Increased Claudin-2 Expression Research Articles

The activation of adenosine monophosphate-activated protein kinase inhibits the migration of tongue squamous cell carcinoma cells by targeting Claudin-1 via epithelial-mesenchymal transition.

The role of Claudin-1 in tongue squamous cell carcinoma (TSCC) metastasis needs further clarification, particularly its impact on cell migration. Herein, our study aims to investigate the role of Claudin-1 in TSCC cell migration and its underlying mechanisms. 36 TSCC tissue samples underwent immunohistochemical staining for Claudin-1. Western blotting and immunofluorescence analyses were conducted to evaluate Claudin-1 expression and distribution in TSCC cells. Claudin-1 knockdown cell lines were established using short hairpin RNA transfection. Migration effects were assessed through wound healing assays. Furthermore, the expression of EMT-associated molecules was measured via western blotting. Claudin-1 expression decreased as TSCC malignancy increased. Adenosine monophosphate-activated protein kinase (AMPK) activation led to increased Claudin-1 expression and membrane translocation, inhibiting TSCC cell migration and epithelial-mesenchymal transition (EMT). Conversely, Claudin-1 knockdown reversed these inhibitory effects on migration and EMT caused by AMPK activation. Our results indicated that AMPK activation suppresses TSCC cell migration by targeting Claudin-1 and EMT pathways.

Dietary rutin improves the antidiarrheal capacity of weaned piglets by improving intestinal barrier function, antioxidant capacity and cecal microbiota composition.

Early weaning is prone to damage intestinal barrier function, resulting in diarrhea, whereas rutin, as a natural flavonoid with multiple biological functions, shows potential in piglets. Therefore, the effects of dietary rutin on growth, antidiarrheal, barrier function, antioxidant status and cecal microbiota of weaned piglets were investigated with the control group (CON) (basal diet) and Rutin (basal diet+500 mg kg-1 rutin) groups fed for 14 days. The results showed that dietary 500 mg kg-1 rutin significantly decreased diarrhea index, serum diamine oxidase activity and total aerobic bacterial population in mesenteric lymph nodes, whereas it significantly increased the gain-to-feed ratio (G:F) and serum growth hormone content, jejunal villus height and villus height to crypt depth ratio, and also enhanced jejunal claudin-1 and zonula occludens-1 mRNA and protein expression. Meanwhile, dietary rutin significantly decreased inflammation-associated mRNA expression, malondialdehyde (MDA) content, swollen mitochondrial number and mitochondrial area in the jejunum, whereas it increased the total superoxide dismutase (T-SOD) and glutathione peroxidase activities and activated the Nrf2 signaling pathway. Moreover, dietary rutin significantly increased Firmicutes abundance and decreased Campylobacterota abundance, which were closely associated with the decreased diarrhea index and MDA content or increased Claudin-1 expression and T-SOD activity. Dietary 500 mg kg-1 rutin increased G:F by improving intestinal morphology, and alleviated diarrhea by enhancing intestinal barrier, which might be associated with the enhanced antioxidant capacity via activating the Nrf2/Keap1 signaling pathway and the improved cecal microbial composition in weaned piglets. © 2024 Society of Chemical Industry.

Near-Infrared In Vivo Imaging of Claudin-1 Expression by Orthotopically Implanted Patient-Derived Colonic Adenoma Organoids

Background: Claudin-1 becomes overexpressed during the transformation of normal colonic mucosa to colorectal cancer (CRC). Methods: Patient-derived organoids expressed clinically relevant target levels and genetic heterogeneity, and were established from human adenoma and normal colons. Colonoids were implanted orthotopically in the colons of immunocompromised mice. This pre-clinical model of CRC provides an intact microenvironment and representative vasculature. Colonoid growth was monitored using white light endoscopy. A peptide specific for claudin-1 was fluorescently labeled for intravenous administration. NIR fluorescence images were collected using endoscopy and endomicroscopy. Results: NIR fluorescence images collected using wide-field endoscopy showed a significantly greater target-to-background (T/B) ratio for adenoma versus normal (1.89 ± 0.35 and 1.26 ± 0.06) colonoids at 1 h post-injection. These results were confirmed by optical sections collected using endomicroscopy. Optical sections were collected in vivo with sub-cellular resolution in vertical and horizontal planes. Greater claudin-1 expression by individual epithelial cells in adenomatous versus normal crypts was visualized. A human-specific cytokeratin stain ex vivo verified the presence of human tissues implanted adjacent to normal mouse colonic mucosa. Conclusions: Increased claudin-1 expression was observed from adenoma versus normal colonoids in vivo using imaging with wide field endoscopy and endomicrosopy.

