Occludin and collagen IV degradation mediated by the T9SS effector SspA contributes to blood-brain barrier damage in ducks during Riemerella anatipestifer infection.

Riemerella anatipestifer infection is characterized by meningitis with neurological symptoms in ducklings and has adversely affected the poultry industry. R. anatipestifer strains can invade the duck brain to cause meningitis and neurological symptoms, but the underlying mechanism remains unknown. In this study, we showed that obvious clinical symptoms, an increase in blood‒brain barrier (BBB) permeability, and the accumulation of inflammatory cytokines occurred after intravenous infection with the Yb2 strain but not the mutant strain Yb2ΔsspA, indicating that Yb2 infection can lead to cerebrovascular dysfunction and that the type IX secretion system (T9SS) effector SspA plays a critical role in this pathological process. In addition, we showed that Yb2 infection led to rapid degradation of occludin (a tight junction protein) and collagen IV (a basement membrane protein), which contributed to endothelial barrier disruption. The interaction between SspA and occludin was confirmed by coimmunoprecipitation. Furthermore, we found that SspA was the main enzyme mediating occludin and collagen IV degradation. These data indicate that R. anatipestifer SspA mediates occludin and collagen IV degradation, which functions in BBB disruption in R. anatipestifer-infected ducks. These findings establish the molecular mechanisms by which R. anatipestifer targets duckling endothelial cell junctions and provide new perspectives for the treatment and prevention of R. anatipestifer infection.

Atorvastatin-induced intracerebral hemorrhage is inhibited by berberine in zebrafish.

Intracerebral hemorrhage (ICH), for which there are currently no effective preventive or treatment methods, has a very high fatality rate. Statins, such as atorvastatin (ATV), are the first-line drugs for regulating blood lipids and treating hyperlipidemia-related cardiovascular diseases. However, ATV-associated ICH has been reported, although its incidence is rare. In this study, we aimed to investigate the protective action and mechanisms of berberine (BBR) against ATV-induced brain hemorrhage. We established an ICH model in zebrafish induced by ATV (2 µM) and demonstrated the effects of BBR (10, 50, and 100 µM) on ICH via protecting the vascular network using hemocyte staining and three transgenic zebrafish. BBR was found to reduce brain inflammation and locomotion injury in ICH-zebrafish. Mechanism research showed that ATV increased the levels of VE-cadherin and occludin proteins but disturbed their localization at the cell membrane by abnormal phosphorylation, which decreased the number of intercellular junctions between vascular endothelial cells (VECs), disrupting the integrity of vascular walls. BBR reversed the effects of ATV by promoting autophagic degradation of phosphorylated VE-cadherin and occludin in ATV-induced VECs examined by co-immunoprecipitation (co-IP). These findings provide crucial insights into understanding the BBR mechanisms involved in the maintenance of vascular integrity and in mitigating adverse reactions to ATV.

The FAK/occludin/ZO-1 complex is critical for cadmium-induced testicular damage by disruption of the integrity of the blood-testis barrier in chickens.

Cadmium (Cd) is a well-known testis toxicant. The blood-testis barrier (BTB) is a crucial component of the testis. Cd can disrupt the integrity of the BTB and reproductive function. However, the mechanism of Cd-induced disruption of BTB and testicular damage has not been fully elucidated. Here, our study investigates the effects of Cd on BTB integrity and testicular dysfunction. 80 (aged 1 day) Hy-Line white variety chickens were randomly designed into 4 groups and treated for 90 days, as follows: control group (essential diet), 35 Cd, 70 Cd and 140 Cd groups (35, 70 and 140 mg/kg Cd). The results found that Cd exposure diminished volume of the testes and induced histopathological lesions in the testes. Exposure to Cd induced an inflammatory response, disrupted the structure and function of the FAK/occludin/ZO-1 protein complex and disrupted the tight junction and adherens junction in the BTB. In addition, Cd exposure reduced the expression of steroid-related proteins and inhibited testosterone synthesis. Taken together, these data elucidate that Cd disrupts the integrity of the BTB and further inhibits spermatogenesis by dissociating the FAK/occludin/ZO-1 complex, which provides a basis for further investigation into the mechanisms of Cd-induced impairment of male reproductive function and pharmacological protection.

Gingipain-carrying outer membrane vesicles from Porphyromonas gingivalis cause barrier dysfunction of Caco-2 cells by releasing gingipain into the cytosol.

