Intercellular Tight Junctions Research Articles

Expert consensus on the morphological evaluation of human cleavage-stage embryos and blastocysts.

Expert consensus on the morphological evaluation of human cleavage-stage embryos and blastocysts.

Gasdermin A Is Required for Epidermal Cornification during Skin Barrier Regeneration and in an Atopic Dermatitis-Like Model

Atopic dermatitis (AD) is featured with impaired skin barrier. The stratum corneum (SC) and the intercellular tight junctions (TJs) constitute the permeability barrier, which is essential to protect the host water loss and pathogen entrance. The epidermal barrier is constantly renewed by differentiating keratinocytes through cornification, during which autophagy contributes to organelles and nucleus elimination. The human GSDMA and its mouse homologs Gsdma1-3 are expressed in the suprabasal epidermis. Although a pyroptotic role for GSDMA/Gsdma1 in host defense against Streptococcus pyogenes has been reported, the physiological function of Gsdma1/a2/a3 in epidermal homeostasis remains elusive. Herein, through repeated epidermal barrier disruption, we found that TJ formation and SC maturation were defective in the Gsdma1/a3-deficient epidermis. Using comparative gene profiling analysis, mitochondrial respiration measurement, and in vivo tracing of mitophagy, our data indicate that Gsdma1/a3 activation leads to mitochondrial dysfunction and subsequently facilitates mitochondrial turnover and epidermal cornification. In calcipotriol (MC903)-induced AD-like animal model, we demonstrated that Gsdma1/a3-deficiency selectively enhanced the Th2 response. Remarkably, the GSDMA expression is reduced in the epidermis of patients with AD compared to normal individuals. Gsdma1/a3-deficiency might be involved in AD pathogenesis, likely through GSDMA-mediated epidermal differentiation and cornification.

Analysis of Communal Molecular Mechanism Between Chronic Obstructive Pulmonary Disease and Osteoporosis.

Chronic obstructive pulmonary disease (COPD) patients with osteoporosis (OP) usually experience more frequent exacerbations, worse quality of life, and heavier economic burden, however, few studies have investigated common molecular mechanisms of COPD and OP. To explore the relationship between COPD and OP through bioinformatics analysis. The miRNA microarray data of COPD and OP were retrieved from the Gene Expression Database (GEO), and the differentially expressed microRNAs (DEmiRNAs) were screened and the intersection was obtained. The Targetscan, miRDB, and miRWalk databases were used to predict the target genes of DEmiRNA, and the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the R package clusterProfiler, the STRING database was used to analyze the target protein-protein interaction network (PPI) and screens to determine the core modules and core genes. Two DEmiRNAs (miR-23a-5p, miR-194-3p) have been found in COPD and OP, which have predicted 76 and 114 target genes, respectively. GO functional annotations of miR-23a-5p were significantly enriched in CD40 signaling pathway, ubiquitin-conjugating enzyme activity, etc; KEGG pathways of miR-23a-5p were significantly enriched in ubiquitin-mediated proteolysis, folate biosynthesis, and regulation of actin cytoskeleton. GO function annotations of miR-194-3p were significantly enriched in T cell activation regulation, ubiquitin protein ligase activity, and DNA transcription factor binding; KEGG pathways of miR-194-3p were significantly enriched in cell adhesion molecules, intercellular tight junctions, and lysosomal pathway. PPI analysis found target coding proteins formed complex regulatory networks. Ten core genes (TP53, SRC, PXN, CHD4, SYK, TNRC6B, PML, KAT5, BRD1 and IGF2) were picked out among them, then we used the MCODE plugin found three core subnetworks. Two identical DEmiRNAs (miR-23a-5p, miR-194-3p) exist in the peripheral blood of COPD and OP patients, which are important biomarkers for COPD patients with OP and may represent novel targets for diagnosis and treatment of COPD patients with OP.