Teriflunomide Promotes Blood-Brain Barrier Integrity by Upregulating Claudin-1 via the Wnt/β-catenin Signaling Pathway in Multiple Sclerosis.

The blood-brain barrier (BBB) and tight junction (TJ) proteins maintain the homeostasis of the central nervous system (CNS). The dysfunction of BBB allows peripheral T cells infiltration into CNS and contributes to the pathophysiology of multiple sclerosis (MS). Teriflunomide is an approved drug for the treatment of MS by suppressing lymphocytes proliferation. However, whether teriflunomide has a protective effect on BBB in MS is not understood. We found that teriflunomide restored the injured BBB in the EAE model. Furthermore, teriflunomide treatment over 6months improved BBB permeability and reduced peripheral leakage of CNS proteins in MS patients. Teriflunomide increased human brain microvascular endothelial cell (HBMEC) viability and promoted BBB integrity in an in vitro cell model. The TJ protein claudin-1 was upregulated by teriflunomide and responsible for the protective effect on BBB. Furthermore, RNA sequencing revealed that the Wnt signaling pathway was affected by teriflunomide. The activation of Wnt signaling pathway increased claudin-1 expression and reduced BBB damage in cell model and EAE rats. Our study demonstrated that teriflunomide upregulated the expression of the tight junction protein claudin-1 in endothelial cells and promoted the integrity of BBB through Wnt signaling pathway.

Claudin-2 in hyperproliferative migrating keratinocytes and migration inhibition via siRNA knockdown.

Claudin-2 is a tight junction protein found in various tissues including the epidermis of the skin. Intracellular signalling via claudin-2 may have an effect on cell proliferation and migration. While the role of claudin-2 in the epidermis has not been established, here we show an increase in claudin-2 expression in hyperproliferative archival skin samples. To further examine the role of claudin-2 in cell migration we examined its expression in cultured keratinocytes and found it was increased in wound margins in an in vitro scratch test assay. We then used a claudin-2 knockdown assay using small interfering ribonucleic acid (siRNA) with a 77% transfection efficiency and decrease in claudin-2 protein via Western blot analysis to examine cell migration, which was inhibited following claudin-2 knockdown over a 5-day period. Cells transfected with claudin-2 siRNA also showed a decreased size compared to controls and a more diffuse staining pattern. Lastly we examined claudin-2 expression in migrating keratinocytes by Western blot analysis and found a significant decrease in protein staining in scratch-test assay cultures after 4 h, followed by a significant increase in claudin-2 protein after 24 h. Taken together these results indicate a role for claudin-2 signalling in proliferation and cell migration in the epidermis of the skin.

Intestinal Epithelial Cell PTPN2 Restricts Intestinal Permeability and Tight-Junction Remodelling Caused by Adherent-invasive E. coli in Mice

Background: Alterations in epithelial tight junction proteins can compromise the ability of the intestinal mucosa to act as a barrier between luminal contents and underlying tissues. The resulting increase in intestinal permeability can give rise to conditions such as inflammatory bowel disease (IBD). Additionally, expansion of pathobionts, such as adherent-invasive Escherichia coli (AIEC), are associated with IBD. Mice deficient in the IBD gene candidate, Ptpn2, exhibit increased intestinal permeability and a pronounced expansion of a novel murine AIEC ( mAIEC). This study aimed to investigate how PTPN2 expression in intestinal epithelial cells protects intestinal barrier properties after mAIEC infection. Methods: Tamoxifen-inducible, intestinal epithelial cell (IEC)-specific knockout mice ( Ptpn2 ΔIEC ) and control littermates ( Ptpn2 fl/fl ) were infected with either PBS, non-invasive E. coli K12, or mcherry-tagged mAIEC ( mAIEC red ). FITC-Dextran (FD4) and Rhodamine-Dextran (RD70) permeability probes were administered by oral-gavage and serum collected after 5hrs. Results: Ptpn2 ΔIEC mice exhibited intestinal region-specific higher mAIEC red - but not K12 - bacterial load compared to Ptpn2 fl/fl mice ( P=0.038). E. coli K12 increased FD4 but not RD70 permeability in Ptpn2 ΔIEC mice compared with Ptpn2 fl/fl mice ( P=0.0025). mAIEC red also had no effect on RD70, but caused an even greater increase in FD4 permeability in Ptpn2 ΔIEC vs. Ptpn2 fl/fl mice ( P< 0.0001). Ptpn2 fl/fl mice infected with mAIEC red also exhibited increased FD4 permeability compared to PBS-treated littermates ( P =0.0210). To identify if increased FD4 permeability was due to changes in expression of relevant tight-junction proteins, we performed western blot analysis on isolated IECs. Ptpn2 ΔIEC + mAIEC red mice had decreased expression of occludin ( P=0.0030) and the adherens junction protein, E-cadherin ( P=0.0142). Expression of the cation pore, claudin-2, was increased in PBS and K12-infected Ptpn2 ΔIEC vs. Ptpn2 fl/fl mice ( P=0.015). Interestingly, mAIEC red increased claudin-2 expression in Ptpn2 fl/fl mice compared to PBS controls ( P=0.0105) however, Ptpn2 ΔIEC + mAIEC red mice exhibited decreased claudin-2 levels compared to Ptpn2 fl/fl + mAIEC red mice ( P=0.0436). Immunofluorescence staining of claudin-2 confirmed reduced claudin-2 in Ptpn2 ΔIEC + mAIEC red mice. Furthermore, we observed gaps in ZO-1 membrane localization in Ptpn2 ΔIEC mice treated with PBS and K12 but not in their Ptpn2 fl/fl controls. ZO-1 membrane localization was dramatically depleted in Ptpn2 ΔIEC + mAIEC red mice compared to the Ptpn2 fl/fl + mAIEC red controls. Conclusion: Intestinal epithelial PTPN2 exhibits a major role in maintaining intestinal homeostasis by preserving intestinal barrier function and the integrity of tight-junction protein localization. Given that loss of PTPN2 in vivo also provokes expansion of adherent-invasive E. coli, these studies indicate that PTPN2 is an important mediator of host-microbiota crosstalk. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alleviation of cholestatic liver injury and intestinal permeability by lubiprostone treatment in bile duct ligated rats: role of intestinal FXR and tight junction proteins claudin-1, claudin-2, and occludin.