Ingestion of Porphyromonas gingivalis, a periodontal pathogen, disrupts the intestinal barrier in mice. However, the involvement of outer membrane vesicles (OMVs) secreted from P. gingivalis in the destruction of the intestinal barrier remains unclear. In this study, we tested the hypothesis that OMVs carrying gingipains, the major cysteine proteases produced by P. gingivalis, affects the intestinal barrier function. OMVs increased the permeability of the Caco-2 cell monolayer, a human intestinal epithelial cell line, accompanied by degradation of the tight junction protein occludin. In contrast, OMVs prepared from mutant strains devoid of gingipains failed to induce intestinal barrier dysfunction or occludin degradation in Caco-2 cells. A close histological examination revealed the intracellular localization of gingipain-carrying OMVs. Gingipain activity was detected in the cytosolic fraction of Caco-2 cells after incubation with OMVs. These results suggest that gingipains were internalized into intestinal cells through OMVs and transported into the cytosol, where they then directly degraded occludin from the cytosolic side. Thus, P. gingivalis OMVs might destroy the intestinal barrier and induce systemic inflammation via OMV itself or intestinal substances leaked into blood vessels, causing various diseases.

Streptococcus pneumoniae extracellular vesicles aggravate alveolar epithelial barrier disruption via autophagic degradation of OCLN (occludin).

Streptococcus pneumoniae (S. pneumoniae) represents a major human bacterial pathogen leading to high morbidity and mortality in children and the elderly. Recent research emphasizes the role of extracellular vesicles (EVs) in bacterial pathogenicity. However, the contribution of S. pneumoniae EVs (pEVs) to host-microbe interactions has remained unclear. Here, we observed that S. pneumoniae infections in mice led to severe lung injuries and alveolar epithelial barrier (AEB) dysfunction. Infections of S. pneumoniae reduced the protein expression of tight junction protein OCLN (occludin) and activated macroautophagy/autophagy in lung tissues of mice and A549 cells. Mechanically, S. pneumoniae induced autophagosomal degradation of OCLN leading to AEB impairment in the A549 monolayer. S. pneumoniae released the pEVs that could be internalized by alveolar epithelial cells. Through proteomics, we profiled the cargo proteins inside pEVs and found that these pEVs contained many virulence factors, among which we identified a eukaryotic-like serine-threonine kinase protein StkP. The internalized StkP could induce the phosphorylation of BECN1 (beclin 1) at Ser93 and Ser96 sites, initiating autophagy and resulting in autophagy-dependent OCLN degradation and AEB dysfunction. Finally, the deletion of stkP in S. pneumoniae completely protected infected mice from death, significantly alleviated OCLN degradation in vivo, and largely abolished the AEB disruption caused by pEVs in vitro. Overall, our results suggested that pEVs played a crucial role in the spread of S. pneumoniae virulence factors. The cargo protein StkP in pEVs could communicate with host target proteins and even hijack the BECN1 autophagy initiation pathway, contributing to AEB disruption and bacterial pathogenicity.Abbreviations: AEB: alveolarepithelial barrier; AECs: alveolar epithelial cells; ATG16L1: autophagy related 16 like 1; ATP:adenosine 5'-triphosphate; BafA1: bafilomycin A1; BBB: blood-brain barrier; CFU: colony-forming unit; co-IP: co-immunoprecipitation; CQ:chloroquine; CTRL: control; DiO: 3,3'-dioctadecylox-acarbocyanineperchlorate; DOX: doxycycline; DTT: dithiothreitol; ECIS: electricalcell-substrate impedance sensing; eGFP: enhanced green fluorescentprotein; ermR: erythromycin-resistance expression cassette; Ery: erythromycin; eSTKs: eukaryotic-like serine-threoninekinases; EVs: extracellular vesicles; HA: hemagglutinin; H&E: hematoxylin and eosin; HsLC3B: human LC3B; hpi: hours post-infection; IP: immunoprecipitation; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LC/MS: liquid chromatography-mass spectrometry; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MVs: membranevesicles; NC:negative control; NETs:neutrophil extracellular traps; OD: optical density; OMVs: outer membrane vesicles; PBS: phosphate-buffered saline; pEVs: S.pneumoniaeextracellular vesicles; protK: proteinase K; Rapa: rapamycin; RNAi: RNA interference; S.aureus: Staphylococcusaureus; SNF:supernatant fluid; sgRNA: single guide RNA; S.pneumoniae: Streptococcuspneumoniae; S.suis: Streptococcussuis; TEER: trans-epithelium electrical resistance; moi: multiplicity ofinfection; TEM:transmission electron microscope; TJproteins: tight junction proteins; TJP1/ZO-1: tight junction protein1; TSA: tryptic soy agar; WB: western blot; WT: wild-type.

