Effect of sodium butyrate treatment at the basolateral membranes on the tight junction barrier function via a monocarboxylate transporter in goat mammary epithelial cells.

In lactating mammary glands, tight junctions (TJs) prevent blood from mixing with milk and maintain epithelial cell polarity, which is important for milk production. This study aimed to investigate the effect of sodium acetate and sodium butyrate (SB) stimulation direction on the TJ barrier function, which is measured with regard to transepithelial electrical resistance and fluorescein flux, in goat mammary epithelial cells. The expression and localization of the TJ proteins claudin-3 and claudin-4 were examined using Western blotting and immunofluorescence. SB treatment in the lower chamber of cell culture inserts adversely affected the TJ barrier function, whereas sodium acetate barely had any effect, regardless of stimulation direction. In addition, SB treatment in the lower chamber significantly upregulated claudin-3 and claudin-4, whereas TJ proteins showed intermittent localization. Moreover, SB induced endoplasmic reticulum (ER) stress. ARC155858, a monocarboxylate transporter-1 inhibitor, alleviated the adverse impact of SB on TJs and the associated ER stress. Interestingly, sodium ß-hydroxybutyrate, a butyrate metabolite, did not affect the TJ barrier function. Our findings indicate that sodium acetate and SB influence the TJ barrier function differently, and excessive cellular uptake of SB can disrupt TJs and induce ER stress.

Disruption of pulmonary microvascular endothelial barrier by dysregulated claudin-8 and claudin-4: uncovered mechanisms in porcine reproductive and respiratory syndrome virus infection.

The pulmonary endothelium is a dynamic and metabolically active monolayer of endothelial cells. Dysfunction of the pulmonary endothelial barrier plays a crucial role in the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), frequently observed in the context of viral pneumonia. Dysregulation of tight junction proteins can lead to the disruption of the endothelial barrier and subsequent leakage. Here, the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) served as an ideal model for studying ALI and ARDS. The alveolar lavage fluid of pigs infected with HP-PRRSV, and the supernatant of HP-PRRSV infected pulmonary alveolar macrophages were respectively collected to treat the pulmonary microvascular endothelial cells (PMVECs) in Transwell culture system to explore the mechanism of pulmonary microvascular endothelial barrier leakage caused by viral infection. Cytokine screening, addition and blocking experiments revealed that proinflammatory cytokines IL-1ß and TNF-α, secreted by HP-PRRSV-infected macrophages, disrupt the pulmonary microvascular endothelial barrier by downregulating claudin-8 and upregulating claudin-4 synergistically. Additionally, three transcription factors interleukin enhancer binding factor 2 (ILF2), general transcription factor III C subunit 2 (GTF3C2), and thyroid hormone receptor-associated protein 3 (THRAP3), were identified to accumulate in the nucleus of PMVECs, regulating the transcription of claudin-8 and claudin-4. Meanwhile, the upregulation of ssc-miR-185 was found to suppress claudin-8 expression via post-transcriptional inhibition. This study not only reveals the molecular mechanisms by which HP-PRRSV infection causes endothelial barrier leakage in acute lung injury, but also provides novel insights into the function and regulation of tight junctions in vascular homeostasis.

Autoinducer-2 promotes the colonization of Lactobacillus rhamnosus GG to improve the intestinal barrier function in a neonatal mouse model of antibiotic-induced intestinal dysbiosis.

