Junction Permeability Research Articles

Biophysical Basis of Paracellular Barrier Modulation by a Pan-Claudin-Binding Molecule.

Claudins are a 27-member protein family that form and fortify specialized cell contacts in endothelium and epithelium called tight junctions. Tight junctions restrict paracellular transport across tissues by forming molecular barriers between cells. Claudin-binding molecules thus hold promise for modulating tight junction permeability to deliver drugs or as therapeutics to treat tight junction-linked disease. The development of claudin-binding molecules, however, is hindered by their intractability and small targetable surfaces. Here, we determine that a synthetic antibody fragment (sFab) we developed binds directly to 10 claudin subtypes with nanomolar affinity by targeting claudin's paracellular-exposed surface. Application of this sFab to cells that model intestinal epithelium show that it opens the paracellular barrier comparable to a known, but application limited, tight junction modulator. This novel pan-claudin-binding molecule can probe claudin or tight junction structure and holds potential as a broad modulator of tight junction permeability for basic or translational applications.

Robotic manipulation of cardiomyocytes to identify gap junction modifiers for arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is a leading cause of sudden cardiac death among young adults. Aberrant gap junction remodeling has been linked to disease-causative mutations in plakophilin-2 (PKP2). Although gap junctions are a key therapeutic target, measurement of gap junction function in preclinical disease models is technically challenging. To quantify gap junction function with high precision and high consistency, we developed a robotic cell manipulation system with visual feedback from digital holographic microscopy for three-dimensional and label-free imaging of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The robotic system can accurately determine the dynamic height changes in the cells' contraction and resting phases, microinject drug-treated healthy and diseased iPSC-CMs in their resting phase with constant injection depth across all cells, and deposit a membrane-impermeable dye that solely diffuses between cells through gap junctions for measuring the gap junction diffusion function. The robotic system was applied toward a targeted drug screen to identify gap junction modulators and potential therapeutics for ACM. Five compounds were found to dose-dependently enhance gap junction permeability in cardiomyocytes with PKP2 knockdown. In addition, PCO 400 (pinacidil) reduced beating irregularity in a mouse model of ACM expressing mutant PKP2 (R735X). These results highlight the utility of the robotic cell manipulation system to efficiently assess gap junction function in a relevant preclinical disease model, thus providing a technique to advance drug discovery for ACM and other gap junction-mediated diseases.

Assessment of equine intestinal epithelial junctional complexes and barrier permeability using a monolayer culture system.

Gastrointestinal disease is a leading cause of death in mature horses. A lack of in vitro modeling has impeded the development of novel therapeutics. The objectives of this study were to develop and further characterize a small intestinal monolayer cell culture derived from equine jejunum including establishing normal measurements of intestinal permeability and restitution. Three-dimensional enteroids, derived from postmortem sampling of equine jejunum, were utilized to develop confluent epithelial monolayers. The presence of differentiated intestinal epithelial cell types and tight junctions were confirmed using histology, reverse transcription PCR (RT-PCR), RNAscope, protein immunofluorescence and transmission electron microscopy. Transepithelial resistance (TER) and macromolecule flux were assessed as measurements of paracellular and transcellular permeability. Scratch assays were utilized to model and assess intestinal restitution. Monolayer cell cultures reached 100% confluency by ~5-7 days. Equine jejunum monolayers were confirmed as epithelial in origin, with identification of differentiated intestinal epithelial cell types and evidence of tight junction proteins. Function of the intestinal barrier was supported by acquisition of physiologically normal TER values (179.9 ± 33.7 ohms*cm2) and limited macromolecule flux (22 ± 8.8% at 60 min). Additionally, following a scratch wound, epithelial cell monolayers migrated to close gap defects within 24 h. In conclusion, this study describes the development of a novel intestinal epithelial monolayer cell culture for equine jejunum, and provides evidence of intestinal epithelial cell differentiation, formation of physiologically relevant barrier function and use as a model of intestinal restitution to test potential therapeutics for equine colic.

The Scribble-SGEF-Dlg1 complex regulates E-cadherin and ZO-1 stability, turnover and transcription in epithelial cells.