Altered Expression of Intestinal Tight Junctions in Patients with Chronic Kidney Disease: A Pathogenetic Mechanism of Intestinal Hyperpermeability.

BACKGROUND: Systemic inflammation in chronic kidney disease (CKD) is associated (as a cause or effect) with intestinal barrier dysfunction and increased gut permeability, with mechanisms not yet fully understood. This study investigated different parameters of the intestinal barrier in CKD patients, especially tight junction (TJ) proteins and their possible association with systemic endotoxemia and inflammation. METHODS: Thirty-three patients with stage I-IV CKD (n = 17) or end-stage kidney disease (ESKD) (n = 16) and 11 healthy controls underwent duodenal biopsy. Samples were examined histologically, the presence of CD3+ T-lymphocytes and the expression of occludin and claudin-1 in the intestinal epithelium was evaluated by means of immunohistochemistry, circulating endotoxin concentrations were determined by means of ELISA and the concentrations of the cytokines IL-1ß, IL-6, IL-8, IL-10 and TNF-α in serum were measured using flow cytometry. RESULTS: Patients with stage I-IV CKD or ESKD had significantly higher serum endotoxin, IL-6, IL-8 and IL-10 levels compared to controls. Intestinal occludin and claudin-1 were significantly decreased, and their expression was inversely correlated with systemic endotoxemia. Regarding occludin, a specific expression pattern was observed, with a gradually increasing loss of its expression from the crypt to the tip of the villi. CONCLUSION: The expression of occludin and claudin-1 in enterocytes is significantly reduced in patients with CKD, contributing to systemic endotoxemia and inflammatory responses in these patients.

Lipopolysaccharide, VE-cadherin, HMGB1, and HIF-1α levels are elevated in the systemic circulation in chronic migraine patients with medication overuse headache: evidence of leaky gut and inflammation.

OBJECTIVE: Medication overuse headache (MOH) was recently shown to be associated with leaky gut in rodents. We aimed to investigate whether chronic migraine (CM) patients with MOH have elevated lipopolysaccharide levels and inflammatory molecules in blood circulation. MATERIALS AND METHODS: The study included women participants (40 CM patients with NSAID overuse headache, 35 episodic migraine (EM) patients, and 20 healthy non-headache sufferers). Migraine duration, monthly migraine headache days, MigSCog, HADS-D, HADS-A, and HIT-6 scores were recorded. Serum samples were collected to measure circulating LPS, LPS binding protein (LBP), tight junction protein occludin, adherens junction protein vascular endothelial cadherin (VE-cadherin), CGRP, HMGB1, HIF-1α, IL-6, and IL-17 levels. RESULTS: Serum LPS, VE-Cadherin, CGRP, HIF-1α, and IL-6 levels were significantly higher in the CM + MOH group compared to the EM group and healthy controls while serum LBP and HMGB1 were higher in the CM + MOH group compared to healthy controls. IL-17 and occludin levels were comparable between the three groups. Serum HMGB1 levels in EM patients were higher compared to the control group. Mig-SCog and HIT-6 scores were higher in the CM + MOH group compared to EM patients. HADS-A and HADS-D scores were significantly higher in the CM + MOH group compared to EM patients and healthy controls, and they were also higher in EM patients compared to healthy subjects. LPS levels were correlated with VE-cadherin and occludin levels. The number of monthly migraine headache days was positively correlated with serum LPS, HIF-1α, VE-cadherin, and IL-6 levels, HADS-A, HADS-D, HIT-6, and MigSCog scores. CONCLUSION: We have evidence for the first time that CM + MOH is associated with elevated serum LPS and LBP levels suggestive of LPS leak into the systemic circulation. Higher levels of nociceptive and/or pro-inflammatory molecules such as HMGB1, HIF-1α, IL-6, and CGRP may play a role in trigeminal sensitization and neurobiology of MOH. Intestinal hyperpermeability and consequent inflammatory response should be considered as a potential contributory factor in patients with MOH.

Campylobacter jejuni Surface-Bound Protease HtrA, but Not the Secreted Protease nor Protease in Shed Membrane Vesicles, Disrupts Epithelial Cell-to-Cell Junctions.