BACKGROUND: Human health is seriously threatened by antibiotic-induced intestinal disorders. Herein, we aimed to determine the effects of Autoinducer-2 (AI-2) combined with Lactobacillus rhamnosus GG (LGG) on the intestinal barrier function of antibiotic-induced intestinal dysbiosis neonatal mice. METHODS: An antibiotic-induced intestinal dysbiosis neonatal mouse model was created using antibiotic cocktails, and the model mice were randomized into the control, AI-2, LGG, and LGG + AI-2 groups. Intestinal short-chain fatty acids and AI-2 concentrations were detected by mass spectrometry and chemiluminescence, respectively. The community composition of the gut microbiota was analyzed using 16S rDNA sequencing, and biofilm thickness and bacterial adhesion in the colon were assessed using scanning electron microscopy. Transcriptome RNA sequencing of intestinal tissues was performed, and the mRNA and protein levels of HCAR2 (hydroxycarboxylic acid receptor 2), claudin3, and claudin4 in intestinal tissues were determined using quantitative real-time reverse transcription PCR and western blotting. The levels of inflammatory factors in intestinal tissues were evaluated using enzyme-linked immunosorbent assays (ELISAs). D-ribose, an inhibitor of AI-2, was used to treat Caco-2 cells in vitro. RESULTS: Compared with the control, AI-2, and LGG groups, the LGG + AI-2 group showed increased levels of intestinal AI-2 and proportions of Firmicutes and Lacticaseibacillus, but a reduced fraction of Proteobacteria. Specifically, the LGG + AI-2 group had considerably more biofilms and LGG on the colon surface than those of other three groups. Meanwhile, the combination of AI-2 and LGG markedly increased the concentration of butyric acid and promoted Hcar2, claudin3 and claudin4 expression levels compared with supplementation with LGG or AI-2 alone. The ELISAs revealed a significantly higher tumor necrosis factor alpha (TNF-α) level in the control group than in the LGG and LGG + AI-2 groups, whereas the interleukin 10 (IL-10) level was significantly higher in the LGG + AI-2 group than in the other three groups. In vitro, D-ribose treatment dramatically suppressed the increased levels of Hcar2, claudin3, and claudin4 in Caco-2 cells induced by AI-2 + LGG. CONCLUSIONS: AI-2 promotes the colonization of LGG and biofilm formation to improve intestinal barrier function in an antibiotic-induced intestinal dysbiosis neonatal mouse model.

Structural basis for Clostridium perfringens enterotoxin targeting of claudins at tight junctions in mammalian gut.

The bacterium Clostridium perfringens causes severe, sometimes lethal gastrointestinal disorders in humans, including enteritis and enterotoxemia. Type F strains produce an enterotoxin (CpE) that causes the third most common foodborne illness in the United States. CpE induces gut breakdown by disrupting barriers at cell-cell contacts called tight junctions (TJs), which are formed and maintained by claudins. Targeted binding of CpE to specific claudins, encoded by its C-terminal domain (cCpE), loosens TJ barriers to trigger molecular leaks between cells. Cytotoxicity results from claudin-bound CpE complexes forming pores in cell membranes. In mammalian tissues, ∼24 claudins govern TJ barriers-but the basis for CpE's selective targeting of claudins in the gut was undetermined. We report the structure of human claudin-4 in complex with cCpE, which reveals that enterotoxin targets a motif conserved in receptive claudins and how the motif imparts high-affinity CpE binding to these but not other subtypes. The structural basis of CpE targeting is supported by binding affinities, kinetics, and half-lives of claudin-enterotoxin complexes and by the cytotoxic effects of CpE on claudin-expressing cells. By correlating the binding residence times of claudin-CpE complexes we determined to claudin expression patterns in the gut, we uncover that the primary CpE receptors differ in mice and humans due to sequence changes in the target motif. These findings provide the molecular and structural element CpE employs for subtype-specific targeting of claudins during pathogenicity of C. perfringens in the gut and a framework for new strategies to treat CpE-based illnesses in domesticated mammals and humans.

Expression Patterns of Grainyhead-Like 2 and Ovo-Like 2 in Mouse Mammary Gland Development During Pregnancy, Lactation, and Weaning.