Effects of Volatile Anesthetics versus Ketamine on Blood-Brain Barrier Permeability via Lipid-Mediated Alterations of Endothelial Cell Membranes

The purpose of this study was to investigate the effects of the volatile anesthetic agents isoflurane and sevoflurane, at clinically relevant concentrations, on the fluidity of lipid membranes and permeability of the blood-brain barrier (BBB). We analyzed the in vitro effects of isoflurane or ketamine using erythrocyte ghosts (sodium fluorescein permeability), monolayers of brain microvascular endothelial cells ([13C]sucrose and fluorescein permeability), or liposomes (fluorescence anisotropy). Additionally, we determined the effects of 30-minute exposure of mice to isoflurane on the brain tight junction proteins. Finally, we investigated in vivo brain uptake of [13C]mannitol and [13C]sucrose after intravenous administration in mice under anesthesia with isoflurane, sevoflurane, or ketamine/xylazine in addition to the awake condition. Isoflurane at 1-mM and 5-mM concentrations increased fluorescein efflux from the erythrocyte ghosts in a concentration-dependent manner. Similarly, in endothelial cell monolayers exposed to 3% (v/v) isoflurane, permeability coefficients rose by about 25% for fluorescein and 40% for [13C]sucrose, whereas transendothelial resistance and cell viability remained unaffected. Although isoflurane caused a significant decrease in liposomes anisotropy values, ketamine/xylazine did not show any effects. Brain uptake clearance (apparent Kin) of the passive permeability markers in vivo in mice approximately doubled under isoflurane or sevoflurane anesthesia compared with either ketamine/xylazine anesthesia or the awake condition. In vivo exposure of mice to isoflurane did not change any of the brain tight junction proteins. Our data support membrane permeabilization rather than loosening of intercellular tight junctions as an underlying mechanism for increased permeability of the endothelial cell monolayers and the BBB in vivo. SIGNIFICANCE STATEMENT: The blood-brain barrier controls the entry of endogenous substances and xenobiotics from the circulation into the central nervous system. Volatile anesthetic agents like isoflurane alter the lipid structure of cell membranes, transiently facilitating the brain uptake of otherwise poorly permeable, hydrophilic small molecules. Clinical implications may arise when potentially neurotoxic drugs gain enhanced access to the central nervous system under inhalational anesthetics.

The Ultrastructure of Hepatic Stellate Cell-Macrophage Intercellular Crosstalk as a New Morphological Insight into Phenomenon of Fibrogenesis in Pediatric Autoimmune Hepatitis.

Ultrastructural assessment of the HSC population and NPCs was performed in transmission electron microscopy (TEM) using pretreatment liver biopsies from 25 children (8 boys and 17 girls) aged 4-17 with clinic-pathologically diagnosed untreated AIH. Submicroscopic evaluation allowed easy identification of numerous HSCs in the form of transitory cells, i.e., T-HSCs, accompanied by signs of fibrosis. T-HSCs included cells with features of activation initiation (iHSCs) and activation perpetuation (pHSCs), indicating high HSC activation plasticity. The pHSCs were markedly elongated and mainly showed a distinct loss of lipid cytoplasmic material, expanded and dilated channels of granular endoplasmic reticulum, and linear bundles of microfilaments beneath the cell membrane. They were surrounded by usually mature collagen fibers. Frequently activated KCs/MPs adhered directly to T-HSCs. Between them, tight intercellular junctions were formed by means of point desmosomes. Our qualitative TEM observations indicate a key role of T-HSCs in liver fibrogenesis in pediatric AIH, with the essential involvement of activated KCs/MPs that directly adhere to them. Tight intercellular junctions, being the ultrastructural exponent of the specific cellular mechanisms of the crosstalk between NPCs, can play a vital role in hepatic collagen fibroplasia. A better understanding of HSC population morphology at the ultrastructural level in AIH seems important not only to improve the disease morphological diagnostics but to also provide new insights into therapeutic interventions for the phenomenon of liver fibrogenesis.