Gut barrier disintegrity and endotoxin translocation to the liver and systemic circulation are serious clinical complications associated with the stoppage of intestinal bile flow. There is no precise pharmacological option to prevent increased intestinal permeability after bile duct ligation (BDL). Lubiprostone, a chloride channel-2 agonist, has been shown to accelerate restoration of epithelial barrier dysfunction caused by injury, but the exact mechanisms underlying the beneficial effects of lubiprostone on intestine barrier integrity remain unknown. Here, we assessed the beneficial effect of lubiprostone on cholestasis caused by BDL and relevant mechanisms. Male rats were subjected to BDL for 21days. Seven days after BDL induction, lubiprostone was administered twice daily (10µg/kg of body weight). Intestinal permeability was assessed through measurements of serum lipopolysaccharide (LPS) concentration. Real-time PCR was conducted to assess expression of intestinal claudin-1 occludin and FXR genes, which are important in preserving the intestinal epithelial barrier integrity, as well as claudin-2 being involved in a leaky gut barrier. Histopathological alterations were alsomonitored for liver injury. Lubiprostone significantly decreased BDL-induced systemic LPS elevation in rats. BDL induced a significant reduction in FXR, occludin, and claudin-1 genes expression, while increased claudin-2 expression in rat colon. Treatment with lubiprostone significantly restored expression of these genes to the control values. BDL also increased the level of hepatic enzymes ALT, ALP, AST, and total bilirubin, while lubiprostone could preserve the hepatic enzymes and total bilirubin in the treated BDL rats. Lubiprostone also caused a significant reduction in BDL-induced liver fibrosis and intestinal damage in rats. Our results suggest thatlubiprostone favorably prevents BDL-induced alterations in intestinal epithelial barrier integrity possibly via modulating intestinal FXRs and tight junction gene expression.

Fermented Glutinous Rice Extract Mitigates DSS-Induced Ulcerative Colitis by Alleviating Intestinal Barrier Function and Improving Gut Microbiota and Inflammation.

Ulcerative colitis (UC) is an inflammatory bowel disease caused by various factors, including intestinal inflammation and barrier dysfunction. Herein, we determined the effects of fermented glutinous rice (FGR) on the expression of tight junction proteins and levels of inflammation and apoptosis in the dextran sodium sulfate (DSS)-induced acute colitis model. FGR was orally administered once per day to C57BL/6J mice with colitis induced by 5% DSS in drinking water. FGR administration recovered DSS-induced body weight loss and irregularly short colon lengths. FGR inhibited the DSS-induced decrease in FITC-dextran (FD)-4 permeability and myeloperoxidase activity. Moreover, FGR treatment repaired the reduction of zonula occluden-1 (ZO-1) and occludin expression and the increase in claudin-2 expression in colonic tissue relative to that following DSS administration. FGR treatment significantly recovered expression of cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β, in serum or respective mRNA expression in colonic tissue relative to that following DSS administration. FGR regulated levels of oxidative stress-related factors, such as malondialdehyde and glutathione, and the activity of catalase and superoxide dismutase in the colon tissue of the DSS-induced acute colitis mice model. Furthermore, FGR treatment inhibited apoptosis by reducing the activity of caspase-3 and the ratio of Bcl-2 associated X (Bax)/B-cell lymphoma 2 (Bcl-2). Collectively, FGR treatment protected the intestinal barrier from dysfunction and inhibited inflammation and apoptosis in DSS-induced colitis. Therefore, FGR may decrease the inflammatory response and be a candidate for treating and prevention inflammatory bowel disease by protecting the intestinal integrity.

