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Objective To investigate the expression of P504s and claudin-4 in prostate cancer tissues and assess the value of their combined detection in prognostic evaluation of prostate cancer. Methods We analyzed a total of 154 surgical specimens or biopsy samples of prostate tissues collected between January, 2013 and July, 2015 in the pathological tissue bank of our hospital. These specimens, which consisted of 84 prostate cancer tissues from patients aged 52 to 83 years (mean 70.9±7.3 years) and 70 benign prostatic hyperplasia tissues from patients aged 51 to 85 years (mean 68.2±7.4 years), were used to construct 60-dot matrix paraffin tissue chips for detection of P504s and claudin-4 proteins using immunohistochemistry with SP method. The correlation of P504s and claudin-4 expression with the prognosis of the patients with prostate cancer was analyzed. Results The positive expression rates of P504s and claudin-4 protein were significantly lower in benign prostatic hyperplasia tissues than in prostate cancer tissues (P < 0.01). The tissue microarray data did not reveal significant difference in the positive expression rate of P504s in prostate cancer tissues with different Gleason scores or in different stages. The positive expression rates of claudin-4 protein was significantly lower in prostate cancer with a Gleason score ≤6 than in that with a score ≥7 (60.98% vs 74.42%, P < 0.05), and was also lower in prostate cancer in T2a stage or lower than that in T2b stage or higher (55.26% vs 78.26%, P < 0.01). Conclusion Abnormal expression of P504s and claudin-4 proteins may be related with the occurrence and progression of prostate cancer, and prostate cancer with a higher level of malignancy expresses higher levels of claudin-4. Combined detection of P504s and claudin-4 can improve the detection rate of prostate cancer and can be of value in prognostic evaluation of the patients.

Intestinal oxidative stress produces pro-inflammatory cytokines, which increase tight junction (TJ) permeability, leading to intestinal and systemic inflammation. Cystine (Cys2) is a substrate of glutathione (GSH) and inhibits inflammation, however, it is unclear whether Cys2 locally improves intestinal barrier dysfunction. Thus, we investigated the local effects of Cys2 on oxidative stress-induced TJ permeability and intestinal inflammatory responses. Caco-2 cells were cultured in a Cys2-supplemented medium for 24 h and then treated with H2O2 for 2 h. We assessed TJ permeability by measuring transepithelial electrical resistance and the paracellular flux of fluorescein isothiocyanate–dextran 4 kDa. We measured the concentration of Cys2 and GSH after Cys2 pretreatment. The mRNA expression of pro-inflammatory cytokines was assessed. In addition, the levels of TJ proteins were assessed by measuring the expression of TJ proteins in the whole cells and the ratio of TJ proteins in the detergent-insoluble fractions to soluble fractions (IS/S ratio). Cys2 treatment reduced H2O2-induced TJ permeability. Cys2 did not change the expression of TJ proteins in the whole cells, however, suppressed the IS/S ratio of claudin-4. Intercellular levels of Cys2 and GSH significantly increased in cells treated with Cys2. Cys2 treatment suppressed the mRNA expression of pro-inflammatory cytokines, and the mRNA levels were significantly correlated with TJ permeability. In conclusion, Cys2 treatment locally reduced oxidative stress-induced intestinal barrier dysfunction possively due to the mitigation of claudin-4 dislocalization. Furthermore, the effect of Cys2 on the improvement of intestinal barrier function is related to the local suppression of oxidative stress-induced pro-inflammatory responses. [ABSTRACT FROM AUTHOR]

To investigate the effects of radiation on rat submandibular glands and the possible protective effects of ischemic preconditioning, the submandibular glands of Wistar rats were subjected to in situ radiation after ischemic preconditioning. The glands were exposed to X‐radiation at a single dose of 20 Gy. Ischemic preconditioning was achieved by three min of ischemia and three min of reperfusion, repeated three times before irradiation. Salivary secretion, histological changes, alterations in tight junctions, and the levels of oxidative stress, pro‐inflammatory cytokines, and water secretion proteins mediated by the muscarinic acetylcholine M3 subtype receptor were determined at 1 and 12 weeks post‐irradiation. In glands subjected to irradiation only, the secretion, superoxide dismutase activity, tight junction width, acinar cell number, and M3 receptor and aquaporin‐5 levels were lower at 1 and 12 weeks than seen in the ischemically preconditioned irradiated glands. In contrast, tumor necrosis factor‐α, malondialdehyde, myeloperoxidase activity, and the expression of the tight junction protein claudin‐4 were significantly higher in the irradiated only glands. Our study revealed that radiation caused a series of injury‐stress responses, especially damage to the water secretion pathway mediated by the M3 receptor that ultimately led to hyposecretion, which might play an important role in the dysfunction of the irradiated only glands. Ischemic preconditioning reduced the radiation‐induced injury to submandibular glands and ameliorated salivary hyposecretion. [ABSTRACT FROM AUTHOR]