Fundamental functions of the intestinal epithelium include the digestion of food, absorption of nutrients, and its ability to act as the first barrier against intruding microbes. Campylobacter jejuni is a major zoonotic pathogen accounting for a substantial portion of bacterial foodborne illnesses. The germ colonizes the intestines of birds and is mainly transmitted to humans through the consumption of contaminated poultry meat. In the human gastrointestinal tract, the bacterium triggers campylobacteriosis that can progress to serious secondary disorders, including reactive arthritis, inflammatory bowel disease and Guillain-Barré syndrome. We recently discovered that C. jejuni serine protease HtrA disrupts intestinal epithelial barrier functions via cleavage of the tight and adherens junction components occludin, claudin-8 and E-cadherin. However, it is unknown whether epithelial damage is mediated by the secreted soluble enzyme, by HtrA contained in shed outer-membrane vesicles (OMVs) or by another mechanism that has yet to be identified. In the present study, we investigated whether soluble recombinant HtrA and/or purified OMVs induce junctional damage to polarized intestinal epithelial cells compared to live C. jejuni bacteria. By using electron and confocal immunofluorescence microscopy, we show that HtrA-expressing C. jejuni bacteria trigger efficient junctional cell damage, but not soluble purified HtrA or HtrA-containing OMVs, not even at high concentrations far exceeding physiological levels. Instead, we found that only bacteria with active protein biosynthesis effectively cleave junctional proteins, which is followed by paracellular transmigration of C. jejuni through the epithelial cell layer. These findings shed new light on the pathogenic activities of HtrA and virulence strategies of C. jejuni.

Canonical and Non-Canonical Localization of Tight Junction Proteins during Early Murine Cranial Development.

This study investigates the intricate composition and spatial distribution of tight junction complex proteins during early mouse neurulation. The analyses focused on the cranial neural tube, which gives rise to all head structures. Neurulation brings about significant changes in the neuronal and non-neuronal ectoderm at a cellular and tissue level. During this process, precise coordination of both epithelial integrity and epithelial dynamics is essential for accurate tissue morphogenesis. Tight junctions are pivotal for epithelial integrity, yet their complex composition in this context remains poorly understood. Our examination of various tight junction proteins in the forebrain region of mouse embryos revealed distinct patterns in the neuronal and non-neuronal ectoderm, as well as mesoderm-derived mesenchymal cells. While claudin-4 exhibited exclusive expression in the non-neuronal ectoderm, we demonstrated a neuronal ectoderm specific localization for claudin-12 in the developing cranial neural tube. Claudin-5 was uniquely present in mesenchymal cells. Regarding the subcellular localization, canonical tight junction localization in the apical junctions was predominant for most tight junction complex proteins. ZO-1 (zona occludens protein-1), claudin-1, claudin-4, claudin-12, and occludin were detected at the apical junction. However, claudin-1 and occludin also appeared in basolateral domains. Intriguingly, claudin-3 displayed a non-canonical localization, overlapping with a nuclear lamina marker. These findings highlight the diverse tissue and subcellular distribution of tight junction proteins and emphasize the need for their precise regulation during the dynamic processes of forebrain development. The study can thereby contribute to a better understanding of the role of tight junction complex proteins in forebrain development.

The combination model of serum occludin and clinical risk factors improved the efficacy for predicting hemorrhagic transformation in stroke patients with recanalization.

Background: and Purpose: Hemorrhagic transformation (HT) is one of the severe complications in acute ischemic stroke, especially for the patients who undergo recanalization treatment. It is crucial to screen patients who have high risk of HT before recanalization. However, current prediction models based on clinical factors are not ideal for clinical practice. Serum occludin, a biomarker for cerebral ischemia-induced blood-brain barrier disruption, has potential for predicting HT. This study was to investigate whether the combination of serum occludin and clinical risk factors improved the efficacy of predicting HT. Methods: This was a single-center prospective observational study. Baseline clinical data and blood samples of recanalization patients were collected upon admission to our hospital. The level of serum occludin was measured using enzyme-linked immunosorbent assay. The diagnosis of HT was confirmed by CT scans within 36 h post recanalization. Results: A total of 324 patients with recanalization were enrolled and 68 patients presented HT occurrence. HT patients had the higher level of baseline occludin than patients without HT (p < 0.001). Multivariate regression analysis showed that serum occludin level, Alberta Stroke Program Early CT Scores and endovascular therapy were independent risk factors (p < 0.05) for HT after adjusting potential confounders. The combination of serum occludin and clinical risk factors significantly improved the accuracy of predicting HT [area under the curve (AUC, 0.821 vs 0.701, p < 0.001), and net reclassification improvement (31.1 %), integrated discrimination improvement (21.5 %), p < 0.001] compared to a model employing only clinical risk factors. The modified AUC (0.806) of combined model based on 10-fold-cross-validation was still higher than clinical risk model (0.701). Conclusion: The combination of serum occludin and clinical risk factors significantly improved the prediction efficacy for HT, providing a novel potential prediction model to screen for patients with high risk of HT before recanalization in acute ischemic stroke.