SGEF (also known as ARHGEF26), a RhoG specific GEF, can form a ternary complex with the Scribble polarity complex proteins Scribble and Dlg1, which regulates the formation and maintenance of adherens junctions and barrier function of epithelial cells. Notably, silencing SGEF results in a dramatic downregulation of both E-cadherin and ZO-1 (also known as TJP1) protein levels. However, the molecular mechanisms involved in the regulation of this pathway are not known. Here, we describe a novel signaling pathway governed by the Scribble-SGEF-Dlg1 complex. Our results show that the three members of the ternary complex are required to maintain the stability of the apical junctions, ZO-1 protein levels and tight junction (TJ) permeability. In contrast, only SGEF is necessary to regulate E-cadherin levels. The absence of SGEF destabilizes the E-cadherin-catenin complex at the membrane, triggering a positive feedback loop that exacerbates the phenotype through the repression of E-cadherin transcription in a process that involves the internalization of E-cadherin by endocytosis, β-catenin signaling and the transcriptional repressor Slug (also known as SNAI2).

Na-caprate-induced increase in MDCK II epithelial cell leak pathway permeability and opening number is associated with disruption of basal F-actin organization.

Confluent populations of the epithelial cell line, MDCK II, develop circumferential tight junctions joining adjacent cells to create a barrier to the paracellular movement of solutes and water. Treatment of MDCK II cell populations from the apical surface with 1 mM Na-caprate increased permeability to macromolecules (Leak Pathway) without increasing monolayer disruption or cell death. Graphical analysis of the apparent permeability versus solute Stokes radius for a size range of fluorescein-dextran species indicates apical 1 mM Na-caprate enhances Leak Pathway permeability by increasing the number of Leak Pathway openings without significantly affecting opening size. Na-caprate treatment did not alter the content of any tight junction protein examined. Treatment of MDCK II cell populations with apical 1 mM Na-caprate disrupted basal F-actin stress fibers and decreased the tortuosity of the tight junctions. Treatment of MDCK II cell populations with blebbistatin, a myosin ATPase inhibitor, alone had little effect on Leak Pathway permeability but synergistically increased Leak Pathway permeability when added with 1 mM Na-caprate. Na-caprate exhibited a similar ability to increase Leak Pathway permeability in wild-type MDCK II cell monolayers and ZO-1 knockdown MDCK II cell monolayers but an enhanced ability to increase Leak Pathway permeability in monolayers of TOCA-1 knockout MDCK II cells. These results demonstrate that Na-caprate increases MDCK II cell population Leak Pathway permeability by increasing the number of Leak Pathway openings. This action is likely mediated by alterations in F-actin organization, primarily involving disruption of basal F-actin stress fibers.NEW & NOTEWORTHY This study determines the underlying change in the openings in the epithelial tight junction permeability barrier structure that leads to a change in the paracellular permeability to macromolecules (the Leak Pathway) and connects this to disruption of specific F-actin structures within the cells. It provides important and novel insights into how tight junction permeability to macromolecules is modulated by specific changes to cellular and tight junction composition/organization.

Katanin regulatory subunit B1 (KATNB1) regulates BTB dynamics through changes in cytoskeletal organization.

In this study, we have explored the role of the KATNB1 gene, a microtubule-severing protein, in the seminiferous epithelium of the rat testis. Our data have shown that KATNB1 expressed in rat brain, testes, and Sertoli cells. KATNB1 was found to co-localize with α-tubulin showing a unique stage-specific distribution across the seminiferous epithelium. Knockdown of KATNB1 by RNAi led to significant disruption of the tight junction (TJ) permeability barrier function in primary Sertoli cells cultured invitro with an established functional TJ-barrier, as well as perturbations in the microtubule and actin cytoskeleton organization. The disruption in these cytoskeletal structures, in turn, led to improper distribution of TJ and basal ES proteins essential for maintaining the Sertoli TJ function. More importantly, overexpression of KATNB1 in the testis invivo was found to block cadmium-induced blood-testis barrier (BTB) disruption and testis injury. KATNB1 exerted its promoting effects on BTB and spermatogenesis through corrective spatiotemporal expression of actin- and microtubule-based regulatory proteins by maintaining the proper organization of cytoskeletons in the testis, illustrating its plausible therapeutic implication. In summary, Katanin regulatory subunit B1 (KATNB1)plays a crucial role in BTB and spermatogenesis through its effects on the actin- and microtubule-based cytoskeletons in Sertoli cells and testis, providing important insights into male reproductive biology.

Increased activity of epithelial Cdc42 Rho GTPase and tight junction permeability in the Cftr knockout intestine.

