The Role of ZO-2 in Modulating JAM-A and γ-Actin Junctional Recruitment, Apical Membrane and Tight Junction Tension, and Cell Response to Substrate Stiffness and Topography.

This work analyzes the role of the tight junction (TJ) protein ZO-2 on mechanosensation. We found that the lack of ZO-2 reduced apical membrane rigidity measured with atomic force microscopy, inhibited the association of γ-actin and JAM-A to the cell border, and instead facilitated p114RhoGEF and afadin accumulation at the junction, leading to an enhanced mechanical tension at the TJ measured by FRET, with a ZO-1 tension probe, and increased tricellular TJ tension. Simultaneously, adherens junction tension measured with an E-cadherin probe was unaltered. The stability of JAM-A and ZO-2 binding was assessed by a collaborative in silico study. The absence of ZO-2 also impacted the cell response to the substrate, as monolayers plated in 20 kPa hydrogels developed holes not seen in parental cultures and displayed a retarded elongation and formation of cell aggregates. The absence of ZO-2 was sufficient to induce YAP and Snail nuclear accumulation in cells cultured over glass, but when ZO-2 KD cells were plated in nanostructured ridge arrays, they displayed an increased abundance of nuclear Snail and conspicuous internalization of claudin-4. These results indicate that the absence of ZO-2 also impairs the response of cells to substrate stiffness and exacerbates transformation triggered by substrate topography.

Disheveled-1 Interacts with Claudin-5 and Contributes to Norrin-Induced Endothelial Barrier Restoration.

Previous studies have revealed that norrin can reverse vascular endothelial-growth-factor (VEGF)-induced permeability in a ß-catenin-dependent pathway. Here, we have explored the contribution of disheveled-1 (DVL1) in norrin-induced blood-retinal barrier (BRB) restoration. We provide evidence that in addition to canonical signaling, DVL1 promotes tight junction (TJ) stabilization through a novel, non-canonical signaling pathway involving direct claudin-5 (CLDN5) binding. Immunofluorescence staining of rat retinal cross-sections showed enriched expression of DVL1 and 3 at endothelial capillaries and co-localization with CLDN5 and ZO-1 at the TJ complex in primary bovine retinal endothelial cells (BRECs). Barrier properties of BRECs were determined via measurements of trans-endothelial electrical resistance (TEER) or permeability to 70 kDa RITC-dextran. These studies demonstrated that norrin restoration of barrier properties after VEGF treatment required DVL1 as an siRNA knockdown of Dvl1 but not Dvl2 or Dvl3, reduced basal barrier properties and ablated norrin-induced barrier restoration. However, loss of Dvl1 did not decrease ß-catenin signaling activity as measured by Axin2 mRNA expression, suggesting the contribution of a non-canonical pathway. DVL and TJ protein interactions were analyzed via co-immunoprecipitation of endogenous protein in BRECs, which demonstrated that DVL1 interacts with both CLDN5 and ZO-1, while DVL3 interacts only with ZO-1. These interactions were most abundant after inducing BRB restoration by treating BRECs with VEGF and norrin. DVL has previously been shown to form intramolecular bindings between the C-terminal PDZ-binding motif (PDZ-BM) with an internal PDZ domain. Co-transfection of HEK293 cells with DVL1 and CLDN5 or relevant mutants revealed that DVL1 interacts with CLDN5 through the DVL PDZ domain binding, CLDN5 PDZ-BM, in competition with DVL1 PDZ-BM, since DVL/CLDN5 interaction increases with deletion of the DVL1 PDZ-BM and decreases by co-expressing the C-terminal fragment of DVL1 containing the PDZ-BM or through deletion of CLDN5 PDZ-BM. In BREC cells, transfection of the C-terminal fragment of DVL1 downregulates the expression of CLDN5 but does not affect the expression of other proteins of the TJs, including ZO-1, occludin, CLDN1 or VE-cadherin. Blocking DVL1/CLDN5 interaction increased basal permeability and prevented norrin induction of barrier properties after VEGF. Combined with previous data, these results demonstrate that norrin signals through both a canonical ß-catenin pathway and a non-canonical signaling pathway by which DVL1 directly binds to CLDN5 to promote barrier properties.

What do we know about blood-testis barrier? current understanding of its structure and physiology.