The role of Nrf2 in the alteration of tight junction protein expression in choroid plexus epithelial cells created by lanthanum-activated MMP9

Objective: To investigate the effect of nuclear factor erythroid 2-related factor 2 (Nrf2) in the alteration of tight junction protein expression in choroid plexus epithelial cells created by lanthanum-activated matrix metalloproteinase 9 (MMP9) . Methods: In October 2020, immortalized rat choroid plexus epithelial cell line (Z310) cells were used as the blood-cerebrospinal fluid barrier in vitro, and were divided into control group and 0.125, 0.25, 0.5 mmol/L lanthanum chloride (LaCl(3)) treatment group. After treating Z310 cells with different concentrations of LaCl(3) for 24 hours, the morphological changes of Z310 cells were observed under inverted microscope, the protein expression levels of MMP9, occludin and zonula occludens-1 (ZO-1) were observed by cellular immunofluorescence method, and the protein expression levels of MMP9, tissue inhibitors of metalloproteinase1 (TIMP1) , occludin, ZO-1 and Nrf2 were detected by Western blotting. The level of reactive oxygen species (ROS) in cells was detected by flow cytometry. Results: Compared with the control group, Z310 cells in the LaCl(3) treatment group were smaller in size, with fewer intercellular junctions, and more dead cells and cell fragments. The expression level of MMP9 protein in cells treated with 0.25 and 0.5 mmol/L LaCl(3) was significantly higher than that in the control group (P<0.05) , and the expression level of TIMP1 and tight junction proteins occudin and ZO-1 was significantly lower than that in the control group (P<0.05) . Compared with the control group, the ROS production level in the 0.25, 0.5 mmol/L LaCl(3) treatment group was significantly increased (P<0.05) , and the Nrf2 protein expression level in the 0.125, 0.25, 0.5 mmol/L LaCl(3) treatment group was significantly decreased (P<0.05) . Conclusion: Lanthanum may increase the level of ROS in cells by down regulating the expression of Nrf2, thus activating MMP9 to reduce the expression level of intercellular tight junction proteins occludin and ZO-1.

Morphological and Functional Effects of Ultrasound on Blood–Brain Barrier Transitory Opening: An In Vitro Study on Rat Brain Endothelial Cells

With the recent advances in medicine, human life expectancy is increasing; however, the extra years of life are not necessarily spent in good health or free from disability, resulting in a significantly higher incidence of age-associated pathologies. Among these disorders, neurodegenerative diseases have a significant impact. To this end, the presence of the protective blood-brain barrier (BBB) represents a formidable obstacle to the delivery of therapeutics. Thus, this makes it imperative to define strategies to bypass the BBB in order to successfully target the brain with the appropriate drugs. It has been demonstrated that targeting the BBB by ultrasound (US) can transiently make this anatomical barrier permeable and in so doing, allow the delivery of therapeutics. Thus, our aim was to carry out an in depth in vitro molecular and morphological study on the effects of US treatment on the BBB. The rat brain endothelial (RBE4) cell line was challenged with exposure to 12 MHz diagnostic US treatment for 10, 20, and 30 min. Cell viability assays, Western blotting analysis on the endoplasmic reticulum (ER), and oxidative stress marker evaluation were then performed, along with cytological and immunofluorescence staining, in order to evaluate the effects of US on the intercellular spaces and tight junction distribution of the brain endothelial cells. We observed that the US treatment exerted no toxic effects on either RBE4 cell viability or the upregulation/dislocation of the ER and oxidative stress marker (GRP78 and cytochrome C, respectively). Further, we observed that the application of US induced an increase in the intercellular spaces, as shown by Papanicolaou staining, mainly due to the altered distribution of the tight junction protein zonula occludens-1 (ZO-1). This latter US-dependent effect was transient and disappeared 20 min after the removal of the stimulus. In conclusion, our results show that US induces a transient alteration of the BBB, without altering the intracellular signaling pathways such as the ER and oxidative stress that could potentially be toxic for endothelial cells. These results suggested that US treatment could represent a potential strategy for improving drug delivery to the brain.

Experimental Study on the Treatment of Ulcerative Proctitis in Rats by Combining Baitouweng Decoction with Mesalamine Retention Enema