Grainyhead-like 2 (Grhl2) is a transcription factor that regulates cell adhesion genes in mammary ductal development and serves as a repressor of the epithelial-mesenchymal transition. Conversely, Ovo-like2 (Ovol2) is a target gene of Grhl2 but functions as a substitute in Grhl2-deficient mice, facilitating successful epithelial barrier formation and lumen expansion in kidney-collecting ductal epithelial cells. Our objective was to examine the expression patterns of Grhl2, Ovol2, and their associated genes during the intricate phases of mouse mammary gland development. The mRNA expression of Grhl2 and Ovol2 increased after pregnancy. We observed Grhl2 protein presence in the epithelial cell's region, coinciding with acini formation, and its signal significantly correlated with E-cadherin (Cdh1) expression. However, Ovol2 was present in the epithelial region without a correlation with Cdh1. Similarly, Zeb1, a mesenchymal transcription factor, showed Cdh1-independent expression. Subsequently, we explored the interaction between Rab25, a small G protein, and Grhl2/Ovol2. The expressions of Grhl2 and Ovol2 exhibited a strong correlation with Rab25 and claudin-4, a tight junction protein. These findings suggest that Grhl2 and Ovol2 may collaborate to regulate genes associated with cell adhesion and are crucial for maintaining epithelial integrity during the different phases of mammary gland development.

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.

Quercetin Improves Barrier Properties in Porcine Small Intestine but Not in Peyer's Patches.

Peyer's patches (PPs) are part of the gut-associated lymphatic tissue (GALT) and represent the first line of the intestinal immunological defense. They consist of follicles with lymphocytes and an overlying subepithelial dome with dendritic cells and macrophages, and they are covered by the follicle-associated epithelium (FAE). A sealed paracellular pathway in the FAE is crucial for the controlled uptake of luminal antigens. Quercetin is the most abundant plant flavonoid and has a barrier-strengthening effect on tight junctions (TJs), a protein complex that regulates the paracellular pathway. In this study, we aimed to analyze the effect of quercetin on porcine PPs and the surrounding villus epithelium (VE). We incubated both tissue types for 4 h in Ussing chambers, recorded the transepithelial electrical resistance (TEER), and measured the unidirectional tracer flux of [3H]-mannitol. Subsequently, we analyzed the expression, protein amount, and localization of three TJ proteins, claudin 1, claudin 2, and claudin 4. In the PPs, we could not detect an effect of quercetin after 4 h, neither on TEER nor on the [3H]-mannitol flux. In the VE, quercetin led to a higher TEER value, while the [3H]-mannitol flux was unchanged. The pore-forming claudin 2 was decreased while the barrier-forming claudin 4 was increased and the expression was upregulated. Claudin 1 was unchanged and all claudins could be located in the paracellular membrane by immunofluorescence microscopy. Our study shows the barrier-strengthening effect of quercetin in porcine VE by claudin 4 upregulation and a claudin 2 decrease. Moreover, it underlines the different barrier properties of PPs compared to the VE.

Transcriptional Levels of Intercellular Junction Proteins in an Alveolar Epithelial Cell Line Exposed to Irradiation or Bleomycin.

We performed a comparative study of the effects of X-ray irradiation and bleomycin on the mRNA levels of E-cadherin and tight junction proteins (claudin-3, claudin-4, claudin-18, ZO-2, and occludin) in an alveolar epithelial cell line L2. Irradiation decreased claudin-4 levels and increased occludin levels, while the levels of other mRNAs remained unchanged. Bleomycin increased the expression levels of all proteins examined except claudin-3. Irradiation and bleomycin have different effects on the expression level of intercellular junction proteins, indicating different reactions triggered in alveolar epithelial cells and a great prospects of further comparative studies.

Development, structure, and mechanism of synthetic antibodies that target claudin and Clostridium perfringens enterotoxin complexes.