Blood-testis barrier (BTB) creates a particular compartment in the seminiferous epithelium. Contacting Sertoli cell-Sertoli cell plasma membranes possess specialized junction proteins which present a complex dynamic of formation and dismantling. Thus, these specialized structures facilitate germ cell movement across the BTB. Junctions are constantly rearranged during spermatogenesis while the BTB preserves its barrier function. Imaging methods are essential to studying the dynamic of this sophisticated structure in order to understand its functional morphology. Isolated Sertoli cell cultures cannot represent the multiple interactions of the seminiferous epithelium and in situ studies became a fundamental approach to analyze BTB dynamics. In this review, we discuss the contributions of high-resolution microscopy studies to enlarge the body of morphofunctional data to understand the biology of the BTB as a dynamic structure. The first morphological evidence of the BTB was based on a fine structure of the junctions, which was resolved with Transmission Electron Microscopy. The use of conventional Fluorescent Light Microscopy to examine labelled molecules emerged as a fundamental technique for elucidating the precise protein localization at the BTB. Then laser-scanning confocal microscopy allowed the study of three-dimensional structures and complexes at the seminiferous epithelium. Several junction proteins, like the transmembrane, scaffold and signaling proteins, were identified in the testis using traditional animal models. BTB morphology was analyzed in different physiological conditions as the spermatocyte movement during meiosis, testis development, and seasonal spermatogenesis, but also structural elements, proteins, and BTB permeability were studied. Under pathological, pharmacological, or pollutant/toxic conditions, there are significant studies that provide high-resolution images which help to understand the dynamic of the BTB. Notwithstanding the advances, further research using new technologies is required to gain information on the BTB. Super-resolution light microscopy is needed to provide new research with high-quality images of targeted molecules at a nanometer-scale resolution. Finally, we highlight research areas that warrant future studies, pinpointing new microscopy approaches and helping to improve our ability to understand this barrier complexity.

Bisphenols as promoters of the dysregulation of cellular junction proteins of the blood-testis barrier in experimental animals: A systematic review of the literature.

Daily, people are exposed to chemicals and environmental compounds such as bisphenols (BPs). These substances are present in more than 80% of human fluids. Human exposure to BPs is associated with male reproductive health disorders. Some of the main targets of BPs are intercellular junction proteins of the blood-testis barrier (BTB) in Sertoli cells because BPs alter the expression or induce aberrant localization of these proteins. In this systematic review, we explore the effects of BP exposure on the expression of BTB junction proteins and the characteristics of in vivo studies to identify potential gaps and priorities for future research. To this end, we conducted a systematic review of articles. Thirteen studies met our inclusion criteria. In most studies, animals treated with bisphenol-A (BPA) showed decreased occludin expression at all tested doses. However, bisphenol-AF treatment did not alter occludin expression. Cx43, ZO-1, ß-catenin, nectin-3, cortactin, paladin, and claudin-11 expression also decreased in some tested doses of BP, while N-cadherin and FAK expression increased. BP treatment did not alter the expression of α and γ catenin, E-cadherin, JAM-A, and Arp 3. However, the expression of all these proteins was altered when BPA was administered to neonatal rodents in microgram doses. The results show significant heterogeneity between studies. Thus, it is necessary to perform more research to characterize the changes in BTB protein expression induced by BPs in animals to highlight future research directions that can inform the evaluation of risk of toxicity in humans.

Regionalized analysis of gut barrier components in the small intestine of BALB/c mice following chronic immobilization stress.

BACKGROUND: Microbiota and tight junction proteins (TJPs) are components of the gut barrier, and are considered stress targets that have deleterious effects on intestinal homeostasis. OBJECTIVES: This study aimed to evaluate the effects of chronic immobilization stress on selected small intestine homeostasis parameters. MATERIAL AND METHODS: Female BALB/c mice were divided into a stress group that underwent short-term immobilization for 2 h per day for 4 consecutive days, and a non-stressed control group (n = 6 per group). Proximal and distal small intestine samples were excised to assess colony-forming units per gram (CFU/g) of total bifidobacteria in selective agar plates, luminal albumin was assessed using immune-enzymatic assay, pro-inflammatory cytokines were evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and TJPs (pore-forming, claudin (Cld)-2; pore-sealing, Cld-4; ambiguous, Cld-7, -12 and -15) were assessed with RT-qPCR and western blotting. RESULTS: Compared with the control group, the stress group had lower body weight and energy intake. In the distal region, the stressed mice had lower bifidobacteria count and messenger ribonucleic acid (mRNA) expression of Cld-2, Cld-4 and Cld-12, though they had more albumin and higher interleukin (IL)-6 mRNA expression. Within the proximal region, the stressed mice had higher mRNA expression of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), IL-6, Cld-7, Cld-12, and Cld-15, along with lower levels of IL-10 and Cld-4. However, mRNA and protein expression of TJPs were discordant. CONCLUSIONS: These findings indicate divergent stress-induced outcomes in the small intestine, evidenced by the elicitation of a pro-inflammatory response and decreased anti-inflammatory response in the duodenum, and by increased albumin transudation and decreased bifidobacterial growth in the distal region.