Clinical Significance of Claudin Expression in Oral Squamous Cell Carcinoma.

A change in claudin expression has been demonstrated in various tumors. The present study specifically compares claudin expression in oral squamous cell carcinoma (OSCC) with healthy oral epithelium from the same individual and analyzes the association between claudin expression and the clinically relevant course parameters. Our study includes tissue samples and clinically relevant follow-up data from 60 patients with primary and untreated OSCC. The oral mucosa was analyzed via Western blot for the expression of claudin-1, -2, -3, -4, -5, and -7. Importantly, the tumor and healthy tissues were obtained pairwise from patients, allowing for intraindividual comparisons. Both the healthy and tumor epithelium from the oral cavity did not express the claudin-3 protein. The intraindividual comparison revealed that, in OSCC, claudin-2 expression was higher, and the expression of claudin-4, -5, and -7 was lower than in healthy epithelium. An association was found between increased claudin-2 expression and shorter relapse-free survival. In addition, the reduced expression of claudin-4 had a negative impact on relapse-free survival. Furthermore, associations between the reduced expression of claudin-7 and the stage of a tumor, or the presence of lymph node metastases, were found. Thus, the expression level of claudin-2, -4, and -7 appears to be predictive of the diagnosis and prognosis of OSCC.

Loss of claudin-4 reduces DNA damage repair and increases sensitivity to PARP inhibitors (228)

Objectives: Claudin-4 is aberrantly expressed in nearly 70% of all ovarian cancer tumors, and increased claudin-4 expression conveys a worse overall survival. Poly (ADP-ribose) polymerase (PARP) inhibitors are a therapeutic option for patients with ovarian cancer. PARP inhibitors exploit deficiencies in DNA damage repair (e.g., BRCA-mutation) by inducing replication stress and DNA damage. This study aimed to define the relationship between claudin-4 expression and response to PARP inhibitors. Results: Elevated claudin-4 mRNA expression is associated with ovarian cancer, DNA damage repair pathways, and resistance to platinum-based chemotherapies and PARP inhibitors. Claudin-4 knockdown significantly sensitized ovarian cancer cells to PARP inhibitors. Specifically, loss of claudin-4 reduced the 50% inhibitory concentration (IC50) of olaparib by 70% (IC50 - 378 nM to 88 nM) and rucaparib by 85% (IC50 - 189 nM to 27.5 nM). Furthermore, loss of claudin-4 expression sensitized ovarian cancer cells to several FDA-approved chemotherapies that induce DNA damage. Claudin-4 downregulation resulted in the decrease of several DNA damage repair effectors, including 53BP1 and XRCC1. Claudin-4/53BP1/XRCC1 expression was mainly confined to the tumor cells. Claudin-4 knockdown did not change homology-directed repair but reduced both non-homologous end-joining activity by 41% (p<0.001) and 53BP1 foci formation by an average of 50% (p<0.05). In the TCGA, lower claudin-4 expression correlated to increased mutational burden (Claudin-4 “High”, 45.2 mutations vs Claudin-4 “Low” 134.6 mutations; p<0.01). In 15 primary ovarian cancer tumors (12 high-grade serous, one low-grade serous, one mucinous, and one granulosa cell), “low”/null claudin-4 expressing tumors had a significant reduction in Ki67 after PARP inhibitor treatment alone (p<0.01). In contrast, “High” claudin-4 tumors only had significantly reduced Ki67 in the claudin-4 inhibitor alone (p<0.0001) and in the CMP/olaparib combination (p<0.0001). Conclusions: Claudin-4 is related to non-homologous end-joining DNA damage repair and 53BP1 foci formation. Claudin-4 inhibition in “high” claudin-4 tumors sensitized tumors to PARP inhibition. Claudin-4 expression in high-grade ovarian cancer tumors could potentially serve as both a marker of PARP inhibitor response and a target to improve PARP inhibitor response. Objectives: Claudin-4 is aberrantly expressed in nearly 70% of all ovarian cancer tumors, and increased claudin-4 expression conveys a worse overall survival. Poly (ADP-ribose) polymerase (PARP) inhibitors are a therapeutic option for patients with ovarian cancer. PARP inhibitors exploit deficiencies in DNA damage repair (e.g., BRCA-mutation) by inducing replication stress and DNA damage. This study aimed to define the relationship between claudin-4 expression and response to PARP inhibitors. Results: Elevated claudin-4 mRNA expression is associated with ovarian cancer, DNA damage repair pathways, and resistance to platinum-based chemotherapies and PARP inhibitors. Claudin-4 knockdown significantly sensitized ovarian cancer cells to PARP inhibitors. Specifically, loss of claudin-4 reduced the 50% inhibitory concentration (IC50) of olaparib by 70% (IC50 - 378 nM to 88 nM) and rucaparib by 85% (IC50 - 189 nM to 27.5 nM). Furthermore, loss of claudin-4 expression sensitized ovarian cancer cells to several FDA-approved chemotherapies that induce DNA damage. Claudin-4 downregulation resulted in the decrease of several DNA damage repair effectors, including 53BP1 and XRCC1. Claudin-4/53BP1/XRCC1 expression was mainly confined to the tumor cells. Claudin-4 knockdown did not change homology-directed repair but reduced both non-homologous end-joining activity by 41% (p<0.001) and 53BP1 foci formation by an average of 50% (p<0.05). In the TCGA, lower claudin-4 expression correlated to increased mutational burden (Claudin-4 “High”, 45.2 mutations vs Claudin-4 “Low” 134.6 mutations; p<0.01). In 15 primary ovarian cancer tumors (12 high-grade serous, one low-grade serous, one mucinous, and one granulosa cell), “low”/null claudin-4 expressing tumors had a significant reduction in Ki67 after PARP inhibitor treatment alone (p<0.01). In contrast, “High” claudin-4 tumors only had significantly reduced Ki67 in the claudin-4 inhibitor alone (p<0.0001) and in the CMP/olaparib combination (p<0.0001). Conclusions: Claudin-4 is related to non-homologous end-joining DNA damage repair and 53BP1 foci formation. Claudin-4 inhibition in “high” claudin-4 tumors sensitized tumors to PARP inhibition. Claudin-4 expression in high-grade ovarian cancer tumors could potentially serve as both a marker of PARP inhibitor response and a target to improve PARP inhibitor response.