Increased intestinal permeability is a manifestation of cystic fibrosis (CF) in people with CF (pwCF) and in CF mouse models. CF transmembrane conductance regulator knockout (Cftr KO) mouse intestine exhibits increased proliferation and Wnt/β-catenin signaling relative to wild-type mice (WT). Since the Rho GTPase Cdc42 plays a central role in intestinal epithelial proliferation and tight junction remodeling, we hypothesized that Cdc42 may be altered in the Cftr KO crypts. Immunofluorescence showed distinct tight junction localization of Cdc42 in Cftr KO fresh crypts and enteroids, the latter indicating an epithelial-autonomous feature. Quantitative PCR and immunoblots revealed similar expression of Cdc42 in the Cftr KO crypts/enteroids relative to WT, whereas pulldown assays showed increased GTP-bound (active) Cdc42 in proportion to total Cdc42 in Cftr KO enteroids. Cdc42 activity in the Cftr KO and WT enteroids could be reduced by inhibition of the Wnt transducer Disheveled. With the use of a dye permeability assay, Cftr KO enteroids exhibited increased paracellular permeability to 3 kDa dextran relative to WT. Leak permeability and Cdc42 tight junction localization were reduced to a greater extent by inhibition of Wnt/β-catenin signaling with endo-IWR1 in Cftr KO relative to WT enteroids. Increased proliferation or inhibition of Cdc42 activity with ML141 in WT enteroids had no effect on permeability. In contrast, inhibition of Cdc42 with ML141 increased permeability to both 3 kDa dextran and tight junction impermeant 500 kDa dextran in Cftr KO enteroids. These data suggest that increased constitutive Cdc42 activity may alter the stability of paracellular permeability in Cftr KO crypt epithelium.NEW & NOTEWORTHY Increased tight junction localization and GTP-bound activity of the Rho GTPase Cdc42 was identified in small intestinal crypts and enteroids of cystic fibrosis (CF) transmembrane conductance regulator knockout (Cftr KO) mice. The increase in epithelial Cdc42 activity was associated with increased Wnt signaling. Paracellular flux of an uncharged solute (3 kDa dextran) in Cftr KO enteroids indicated a moderate leak permeability under basal conditions that was strongly exacerbated by Cdc42 inhibition. These findings suggest increased activity of Cdc42 in the Cftr KO intestine underlies alterations in intestinal permeability.

Lactobacillus acidophilus inhibits the TNF-α-induced increase in intestinal epithelial tight junction permeability via a TLR-2 and PI3K-dependent inhibition of NF-κB activation.

Defective intestinal epithelial tight junction (TJ), characterized by an increase in intestinal TJ permeability, has been shown to play a critical role in thepathogenesis of inflammatory bowel disease (IBD). Tumor necrosis factor-α (TNF-α) is a key pro-inflammatory cytokine involved in the immunopathology of IBD and has been shown to cause an increase in intestinal epithelial TJ permeability. Although TNF-α antibodies and other biologics have been advanced for use in IBD treatment, these therapies are associated with severe side effects and have limited efficacy, and there is an urgent need for therapies with benign profiles and high therapeutic efficacy. Probiotic bacteria have beneficial effects and are generally safe and represent an important class of potential therapeutic agents in IBD. Lactobacillus acidophilus (LA) is one of the most used probiotics for wide-ranging health benefits, including in gastrointestinal, metabolic, and inflammatory disorders. A specific strain of LA, LA1, was recently demonstrated to have protective and therapeutic effects on the intestinal epithelial TJ barrier. However, the mechanisms of actions of LA1 remain largely unknown. The primary aim of this study was to investigate microbial-epithelial interactions and novel signaling pathways that regulate the effect of LA1 on TNF-α-induced increase in intestinal epithelial TJ permeability, using cell culture and animal model systems. Pre-treatment of filter-grown Caco-2 monolayers with LA1 prevented the TNF-α-induced increase in intestinal epithelial TJ permeability by inhibiting TNF-α-induced activation of NF-κB p50/p65 and myosin light chain kinase (MLCK) gene and kinase activity in a TLR-2-dependent manner. LA1 produced a TLR-2- and MyD88-dependent activation of NF-κB p50/p65 in immune cells; however, LA1, in intestinal cells, inhibited the NF-κB p50/p65 activation in a TLR-2-dependent but MyD88-independent manner. In addition, LA1 inhibition of NF-κB p50/p65 and MLCK gene was mediated by TLR-2 pathway activation of phosphatidylinositol 3-kinase (PI3K) and IKK-α phosphorylation. Our results demonstrated novel intracellular signaling pathways by which LA1/TLR-2 suppresses the TNF-α pathway activation of NF-κB p50/p65 in intestinal epithelial cells and protects against the TNF-α-induced increase in intestinal epithelial TJ permeability.

