Blood-brain barrier (BBB) dysfunction is a key feature in neuroimmunological and neurodegenerative diseases. In this study, we developed a microfluidic human BBB-on-a-chip to model barrier dysfunction and immune cell migration using immortalized TY10 brain endothelial cells, pericytes, and astrocytes. It was found that immortalized TY10 brain endothelial cells developed a microvascular structure under flow. Pericytes were localized on the basal side surrounding the TY10 microvascular structure, showing an in vivo-like structure. Barrier integrity increased under co-culture with pericytes. In addition, both ethylenediaminetetraacetic acid (EDTA) and anti-Claudin-5 (CLDN5) neutralizing antibody caused a decrease in the transendothelial electrical resistance (TEER). EDTA caused the leakage of 20 kDa dextran, suggesting different effects on the BBB based on the mechanism of action, whereas anti-CLDN5 antibody did not cause leakage. In the tri-culture model, human T cells migrated through endothelial vessels towards basal C-X-C motif chemokine ligand 12 (CXCL12). The live-imaging analysis confirmed the extravasation of fluorescence-labelled T cells in a CXCL12-concentration- and time-dependent manner. Our BBB model had an in vivo-like structure and successfully represented barrier dysfunction and transendothelial T cell migration. In addition, our study suggests that the inhibition of CLDN5 attenuates the BBB in humans. This platform has various potential uses in relation to the BBB in both drug discovery research and in elucidating the mechanisms of central nervous system diseases.

The epithelial barrier serves as a critical defense mechanism separating the human body from the external environment, fulfilling both physical and immune functions. This barrier plays a pivotal role in shielding the body from environmental risk factors such as allergens, pathogens, and pollutants. However, since the 19th century, the escalating threats posed by environmental pollution, global warming, heightened usage of industrial chemical products, and alterations in biodiversity have contributed to a noteworthy surge in allergic disease incidences. Notably, allergic diseases frequently exhibit dysfunction in the epithelial barrier. The proposed epithelial barrier hypothesis introduces a novel avenue for the prevention and treatment of allergic diseases. Despite increased attention to the role of barrier dysfunction in allergic disease development, numerous questions persist regarding the mechanisms underlying the disruption of normal barrier function. Consequently, this review aims to provide a comprehensive overview of the epithelial barrier\'s role in allergic diseases, encompassing influencing factors, assessment techniques, and repair methodologies. By doing so, it seeks to present innovative strategies for the prevention and treatment of allergic diseases.

Intestinal epithelia express two long myosin light-chain kinase (MLCK) splice variants, MLCK1 and MLCK2, which differ by the absence of a complete immunoglobulin (Ig)-like domain 3 within MLCK2. MLCK1 is preferentially associated with the perijunctional actomyosin ring at steady state, and this localization is enhanced by inflammatory stimuli including tumor necrosis factor (TNF). Here, we sought to identify MLCK1 domains that direct perijunctional MLCK1 localization and their relevance to disease. Ileal biopsies from Crohn\'s disease patients demonstrated preferential increases in MLCK1 expression and perijunctional localization relative to healthy controls. In contrast to MLCK1, MLCK2 expressed in intestinal epithelia is predominantly associated with basal stress fibers, and the two isoforms have distinct effects on epithelial migration and barrier regulation. MLCK1(Ig1-4) and MLCK1(Ig1-3), but not MLCK2(Ig1-4) or MLCK1(Ig3), directly bind to F-actin in vitro and direct perijunctional recruitment in intestinal epithelial cells. Further study showed that Ig1 is unnecessary, but that, like Ig3, the unstructured linker between Ig1 and Ig2 (Ig1/2us) is essential for recruitment. Despite being unable to bind F-actin or direct recruitment independently, Ig3 does have dominant negative functions that allow it to displace perijunctional MLCK1, increase steady-state barrier function, prevent TNF-induced MLCK1 recruitment, and attenuate TNF-induced barrier loss. These data define the minimal domain required for MLCK1 localization and provide mechanistic insight into the MLCK1 recruitment process. Overall, the results create a foundation for development of molecularly targeted therapies that target key domains to prevent MLCK1 recruitment, restore barrier function, and limit inflammatory bowel disease progression.