Objective This study aims to investigate the effects of ionizing radiation on the secretion of the paracellular pathway in rat submandibular glands (SMGs) and reveal the changes in the tight junction (TJ) protein claudin-4. Methods A total of 24 Wistar rats were randomly divided into control and irradiation groups. The irradiation groups were further divided into 1, 4, and 12 weeks groups after irradiation. One-time 20 Gy irradiation was given to the SMG area on the experimental side of the irradiation group. At 1, 4, and 12 weeks after irradiation, the secretion of SMGs was measured using the Schirmer's test. The pathological changes in the gland tissues were observed under light microscopy after hematoxylin-eosin (HE) staining. The changes in the TJ ultrastructure were observed under transmission electron microscopy. The immunofluorescence staining and Western blot were used to detect the expression levels of muscarinic acetylcholine M3 receptor, aquaporin 5 (AQP5), and claudin-4 protein. Results At 1, 4, and 12 weeks after irradiation, the secretion of SMGs in the irradiation group was significantly decreased and lower than that in the control group (P<0.01). At 1 week, the interstitial edema was observed in SMG tissues. Nuclear pyknosis, decreased number of acinar cells, and small focal necrosis with inflammatory infiltration were also observed over time. However, these changes were most evident at 12 weeks after irradiation. In the irradiation group, the TJ ultrastructure of glands at different times appeared to be fuzzy, collapsed, and had decreased electron density. Moreover, the width of TJs was remarkably decreased (P<0.01). The expression levels of M3 and AQP5 were decreased in a time-dependent manner, and the fluorescence intensity was significantly reduced after irradiation. However, the expression levels and fluorescence intensity of claudin-4 were enhanced in different degrees. Conclusion The changes in the TJ structure, the upregulation of the claudin-4 expression, and the damage in the paracellular pathway were involved in the hyposecretion of SMGs after irradiation. than that in the control group (P<0.01). At 1 week, the interstitial edema was observed in SMG tissues. Nuclear pyknosis, decreased number of acinar cells, and small focal necrosis with inflammatory infiltration were also observed over time. However, these changes were most evident at 12 weeks after irradiation. In the irradiation group, the TJ ultrastructure of glands at different times appeared to be fuzzy, collapsed, and had decreased electron density. Moreover, the width of TJs was remarkably decreased (P<0.01). The expression levels of M3 and AQP5 were decreased in a time-dependent manner, and the fluorescence intensity was significantly reduced after irradiation. However, the expression levels and fluorescence intensity of claudin-4 were enhanced in different degrees. Conclusion The changes in the TJ structure, the upregulation of the claudin-4 expression, and the damage in the paracellular pathway were involved in the hyposecretion of SMGs after irradiation. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress that disrupts ER function can occur in response to a wide variety of cellular stress factors leads to the accumulation of unfolded and misfolded proteins in the ER. Many studies have shown that ER stress amplified inflammatory reactions and was involved in various inflammatory diseases. However, little is known regarding the role of ER stress in hyperoxia-induced acute lung injury (HALI). This study investigated the influence of ER stress inhibitor, 4-phenyl butyric acid (4-PBA), in mice with HALI. Treatment with 4-PBA in the hyperoxia groups significantly prolonged the survival, decreased lung edema, and reduced the levels of inflammatory mediators, lactate dehydrogenase, and protein in bronchoalveolar lavage fluid, and increased claudin-4 protein expression in lung tissue. Moreover, 4-PBA reduced the ER stress-related protein expression, NF-κB activation, and apoptosis in the lung tissue. In in vitro study, 4-PBA also exerted a similar effect in hyperoxia-exposed mouse lung epithelial cells (MLE-12). However, when claudin-4 siRNA was administrated in mice and MLE-12 cells, the protective effect of 4-PBA was abrogated. These results suggested that 4-PBA protected against hyperoxia-induced ALI via enhancing claudin-4 expression.

Endoplasmic reticulum (ER) stress that disrupts ER function can occur in response to a wide variety of cellular stress factors leads to the accumulation of unfolded and misfolded proteins in the ER. Many studies have shown that ER stress amplified inflammatory reactions and was involved in various inflammatory diseases. However, little is known regarding the role of ER stress in hyperoxia-induced acute lung injury (HALI). This study investigated the influence of ER stress inhibitor, 4-phenyl butyric acid (4-PBA), in mice with HALI. Treatment with 4-PBA in the hyperoxia groups significantly prolonged the survival, decreased lung edema, and reduced the levels of inflammatory mediators, lactate dehydrogenase, and protein in bronchoalveolar lavage fluid, and increased claudin-4 protein expression in lung tissue. Moreover, 4-PBA reduced the ER stress-related protein expression, NF-κB activation, and apoptosis in the lung tissue. In in vitro study, 4-PBA also exerted a similar effect in hyperoxia-exposed mouse lung epithelial cells (MLE-12). However, when claudin-4 siRNA was administrated in mice and MLE-12 cells, the protective effect of 4-PBA was abrogated. These results suggested that 4-PBA protected against hyperoxia-induced ALI via enhancing claudin-4 expression. [ABSTRACT FROM AUTHOR]