Deficient Gap Junction Coupling in Two Common Hearing Loss-Related Variants of GJB2.

The aim of this study was to explore the functional consequences of two common variants, p.V37I and c.299-300delAT, in the hearing loss-associated gene GJB2. Connexin 26 expression and gap junctional permeability were studied in HEK 293T cells transfected with plasmids expressing GJB2 wild-type, p.V37I, or c.299-300delAT CX26 proteins tagged with fluorescent markers. Functional analyses of various GJB2 haplotypes were conducted to thoroughly evaluate alterations in ionic and small-molecule coupling. The p.V37I protein was localized at the plasma membrane, but it failed to effectively transport intercellular propidium iodide or Ca2+ efficiently, indicating an impairment in both biochemical and ionic coupling. The presence of GJB2 p.V37I seemed to increase the cells' sensitivity to H2O2 treatment. In contrast, the known variant c.299-300delAT protein was not transported to the cell membrane and was unable to form gap junctions, remaining confined to the cytoplasm. Both ionic and biochemical coupling were defective in cells transfected with c.299-300delAT. The p.V37I and c.299-300delAT GJB2 mutations resulted in deficient gap junction-mediated coupling. Additionally, environmental factors could influence the functional outcomes of the GJB2 p.V37I mutation. These findings could pave the way for the development of molecular therapies targeting GJB2 mutations to treat hearing loss.

Bifidobacterium bifidum Strain BB1 Inhibits Tumor Necrosis Factor-α–Induced Increase in Intestinal Epithelial Tight Junction Permeability via Toll-Like Receptor-2/Toll-Like Receptor-6 Receptor Complex–Dependent Stimulation of Peroxisome Proliferator-Activated Receptor γ and Suppression of NF-κB p65

Bifidobacterium bifidum (BB) strain BB1 causes a strain-specific enhancement in intestinal epithelial tight junction (TJ) barrier. TNF-α induces an increase in intestinal epithelial TJ permeability and promotes intestinal inflammation. The major purpose of this study was to delineate the protective effect of BB1 against the TNF-α induced increase in intestinal TJ permeability and to unravel the intracellular mechanisms involved. Previously reported, TNF-α produces an increase in intestinal epithelial TJ permeability in Caco-2 monolayers and in mice. The addition of BB1 inhibited the TNF-α increase in Caco-2 intestinal TJ permeability and mouse intestinal permeability in a strain-specific manner. BB1 inhibited the TNF-α induced increase in intestinal TJ permeability by interfering the with TNF-α induced enterocyte NF-κB p50/p65 and MLCK gene activation. The BB1 protective effect against the TNF-α induced increase in intestinal permeability was mediated by TLR-2/TLR-6 heterodimer complex activation of PPAR-γ and PPAR-γ pathway inhibition of TNF-α induced IKK-α activation, which in turn resulted in a step-wise inhibition of NF-κB p50/p65, MLCK gene, MLCK kinase activity, MLCK-induced opening of the TJ barrier. In conclusion, these studies unravel novel intracellular mechanisms of BB1 protection against the TNF-α induced increase in intestinal TJ permeability. Our data show that BB1 protects against the TNF-α induced increase in intestinal epithelial TJ permeability via a PPAR-γ dependent inhibition of NF-κB p50/p65 and MLCK gene activation.

The Planar Cell Polarity Protein Fat1 in Sertoli Cell Function.

Fat (FAT atypical cadherin) and Dchs (Dachsous cadherin-related protein) in adjacent Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid interfaces create an important intercellular bridge whose adhesive function is in turn supported by Fjx1, a nonreceptor Ser/Thr protein kinase. This concept is derived from earlier studies of Drosophila, which has been confirmed in this and earlier reports as well. Herein, we use the approach of knockdown of Fat1 by RNAi using primary cultures of Sertoli cells that mimicked the blood-testis barrier (BTB) in vivo, and a series of coherent experiments including functional assays to monitor the Sertoli cell tight junction (TJ) permeability barrier and a functional in vitro TJ integrity assay to assess the role of Fat1 in the testis. It was shown that planar cell polarity (PCP) protein Fat1 affected Sertoli cell function through its modulation of actin and microtubule cytoskeletal function, altering their polymerization activity through the Fat1/Fjx1 complex. Furthermore, Fat1 is intimately associated with β-catenin and α-N-catenin, as well as with Prickle 1 of the Vangl1/Prickle 1 complex, another PCP core protein to support intercellular interactions to confer PCP. In summary, these findings support the notion that the Fat:Dchs and the Vangl2:Fzd PCP intercellular bridges are tightly associated with basal ES/TJ structural proteins to stabilize PCP function at the Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid interface to sustain spermatogenesis.