Claudin-1-Targeted Nanoparticles for Delivery to Aging-Induced Alterations in the Blood-Brain Barrier.

Aging-induced alterations to the blood-brain barrier (BBB) are increasingly being seen as a primary event in chronic progressive neurological disorders that lead to cognitive decline. With the goal of increasing delivery into the brain in hopes of effectively treating these diseases, a large focus has been placed on developing BBB permeable materials. However, these strategies have suffered from a lack of specificity toward regions of disease progression. Here, we report on the development of a nanoparticle (C1C2-NP) that targets regions of increased claudin-1 expression that reduces BBB integrity. Using dynamic contrast enhanced magnetic resonance imaging, we find that C1C2-NP accumulation and retention is significantly increased in brains from 12 month-old mice as compared to nontargeted NPs and brains from 2 month-old mice. Furthermore, we find C1C2-NP accumulation in brain endothelial cells with high claudin-1 expression, suggesting target-specific binding of the NPs, which was validated through fluorescence imaging, in vitro testing, and biophysical analyses. Our results further suggest a role of claudin-1 in reducing BBB integrity during aging and show altered expression of claudin-1 can be actively targeted with NPs. These findings could help develop strategies for longitudinal monitoring of tight junction protein expression changes during aging as well as be used as a delivery strategy for site-specific delivery of therapeutics at these early stages of disease development.

A Claudin-Based Molecular Signature Identifies High-Risk, Chemoresistant Colorectal Cancer Patients.

Identifying molecular characteristics that are associated with aggressive cancer phenotypes through gene expression profiling can help predict treatment responses and clinical outcomes. Claudins are deregulated in colorectal cancer (CRC). In CRC, increased claudin-1 expression results in epithelial-to-mesenchymal transition and metastasis, while claudin-7 functions as a tumor suppressor. In this study, we have developed a molecular signature based on claudin-1 and claudin-7 associated with poor patient survival and chemoresistance. This signature was validated using an integrated approach including publicly available datasets and CRC samples from patients who either responded or did not respond to standard-of-care treatment, CRC cell lines, and patient-derived rectal and colon tumoroids. Transcriptomic analysis from a patient dataset initially yielded 23 genes that were differentially expressed along with higher claudin-1 and decreased claudin-7. From this analysis, we selected a claudins-associated molecular signature including PIK3CA, SLC6A6, TMEM43, and ASAP-1 based on their importance in CRC. The upregulation of these genes and their protein products was validated using multiple CRC patient datasets, in vitro chemoresistant cell lines, and patient-derived tumoroid models. Additionally, blocking these genes improved 5-FU sensitivity in chemoresistant CRC cells. Our findings propose a new claudin-based molecular signature that associates with poor prognosis as well as characteristics of treatment-resistant CRC including chemoresistance, metastasis, and relapse.