Plasmodium falciparum rhoptry neck protein 4 has conserved regions mediating interactions with receptors on human erythrocytes and hepatocyte membrane.

Plasmodium falciparum-related malaria represents a serious worldwide public health problem due to its high mortality rates. P. falciparum expresses rhoptry neck protein 4 (PfRON4) in merozoite and sporozoite rhoptries, it participates in tight junction-TJ formation via the AMA-1/RON complex and is refractory to complete genetic deletion. Despite this, which PfRON4 key regions interact with host cells remain unknown; such information would be useful for combating falciparum malaria. Thirty-two RON4 conserved region-derived peptides were chemically synthesised for determining and characterising PfRON4 regions having high host cell binding affinity (high activity binding peptides or HABPs). Receptor-ligand interaction/binding assays determined their specific binding capability, the nature of their receptors and their ability to inhibit in vitro parasite invasion. Peptides 42477, 42479, 42480, 42505 and 42513 had greater than 2% erythrocyte binding activity, whilst peptides 42477 and 42480 specifically bound to HepG2 membrane, both of them having micromolar and submicromolar range dissociation constants (Kd). Cell-peptide interaction was sensitive to treating erythrocytes with trypsin and/or chymotrypsin and HepG2 with heparinase I and chondroitinase ABC, suggesting protein-type (erythrocyte) and heparin and/or chondroitin sulphate proteoglycan receptors (HepG2) for PfRON4. Erythrocyte invasion inhibition assays confirmed HABPs' importance during merozoite invasion. PfRON4 800-819 (42477) and 860-879 (42480) regions specifically interacted with host cells, thereby supporting their inclusion in a subunit-based, multi-antigen, multistage anti-malarial vaccine.

Cooperative Binding of SRSF3 to Structured 3'ss-α Exon RNA during α Exon Inclusion in the ZO-1 mRNA.

ZO-1α+ and ZO-1α- proteins are expressed in hermetic and leaky tight junctions, respectively. Two cis-acting distant exonic elements partly activate the 240 nucleotide-long α exon producing the ZO-1α+ isoform. However, the elements within and around the α exon and their respective factors involved in its splicing are unknown. To study the dynamic interaction between SRSF3 and its bioinformatically predicted target sites around the 3'ss upstream of the α exon during its activation, we performed EMSA, crosslinking, and in vivo splicing assays by ZO-1 minigene expression and siRNA-mediated silencing in transfected cells. Using V1 RNase, we probed the possible formation of a hairpin RNA structure between the intronic and proximal exonic SRSF3 binding sites. The hairpin sufficed for complex formations in the EMSA. The interaction of SRSF3 with the intronic site promoted the cooperative binding of SRSF3 to the exonic site. Finally, SRSF3 restored α exon activation in SRSF3 knockdown transfectants. Altogether, our results show that SRSF3-hairpin RNA interaction is crucial in the early recognition of 3'ss for α exon activation. It remains to be explored whether SRSF3 recruits or stabilizes the binding of other factors or brings separate splice sites into proximity.

Consumption of a multi-deficient diet causes dynamic changes in the intestinal morphofunctional barrier, body composition and impaired physical development in post-weaning mice.