Strains of Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflicts humans and domesticated animals, causing prevalent gastrointestinal illnesses. CpE's C-terminal domain (cCpE) binds cell surface receptors, followed by a restructuring of its N-terminal domain to form a membrane-penetrating ß-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are known receptors for CpE and also control the architecture and function of cell-cell contacts (tight junctions) that create barriers to intercellular molecular transport. CpE binding and assembly disables claudin barrier function and induces cytotoxicity via ß-pore formation, disrupting gut homeostasis; however, a structural basis of this process and strategies to inhibit the claudin-CpE interactions that trigger it are both lacking. Here, we used a synthetic antigen-binding fragment (sFab) library to discover two sFabs that bind claudin-4 and cCpE complexes. We established these sFabs' mode of molecular recognition and binding properties and determined structures of each sFab bound to claudin-4-cCpE complexes using cryo-EM. The structures reveal that the sFabs bind a shared epitope, but conform distinctly, which explains their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in binding changes, validating the structures, and uncovering the sFab's targeting mechanism. From these insights, we generated a model for CpE's claudin-bound ß-pore that predicted sFabs would not prevent cytotoxicity, which we then verified in vivo. Taken together, this work demonstrates the development and mechanism of claudin/cCpE-binding sFabs that provide a framework and strategy for obstructing claudin/CpE assembly to treat CpE-linked gastrointestinal diseases.

Claudin-4-adhesion signaling drives breast cancer metabolism and progression via liver X receptor ß.

BACKGROUND: Cell adhesion is indispensable for appropriate tissue architecture and function in multicellular organisms. Besides maintaining tissue integrity, cell adhesion molecules, including tight-junction proteins claudins (CLDNs), exhibit the signaling abilities to control a variety of physiological and pathological processes. However, it is still fragmentary how cell adhesion signaling accesses the nucleus and regulates gene expression. METHODS: By generating a number of knockout and rescued human breast cell lines and comparing their phenotypes, we determined whether and how CLDN4 affected breast cancer progression in vitro and in vivo. We also identified by RNA sequencing downstream genes whose expression was altered by CLDN4-adhesion signaling. Additionally, we analyzed by RT-qPCR the CLDN4-regulating genes by using a series of knockout and add-back cell lines. Moreover, by immunohistochemistry and semi-quantification, we verified the clinicopathological significance of CLDN4 and the nuclear receptor LXRß (liver X receptor ß) expression in breast cancer tissues from 187 patients. RESULTS: We uncovered that the CLDN4-adhesion signaling accelerated breast cancer metabolism and progression via LXRß. The second extracellular domain and the carboxy-terminal Y197 of CLDN4 were required to activate Src-family kinases (SFKs) and the downstream AKT in breast cancer cells to promote their proliferation. Knockout and rescue experiments revealed that the CLDN4 signaling targets the AKT phosphorylation site S432 in LXRß, leading to enhanced cell proliferation, migration, and tumor growth, as well as cholesterol homeostasis and fatty acid metabolism, in breast cancer cells. In addition, RT-qPCR analysis showed the CLDN4-regulated genes are classified into at least six groups according to distinct LXRß- and LXRßS432-dependence. Furthermore, among triple-negative breast cancer subjects, the "CLDN4-high/LXRß-high" and "CLDN4-low and/or LXRß-low" groups appeared to exhibit poor outcomes and relatively favorable prognoses, respectively. CONCLUSIONS: The identification of this machinery highlights a link between cell adhesion and transcription factor signalings to promote metabolic and progressive processes of malignant tumors and possibly to coordinate diverse physiological and pathological events.

RBM15 suppresses hepatic insulin sensitivity of offspring of gestational diabetes mellitus mice via m6A-mediated regulation of CLDN4.