To study the effect and mechanism of Baitouweng decoction, mesalamine and its combination with an enema in the treatment of ulcerative proctitis in rats is the objective of the study. The rats were divided into seven groups: Control (Normal), model (ulcerative proctitis), mesalamine, Baitouweng decoction, lowdose Baitouweng decoction combined with mesalamine, medium-dose Baitouweng decoction combined with mesalamine, high-dose Baitouweng decoction combined with mesalamine group. Except for the control group, all rats were induced with a 5 % acetic acid solution for ulcerative proctitis. The severity of ulcerative proctitis was measured by the disease activity index score; morphological changes of rectal tissue were observed by hematoxylin and eosin staining; expression of cytokines tumor necrosis factor alpha, interleukin-1 beta, interleukin-6, interferon‐gamma, interleukin-10 messenger ribonucleic acid was detected by reverse transcription-polymerase chain reaction; Western blot analysis of signaling pathway proteins nuclear factor kappa B p65 and phosphorylated-nuclear factor kappa B p65 and the expression of tight junction proteins claudin-1, occludin and zonula occludens-1 was detected by Western blot and immunohistochemistry. The results revealed that the disease activity index scores of rats in each dosing group were significantly lower than those in the ulcerative proctitis group. After treatment, the expression of pro-inflammatory factors tumor necrosis factor alpha, interleukin-1 beta, interleukin-6 and interferon‐gamma was inhibited and the expression of the anti-inflammatory factor interleukin-10 was up-regulated. In conclusion, Baitouweng decoction combined with mesalamine had a synergistic effect on the treatment of ulcerative proctitis. The mechanism of action may be to regulate the synthesis and release of inflammatory factors by inhibiting the over-activation of nuclear factor kappa B p65, reducing the damage of pro-inflammatory factors to rectal mucosa and repairing the intercellular tight junction structure, thus improving the intestinal barrier and reducing the rectal inflammatory response.

Framework Nucleic Acids as Blood-Retinal-Barrier-Penetrable Nanocarrier for Periocular Administration.

Designing an ocular drugs delivery system that can permeate the outer blood-retinal barrier (oBRB) is crucial for the microinvasive or noninvasive treatment of ocular fundus diseases. However, due to the lack of a nanocarrier that can maintain structure and composition at the oBRB, only intravitreal injection at the eyeball can deliver therapeutics directly to the ocular fundus via paracellular and intercellular routes, despite the intraocular operations risks. Here, we demonstrated tetrahedral framework nucleic acids (tFNAs) can penetrate the oBRB and deliver therapeutic nucleic acids to the retina of the rat eye in vivo following subconjunctival injection. We also discovered that tFNAs were transported via a paracellular route across the intercellular tight junctions at the oBRB. The histology analysis for ocular layers indicated that individual and aptamer/doxorubicin-loaded tFNAs penetrated all layers of the posterior segment of the eyeball to reach the innermost retina and persisted for over 3 days with minimal systemic biodistribution. We expect that the programmability and penetrability of tFNAs will provide a promising method for drug delivery across oBRB and long-term sustenance at the target site via periocular administration to various tissues.

Recombinant human plasma gelsolin reverses increased permeability of the blood–brain barrier induced by the spike protein of the SARS-CoV-2 virus

BackgroundPlasma gelsolin (pGSN) is an important part of the blood actin buffer that prevents negative consequences of possible F-actin deposition in the microcirculation and has various functions during host immune response. Recent reports reveal that severe COVID-19 correlates with reduced levels of pGSN. Therefore, using an in vitro system, we investigated whether pGSN could attenuate increased permeability of the blood–brain barrier (BBB) during its exposure to the portion of the SARS-CoV-2 spike protein containing the receptor binding domain (S1 subunit).Materials and methodsTwo- and three-dimensional models of the human BBB were constructed using the human cerebral microvascular endothelial cell line hCMEC/D3 and exposed to physiologically relevant shear stress to mimic perfusion in the central nervous system (CNS). Trans-endothelial electrical resistance (TEER) as well as immunostaining and Western blotting of tight junction (TJ) proteins assessed barrier integrity in the presence of the SARS-CoV-2 spike protein and pGSN. The IncuCyte Live Imaging system evaluated the motility of the endothelial cells. Magnetic bead-based ELISA was used to determine cytokine secretion. Additionally, quantitative real-time PCR (qRT-PCR) revealed gene expression of proteins from signaling pathways that are associated with the immune response.ResultspGSN reversed S1-induced BBB permeability in both 2D and 3D BBB models in the presence of shear stress. BBB models exposed to pGSN also exhibited attenuated pro-inflammatory signaling pathways (PI3K, AKT, MAPK, NF-κB), reduced cytokine secretion (IL-6, IL-8, TNF-α), and increased expression of proteins that form intercellular TJ (ZO-1, occludin, claudin-5).ConclusionDue to its anti-inflammatory and protective effects on the brain endothelium, pGSN has the potential to be an alternative therapeutic target for patients with severe SARS-CoV-2 infection, especially those suffering neurological complications of COVID-19.