Few studies have focused on nutrient-deficient diets and associated pathobiological dynamics of body composition and intestinal barrier function. This study evaluated the impact of a nutrient-deficient diet on physical development and intestinal morphofunctional barrier in mice. C57BL/6 (21 days of age) mice were fed a Northeastern Brazil regional basic diet (RBD) or a control diet for 21 d. The animals were subjected to bioimpedance analysis, lactulose test, morphometric analysis and quantitative reverse transcription-PCR to evaluate tight junctions and intestinal transporters. RBD feeding significantly reduced weight (P < 0·05) from day 5, weight gain from day 3 and tail length from day 14. The intake of RBD reduced total body water, extracellular fluid, fat mass and fat-free mass from day 7 (P < 0·05). RBD induced changes in the jejunum, with an increase in the villus:crypt ratio on day 7, followed by reduction on days 14 and 21 (P < 0·05). Lactulose:mannitol ratio increased on day 14 (P < 0·05). Changes in intestinal barrier function on day 14 were associated with reductions in claudin-1 and occludin, and on day 21, there was a reduction in the levels of claudin-2 and occludin. SGLT-1 levels decreased on day 21. RBD compromises body composition and physical development with dynamic changes in intestinal barrier morphofunctional. RBD is associated with damage to intestinal permeability, reduced levels of claudin-1 and occludin transcripts and return of bowel function in a chronic period.

Potential pharmacological target of tight junctions to improve the BBB permeability in neonatal Hypoxic-Ischemic encephalopathy Diseases.

Neonatal encephalopathy (NE) is a pathological condition that describes a neurocognitive malfunction in the newborn that arises from fetal, peripartum, or intrapartum events of multifactorial nature, having a poor prognosis and accounting for an incidence of 5-8 per 1000 live births. Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the most studied paradigms of NE, caused by a scarce cerebral perfusion and oxygen supply during perinatal life. The cerebral hypoxic-ischemic insult promotes a loss of permeability of the blood-brain barrier (BBB), an essential structural intermediary of blood-brain communication. This permeability disruption is associated with an increase in inflammatory cytokines, an increase of adhesion molecules, and oxidative stress which disturb the tight junction (TJ) performance and enable transcytosis and paracellular leakage, ultimately leading to death from brain cells. In this context, TJs proteins are essential to preserving the barrier mechanical stability and signaling that modulates the brain-blood vessel multicellular domains, known as neurovascular units (NVU). Recent studies have proposed different strategies with neuroprotective effects that allow for maintaining or restoring the integrity and permeability of the BBB. This review identifies and discusses regulator mechanisms and novel aspects of TJs in the BBB disruption induced by cerebral hypoxic insults during the perinatal period, evaluating potential pharmacological strategies to safeguard BBB integrity.

A one month high fat diet disrupts the gut microbiome and integrity of the colon inducing adiposity and behavioral despair in male Sprague Dawley rats.

High-fat diet (HFD) is associated with gut microbiome dysfunction and mental disorders. However, the time-dependence as to when this occurs is unclear. We hypothesized that a short-term HFD causes colonic tissue integrity changes resulting in behavioral changes. Rats were fed HFD or low-fat diet (LFD) for a month and gut microbiome, colon, and behavior were evaluated. Behavioral despair was found in the HFD group. Although obesity was absent, the HFD group showed increased percent weight gain, epididymal fat tissue, and leptin expression. Moreover, the HFD group had increased colonic damage, decreased expression of the tight junction proteins, and higher lipopolysaccharides (LPS) in serum. Metagenomic analysis revealed that the HFD group had more Bacteroides and less S24-7 which correlated with the decreased claudin-5. Finally, HFD group showed an increase of microglia percent area, increased astrocytic projections, and decreased phospho-mTOR. In conclusion, HFD consumption in a short period is still sufficient to disrupt gut integrity resulting in LPS infiltration, alterations in the brain, and behavioral despair even in the absence of obesity.

Characterization and evaluation of Colombian propolis on the intestinal integrity of broilers.

Nutritional additives such as propolis seek to improve intestinal health as an alternative to the global ban on in-feed antibiotics used as growth promoters (AGP). The objective of this study was to evaluate the effect of propolis supplementation in diet of broilers. Four hundred and fifty straight-run Ross 308 AP broilers were fed with a basal diet (BD) throughout the whole experimental period. Birds were randomly distributed into 5 groups at d 14: negative control without antibiotics nor propolis (AGP-), positive control 500 ppm of Zinc Bacitracin as growth promoter (AGP+), and 3 groups supplemented with 150, 300, and 450 ppm of propolis. Every group included 6 replicates of 15 birds each. Propolis concentration was increased from d 22 to 42, in experimental groups to 300, 600, and 900 ppm of propolis, and 10% of raw soybean was included as a challenge in all groups during the same period. Analysis of productive parameters, intestinal morphometry, and relative quantification of genes associated with epithelial integrity by qPCR were performed at 21 and 42 d. The groups with the greatest weights were those that consumed diets including 150 (21 d) and 900 ppm (42 d) of propolis compared with all treatments. The lowest score of ISI was found at 300 (21 d) and 600 ppm (42 d). A lower degree of injury in digestive system was seen with the inclusion of 300 ppm (21 d) and 900 ppm (42 d). Up-regulation of zonula occludens-1 (ZO-1) was observed in jejunum of broilers supplemented with 150 and 300 ppm at 21 d. Up-regulation of ZO-1 and TGF-ß was also evidenced in ileum at all propolis inclusion levels at 42-day-old compared to AGP+ and AGP-. The beneficial effects were evidenced at inclusion levels of 150 ppm in the starter and 900 ppm in the finisher. According to the results, the Colombian propolis inclusion can improve productive performance, physiological parameters, and gene expression associated with intestinal integrity.