Atopic dermatitis (AD) is the most common chronic inflammatory skin disease and presents a major public health problem worldwide. It is characterized by a recurrent and/or chronic course of inflammatory skin lesions with intense pruritus. Its pathophysiologic features include barrier dysfunction, aberrant immune cell infiltration, and alterations in the microbiome that are associated with genetic and environmental factors. There is a complex crosstalk between these components, which is primarily mediated by cytokines. Epidermal barrier dysfunction is the hallmark of AD and is caused by the disruption of proteins and lipids responsible for establishing the skin barrier. To better define the role of cytokines in stratum corneum lipid abnormalities related to AD, we conducted a systematic review of biomedical literature in PubMed from its inception to 5 September 2023. Consistent with the dominant TH2 skewness seen in AD, type 2 cytokines were featured prominently as possessing a central role in epidermal lipid alterations in AD skin. The cytokines associated with TH1 and TH17 were also identified to affect barrier lipids. Considering the broad cytokine dysregulation observed in AD pathophysiology, understanding the role of each of these in lipid abnormalities and barrier dysfunction will help in developing therapeutics to best achieve barrier homeostasis in AD patients.

Quercetin (Que) is a flavonol compound found in plants, which has a variety of biological activities. Necroptosis, a special form of programmed cell death, plays a vital role in the development of many gastrointestinal diseases. This study aimed to explore whether Que could attenuate the intestinal injury and barrier dysfunction of piglets after deoxynivalenol (DON) exposure through modulating the necroptosis signaling pathway. Firstly, twenty-four weaned piglets were used in a 2 × 2 factorial design and the main factors, including Que (basal diet or diet supplemented with 100 mg/kg Que) and DON exposure (control feed or feed contaminated with 4 mg/kg DON). After feeding for 21 d, piglets were killed for samples. Next, the intestinal porcine epithelial cell line (IPEC-1) was pretreated with or without Que (10 μmol/mL) in the presence or absence of a DON challenge (0.5 μg/mL). Dietary Que increased the body weight, average daily gain, and average daily feed intake (p < 0.05) through the trial. Que supplementation improved the villus height, and enhanced the intestinal barrier function (p < 0.05) indicated by the higher protein expression of occludin and claudin-1 (p < 0.05) in the jejunum of the weaned piglets after DON exposure. Dietary Que also down-regulated the protein abundance of total receptor interacting protein kinase 1 (t-RIP1), phosphorylated RIP1 (p-RIP1), p-RIP3, total mixed lineage kinase domain-like protein (t-MLKL), and p-MLKL (p < 0.05) in piglets after DON exposure. Moreover, Que pretreatment increased the cell viability and decreased the lactate dehydrogenase (LDH) activity (p < 0.05) in the supernatant of IPEC-1 cells after DON challenge. Que treatment also improved the epithelial barrier function indicated by a higher transepithelial electrical resistance (TEER) (p < 0.001), lower fluorescein isothiocyanate-labeled dextran (FD4) flux (p < 0.001), and better distribution of occludin and claudin-1 (p < 0.05) after DON challenge. Additionally, pretreatment with Que also inhibited the protein abundance of t-RIP1, p-RIP1, t-RIP3, p-RIP3, t-MLKL, and p-MLKL (p < 0.05) in IPEC-1 cells after DON challenge. In general, our data suggest that Que can ameliorate DON-induced intestinal injury and barrier dysfunction associated with suppressing the necroptosis signaling pathway.