Interleukin 17A Contributes to Blood-Brain Barrier Disruption of Hypothalamic Paraventricular Nucleus in Rats With Myocardial Infarction.

BACKGROUND: Elevated inflammatory cytokines in the periphery have been identified as active contributors to neuroinflammation and sympathetic overactivity in heart failure (HF). Yet, the exact mechanisms by which these cytokines breach the blood-brain barrier (BBB) to exert their effects on the brain remain elusive. Interleukin 17A has been linked to BBB disruption in various neurologic disorders, and its levels were significantly augmented in circulation and the brain in HF. The present study aimed to determine whether the BBB integrity was compromised within the hypothalamic paraventricular nucleus (PVN), and if so, whether interleukin 17A contributes to BBB disruption in myocardial infarction-induced HF. METHODS AND RESULTS: Male Sprague-Dawley rats underwent coronary artery ligation to induce HF or sham surgery. Some HF rats received bilateral PVN microinjections of an interleukin 17 receptor A small interfering RNA or a scrambled small interfering RNA adeno-associated virus. Four weeks after coronary artery ligation, the permeability of the BBB was evaluated by intracarotid injection of fluorescent dyes (fluorescein isothiocyanate-dextran 10 kDa+rhodamine-dextran 70 kDa). Compared with sham-operated rats, HF rats exhibited an elevated extravasation of fluorescein isothiocyanate-dextran 10 kDa within the PVN but not in the brain cortex. The plasma interleukin 17A levels were positively correlated with fluorescein isothiocyanate 10 kDa extravasation in the PVN. The expression of caveolin-1, a transcytosis marker, was augmented, whereas the expression of tight junction proteins was diminished in HF rats. Interleukin 17 receptor A was identified within the endothelium of PVN microvessels. Treatment with interleukin 17 receptor A small interfering RNA led to a significant attenuation of fluorescein isothiocyanate 10 kDa extravasation in the PVN and reversed expression of caveolin-1 and tight junction-associated proteins in the PVN. CONCLUSIONS: Collectively, these data indicate that BBB permeability within the PVN is enhanced in HF and is likely attributable to increased interleukin 17A/interleukin 17 receptor A signaling in the BBB endothelium, by promoting caveolar transcytosis and degradation of tight junction complexes.

Tight junction and kidney stone disease.

Defects of tight junction (TJ) are involved in many diseases related to epithelial cell functions, including kidney stone disease (KSD), which is a common disease affecting humans for over a thousand years. This review provides brief overviews of KSD and TJ, and summarizes the knowledge on crystal-induced defects of TJ in renal tubular epithelial cells (RTECs) in KSD. Calcium oxalate (CaOx) crystals, particularly COM, disrupt TJ via p38 MAPK and ROS/Akt/p38 MAPK signaling pathways, filamentous actin (F-actin) reorganization and α-tubulin relocalization. Stabilizing p38 MAPK signaling, reactive oxygen species (ROS) production, F-actin and α-tubulin by using SB239063, N-acetyl-L-cysteine (NAC), phalloidin and docetaxel, respectively, successfully prevent the COM-induced TJ disruption and malfunction. Additionally, genetic disorders of renal TJ, including mutations and single nucleotide polymorphisms (SNPs) of CLDN2, CLDN10b, CLDN14, CLDN16 and CLDN19, also affect KSD. Finally, the role of TJ as a potential target for KSD therapeutics and prevention is also discussed.

Association of serum occludin levels and perihematomal edema volumes in intracranial hemorrhage patients.

BACKGROUND AND PURPOSE: Perihematomal edema (PHE) is one of the severe secondary damages following intracranial hemorrhage (ICH). Studies showed that blood-brain barrier (BBB) injury contributes to the development of PHE. Previous studies showed that occludin protein is a potential biomarker of BBB injury. In the present study, we investigated whether the levels of serum occludin on admission are associated with PHE volumes in ICH patients. METHODS: This cross-sectional study included 90ICH patients and 32 healthy controls.The volumes of hematoma and PHE were assessed using non-contrast cranial CT within 30 min of admission. Blood samples were drawn on admission, and the levels of baseline serum occludin were detected using enzyme-linked immunosorbent assay. Partial correlation analysis and multiple linear regression analysis were performed to evaluate the association between serum occludin levels and PHE volumes in ICH patients. RESULTS: The serum occludin levels in ICH patients were much higher than health controls (median 0.27 vs. 0.13 ng/mL, p < 0.001). At admission, 34 ICH patients (37.78%) had experienced a severe PHE (≥30 mL), and their serum occludin levels were higher compared to those with mild PHE (<30 mL) (0.78 vs. 0.21 ng/mL, p < 0.001). The area under the receiver operating characteristics curve (ROC) of serum occludin level in predicting severe PHE was 0.747 (95% confidence interval CI 0.644-0.832, p < 0.001). There was a significant positive correlation between serum occludin levels and PHE volumes (partial correlation r = 0.675, p < 0.001). Multiple linear regression analysis showed that serum occludin levels remained independently associated with the PHE volumes after adjusting other confounding factors. CONCLUSION: The present study showed that serum occludin levels at admission were independently correlated with PHE volumes in ICH patients, which may provide a biomarker indicating PHE volume change.