Polyubiquitination and SUMOylation Sites Regulate the Stability of ZO-2 Protein and the Sealing of Tight Junctions.

Tight junctions (TJs) regulate the transit of ions and molecules through the paracellular pathway in epithelial cells. Zonula occludens 2 (ZO-2) is a cytoplasmic TJ protein. Here, we studied the ubiquitination of hZO-2 employing mutants of SUMOylation site K730 present in the GuK domain and the putative ubiquitination residues K759 and K992 located at the GuK domain and proline-rich region, respectively. In immunoprecipitation experiments done with MDCK cells transfected with wild-type (WT) hZO-2 or the ubiquitination-site mutants hZO-2-K759R or -K992R, we observed diminished ubiquitination of the mutants, indicating that residues K759 and K992 in hZO-2 are acceptors for ubiquitination. Moreover, using TUBES, we found that residues K759 and K992 of hZO-2 are targets of K48 polyubiquitination, a signal for proteasomal degradation. Accordingly, compared to WT hZO-2, the half-life of hZO-2 mutants K759R and K992R augmented from 19.9 to 37.3 and 23.3 h, respectively. Instead, the ubiquitination of hZO-2 mutant K730R increased, and its half-life diminished to 6.7 h. The lack of these lysine residues in hZO-2 affects TJ sealing as the peak of TER decreased in monolayers of MDCK cells transfected with any of these mutants. These results highlight the importance of ZO-2 ubiquitination and SUMOylation to maintain a healthy and stable pool of ZO-2 molecules at the TJ.

Oxygen-dependent regulation of permeability in low resistance intestinal epithelial cells infected with Giardia lamblia.

Intestinal epithelial cells (IECs) reside in a highly anaerobic environment that is subject to daily fluctuations in partial oxygen pressure (pO2), depending on intestinal tissue perfusion. This condition, known as physiological hypoxia, has a major impact on the maintenance of gut homeostasis, such as effects on the integrity and function of the intestinal epithelial barrier. Giardia lamblia is a microaerophilic protozoan parasite that infects and colonizes the small intestine of its host, causing watery diarrhea. The disease, known as giardiasis, is associated with enhanced intestinal permeability and disruption or reorganization of tight junction (TJ) proteins between IECs. Given the central role of oxygen in gut homeostasis, in this study, we aimed to evaluate whether pO2 affects intestinal permeability (flux of ions and macromolecules) and TJ protein expression in human IECs during G. lamblia infection. Using human cell lines HuTu-80 and Caco-2 as models of "loose" (low resistance) and "tight" (high resistance) intestines, respectively, we elucidated that low pO2 drives intestinal barrier dysfunction in IECs infected with trophozoites through dephosphorylation of protein kinase C (PKC α/ß II). Additionally, we demonstrated that IECs infected with trophozoites in the presence of a pharmacological PKC activator (phorbol 12-myristate 13-acetate) partially restored the barrier function, which was correlated with increased protein expression levels of zonula occludens (ZO)-2 and occludin. Collectively, these results support the emerging theory that molecular oxygen impacts gut homeostasis during Giardia infection via direct host signaling pathways. These findings further our knowledge regarding Giardia-host interactions and the pathophysiological mechanisms of human giardiasis.

Lactobacillus delbrueckii CIDCA 133 Ameliorates Chemotherapy-Induced Mucositis by Modulating Epithelial Barrier and TLR2/4/Myd88/NF-κB Signaling Pathway.