Liver X receptor activation mitigates oxysterol-induced dysfunction in fetoplacental endothelial cells

Maintaining the homeostasis of the placental vasculature is of paramount importance for ensuring normal fetal growth and development. Any disruption in this balance can lead to perinatal morbidity. Several studies have uncovered an association between high levels of oxidized cholesterol (oxysterols), and complications during pregnancy, including gestational diabetes mellitus (GDM) and preeclampsia (PE). These complications often coincide with disturbances in placental vascular function. Here, we investigate the role of two oxysterols (7-ketocholesterol, 7β-hydroxycholesterol) in (dys)function of primary fetoplacental endothelial cells (fpEC). Our findings reveal that oxysterols exert a disruptive influence on fpEC function by elevating the production of reactive oxygen species (ROS) and interfering with mitochondrial transmembrane potential, leading to its depolarization. Moreover, oxysterol-treated fpEC exhibited alterations in intracellular calcium (Ca2+) levels, resulting in the reorganization of cell junctions and a corresponding increase in membrane stiffness and vascular permeability. Additionally, we observed an enhanced adhesion of THP-1 monocytes to fpEC following oxysterol treatment. We explored the influence of activating the Liver X Receptor (LXR) with the synthetic agonist T0901317 (TO) on oxysterol-induced endothelial dysfunction in fpEC. Our results demonstrate that LXR activation effectively reversed oxysterol-induced ROS generation, monocyte adhesion, and cell junction permeability in fpEC. Although the effects on mitochondrial depolarization and calcium mobilization did not reach statistical significance, a strong trend towards stabilization of calcium mobilization was evident in LXR-activated cells. Taken together, our results suggest that high levels of systemic oxysterols link to placental vascular dysfunction and LXR agonists may alleviate their impact on fetoplacental vasculature.

Sustained Extracellular Electrical Stimulation Modulates the Permeability of Gap Junctions in rd1 Mouse Retina with Photoreceptor Degeneration.

Neurons build vast gap junction-coupled networks (GJ-nets) that are permeable to ions or small molecules, enabling lateral signaling. Herein, we investigate (1) the effect of blinding diseases on GJ-nets in mouse retinas and (2) the impact of electrical stimulation on GJ permeability. GJ permeability was traced in the acute retinal explants of blind retinal degeneration 1 (rd1) mice using the GJ tracer neurobiotin. The tracer was introduced via the edge cut method into the GJ-net, and its spread was visualized in histological preparations (fluorescent tagged) using microscopy. Sustained stimulation was applied to modulate GJ permeability using a single large electrode. Our findings are: (1) The blind rd1 retinas displayed extensive intercellular coupling via open GJs. Three GJ-nets were identified: horizontal, amacrine, and ganglion cell networks. (2) Sustained stimulation significantly diminished the tracer spread through the GJs in all the cell layers, as occurs with pharmaceutical inhibition with carbenoxolone. We concluded that the GJ-nets of rd1 retinas remain coupled and functional after blinding disease and that their permeability is regulatable by sustained stimulation. These findings are essential for understanding molecular signaling in diseases over coupled networks and therapeutic approaches using electrical implants, such as eliciting visual sensations or suppressing cortical seizures.

Umbilical cord mesenchymal stem cells from gestational diabetes show impaired ability to up-regulate paracellular permeability from sub-endothelial niche.

In vitro studies have shown that Wharton's jelly mesenchymal stem cells (WJ-MSCs) can cross umbilical and uterine endothelial barriers and up-regulate endothelial junctional integrity from sub-endothelial niches. This pericytic behaviour may be lost in pregnancies complicated by gestational diabetes (GDM), where increased vascular permeability and junctional disruption are reported. The aim of the present study was to investigate whether WJ-MSCs isolated from GDM pregnancies displayed any changes in morphology, proliferation, VEGF-A secretion, and their ability to influence paracellular junctional composition and permeability. WJ-MSCs were isolated from human umbilical cords from normal pregnancies (nWJ-MSCs, n=13) and those complicated by GDM (gWJ-MSCs), either diet-controlled (d-GDM, n=13) or metformin-treated (m-GDM, n=9). We recorded that 4-fold more WJ-MSCs migrated from m-GDM, and 2.5-fold from d-GDM cord samples compared with the normal pregnancy. gWJ-MSCs showed a less predominance of spindle-shaped morphology and secreted 3.8-fold more VEGF-A compared with nWJ-MSCs. The number of cells expressing CD105 (Endoglin) was higher in gWJ-MSCs compared with nWJ-MSCs (17%) at P-2. The tracer leakage after 24 h across the HUVEC + gWJ-MSCs bilayer was 22.13% and 11.2% higher in the m-GDM and d-GDM, respectively, HUVEC + nWJ-MSCs. Transfection studies with siRNAs that target Endoglin were performed in n-WJ-MSCs; transfected cells were co-cultured with HUVEC followed by permeability studies and VE-cadherin analyses. Loss of Endoglin also led to increased VEGF-A secretion, increased permeability and affected endothelial stabilization. These results reinforce the pericytic role of nWJ-MSCs to promote vascular repair and the deficient ability of gWJ-MSCs to maintain endothelial barrier integrity.