Evaluation of a connexin-based peptide for the treatment of age-related macular degeneration

A critical target in age-related macular degeneration (AMD) is the retinal pigment epithelium (RPE), which forms the outer blood-retina barrier (BRB). RPE-barrier dysfunction might result from the disruption of intercellular tight junctions (TJs). A Connexin43 (Cx43)-based peptide, aCT1, has been shown to prevent VEGF-induced loss of transepithelial resistance, choroidal neovascularization (CNV) and RPE-cell damage via the stabilization of TJs. Here, we probe the relative efficacies of aCT1 alone, anti-VEGF alone, and aCT1 with anti-VEGF in treating AMD pathologies. aCT1 monotherapy administered as topical eye drops with and without a VEGF blocking antibody administered systemically was tested in a mouse model of laser-induced CNV. The CNV mouse is the standard neovascular AMD model, reproducing hallmarks of its pathology. CNV lesion size and fluid accumulation were assessed using optical coherence tomography. During the angiogenesis phase of CNV lesion development, single applications of anti-VEGF or aCT1 reduced lesion and fluid dome size equally. The combinatorial aCT1/anti-VEGF strategy demonstrated lack of additive effects in this model. These data suggest that TJ stabilization by aCT1 is effective in ameliorating RPE dysfunction in a model of AMD-like angiogenesis, and that this strategy is as effective as the current clinical standard of care, anti-VEGF therapy. Critically, aCT1 holds potential as a new neovascular AMD treatment that can be administered using eye drops, which is preferable to the intravitreal injections required for standard anti-VEGF therapy.

The Role of Essential Oils on Improving Elderly Skin Hydration

Skin hydration has an important role in various diseases of the elderly. Skin hydration is determined by two factors, namely the ability of the stratum corneum (SC) to retain water due to natural moisturizing factors and the barrier function of the intercellular lipid lamellar structure and tight junctions. Several parameters are usually used for skin hydration, such as surface water content (SWC) and trans-epidermal water loss (TEWL). With age, several changes in the skin affect hydration, including changes in the cellular and intercellular lipid matrix, skin pH, SC protease enzymes, decreased sebaceous and sweat gland activity, and decreased estrogen levels in women. Therefore, a moisturizer is needed to minimize the unwanted effects of some of these changes. Selection of the type of moisturizer is very important for the elderly, especially moisturizers with antiseptic, antibacterial, and antioxidant effects are highly recommended. The ability of essential oils as components of moisturizers has been proven in various studies. Essential oils can improve the function of the epidermal barrier and help prevent transepidermal water loss. Therefore, this literature review discusses the role of various essential oils, namely green tea oils (Camellia sinensis or Camellia assamica), virgin coconut oils (Cocos nucifera L.), evening primrose oils (Oenothera paradoxa), grape seed oils (Vitis vinifera L.) .), rosemary oils (Rosmarinus officinalis L.), immortelle oils (Helichrysum italicum), and cannabis seeds oils (Cannabis sativa L.) on improving skin hydration in the elderly.

Systemic effect of arecoline on the gastrointestinal system in oral submucous fibrosis affected wistar rats.

The intestine plays an important role in the digestion and absorption of ingested food and the elimination of undigested food, microbes, and microbial products. The functional reliability of the intestinal mucosal epithelial cells depends on the organised regulation of the epithelial cells, mucus layer, the intercellular tight junction, host innate and acquired immune response. The mucus layer of the gastrointestinal tract is the first line of innate host defense, essentially because of the secretory products of intestinal cells. Present study was conducted to evaluate the effect of arecoline on the gastrointestinal system due to systemic absorption of the drug during the induction period of oral submucous fibrosis (OSMF) in Wistar rats. Oral submucous fibrosis was induced by submucosal injection of arecoline in the buccal mucosa. Arecoline hydrochloride at a dosage of 10 mg/kg was injected into the submucosa of right buccal mucosa in experimental animals over a period of 3 months on every alternate day. After which, right buccal mucosa, gastrointestinal tract organs like stomach, large intestine, small intestine and liver were dissected, subjected to histopathological evaluation of the healthy and experimental Wistar rats were subjected to histopathological evaluation. On histological evaluation, OSMF was seen to affect Wistar rats showed significant changes in oral mucosa, decrease number of goblet cells in the small intestine as well in the large intestine and deranged hepatocytes. These marked changes indicated a definite effect on the gastro intestinal system by arecoline. The study has highlighted the effect of arecoline due to systemic absorption during the induction process of OSMF in Wistar rats.