Intestinal mucositis promoted by the use of anticancer drugs is characterized by ulcerative inflammation of the intestinal mucosa, a debilitating side effect in cancer patients undergoing treatment. Probiotics are a potential therapeutic option to alleviate intestinal mucositis due to their effects on epithelial barrier integrity and anti-inflammatory modulation. This study investigated the health-promoting impact of Lactobacillus delbrueckii CIDCA 133 in modulating inflammatory and epithelial barrier markers to protect the intestinal mucosa from 5-fluorouracil-induced epithelial damage. L. delbrueckii CIDCA 133 consumption ameliorated small intestine shortening, inflammatory cell infiltration, intestinal permeability, villus atrophy, and goblet cell count, improving the intestinal mucosa architecture and its function in treated mice. Upregulation of Muc2, Cldn1, Hp, F11r, and Il10, and downregulation of markers involved in NF-κB signaling pathway activation (Tlr2, Tlr4, Nfkb1, Il6, and Il1b) were observed at the mRNA level. This work suggests a beneficial role of L. delbrueckii strain CIDCA 133 on intestinal damage induced by 5-FU chemotherapy through modulation of inflammatory pathways and improvement of epithelial barrier function.

Is type 2 diabetes mellitus another intercellular junction-related disorder?

Type 2 diabetes mellitus (T2D) is nowadays a worldwide epidemic and has become a major challenge for health systems around the world. It is a multifactorial disorder, characterized by a chronic state of hyperglycemia caused by defects in the production as well as in the peripheral action of insulin. This minireview highlights the experimental and clinical evidence that supports the novel idea that intercellular junctions (IJs)-mediated cell-cell contacts play a role in the pathogenesis of T2D. It focuses on IJs repercussion for endocrine pancreas, intestinal barrier, and kidney dysfunctions that contribute to the onset and evolution of this metabolic disorder.

The Arp2/3 Inhibitory Protein Arpin Is Required for Intestinal Epithelial Barrier Integrity.

The intestinal epithelial barrier (IEB) depends on stable interepithelial protein complexes such as tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton. During inflammation, the IEB is compromised due to TJ protein internalization and actin remodeling. An important actin regulator is the actin-related protein 2/3 (Arp2/3) complex, which induces actin branching. Activation of Arp2/3 by nucleation-promoting factors is required for the formation of epithelial monolayers, but little is known about the relevance of Arp2/3 inhibition and endogenous Arp2/3 inhibitory proteins for IEB regulation. We found that the recently identified Arp2/3 inhibitory protein arpin was strongly expressed in intestinal epithelial cells. Arpin expression decreased in response to tumor necrosis factor (TNF)α and interferon (IFN)γ treatment, whereas the expression of gadkin and protein interacting with protein C-kinase α-subunit 1 (PICK1), other Arp2/3 inhibitors, remained unchanged. Of note, arpin coprecipitated with the TJ proteins occludin and claudin-1 and the AJ protein E-cadherin. Arpin depletion altered the architecture of both AJ and TJ, increased actin filament content and actomyosin contractility, and significantly increased epithelial permeability, demonstrating that arpin is indeed required for maintaining IEB integrity. During experimental colitis in mice, arpin expression was also decreased. Analyzing colon tissues from ulcerative colitis patients by Western blot, we found different arpin levels with overall no significant changes. However, in acutely inflamed areas, arpin was significantly reduced compared to non-inflamed areas. Importantly, patients receiving mesalazine had significantly higher arpin levels than untreated patients. As arpin depletion (theoretically meaning more active Arp2/3) increased permeability, we wanted to know whether Arp2/3 inhibition would show the opposite. Indeed, the specific Arp2/3 inhibitor CK666 ameliorated TNFα/IFNγ-induced permeability in established Caco-2 monolayers by preventing TJ disruption. CK666 treatment also attenuated colitis development, colon tissue damage, TJ disruption, and permeability in dextran sulphate sodium (DSS)-treated mice. Our results demonstrate that loss of arpin triggers IEB dysfunction during inflammation and that low arpin levels can be considered a novel hallmark of acute inflammation.

The blood-brain barrier dysfunction in sepsis.