Immunofluorescence study of cytoskeleton in endothelial cells induced with malaria sera

BackgroundEndothelial cells (ECs) play a major role in malaria pathogenesis, as a point of direct contact of parasitized red blood cells to the blood vessel wall. The study of cytoskeleton structures of ECs, whose main functions are to maintain shape and provide strength to the EC membrane is important in determining the severe sequelae of Plasmodium falciparum malaria. The work investigated the cytoskeletal changes (microfilaments-actin, microtubules-tubulin and intermediate filaments-vimentin) in ECs induced by malaria sera (Plasmodium vivax, uncomplicated P. falciparum and complicated P. falciparum), in relation to the levels of pro-inflammatory cytokines.MethodsMorphology and fluorescence intensity of EC cytoskeleton stimulated with malaria sera were evaluated using immunofluorescence technique. Levels of tumour necrosis factor (TNF) and interferon (IFN)-gamma (γ) were determined using enzyme-linked immunosorbent assay (ELISA). Control experimental groups included ECs incubated with media alone and non-malaria patient sera. Experimental groups consisted of ECs incubated with malaria sera from P. vivax, uncomplicated P. falciparum and complicated P. falciparum. Morphological scores of cytoskeletal alterations and fluorescence intensity were compared across each experiment group, and correlated with TNF and IFN-γ.ResultsThe four morphological changes of cytoskeleton included (1) shrinkage of cytoskeleton and ECs with cortical condensation, (2) appearance of eccentric nuclei, (3) presence of “spiking pattern” of cytoskeleton and EC membrane, and (4) fragmentation and discontinuity of cytoskeleton and ECs. Significant damages were noted in actin filaments compared to tubulin and vimentin filaments in ECs stimulated with sera from complicated P. falciparum malaria. Morphological damages to cytoskeleton was positively correlated with fluorescence intensity and the levels of TNF and IFN-γ.ConclusionsECs stimulated with sera from complicated P. falciparum malaria showed cytoskeletal alterations and increased in fluorescence intensity, which was associated with high levels of TNF and IFN-γ. Cytoskeletal changes of ECs incubated with complicated P. falciparum malaria sera can lead to EC junctional alteration and permeability changes, which is mediated through apoptotic pathway. The findings can serve as a basis to explore measures to strengthen EC cytoskeleton and alleviate severe malaria complications such as pulmonary oedema and cerebral malaria. In addition, immunofluorescence intensity of cytoskeleton could be investigated as potential prognostic indicator for malaria severity.

Assessment of Serum Zonulin Levels in Individuals Diagnosed With Chronic Spontaneous Urticaria.

There have been reports indicating a correlation between heightened intestinal permeability and many autoimmune and chronic inflammatory disorders. The involvement of autoimmunity is now recognized as a significant factor in the development of chronic spontaneous urticaria (CSU). Zonulin is an important biomarker that regulates tight junction permeability within cells in the gastrointestinal tract, hence facilitating intestinal permeability. To evaluate the correlation of CSU with intestinal permeability by measuring the serum levels of zonulin in patients diagnosed with CSU. The study included 60 patients diagnosed with CSU and 64 age- and sex-matched healthy individuals as controls. Levels of serum zonulin were determined using the ELISA method. Although the serum zonulin value of the patients was higher compared to the controls, the difference did not reach a significant level (24.65±8.49 ng/ml vs. 21.03±7.36 ng/ml, p=0.077). The serum zonulin level had a significant correlation with the urticaria activity score in the CSU group (p=0.013). The results of the current study revealed that serum zonulin values significantly differed between patients with CSU and healthy controls. This study is important in terms of being the first to investigate the serum zonulin levels in CSU. However, there is a need for further studies with larger patient groups.