Immunoexpression of claudin-4 and correlation with estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2-neu in breast cancer.

Claudins are important transmembrane proteins in tight junction. The role of intercellular tight junctions in breast epithelial cells is traditionally thought to be in maintaining polarity and barrier function. However, claudin-4, a tight junction protein, is overexpressed in breast tumor cells compared to normal epithelial cells, which generally corresponds to loss in polarity and can provide valuable information about biology of the tumor. A prospective clinical study was conducted to assess the expression claudin-4 in patients with breast cancer and its correlation with hormone receptors - estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)-neu. The study included 102 biopsy-proven breast carcinoma patients. The biopsy samples were evaluated immunohistochemistry for expression of ER, PR, HER2-neu, and claudin-4. The expression of claudin-4 was correlated with ER, PR, and HER2-neu. In the study, we found that out of 26 cases of high claudin-4, 25 cases (96.15%) were ER negative and P < 0.001, which was significant. Similar results were found with PR-negative cases. Whereas, out of 76 cases with low claudin-4, 54 cases (71.05%) were HER2-neu negative and P = 0.022, which was significant. Claqudin-4 expression has a negative correlation with ER and PR and has a positive correlation with HER2-neu. Hence, it can be effectively utilized as a prognostic and therapeutic marker in breast cancer in the future.

A doxycycline-inducible CYP3A4-Caco-2 cell line as a model for evaluating safety of aflatoxin B1 in the human intestine

Exposure of humans to aflatoxin B1 (AFB1) via ingestion of contaminated agricultural products is a major concern for human health throughout the world because epoxidized AFB1, biotransformed from AFB1 by hepatic CYP3A4, is strongly hepatotoxic and hepatocarcinogenic. Intestinal epithelial cells serve as a physical and physiological barrier against xenobiotics via their intercellular tight junction (TJ) seals and the metabolizing enzyme CYP3A4. However, the effect of AFB1 on the intestinal barrier remains unclear. Here, we investigated the influence of AFB1 on these physical and physiological intestinal barriers by means of an in vitro human intestinal model utilizing doxycycline-inducible CYP3A4-expressing Caco-2 cells, in which CYP3A4 activity is comparable to that in the adult human intestine. Cellular toxicity of AFB1 in induced Caco-2 cells (i.e., cells in which expression of CYP3A4 is induced by doxycycline) was approximately 5 times that of uninduced Caco-2 cells. Exposure to 16 µM AFB1 did not decrease the transepithelial electric resistance (TEER; a measure of TJ barrier integrity) in monolayers of uninduced Caco-2 cells to 95.8 % of that in vehicle-treated cells; in contrast, in induced Caco-2 cells, TEER was reduced to 28.8 %. Exposure to 16 µM AFB1 increased paracellular permeation of 4- and 20-kDa dextrans (paracellular permeation markers) through monolayers of induced Caco-2 cells to 5.4 and 5.2 times that through uninduced Caco-2 cells. These results together show that ingested AFB1 can modulate the intestinal barrier, and that inducible CYP3A4-expressing Caco-2 cells are a promising tool for evaluating the safety of food contaminants in the human intestine.