Opioid crisis is an ongoing epidemic since the past several decades in the United States. Opioid use-associated microbial dysbiosis is emerging as a key regulator of intestinal homeostasis and behavioral responses to opioid. However, the mechanistic insight into the role of microbial community in modulating host response is unavailable. To uncover the role of opioid-induced dysbiosis in disrupting intestinal homeostasis we utilized whole genome sequencing, untargeted metabolomics, and mRNA sequencing to identify changes in microbiome, metabolome, and host transcriptome respectively. Morphine treatment resulted in significant expansion of Parasuterella excrementihominis, Burkholderiales bacterium 1_1_47, Enterococcus faecalis, Enterorhabdus caecimuris and depletion of Lactobacillus johnsonii. These changes correlated with alterations in lipid metabolites and flavonoids. Significant alteration in microbial metabolism (metabolism of lipids, amino acids, vitamins and cofactors) and increased expression of virulence factors and biosynthesis of lipopolysaccharides (LPS) and lipoteichoic acid (LTA) were observed in microbiome of morphine-treated animals. In concurrence with changes in microbiome and metabolome extensive changes in innate and adaptive immune response, lipid metabolism, and gut barrier dysfunction were observed in the host transcriptome. Microbiome depleted mice displayed lower levels of inflammation, immune response and tissue destruction compared to mice harboring a dysbiotic microbiome in response to morphine treatment, thus establishing dysbiotic microbiome as mediator of morphine gut pathophysiology. Integrative analysis of multi-omics data highlighted the associations between Parasutterella excrementihominis, Burkholderiales bacterium 1_1_47, Enterococcus faecalis, Enterorhabdus caecimuris and altered levels of riboflavin, flavonoids, and lipid metabolites including phosphocholines, carnitines, bile acids, and ethanolamines with host gene expression changes involved in inflammation and barrier integrity of intestine. Omic analysis also highlighted the role of probiotic bacteria Lactobacillus johnsonii, metabolites flavonoids and riboflavin that were depleted with morphine as important factors for intestinal homeostasis. This study presents for the first time ever an interactive view of morphine-induced changes in microbial metabolism, strain level gut microbiome analysis and comprehensive view of changes in gut transcriptome. We also identified areas of potential therapeutic interventions to limit microbial dysbiosis and present a unique resource to the opioid research community.

Large-scale use of antimicrobials in agriculture and medicine contributes to antibiotic residues in raw foods, the spread of antimicrobial resistance (AMR) and drug pollution, which seriously threatens human health and imposes significant economic burdens on society, suggesting the need for novel therapeutic options that prevent or control zoonoses. In this study, four probiotics were selected to assess their capability to alleviate pathogen-induced damage. Results showed that a simulated gastrointestinal juice and bile tolerated L. plantarum Lac16 with high lactic acid secretion can significantly inhibit the growth of multiple zoonotic pathogens. Lac16 also significantly inhibited the biofilm formation and mRNA expression of virulence traits (genes related to virulence, toxins, flagella biogenesis and motility, antibiotic resistance, biofilm formation and AI-2 quorum sensing) of enterohemorrhagic E. coli O157:H7 (EHEC). Furthermore, Lac16 and Lac26 significantly protected C. elegans against zoonotic pathogen-induced (EHEC, S. typhimurium, C. perfringens) deaths. Moreover, Lac16 significantly promoted epithelial repair and ameliorated lipopolysaccharide (LPS)-induced intestinal epithelial apoptosis and barrier dysfunction by activating the Wnt/β-catenin signaling pathway, and markedly reduced LPS-induced inflammatory responses by inhibiting the TLR4/MyD88 signaling pathway. The present results indicate that Lac16 attenuates enterohemorrhagic E. coli infection-induced damage by inhibiting key virulence traits of E. coli, promoting epithelial repair and improving intestinal epithelial barrier function, which may be mediated by the activated Wnt/β-catenin signaling pathway and the inhibited TLR4/MyD88 signaling pathway of the intestinal epithelium.