Fenofibrate reduces glucose-induced barrier dysfunction in feline enteroids

Diabetes mellitus (DM) is a common chronic metabolic disease in humans and household cats that is characterized by persistent hyperglycemia. DM is associated with dysfunction of the intestinal barrier. This barrier is comprised of an epithelial monolayer that contains a network of tight junctions that adjoin cells and regulate paracellular movement of water and solutes. The mechanisms driving DM-associated barrier dysfunction are multifaceted, and the direct effects of hyperglycemia on the epithelium are poorly understood. Preliminary data suggest that fenofibrate, An FDA-approved peroxisome proliferator-activated receptor-alpha (PPARα) agonist drug attenuates intestinal barrier dysfunction in dogs with experimentally-induced DM. We investigated the effects of hyperglycemia-like conditions and fenofibrate treatment on epithelial barrier function using feline intestinal organoids. We hypothesized that glucose treatment directly increases barrier permeability and alters tight junction morphology, and that fenofibrate administration can ameliorate these deleterious effects. We show that hyperglycemia-like conditions directly increase intestinal epithelial permeability, which is mitigated by fenofibrate. Moreover, increased permeability is caused by disruption of tight junctions, as evident by increased junctional tortuosity. Finally, we found that increased junctional tortuosity and barrier permeability in hyperglycemic conditions were associated with increased protein kinase C-α (PKCα) activity, and that fenofibrate treatment restored PKCα activity to baseline levels. We conclude that hyperglycemia directly induces barrier dysfunction by disrupting tight junction structure, a process that is mitigated by fenofibrate. We further propose that counteracting modulation of PKCα activation by increased intracellular glucose levels and fenofibrate is a key candidate regulatory pathway of tight junction structure and epithelial permeability.

Isomerization and phosphorylation significantly influence the effect of beta amyloid on blood‐brain barrier endothelial cells

AbstractBackgroundThe blood‐brain barrier (BBB) is typically compromised in Alzheimer’s disease (AD): its permeability and intracellular parameters of the endothelial cells are affected. AD‐related amyloid isoforms –Aβ42, isoD7‐Aβ42 and pS8‐Aβ42, inducing amyloidogenesis in a different mode – may have peripheral origin and affect the BBB cells. This study aimed to assess this effect.MethodMouse brain endothelial cells bEnd.3 underwent incubation with 10 µM of Aβ42, isoD7‐Aβ42 and pS8‐Aβ42 for 4, 24 and 48 hours to assess nitric oxide (NO), glutathione (GSH), reactive oxygen species (ROS), mitochondrial potential, Ca2+, cell viability by flow cytometry and mitochondrial functioning by Seahorse technology. To analyze the integrity of the cellular monolayer, the expression level of tight junction proteins and permeability of bEnd.3 monolayer to fluorescent tracers were assessed by Western Blotting and transwell‐modeling, correspondingly .ResultpS8‐Aβ42 and isoD7‐Aβ42 effects on the redox status of the cells and mitochondria functioning differ from the effects of Aβ42. Even at 4 hours, Aβ42 and isoD7‐Aβ42 induce immense changes in mitochondrial potential and NO level but pS8‐Aβ42 does not. Prolonged incubation with three isoforms leads to an increase in ROS, and greater increase is observed after incubation with isoD7‐Aβ42. pS8‐Aβ42 induces ROS increase later than other isoforms. Above all, incubation with pS8‐Aβ42 results in a less pronounced NO и GSH growth compared with Aβ42 и isoD7‐Aβ42 which induce activation of mitochondrial respiration and increase in mitochondrial potential, compared with pS8‐Aβ42. IsoD7‐Aβ42 is mostly toxic to bEnd.3 cells. Although Aβ42 and isoD7‐Aβ42 significantly affect redox parameters of bEnd.3 cells, their effects on BBB permeability and tight junction protein expression are less pronounced. Our data demonstrate that inhibition of NO synthases and NMDA receptors induces changes in BBB cells’ response to Aβ peptides.ConclusionAmyloid isoforms differently affect redox parameters of BBB cells significantly modulating mitochondria functioning. Iso‐Aβ42 induces higher cytotoxicity, thus, being a more pathogenic form. Post‐translational modifications of Aβ42 alter the amyloid effect on the BBB cells. It may result in different changes in BBB functioning and its permeability under amyloid isoforms.This research was funded by Russian Science Foundation (Grant No. #19‐74‐30007).