BACKGROUND: Gestational diabetes Mellitus (GDM) is a common pregnancy-specific disease with high morbidity, which is linked to a high risk of obesity and diabetes in offspring. N6-methyladenosine modification of RNA is emerging as an important epigenetic mechanism that is widely manifested in many diseases. This study aimed to investigate the mechanism of m6A methylation in metabolic syndrome in offspring result from intrauterine hyperglycemia. METHODS: GDM mice were established by feeding a high-fat diet 1 weeks before pregnancy. The m6A RNA methylation quantification kit was used to detect liver tissue methylation levels. PCR array was used to determine the expression of the m6A methylation modification enzyme. Immunohistochemistry, qRT-PCR, and western blot were used to examine the expression of RBM15, METTL13, IGF2BP1, and IGF2BP2. Subsequently, methylated RNA immunoprecipitation sequencing combined with mRNA sequencing, followed by dot blot and glucose uptake tests, were performed. RESULTS: In this study, we found that offspring from a GDM mother were more vulnerable to glucose intolerance and insulin resistance. GC-MS revealed significant metabolic changes including saturated fatty acids and unsaturated fatty acids in liver of GDM offspring. We also demonstrated that global mRNA m6A methylation level was significantly increased in the fetal liver of GDM mice, indicating epigenetic change may have a strong relationship with the mechanism of metabolism syndrome. Concordantly, RBM15, the RNA binding methyltransferase, was upregulated in the liver. In vitro, RBM15 suppressed insulin sensitivity and increased insulin resistance through m6A-regulated epigenetic inhabitation of CLDN4. Moreover, MeRIP-sequencing and mRNA-sequencing revealed that differently regulated genes with differential m6A peaks were enriched in metabolic pathways. CONCLUSION: Our study revealed the essential role of RBM15 in insulin resistance and the effect of RBM15-regulated m6A modification in the metabolic syndrome of offspring of GDM mice.

The tight junction protein Claudin-5 limits endothelial cell motility.

Steinberg's differential adhesion hypothesis suggests that adhesive mechanisms are important for sorting of cells and tissues during morphogenesis (Steinberg, 2007). During zebrafish vasculogenesis, endothelial cells sort into arterial and venous vessel beds but it is unknown whether this involves adhesive mechanisms. Claudins are tight junction proteins regulating the permeability of epithelial and endothelial tissue barriers. Previously, the roles of claudins during organ development have exclusively been related to their canonical functions in determining paracellular permeability. Here, we use atomic force microscopy to quantify claudin-5-dependent adhesion and find that this strongly contributes to the adhesive forces between arterial endothelial cells. Based on genetic manipulations, we reveal a non-canonical role of Claudin-5a during zebrafish vasculogenesis, which involves the regulation of adhesive forces between adjacent dorsal aortic endothelial cells. In vitro and in vivo studies demonstrate that loss of claudin-5 results in increased motility of dorsal aorta endothelial cells and in a failure of the dorsal aorta to lumenize. Our findings uncover a novel role of claudin-5 in limiting arterial endothelial cell motility, which goes beyond its traditional sealing function during embryonic development.

Hypothesis: HEG1 and claudin-4 staining will allow a diagnosis of epithelioid and biphasic mesothelioma versus non-small-cell lung carcinoma with only two stains in most cases.

Separation of mesothelioma from metastatic carcinoma requires immunohistochemical support, with small batteries of stains recommended as a starting-point, but these numbers commonly expand to 10, 12 or more stains, a process that is not only expensive but frequently generates anomalous or confounding results, leading to even more stains. Here we review data on HEG1 clone SKM9-2, a new (now commercially available) mesothelioma marker and claudin-4, a broad-spectrum carcinoma marker, to ask whether these two stains are sufficient, by themselves, to separate mesotheliomas from non-small-cell lung (NSCLC) as well as other carcinomas. Data for HEG1, derived from four laboratories, showed membrane staining in 393 of 434 (91%) epithelioid/biphasic mesotheliomas and one of 360 (0.3%) NSCLC (sensitivity 91%, specificity 99.7%). Reports from seven laboratories evaluating claudin-4 in NSCLC showed positivity in 469 of 502 (93%) carcinomas and weak positivity in five of 463 (1.0%) epithelioid/biphasic mesotheliomas (sensitivity 93%, specificity 98.9%). Comparable results were found with carcinomas from other sites, except for serous and thyroid carcinomas, some of which react with HEG1 but are also positive for claudin-4. For sarcomatoid mesotheliomas, HEG1 sensitivity is modest and staining sometimes difficult to interpret. We hypothesise that the combination of HEG1 and claudin-4 immunostaining will potentially allow the separation of epithelioid/biphasic mesotheliomas from NSCLC carcinomas with high accuracy using only two immunostains in most cases. This combination will probably also work for carcinomas from most other sites, but more reports on HEG1 SKM9-2 staining of carcinomas other than NSCLC are needed. This approach would greatly simplify the diagnosis of mesothelioma.