Dysfunctional Epithelial Barrier Is Characterized by Reduced E-Cadherin in Idiopathic Subglottic Stenosis.

OBJECTIVES: To aim of the study was to characterize the molecular profile and functional phenotype of idiopathic subglottic stenosis (iSGS)-scar epithelium. METHODS: Human tracheal biopsies from iSGS scar (n = 6) and matched non-scar (n = 6) regions were analyzed using single-cell RNA sequencing (scRNA-seq). Separate specimens were used for epithelial cell expansion in vitro to assess average growth rate and functional capabilities using transepithelial-electrical resistance (TEER), fluorescein isothiocyanate-dextran flux permeability assay, ciliary coverage, and cilia beating frequency (CBF). Finally, epithelial tight junction protein expression of cultured cells was quantified using immunoblot assay (n = 4) and immunofluorescence (n = 6). RESULTS: scRNA-seq analysis revealed a decrease in goblet, ciliated, and basal epithelial cells in the scar iSGS cohort. Furthermore, mRNA expression of proteins E-cadherin, claudin-3, claudin-10, occludin, TJP1, and TJP2 was also reduced (p < 0.001) in scar epithelium. Functional assays demonstrated a decrease in TEER (paired 95% confidence interval [CI], 195.68-890.83 Ω × cm2 , p < 0.05), an increase in permeability (paired 95% CI, -6116.00 to -1401.99 RFU, p < 0.05), and reduced epithelial coverage (paired 95% CI, 0.1814-1.766, fold change p < 0.05) in iSGS-scar epithelium relative to normal controls. No difference in growth rate (p < 0.05) or CBF was found (paired 95% CI, -2.118 to 3.820 Hz, p > 0.05). Immunoblot assay (paired 95% CI, 0.0367-0.605, p < 0.05) and immunofluorescence (paired 95% CI, 13.748-59.191 mean grey value, p < 0.05) revealed E-cadherin reduction in iSGS-scar epithelium. CONCLUSION: iSGS-scar epithelium has a dysfunctional barrier and reduced structural protein expression. These results are consistent with dysfunctional epithelium seen in other airway pathology. Further studies are warranted to delineate the causality of epithelial dysfunction on the downstream fibroinflammatory cascade in iSGS. LEVEL OF EVIDENCE: NA Laryngoscope, 134:374-381, 2024.

Occludin is overexpressed in tubo-ovarian high-grade serous carcinoma compared to mesothelioma and is a marker of tumor progression and chemoresistance.

The objective of this study was to analyze the expression and prognostic role of the tight junction protein occludin in high-grade serous carcinoma (HGSC). Occludin protein expression by immunohistochemistry was analyzed in 602 HGSC (417 effusions, 185 surgical specimens). Expression in mesothelioma (n = 87; 45 effusions, 42 surgical specimens) was studied for comparative purposes. Occludin protein expression was found in 587/602 (98%) HGSC vs. 40/87 (46%) mesotheliomas and was predominantly limited to < 5% of cells in the latter (p < 0.001). Occludin was additionally overexpressed in HGSC effusions compared to surgical specimens (p < 0.001) and was overexpressed in post-chemotherapy effusions compared to chemo-naive effusions tapped at diagnosis (p = 0.015). Occludin expression in HGSC surgical specimens was associated with poor chemoresponse (p < 0.001) and primary resistance (p = 0.001). Expression in effusions and surgical specimens was unrelated to survival (p > 0.05). In conclusion, occludin expression is higher in HGSC compared to mesothelioma, and this protein is overexpressed in HGSC effusions, possibly reflecting changes in adhesion related to anchorage-independent growth in this microenvironment. Overexpression in post-chemotherapy compared to chemo-naïve effusions suggest a role in disease progression. Occludin expression in surgical specimens may be related to chemoresistance.