Prophylactic function of Garcinia morella dried fruits in preventing intestinal barrier disruption by lowering tight junction permeability and inflammation

The most frequent cause of gastrointestinal disorders characterized by inflammation and disturbance of the gastrointestinal system, is infection from various bacterial strains. Garcinia morella (Gaertn.) Desr. (Clusiaceae) a well-known traditional food is also used in healing digestive disorders for centuries. However, its protective activity in the pathogenesis of pathogen-induced gut disruption remains unclear. This study investigated the beneficial effect of G. morella fruits in remediating pathogen-induced intestinal disruption in Caco-2 cells. The antimicrobial activity of G. morella hydroalcoholic fruit extract with known chemical composition against the opportunistic pathogens, Escherichia coli and Enterococcus faecalis were evaluated. Transepithelial electrical resistance and dextran permeability assay were performed to evaluate the permeability in Caco-2 monolayers. Western blot assays were performed to detect the expression of the intestinal tight junction proteins: zonula occludens-1, claudin-1, occludin, and E-cadherin; and phosphorylation of inflammatory markers: ERK (Extracellular signal-regulated kinase), and JNK (Jun N-terminal kinase). Three bioactive compounds: garcinol, hydroxycitric acid, and hydroxycitric acid lactone were identified, qualitatively and quantitatively assessed with HPLC (high performance liquid chromatography). G. morella hydroalcoholic extract and its compounds prevented the decrease in transepithelial electrical resistance caused by pathogen infection in Caco-2 cells. G. morella and compounds also decreased permeability in Caco-2 monolayer, increased by pathogen infection. G. morella and garcinol enhanced tight junction protein expression. A decrease in extracellular-signal-regulated kinase and c-Jun N-terminal kinase phosphorylation was observed in G. morella and garcinol-treated cells. Overall, the results showed the potential of G. morella and its compounds in maintaining intestinal integrity and validate its traditional claims in ameliorating stomach ailments. The study also provided new guidance in development of nutraceuticals that aid in curing leaky gut.

Honokiol Prevents Intestinal Barrier Dysfunction in Mice with Severe Acute Pancreatitis and Inhibits JAK/STAT1 Pathway and Acetylation of HMGB1.

To investigate the effect of honokiol (HON) and the role of high-mobility group protein B1 (HMGB1) on the pathogenesis of severe acute pancreatitis (SAP). Thirty mice were numbered according to weight, and randomly divided into 5 groups using a random number table, including control, SAP, SAP and normal saline (SAP+NS), SAP and ethyl pyruvate (SAP+EP), or SAP+HON groups, 6 mice in each group. Samples of pancreas, intestine, and blood were collected 12 h after SAP model induction for examination of pathologic changes, immune function alterations by enzyme linked immunosorbent assay (ELISA), and Western blot. In vitro experiments, macrophages were divided into 5 groups, the control, lipopolysaccharide (LPS), LPS+DMSO (DMSO), LPS+anti-HMGB1 monoclonal antibody (mAb), and LPS+ HON groups. The tight connection level was determined by transmission electron microscopy and fluorescein isothiocyanate-labeled. The location and acetylation of HMGB1 were measured by Western blot. Finally, pyridone 6 and silencing signal transducer and activator of the transcription 1 (siSTAT1) combined with honokiol were added to determine whether the Janus kinase (JAK)/ STAT1 participated in the regulation of honokiol on HMGB1. The protein expression levels of HMGB1, JAK, and STAT1 were detected using Western blot. Mice with SAP had inflammatory injury in the pancreas, bleeding of intestinal tissues, and cells with disrupted histology. Mice in the SAP+HON group had significantly fewer pathological changes. Mice with SAP also had significant increases in the serum levels of amylase, lipase, HMGB1, tumor necrosis factor- α, interleukin-6, diamine oxidase, endotoxin-1, and procalcitonin. Mice in the SAP+HON group did not show these abnormalities (P<0.01). Studies of Caco-2 cells indicated that LPS increased the levels of occludin and claudin-1 as well as tight junction permeability, decreased the levels of junctional adhesion molecule C, and elevated intercellular permeability (P<0.01). HON treatment blocked these effects. Studies of macrophages indicated that LPS led to low nuclear levels of HMGB1, however, HON treatment increased the nuclear level of HMGB1 (P<0.01). HON treatment also inhibited the expressions of JAK1, JAK2, and STAT1 (P<0.01) and increased the acetylation of HMGB1 (P<0.05). HON prevented intestinal barrier dysfunction in SAP by inhibiting HMGB1 acetylation and JAK/STAT1 pathway.