Sepsis is a life-threatening organ dysfunction triggered by a dysregulated host immune response attempting to eliminate the infection. After hospital discharge, half of the sepsis survivors recover, one-third of the patients die the following year, and one-sixth have a long-term cognitive impairment, including memory dysfunction, anxiety, depression, and post-traumatic stress disorder. The infection triggers the host immune response, and both can cause vascular endothelial damage, interrupting tight junctions proteins; consequently, the blood-brain barrier (BBB) breaks down, allowing and facilitating the entry of peripheral immune cells into the brain, which triggers or exacerbates the activation of glial cells and neuroinflammation. The focus of this review is to identify biochemical abnormalities induced by sepsis, which is associated with BBB dysfunction; provide evidence of biomarkers involved in the tight junction disruption and BBB damage, and draw attention to the role of the BBB as a bridge between systemic infection and brain inflammation.

Prophylactic and therapeutic supplementation using fructo-oligosaccharide improves the intestinal homeostasis after mucositis induced by 5- fluorouracil.

The beneficial effects of prebiotic, such as fructo-oligosaccharides (FOS), in intestinal inflammation have been demonstrated in several studies. Herein, we evaluate whether joint treatment with FOS, both before and during mucositis, had additional beneficial effects and investigated the mechanisms underlying in the action of FOS on the intestinal barrier. BALB/c mice were randomly divided into five groups: CTR (without mucositis + saline solution), FOS (without mucositis + 6 % FOS), MUC (mucositis + saline solution), PT (mucositis + 6 % FOS supplementation before disease induction), and TT (mucositis + 6 % FOS supplementation before and during disease induction). Mucositis was induced by intraperitoneal injection (300 mg/kg) of 5-fluorouracil (5-FU). After 72 h, the animals were euthanized and intestinal permeability (IP), tight junction, bacterial translocation (BT), histology and morphometry, and immunoglobulin A secretory (sIgA), inflammatory infiltrate, and production of short-chain fatty acids (acetate, butyrate and propionate) were evaluated. The MUC group showed an increase in the IP, BT, and inflammatory infiltrate but a decrease in the tight junction expression and butyrate and propionate levels (P < 0.05). In the PT and TT groups, FOS supplementation maintained the IP, tight junction expression, and propionate concentration within physiologic levels, increased butyrate levels, and reduced BT and inflammatory infiltrate (P < 0.05). Total treatment with FOS (TT group) was more effective in maintaining histological score, morphometric parameters, and sIgA production. Thus, total treatment (prophylactic and therapeutic supplementation) with FOS was more effective than pretreatment alone, in reducing 5-FU-induced damage to the intestinal barrier.

The magnitude of physical exercise-induced hyperthermia is associated with changes in the intestinal permeability and expression of tight junction genes in rats.

We investigated whether the magnitude of exercise-induced hyperthermia influences intestinal permeability and tight junction gene expression. Twenty-nine male Wistar rats were divided into four groups: rest at 24 °C and exercise at 13 °C, 24 °C or 31 °C. The exercise consisted of a 90-min treadmill run at 15 m/min, and different ambient temperatures were used to produce distinct levels of exercise-induced hyperthermia. Before the experimental trials, the rats were treated by gavage with diethylenetriaminepentaacetic acid labeled with technetium-99 metastable as a radioactive probe. The rats' core body temperature (TCORE) was measured by telemetry. Immediately after the trials, the rats were euthanized, and the intestinal permeability was assessed by measuring the radioactivity of blood samples. The mRNA levels of occludin and zonula occludens-1 (ZO-1) genes were determined in duodenum samples. Exercise at 24 °C increased TCORE to values close to 39 °C, without changing permeability compared with the resting trial at the same environment. Meanwhile, rats' TCORE exceeded 40 °C during exercise at 31 °C, leading to greater permeability relative to those observed after exercise in the other ambient temperatures (e.g., 0.0037%/g at 31 °C vs. 0.0005%/g at 13 °C; data expressed as medians; p < 0.05). Likewise, the rats exercised at 31 °C exhibited higher mRNA levels of ZO-1 and occludin genes than the rats exercised at 24 °C or 13 °C. The changes in permeability and gene expression were positively and significantly associated with the magnitude of hyperthermia. We conclude that marked hyperthermia caused by exercise in the warmer environment increases intestinal permeability and mRNA levels of tight junction genes.

Biallelic Mutations in the LSR Gene Cause a Novel Type of Infantile Intrahepatic Cholestasis.

We identified biallelic pathogenic mutations in the Lipolysis-stimulated lipoprotein receptor (LSR) gene in a patient with infantile intrahepatic cholestasis. We established that mutations in the LSR gene, which encodes a protein which is critical for the formation of tricellular tight junctions in the liver, are a novel cause of pediatric cholestasis.