Torreya grandis Kernel Oil Alleviates Loperamide-Induced Slow Transit Constipation via Up-Regulating the Colonic Expressions of Occludin/Claudin-1/ZO-1 and 5-HT3R/5-HT4R in BALB/c Mice.

SCOPE: Torreya grandis kernel has traditionally been used to remove intestinal parasites and increases intestinal motility. However, the effect of Torreya grandis kernel oil (TKO) on constipation has not yet been investigated. Therefore, mouse model is used to investigate the effect of TKO on slow transit constipation (STC) and its possible mechanism. METHODS AND RESULTS: The effects of TKO on intestinal motility of STC mice are evaluated by fecal weight, fecal water content, colon length, defecation test, and intestinal propulsion test. The mechanism of TKO alleviating STC is explored by detecting biochemical analysis, histological analysis, western blot, qRT-PCR, immunohistochemistry, and gut microbiota analysis. The results reveal that TKO effectively promotes defecation and intestinal motility, increases the level of endothelin-1, and restores the histopathological morphology of the colon under LOP pretreatment. The expression levels of occludin, claudin-1, and zonula occludens-1 (ZO-1) mRNA and protein are up-regulated in mice receiving TKO treatment. The colonic 5-hydroxytryptamine 3R/4R (5-HT3R/5-HT4R) expressions are also increased by TKO supplementation. Additionally, TKO rescues LOP-caused disorders of the gut microbiota. CONCLUSION: Consumption of TKO is beneficial to STC recovery, and it can alleviate LOP-induced STC by up-regulating the colonic expressions of Occludin/Claudin-1/ZO-1 and 5-HT3R/5-HT4R.

Berberine decreases S100B generation to regulate gut vascular barrier permeability in mice with burn injury.

Context: Berberine (BBR) can regulate enteric glial cells (EGCs) and the gut vascular barrier (GVB).Objective: To explore whether BBR regulates GVB permeability via the S100B pathway.Materials and methods: GVB hyperpermeability in C57BL/6J mice was induced by burns or S100B enema. BBR (25 or 50 mg/kg/d, 3 d) was gavaged preburn. S100B monoclonal antibody (S100BmAb) was i.v. injected postburn. Mouse intestinal microvascular endothelial cells (MIMECs) were treated with S100B, S100B plus BBR, or Z-IETD-FMK. GVB permeability was assayed by FITC-dextran, S100B by ELISA, caspase-8, ß-catenin, occludin and PV-1 by immunoblot.Results: Burns elevated S100B in serum and in colonic mucosa to a peak (147.00 ± 4.95 ng/mL and 160.30 ± 8.50 ng/mg, respectively) at 36 h postburn, but BBR decreased burns-induced S100B in serum (126.20 ± 6.30 or 90.60 ± 3.78 ng/mL) and in mucosa (125.80 ± 12.40 or 91.20 ± 8.54 ng/mg). Burns raised GVB permeability (serum FITC-dextran 111.40 ± 8.56 pg/mL) at 48 h postburn, but BBR reduced GVB permeability (serum FITC-dextran 89.20 ± 6.98 or 68.60 ± 5.50 ng/mL). S100B enema (1 µM) aggravated burns-raised GVB permeability (142.80 ± 8.07 pg/mL) and PV-1, but the effect of S100B was antagonized by BBR. Z-IETD-FMK (5 µM) increased S100B-induced permeability to FITC-dextran (205.80 ± 9.70 to 263.80 ± 11.04 AUs) while reducing ß-catenin in MIMECs. BBR (5 µM) reduced S100B-induced permeability (104.20 ± 9.65 AUs) and increased caspase-8, ß-catenin and occludin.Discussion and conclusion: BBR decreases burns-induced GVB hyperpermeability via modulating S100B/caspase-8/ß-catenin pathway and may involve EGCs.

Inhibiting Caveolin-1-Related Akt/mTOR Signaling Pathway Protects Against N-methyl-D-Aspartate Receptor Activation-Mediated Dysfunction of Blood-Brain Barrier in vitro.