Increase in Paracellular Leakage of Amino Acids Mediated by Aging-Induced Reduction of Claudin-4 Expression.

BACKGROUND: Claudins (CLDNs), major components of tight junctions, control paracellular permeabilities of mineral ions and wastes. The absorption of nutrients including glucose and amino acids (AAs) is regulated by intestinal epithelial cells. However, the role of CLDNs is not fully understood. OBJECTIVES: The purpose of this study was to clarify the effect of AA deprivation on the expression of AA transporters and CLDNs, as well as the role of CLDNs in the regulation of paracellular AA fluxes. METHODS: The messenger RNA and protein expression of various CLDNs were examined by real-time quantitative polymerase chain reaction and Western blot analyses, respectively. The AA selectivity of CLDNs was estimated using liquid chromatography-tandem mass spectrometry (LC-MS) analysis. RESULTS: The expression levels of some AA transporters, CLDN4, and CLDN15 were increased by AA deprivation in normal mouse colon-derived MCE301 cells. The expression of AA transporters and CLDN15 in the mouse colon was positively correlated with aging but the expression of CLDN4 was not. The AA deprivation-induced elevation of CLDN4 expression was inhibited by MHY1485, a mammalian target of rapamycin (mTOR) activator. Furthermore, CLDN4 expression was increased by rapamycin, an mTOR inhibitor. mTOR may be involved in the transcriptional activation of CLDN4. The fluxes of AAs from the basal to apical compartments were decreased and increased by CLDN4 overexpression and silencing, respectively. LC-MS analysis showed that the fluxes of all AAs, especially Lys, His, and Arg, were enhanced by CLDN4 silencing. CONCLUSIONS: CLDN4 is suggested to form a paracellular barrier to AAs, especially alkaline AAs, which is attenuated with aging.

Medullary thymic epithelial stem cells maintain a functional thymus to ensure lifelong central T cell tolerance.

Medullary thymic epithelial cells (mTECs) are crucial for central T cell self-tolerance. Although progenitors of mTECs have been demonstrated in thymic organogenesis, the mechanism for postnatal mTEC maintenance remains elusive. We demonstrate that implantation of embryonic TECs expressing claudin-3 and claudin-4 (Cld3,4) in a medulla-defective thymic microenvironment restores medulla formation and suppresses multiorgan autoimmunity throughout life. A minor SSEA-1(+) fraction within the embryonic Cld3,4(hi) TECs contained self-renewable clonogenic TECs, capable of preferentially generating mature mTECs in vivo. Adult SSEA-1(+)Cld3,4(hi) TECs retained mTEC reconstitution potential, although the activity decreased. The clonogenicity of TECs also declined rapidly after birth in wild-type mice, whereas it persisted in Rag2(?/?) adult mice with defective thymopoiesis. The results suggest that unipotent mTEC-restricted stem cells that develop in the embryo have the capacity to functionally reconstitute the thymic medulla long-term, thus ensuring lifelong central T cell self-tolerance.

Gastrointestinal consequences of lipopolysaccharide-induced lung inflammation.