It is now longer than half a century, humans, animals, and nature of the world are under the influence of exposure to many newly introduced noxious substances. These exposures are nowadays pushing the borders to be considered as the causative or exacerbating factors for many chronic disorders including allergic, autoimmune/inflammatory, and metabolic diseases. The epithelial linings serve as the outermost body\'s primary physical, chemical, and immunological barriers against external stimuli. The \"epithelial barrier theory\" hypothesizes that these diseases are aggravated by an ongoing periepithelial inflammation triggered by exposure to a wide range of epithelial barrier-damaging insults that lead to \"epithelitis\" and the release of alarmins. A leaky epithelial barrier enables the microbiome\'s translocation from the periphery to interepithelial and even deeper subepithelial areas together with allergens, toxins, and pollutants. Thereafter, microbial dysbiosis, characterized by colonization of opportunistic pathogen bacteria and loss of the number and biodiversity of commensal bacteria take place. Local inflammation, impaired tissue regeneration, and remodeling characterize the disease. The infiltration of inflammatory cells to affected tissues shows an effort to expulse the tissue invading bacteria, allergens, toxins, and pollutants away from the deep tissues to the surface, representing the \"expulsion response.\" Cells that migrate to other organs from the inflammatory foci may play roles in the exacerbation of various inflammatory diseases in distant organs. The purpose of this review is to highlight and appraise recent opinions and findings on epithelial physiology and its role in the pathogenesis of chronic diseases in view of the epithelial barrier theory.

BACKGROUND: Increased cerebrospinal fluid (CSF) protein concentration is a common finding in neurological diseases of dogs. Distinguishing between intrathecally-produced proteins and proteins that have passed the blood-CSF barrier because of barrier disruption facilitates diagnosis. Albumin is a microprotein mainly produced extrathecally that can be used as a reference marker for blood-CSF barrier dysfunction.
OBJECTIVE: Develop a quotient graph based on the CSF/serum quotient of albumin and immunoglobulin A (IgA; Reibergram) to visualize intrathecal IgA synthesis and blood-CSF barrier dysfunction.
METHODS: Retrospective single-center cohort study. A hyperbolic function was developed using data from 6 healthy Beagles and 38 dogs with neurological diseases in which an isolated blood-CSF barrier dysfunction was expected. The function was validated using data from 10 dogs with expected intrathecal IgA synthesis and was visualized as a quotient graph. Finally, the graph was used to evaluate data of 118 dogs with various neurological diseases.
RESULTS: Within the Reibergram, the function QLim IgA = 0.13 QAlb 2 + 11.9 · 10 - 6 - 1.01 · 10 - 3 describes the upper values of physiological IgA quotients. It detects diseases with expected intrathecal IgA synthesis with higher sensitivity (85%) and specificity (89%) than the IgA index. The upper value of the physiological albumin quotient is 2.22 and detects diseases with expected blood-CSF barrier dysfunction (sensitivity: 81%; specificity: 88%).
CONCLUSIONS: The canine Reibergram can detect blood-CSF barrier dysfunction and intrathecal IgA synthesis in the majority of cases. The graphical visualization simplifies data evaluation and makes it a feasible tool in routine CSF diagnostic testing.

The intestinal microbiome is essential to human health and homeostasis, and is implicated in the pathophysiology of disease, including congenital heart disease and cardiac surgery. Improving the microbiome and reducing inflammatory metabolites may reduce systemic inflammation following cardiac surgery with cardiopulmonary bypass (CPB) to expedite recovery post-operatively. Limited research exists in this area and identifying animal models that can replicate changes in the human intestinal microbiome after CPB is necessary. We used a piglet model of CPB with two groups, CPB (n=5) and a control group with mechanical ventilation (n=7), to evaluate changes to the microbiome, intestinal barrier dysfunction and intestinal metabolites with inflammation after CPB. We identified significant changes to the microbiome, barrier dysfunction, intestinal short-chain fatty acids and eicosanoids, and elevated cytokines in the CPB/deep hypothermic circulatory arrest group compared to the control group at just 4 h after intervention. This piglet model of CPB replicates known human changes to intestinal flora and metabolite profiles, and can be used to evaluate gut interventions aimed at reducing downstream inflammation after cardiac surgery with CPB.