escherichia coli cytotoxic necrotizing factor-1 (CNF-1) as a tool to activate constitutively Rho, Rac and Cdc42 signaling in T84 polarized intestinal epithelial monolayers. The biological effects of the toxin were polarized to the basolateral membrane, and included profound reductions in TJ gate function, accompanied by displacement of the TJ proteins occludin and zonula occludens-1 (ZO-1), and reorganization of junction adhesion molecule-1 (JAM-1) away from the TJ membrane. Immunogold electron microscopy revealed occludi...

Tight junctions (TJs) at the blood-brain barrier (BBB) dynamically alter paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS in response to external stressors, such as pain, inflammation, and hypoxia. In this study, we investigated the effect of λ-carrageenan-induced peripheral inflammatory pain (i.e., hyperalgesia) on the oligomeric assembly of the key TJ transmembrane protein, occludin. Oligomerization of integral membrane proteins is a critical step in TJ complex assembly that enables the generation of tightly packed, large multiprotein complexes capable of physically obliterating the interendothelial space to inhibit paracellular diffusion. Intact microvessels isolated from rat brains were fractionated by detergent-free density gradient centrifugation, and gradient fractions were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis/ Western blot. Injection of λ-carrageenan into the rat hind paw produced after 3 h a marked change in the relative amounts of oligomeric, dimeric, and monomeric occludin isoforms associated with different plasma membrane lipid raft domains and intracellular compartments in endothelial cells at the BBB. Our findings suggest that increased BBB permeability (i.e., leak) associated with λ-carrageenan-induced peripheral inflammatory pain is promoted by the disruption of disulfide-bonded occludin oligomeric assemblies, which renders them incapable of forming an impermeant physical barrier to paracellular transport.