BACKGROUND: Respiratory inflammation is the body's response to lung infection, trauma or hypersensitivity and is often accompanied by comorbidities, including gastrointestinal (GI) symptoms. Why respiratory inflammation is accompanied by GI dysfunction remains unclear. Here, we investigate the effect of lipopolysaccharide (LPS)-induced lung inflammation on intestinal barrier integrity, tight-junctions, enteric neurons and inflammatory marker expression. METHODS: Female C57bl/6 mice (6-8 weeks) were intratracheally administered LPS (5 µg) or sterile saline, and assessed after either 24 or 72 h. Total and differential cell counts in bronchoalveolar lavage fluid (BALF) were used to evaluate lung inflammation. Intestinal barrier integrity was assessed via cross sectional immunohistochemistry of tight junction markers claudin-1, claudin-4 and EpCAM. Changes in the enteric nervous system (ENS) and inflammation in the intestine were quantified immunohistochemically using neuronal markers Hu + and nNOS, glial markers GFAP and S100ß and pan leukocyte marker CD45. RESULTS: Intratracheal LPS significantly increased the number of neutrophils in BALF at 24 and 72 h. These changes were associated with an increase in CD45 + cells in the ileal mucosa at 24 and 72 h, increased goblet cell expression at 24 h, and increased expression of EpCAM at 72 h. LPS had no effect on the expression of GFAP, S100ß, nor the number of Hu + neurons or proportion of nNOS neurons in the myenteric plexus. CONCLUSIONS: Intratracheal LPS administration induces inflammation in the ileum that is associated with enhanced expression of EpCAM, decreased claudin-4 expression and increased goblet cell density, these changes may contribute to systemic inflammation that is known to accompany many inflammatory diseases of the lung.

BAP1 and Claudin-4, But Not MTAP, Reliably Distinguish Borderline and Low-grade Serous Ovarian Tumors From Peritoneal Mesothelioma.

Peritoneal mesothelioma (PM) and serous neoplasms can be difficult to differentiate, particularly in small biopsies. BRCA1-associated protein 1 (BAP1) is expressed in benign tissues, but over 50% of PMs demonstrate complete loss of nuclear expression. Claudin-4, a tight junction protein, is expressed in most epithelial tumors but not in mesotheliomas. Methylthioadenosine phosphorylase (MTAP) is frequently co-deleted with cyclin-dependent kinase inhibitor 2a in mesotheliomas. These markers have proven useful in separating mesothelioma from its mimics, particularly when tumors are pleural based. In the peritoneum, BAP1 loss has been rarely reported in high-grade serous carcinomas, but overall, these markers have been minimally evaluated in ovarian serous borderline tumors and low-grade serous carcinomas. Thus, we assessed the utility of BAP1, claudin-4, and MTAP in the differential diagnosis of PM and low-grade serous neoplasms. Eighteen PM (16 epithelioid, 1 biphasic, and 1 sarcomatous), 24 low-grade serous carcinomas, and 25 serous borderline tumors were stained for BAP1, claudin-4, and MTAP. Loss of BAP1 nuclear expression was observed in 12 (67%) PM (11 epithelioid, 1 biphasic) but was retained in all serous tumors. Claudin-4 was positive in all serous tumors and negative in all PM. Complete loss of cytoplasmic MTAP was noted in 3 (17%) PMs and 1 (4%) serous borderline tumor, while all low-grade serous carcinomas showed retained expression. BAP1 loss reliably distinguishes PM from serous tumors, although it lacks sensitivity. Claudin-4 is a reliable marker to exclude PM. MTAP loss may occur in both PM and serous tumors, and thus is not useful in distinguishing these entities.

Early effects of lipoteichoic acid from Staphylococcus aureus on milk production-related signaling pathways in mouse mammary epithelial cells.