Pre-emptive Short-term Nicotinamide Mononucleotide Treatment in a Mouse Model of Diabetic Nephropathy.

The activation of NAD+-dependent deacetylase, Sirt1, by the administration of nicotinamide mononucleotide (NMN) ameliorates various aging-related diseases. Diabetic db/db mice were treated with NMN transiently for 2 weeks and observed for effects on diabetic nephropathy (DN). At 14 weeks after the treatment period, NMN attenuated the increases in urinary albumin excretion in db/db mice without ameliorating hemoglobin A1c levels. Short-term NMN treatment mitigated mesangium expansion and foot process effacement, while ameliorating decreased Sirt1 expression and increased claudin-1 expression in the kidneys of db/db mice. This treatment also improved the decrease in the expression of H3K9me2 and DNMT1. Short-term NMN treatment also increased kidney concentrations of NAD+ and the expression of Sirt1 and nicotinamide phosphoribosyltransferase (Nampt), and it maintained nicotinamide mononucleotide adenyltransferase1 (Nmnat1) expression in the kidneys. In addition, survival rates improved after NMN treatment. Short-term NMN treatment in early-stage DN has remote renal protective effects through the upregulation of Sirt1 and activation of the NAD+ salvage pathway, both of which indicate NMN legacy effects on DN.

Andrographolide Treatment Lessens Localized, Systemic Inflammation and Improves Other Pathophysiological Traits in a Mouse Model of Gulf War Illness

Gulf War Illness (GWI) is a chronic multi-symptom disorder that affected almost one-third of the deployed veterans during the first Persian Gulf War (1990-91). Symptoms include gastrointestinal disturbances, chronic fatigue, cognitive deficiencies, and neuroinflammation. Our group has recently shown a series of different therapeutic strategies that span oral butyrate, anti-viral drug Ribavirin, and TLR4 antagonist Sparstolonin B effectively improving outcomes to murine GWI-linked symptoms. Andrographolide (AG), a labdane diterpenoid produced by the plant Andrographis paniculata, exerts numerous beneficial effects in various cell types. Adult C57BL/6J mice were exposed to the GWI chemicals (GWC) pyridostigmine bromide and permethrin for a week (GWT group) and simultaneously treated with AG (GWT+AG group). In our previous report, virome dysbiosis showed a strong association with GWI inflammation. Next-Gen Sequencing data showed both virome richness and Shannon diversity were markedly altered in GWT+AG mice than GWT mice. The observed increased relative abundance of Siphoviridae, Myoviridae, and decreased Microviridae abundance in the GWT group was reversed by AG treatment, referring to a restored gut environment. Gut leaching marked by decreased Occludin expression and increased Claudin2 expression was reversed by AG treatment, a likely consequence of AG induced decrease in TLR4 and TLR7 expression. AG administration restored gut barrier integrity, decreased TLR4 activation, NFκb phosphorylation, and gut inflammation. Furthermore, systemic inflammation in GWT mice, evidenced by the high level of serum IL-1β and IL-6, was found to be decreased in GWT+AG mice. Blood-brain barrier (BBB) integrity loss and resulting microglial activation, a common feature of GWI murine pathology were reversed by AG treatment. AG restored dysfunctional BBB by enhancing tight junction protein Claudin5 expression as well as decreased microglial activation. Also, AG administration led to high brain-derived neurotrophic factor (BDNF) expression in the frontal cortex, a molecule with a pronounced role in synaptic plasticity and neuronal regeneration. In summary, these results explicitly imply that AG played a key role in decreasing GWI-associated intestinal, systemic, and neuroinflammation whereas it also improved intestinal barrier and BBB integrity in the GWI mouse model.