BACKGROUND: The aim of this study was to further explore the role of caveolin-1 (Cav-1) related Akt/mTOR signaling pathway in blood brain barrier (BBB) dysfunction caused by NMDAR activation. METHODS: The cell localization of NMDAR GluN1 subunit and Cav-1 was observed on human brain microvascular HBEC-5i cells after immunofluorescence double staining. The transendothelial resistance (TEER) of BBB in vitro was measured by Millicell-ERS cell resistance meter. Sodium fluorescein (SF) was used to measure the permeability of BBB in vitro. A stable Cav-1-silenced HBEC-5i cell line was established by infecting the cells with a lentivirus encoding Cav-1 shRNA. The changes of the protein and mRNA of MMP9 and Occludin induced by NMDA were detected by Western blot (WB) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. The phosphorylated proteins of Cav-1, Akt, and mTOR were detected by WB. RESULTS: NMDAR GluN1 was expressed in the cytoplasm and part of the cell membrane of the HBEC-5i cell line. NMDAR activation decreased TEER and increased the SF of BBB in vitro. HBEC-5i cells incubated with NMDA enhanced the phosphorylation of Cav-1, Akt, and mTOR, also promoting the expression of MMP9 along with the degradation of Occludin. These effects could be reversed by pretreatment with NMDAR antagonist (MK801) or Cav-1 antagonist (Daidzein), or Akt antagonist (LY294002), respectively. Further silencing Cav-1 with LV-Cav-1-RNAi also played a similar protective effect. CONCLUSION: Caveolin-1 (Cav-1) related Akt/mTOR signaling probably contributes to BBB dysfunction by activating NMDAR on human brain microvascular cells.

Medication overuse headache is associated with elevated lipopolysaccharide binding protein and pro-inflammatory molecules in the bloodstream.

OBJECTIVE: Medication overuse headache (MOH) is a secondary headache that accompanies chronic migraine. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most frequently used analgesics worldwide and they are known to induce leaky gut. In this study, we aimed to investigate whether NSAID induced MOH is associated with altered circulating lipopolysaccharide binding protein (LBP) levels and inflammatory molecules. MATERIALS AND METHODS: Piroxicam (10 mg/kg/day, po) for 5 weeks was used to induce MOH in female Sprague Dawley rats. Pain behavior was evaluated by periorbital withdrawal thresholds, head-face grooming, freezing, and head shake behavior. Serum samples and brain tissues were collected to measure circulating LBP, tight junction protein occludin, adherens junction protein vascular endothelial (VE)-cadherin, calcitonin gene-related peptide (CGRP), IL-6 levels and brain high mobility group box-1 (HMGB1) and IL-17 levels. RESULTS: Chronic piroxicam exposure resulted in decreased periorbital mechanical withdrawal thresholds, increased head-face grooming, freezing, and head shake behavior compared to vehicle administration. Serum LBP, CGRP, IL-6, IL-17, occludin, VE-cadherin levels and brain IL-17 and HMGB1 levels were significantly higher in piroxicam group compared to controls. Serum LBP was positively correlated with occludin (r = 0.611), VE-cadherin (r = 0.588), CGRP (r = 0.706), HMGB1 (r = 0.618) and head shakes (r = 0.921), and negatively correlated with periorbital mechanical withdrawal thresholds (r = -0.740). CONCLUSION: Elevated serum LBP, VE-cadherin and occludin levels indicating disrupted intestinal barrier function and leakage of LPS into the systemic circulation were shown in female rats with MOH. LPS induced low-grade inflammation and elevated nociceptive and/or pro-inflammatory molecules such as HMGB1, IL-6, IL-17 and CGRP may play a role in the development and maintenance of MOH. Interference with leaky gut and pro-inflammatory nociceptive molecules could also be a target for sustained management of MOH.

Tight Junction Claudins and Occludin Are Differentially Regulated and Expressed in Genomically Defined Subsets of Colon Cancer.

Metastatic colon cancer remains incurable despite improvements in survival outcomes. New therapies based on the discovery of colon cancer genomic subsets could improve outcomes. Colon cancers from genomic studies with publicly available data were examined to define the expression and regulation of the major tight junction proteins claudins and occludin in genomic groups. Putative regulations of the promoters of tight junction genes by colon-cancer-deregulated pathways were evaluated in silico. The effect of claudin mRNA expression levels on survival of colon cancer patients was examined. Common mutations in colon-cancer-related genes showed variable prevalence in genomically identified groups. Claudin genes were rarely mutated in colon cancer patients. Genomically identified groups of colon cancer displayed distinct regulation of claudins and occludin at the mRNA level. Claudin gene promoters possessed clustered sites of binding sequences for transcription factors TCF4 and SMADs, consistent with a key regulatory role of the WNT and TGFß pathways in their expression. Although an effect of claudin mRNA expression on survival of colon cancer patients as a whole was not prominent, survival of genomic subsets was significantly influenced by claudin mRNA expression. mRNA expression of the main tight junction genes showed differential regulation in various genomically defined subgroups of colon cancer. These data pinpoint a distinct role of claudins and pathways that regulate them in these subgroups and suggest that subgroups of colon cancer should be considered in future efforts to therapeutically target claudins.