Lactobacillus rhamnosus GR-1 attenuates foodborne Bacillus cereus-induced NLRP3 inflammasome activity in bovine mammary epithelial cells by protecting intercellular tight junctions

BackgroundBacillus cereus is an important pathogen that causes human food poisoning, specifically diarrhea and vomiting. B. cereus can also induce mastitis in dairy cows and has a strong survival ability in milk, as it cannot be inactivated by high-temperature short-time pasteurization. Therefore, B. cereus can enter the market through pasteurized milk and other dairy products, imposing enormous hidden dangers on food safety and human health.ResultsIn this study, B. cereus 2101 (BC) was isolated from milk samples of cows with mastitis. BC grew rapidly with strong hemolysis, making it difficult to prevent mastitis and ensure food security. MAC-T cells were treated with BC and/or Lactobacillus rhamnosus GR-1 (LGR-1). Pretreatment with LGR-1 protected the integrity of tight junctions and the expression of zonula occludens-1 (ZO-1) and occludin destroyed by BC. Furthermore, LGR-1 pretreatment reduced the expression of NOD-like receptor family member pyrin domain-containing protein 3 (NLRP3), caspase recruitment and activation domain (ASC), Caspase-1 p20, gasdermin D (GSDMD) p30, inflammatory factors (interleukin (IL)-1β and IL-18), and cell death induced by BC. Moreover, LGR-1 pretreatment reduced NLRP3 inflammasome activity and increased expressions of ZO-1 and occludin induced by lipopolysaccharides (LPS) + ATP stimulation. MAC-T cells were transfected with NLRP3 siRNA or MCC950 and/or treated with BC and/or LGR-1. NLRP3-siRNA transfection and MCC950 attenuated BC-induced NLRP3 inflammasome activity. Expression of inflammatory cytokines and cell death suggested that the inflammatory pathway might play an important role in the induction of the NLRP3 inflammasome by BC and the protection of LGR-1.ConclusionsThese results suggest that LGR-1 might be a probiotic alternative to antibiotics and could be administered to prevent mastitis in dairy cows, thus ensuring food security.

Fusobacterium nucleatum Extracellular Vesicles Promote Experimental Colitis by Modulating Autophagy via the miR-574-5p/CARD3 Axis.

Ulcerative colitis (UC) may be exacerbated by Fusobacterium nucleatum (Fn) infection. However, the mechanism underlying Fn-mediated progression of UC has yet to be established. Here, we aimed to establish whether and how Fn-derived extracellular vesicles (Fn-EVs) participate in the development of experimental colitis through microRNAs (miRNAs). EVs were isolated and purified by ultracentrifugation from Fn and Escherichia coli culture supernatants. Differentially expressed miRNAs in control intestinal epithelial cells (IECs) and Fn-EV-treated IECs were identified by miRNA sequencing. EVs were cocultured with IECs or administered to CARD3wt/CARD3-/- mice by gavage to assess inflammatory responses to and the mechanism of action of Fn-EVs. Fn-EVs promoted upregulation of proinflammatory cytokines (interleukin [IL]-1β, IL-6, tumor necrosis factor α), downregulation of anti-inflammatory IL-10 and intercellular tight junction proteins ZO-1 and occludin, and epithelial barrier dysfunction in IECs. Fn-EVs significantly aggravated experimental colitis in mice associated with Fn-EV-mediated downregulation of miR-574-5p expression and autophagy activation. Blockade of autophagy using chloroquine alleviates barrier damage exacerbated by Fn-EVs in vitro and in vivo. Inhibition of the miR-574-5p/CARD3 axis reduced the severity of colitis, epithelial barrier damage, and autophagy activation induced by Fn-EVs. Here, we describe a new mechanism by which Fn-EVs mediate experimental colitis severity through miR-574-5p/CARD3-dependent autophagy activation, providing a novel target for UC monitoring and targeted therapy.

Neutrophil extracellular traps participate in the development of cancer-associated thrombosis in patients with gastric cancer.