Prognostic Significance of the Expression of Claudin-1 in Head and Neck Squamous Cell Carcinoma

Background and Objectives Claudin-1 (CLDN-1) is the major component of tight junctions and functions in controlling cell to cell adhesion. Certain claudins were expressed aberrantly and proved to have prognostic significance in various human cancers. However, its clinical significance has been poorly understood in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to investigate the relationship between CLDN-1 expression and clinicopathologic parameters in HNSCC.Subjects and Method The surgical specimens of primary HNSCCs from a consecutive cohort of 91 patients were retrospectively collected. Immunohistochemical staining for CLDN-1 was performed blindly by two pathologists. CLDN-1 staining intensity was scored semi-quantitatively on a scale of 0 to 3 (0: negative; 1: weak; 2: moderate; 3: strong). For the statistical analysis, the expression levels were classified as low (negative and weak) and high (moderate and strong). Next, the association between CLDN-1 expression and clinicopathological features &amp; clinical outcomes was analyzed.Results The increased CLDN-1 expression was significantly associated with lymphatic invasion (&lt;i&gt;p&lt;/i&gt;=0.019). The expression level of CLDN-1 was not associated with pathological T stage, lymph node metastasis or recurrence. Kaplan-Meier analysis found that 3-year overall survival (OS) rate was 53% in patients with high level CLDN-1 expression and 74% in patients with low level CLDN-1 expression. It also found that 5-year OS rate was 49% in patients with high level CLDN-1 expression and 68% in patients with low level CLDN-1 expression. A significantly poor OS rate was recorded in patients with high level of CLDN-1 expression compared to patients with low level CLDN-1 expression (&lt;i&gt;p&lt;/i&gt;=0.022).Conclusion CLDN-1 may serve as useful prognostic marker in patients with HNSCCs.

Claudin-1 Is a Valuable Prognostic Biomarker in Colorectal Cancer: A Meta-Analysis.

Background Claudin-1 plays an important part in maintaining the mucosal structures and physiological functions. Several studies showed a relationship between claudin-1 and colorectal cancer (CRC), but its prognostic significance is inconsistent. This meta-analysis assessed the prognostic value and clinical significance of claudin-1 in CRC. Materials and Methods We retrieved eligible studies from PubMed, Cochrane Library, Embase, and Web of Science databases before February 10, 2020. The hazard ratio (HR) with 95% confidence interval (CI) was applied to assess the correlation between claudin-1 and prognosis and clinical features. Heterogeneity was assessed by the Cochran Q test and I-square (I2), while publication bias was evaluated by the Begg test and Egger test. Test sequence analysis (TSA) was used to estimate whether the included studies' number is sufficient. The stability of the results was judged by sensitivity analysis. Metaregression was utilized to explore the possible covariance which may impact on heterogeneity among studies. Results Eight studies incorporating 1704 patients met the inclusion criteria. Meta-analysis showed that the high expression of claudin-1 was associated with better overall survival (HR, 0.46; 95% CI, 0.28–0.76; P = 0.002) and disease-free survival (HR, 0.44; 95% CI, 0.29–0.65; P = 0.003) in CRC. In addition, we found that claudin-1 was related to the better tumor type (n = 6; RR, 0.60; 95% CI, 0.49–0.73; P < 0.00001), negative venous invasion (n = 4; RR, 0.81; 95% CI, 0.70–0.95; P = 0.001), and negative lymphatic invasion (n = 4; RR, 0.83; 95% CI, 0.74–0.92; P = 0.0009). Conclusion The increased claudin-1 expression in CRC is associated with better prognosis. In addition, claudin-1 was related to the better tumor type and the less venous invasion and lymphatic invasion.

Barrier Protection and Recovery Effects of Gut Commensal Bacteria on Differentiated Intestinal Epithelial Cells In Vitro.

Alterations in the gut microbiota composition play a crucial role in the pathogenesis of inflammatory bowel disease (IBD) as specific commensal bacterial species are underrepresented in the microbiota of IBD patients. In this study, we examined the therapeutic potential of three commensal bacterial species, Faecalibacterium prausnitzii (F. prausnitzii), Roseburia intestinalis (R. intestinalis) and Bacteroides faecis (B. faecis) in an in vitro model of intestinal inflammation, by using differentiated Caco-2 and HT29-MTX cells, stimulated with a pro-inflammatory cocktail consisting of interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), interferon-γ (IFNγ), and lipopolysaccharide (LPS). Results obtained in this work demonstrated that all three bacterial species are able to recover the impairment of the epithelial barrier function induced by the inflammatory stimulus, as determined by an amelioration of the transepithelial electrical resistance (TEER) and the paracellular permeability of the cell monolayer. Moreover, inflammatory stimulus increased claudin-2 expression and decreased occludin expression were improved in the cells treated with commensal bacteria. Furthermore, the commensals were able to counteract the increased release of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) induced by the inflammatory stimulus. These findings indicated that F. prausnitzii, R. intestinalis and B. faecis improve the epithelial barrier integrity and limit inflammatory responses.