Staphylococcus aureus causes subclinical mastitis; lipoteichoic acid (LTA) from S. aureus causes mastitis-like adverse effects on milk production by mammary epithelial cells (MECs). Here, we investigated the early effects of LTA from S. aureus on mouse MECs using a culture model, in which MECs produced milk components and formed less permeable tight junctions (TJs). In MECs of this model, Toll-like receptor 2 (receptor for LTA), was localized on the apical membrane, similar to MECs in lactating mammary glands. LTA weakened the TJ barrier within 1 h, concurrently with localization changes of claudin 4. LTA treatment for 24 h increased αS1-casein and decreased ß-casein levels. In MECs exposed to LTA, the activation level of signal transducer and activator of transcription 5 (major transcriptional factor for milk production) was low. LTA activated signaling pathways related to cell survival (extracellular signal-regulated kinase, heat shock protein 27, and Akt) and inflammation (p38, c-Jun N-terminal kinase, and nuclear factor κB). Thus, LTA caused abnormalities in casein production and weakened the TJs by affecting multiple signaling pathways in MECs. LTA-induced changes in signaling pathways were not uniform in all MECs. Such complex and semi-negative actions of LTA may contribute to subclinical mastitis caused by S. aureus.

Tetrahydrocurcumin (THC) enhanced the clearance of Cryptococcus deneoformans during infection in vivo.

Cryptococcal species often cause lung infections and are the main cause of fungal meningitis. Claudin-4 appears to be a major structural component that maintains a tight alveolar barrier and prevents fluid and electrolyte leakage into the alveolar space. We aimed to determine whether S7-tetrahydrocurcumin (THC) could clearance of C. deneoformans and regulate claudin-4 expression during C. deneoformans infection. We investigated the effect of THC on C. deneoformans infection and its possible mechanism in vivo. Transmission electron microscopy was used to observe the ultrastructure of the lung tissue and the invasion of Cryptococcus. To clarify the effect of THC, we examined claudin-4, c-Jun, and Smad2 expression. We also measured claudin-4 expression in pulmonary specimens from clinical patients. THC reduced cryptococcal cell invasion in the lungs, improved alveolar exudation, and reduced inflammation. Pretreatment with THC suppressed c-Jun and Smad2 expression, resulting in significantly increased claudin-4 levels. In contrast, the expression of claudin-4 in clinical specimens from patients with cryptococcal infection was higher than that in normal specimens. THC enhanced the clearance of C. deneoformans during infection in vivo. We investigated the expression of claudin-4 and the possible mechanism of THC against C. deneoformans infection.

CLDN4 as a Novel Diagnostic and Prognostic Biomarker and Its Association with Immune Infiltrates in Ovarian Cancer.

Ovarian cancer (OC) is the seventh most prevalent type of cancer in women and the second most common cause of cancer-related deaths in women worldwide. Because of the high rates of relapse, there is an immediate and pressing need for the discovery of innovative sensitive biomarkers for OC patients. Using TCGA and GSE26712 datasets, we were able to identify 17 survival-related DEGs in OC that had differential expression. CLDN4 was the gene that caught our attention the most out of the 17 important genes since its expression was much higher in OC samples than in nontumor samples. The findings of the ROC assays then confirmed the diagnostic utility of the test in screening OC specimens to differentiate them from nontumor specimens. Patients with high CLDN4 expression predicted a shorter overall survival (OS) and disease-specific survival (DSS) than those with low CLDN4 expression, according to clinical research. Patients with low CLDN4 expression predicted longer OS and DSS. Analysis using both univariate and multivariate techniques revealed that CLDN4 expression was an independent factor associated with a poor prognosis for OS and DSS. Based on multivariate analysis, the C-indexes and calibration plots of the nomogram suggested an effective predictive performance for OC patients. After that, we investigated whether or not there was a link between the infiltration of immune cells and the expression of the CLDN4 gene. We found that the expression of CLDN4 was positively associated with Th17 cells, NK CD56bright cells, while negatively associated with Th2 cells, pDC, and T helper cells. In the end, we carried out RT-PCR on our cohort and confirmed that the level of CLDN4 expression was noticeably elevated in OC specimens in comparison to nontumor tissues. The diagnostic usefulness of CLDN4 expression for OC was also validated by the findings of ROC tests. Thus, our findings revealed that CLDN4 may serve as a predictive biomarker in OC to assess both the clinical outcome of OC patients and the level of immune infiltration.