BACKGROUNDThe development of venous thromboembolism (VTE) is associated with high mortality among gastric cancer (GC) patients. Neutrophil extracellular traps (NETs) have been reported to correlate with the prothrombotic state in some diseases, but are rarely reported in GC patients.AIMTo investigate the effect of NETs on the development of cancer-associated thrombosis in GC patients.METHODSThe levels of NETs in blood and tissue samples of patients were analyzed by ELISA, flow cytometry, and immunofluorescence staining. NET generation and hypercoagulation of platelets and endothelial cells (ECs) in vitro were observed by immunofluorescence staining. NET procoagulant activity (PCA) was determined by fibrin formation and thrombin–antithrombin complex (TAT) assays. Thrombosis in vivo was measured in a murine model induced by flow stenosis in the inferior vena cava (IVC).RESULTSNETs were likely to form in blood and tissue samples of GC patients compared with healthy individuals. In vitro studies showed that GC cells and their conditioned medium, but not gastric mucosal epithelial cells, stimulated NET release from neutrophils. In addition, NETs induced a hypercoagulable state of platelets by upregulating the expression of phosphatidylserine and P-selectin on the cells. Furthermore, NETs stimulated the adhesion of normal platelets on glass surfaces. Similarly, NETs triggered the conversion of ECs to hypercoagulable phenotypes by downregulating the expression of their intercellular tight junctions but upregulating that of tissue factor. Treatment of normal platelets or ECs with NETs augmented the level of plasma fibrin formation and the TAT complex. In the models of IVC stenosis, tumor-bearing mice showed a stronger ability to form thrombi, and NETs abundantly accumulated in the thrombi of tumor-bearing mice compared with control mice. Notably, the combination of deoxyribonuclease I, activated protein C, and sivelestat markedly abolished the PCA of NETs.CONCLUSIONGC-induced NETs strongly increased the risk of VTE development both in vitro and in vivo. NETs are potential therapeutic targets in the prevention and treatment of VTE in GC patients.

Establishment of an astrocyte-like cell line from the brain of tilapia (Oreochromis niloticus) for virus pathogenesis and a vitro model of the blood-brain barrier.

A new cell line was established from the brain of a cultured fish, tilapia (Oreochromis niloticus), designated as TA-02 (Tilapia Astrocyte clone 02 cell line). The TA-02 cells are grown for 300 days in an L-15 medium supplemented with 10% fetal bovine serum (FBS). This cell line showed excellent proliferative capacity and expressed various neuroglial cell markers, including SOX2, SOX10, Hes1, Notch1, Occludin, E-cadherin, and GFAP. In addition, TA-02 cells were susceptible to Tilapia Lake Virus (TiLV) as demonstrated by the presence of a severe cytopathic effect (CPE), virus particle in a transmission electron microscope (TEM), and PCR positive signal. Bacterial cytotoxicity studies showed that Streptococcus agalactiae was toxic to TA-02 cells. When co-culture with trans-well, TA-02 exhibited prominent barrier properties, manifested by tight intercellular junctions and increased trans-endothelial electrical resistance (TEER). In addition, the barrier is effective against Escherichia coli (non-meningitis pathogenic bacteria). In contrast, S. agalactiae (meningitis pathogenic bacteria) can pass through the membrane comprising the cells in the trans-well insert. The newly established TA-02 cell line provided a valuable tool for virus pathogenesis and a vitro model of the fish blood-brain barrier.

SARS-CoV-2 productively infects human brain microvascular endothelial cells

BackgroundThe emergence of the novel, pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global health emergency. SARS-CoV-2 is highly contagious and has a high mortality rate in severe patients. However, there is very limited information on the effect of SARS-CoV-2 infection on the integrity of the blood–brain barrier (BBB).MethodsRNA-sequencing profiling was performed to analyze the transcriptomic changes in human brain microvascular endothelial cells (hBMECs) after SARS-CoV-2 infection. Bioinformatic tools were used for differential analysis. Immunofluorescence, real-time quantitative PCR, and Western blotting analysis were used to explore biological phenotypes.ResultsA total of 927 differentially expressed genes were identified, 610 of which were significantly upregulated while the remaining 317 were downregulated. We verified the significant induction of cytokines, chemokines, and adhesion molecules in hBMECs by SARS-CoV-2, suggesting an activation of the vascular endothelium in brain. Moreover, we demonstrated that SARS-CoV-2 infection could increase the BBB permeability, by downregulating as well as remodeling the intercellular tight junction proteins.ConclusionsOur findings demonstrated that SARS-CoV-2 infection can cause BBB dysfunction, providing novel insights into the understanding of SARS-CoV-2 neuropathogenesis. Moreover, this finding shall constitute a new approach for future prevention and treatment of SARS-CoV-2-induced CNS infection.