Idiopathic pulmonary fibrosis is associated with tight junction protein alterations

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by an abnormal healing response to injury of the alveolar epithelium. Tight junctions provide a physical barrier at the apical intercellular space between epithelial cells and regulate paracellular flux. While tight junction alterations are known to contribute to barrier dysfunction in a number of disease states, the role of tight junction proteins in IPF is poorly defined. To determine a potential role for tight junction protein alterations in IPF, we performed immunohistochemical staining for tight junction proteins ZO-1, occludin, claudin-2, claudin-3, claudin-4, claudin-5, and claudin-18. Staining intensity and localization were compared between IPF and control lung tissues. IPF was associated with type II pneumocyte hyperplasia and altered tight junction protein expression. While there was no difference in the expression of ZO-1, claudin-3, or claudin-5, between IPF and normal control, there was an overall increase in claudin-2 expression in bronchiolar and alveolar epithelium and a decrease in claudin-4 expression in type II pneumocytes. There was also increased occludin and decreased claudin-18 expression in pneumocytes overlying fibroblastic foci. These findings suggest that epithelial barrier alterations may be important to the pathogenesis of IPF.

Integrated Imaging Methodology Detects Claudin-1 Expression in Premalignant Nonpolypoid and Polypoid Colonic Epithelium in Mice.

OBJECTIVES:Conventional colonoscopy with white light illumination detects colonic adenomas based on structural changes alone and is limited by a high miss rate. We aim to demonstrate an integrated imaging strategy that combines wide-field endoscopy and confocal endomicroscopy in real time to visualize molecular expression patterns in vivo to detect premalignant colonic mucosa.METHODS:A peptide specific for claudin-1 is labeled with Cy5.5 and administrated intravenously in genetically engineered mice that develop adenomas spontaneously in the distal colon. Wide-field endoscopy is used to identify the presence of nonpolypoid and polypoid adenomas. Anatomic landmarks are used to guide placement of a confocal endomicroscope with side-view optics to visualize claudin-1 expression patterns with subcellular resolution.RESULTS:Wide-field fluorescence images show peak uptake in colon adenoma at ∼1 hour after systemic peptide administration, and lesion margins are clearly defined. Further examination of the lesion using a confocal endomicroscope shows dysplastic crypts with large size, elongated shape, distorted architecture, and variable dimension compared with normal. The mean fluorescence intensity is significantly higher for dysplasia than normal. Increased claudin-1 expression in dysplasia vs normal is confirmed ex vivo, and the binding pattern is consistent with the in vivo imaging results.DISCUSSION:Wide-field endoscopy can visualize molecular expression of claudin-1 in vivo to localize premalignant colonic mucosa, and confocal endomicroscopy can identify subcellular feature to distinguish dysplasia from normal.

Claudin-1 upregulation is associated with favorable tumor features and a reduced risk for biochemical recurrence in ERG-positive prostate cancer.

Claudin-1 is a membrane-tight junction protein and important for the sealing of the paracellular cleft in epithelial and endothelial cells. Differential expression of Claudin-1 is linked to disease outcome in various cancers. To evaluate the potential relevance of Claudin-1 expression in prostate cancer, a tissue microarray containing samples of 17,747 tumors with annotated clinico-pathological and molecular data was immunohistochemically analyzed for Claudin-1 expression. In normal prostate, glandular cells were always Claudin-1-negative while there was a strong staining of gland-surrounding basal cells. In contrast to normal prostatic glands, a positive Claudin-1 immunostaining, was found, however, in 38.7% of 12,441 interpretable cancers and was considered weak in 12.7%, moderate in 13.2%, and strong in 12.8% of cases. Positive Claudin-1 immunostaining was associated with favorable tumor features like low pT (p = 0.0032), low Gleason grade (p< 0.0001), and a reduced risk of PSA recurrence (p = 0.0005). A positive Claudin-1 staining was markedly more frequent in ERG-positive (63%) than in ERG-negative cancers (23%; p < 0.0001). Subset analyses revealed that all associations of Claudin-1 expression and favorable phenotype and prognosis were driven by ERG-positive cancers. Multivariate analyses revealed, however, that even in ERG-positive cancers, the prognostic impact of high Claudin-1 expression was not independent of established clinico-pathological parameters. Comparison with 12 previously analyzed chromosomal deletions identified conspicuous associations with PTEN and 12p13 deletions potentially indicating functional interactions. These data identify a peculiar role for Claudin-1 in prostate cancer. The protein is overexpressed in a fraction of prostate cancers and increased Claudin-1 expression levels predict a favorable prognosis in ERG-positive cancer.