Background: The distinction between mesothelioma with epithelioid features and metastatic carcinoma may be challenging, particularly on cytology. A novel 2‐hit Claudin‐4 and BRCA‐associated protein 1 (BAP1) panel was investigated. Methods: The objective of this study was to determine the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the panel on cytology from pleural effusions and matched biopsies, including 49 malignant mesotheliomas on cytology with 43 matched biopsies, 49 normal/reactive mesothelial proliferations, and 49 pleural metastatic carcinomas from different primaries with 21 matched pleural biopsies. The diagnostic role of the 4 categories obtained by crossing the immunostaining results was analyzed. Results: Claudin‐4 strongly stained all metastatic carcinomas and tested completely negative in normal mesothelium, benign reactive mesothelial hyperplasia, and malignant mesothelioma. All normal and benign mesothelial proliferations and all carcinomas except 1 were immunoreactive for BAP1, whereas BAP1 loss was observed in 88% of malignant mesotheliomas. The expression of Claudin‐4 alone excluded all benign and malignant mesothelial growth, consistently characterizing all metastatic carcinomas. Double negativity was evident in all malignant mesotheliomas, and double positivity was observed in all metastatic carcinomas. BAP1‐positive/Claudin‐4–negative status was observed only in malignant mesotheliomas and benign mesothelial proliferations. A single metastatic anal squamous cell carcinoma had BAP1‐negative/Claudin‐4–positive staining. Conclusions: Claudin‐4 expression was completely specific and sensitive for metastatic carcinoma, excluding mesothelial proliferations. BAP1 staining characterized 98% of metastatic carcinomas and 100% of benign mesothelial proliferations, whereas negativity was observed almost exclusively in mesotheliomas. This 2‐hit panel is probably the best compromise for differentiating malignant mesothelioma and metastatic carcinoma on either cytology or biopsy specimens. Claudin‐4 expression in effusion cytology is completely specific and sensitive for metastatic carcinoma, excluding mesothelial proliferations; otherwise, BRCA1‐associated protein 1 (BAP1) staining characterizes 98% of metastatic carcinomas and 100% of benign mesothelial proliferations, whereas negativity is almost exclusively disclosed in mesothelioma. This 2‐hit Claudin‐4/BAP1 panel is probably the best compromise in differentiating malignant mesothelioma and metastatic carcinoma on either cytology or biopsy specimens. [ABSTRACT FROM AUTHOR]

Despite recent advances in chemotherapy, pancreatic cancer remains a leading cause of cancer-related deaths. Moreover, although targeted therapy has shown promising therapeutic efficacy in many types of cancers, no such effective targeting strategy for treatment of pancreatic cancer, which is in desperate need for new treatment approaches. Here, we developed claudin-4–targeting Clostridium perfringens enterotoxin (CPE) peptide-conjugated polysialic acid nanoparticles (C-SNPs) for pancreatic cancer-targeted therapy. Doxorubicin-loaded C-SNPs (DOX-C-SNPs) higly accumulated in the targeted pancreatic cancer via enhanced peameability and retention (EPR) effect, targeting claudin-4 in pancreatic cancer that becomes superficially exposed owing to the disruption of tight junctions. Notably, DOX-C-SNP accumulation in the non-targeted, normal pancreas was significantly reduced because of hindered access to claudin-4 in tight junctions. As a result, DOX-C-SNPs substantially suppressed tumor growth in an orthotopic pancreatic cancer model while exerting minimal toxicity against non-targeted, normal tissues. Collectively, these findings indicate that claudin-4–targeting DOX-C-SNPs may have promise in treating pancreatic cancers through targeting of exposed claudin-4. Image 1 • Studies have demonstrated differential expression pattern of claudin-4 between pancreatic cancers and normal tissues. • Polysialic acid-based nanoparticles including CPE specifically bound to claudin-4 exposed to pancreatic cancer cell. • Dox-loaded CPE-conjugated polysialic acid nanoparticles showed improved therapeutic efficacy in pancreatic chemotherapy. • Claudin-4 targeting nanoparticles could be promising agents for effective pancreatic cancer therapy. [ABSTRACT FROM AUTHOR]

Claudins are essential components of tight junctions, which are frequently deregulated in breast cancer. The aim of the current study was to assess claudin-3 and -4 expression in bilateral breast cancer (BBC) and unilateral breast cancer (UBC). Immunohistochemical expression of claudin-3 and claudin-4 was evaluated in tissue microarrays containing 174 cases of BBCs paired with 174 cases of solitary tumors. Each case was classified as claudin-high or claudin-low depending on the H-score value. The results were correlated with histopathological features and the expression of basic breast cancer biomarkers. Median H-scores for claudin-3 were significantly higher in the synchronous BBC (sBBC) than in UBC. Claudin-4-high cases were more prevalent than within the whole BBC group, and sBBC and metachronous BBC (mBBC) alone. In the BBC group negative ER, high Ki-67 and high claudin-3 were independent factors correlated with high claudin-4. In the UBC group, Ki-67 >14% and high claudin-3 were associated with high claudin-4. Our study demonstrates that the expression of claudin-4 is significantly higher in UBC compared to BBC tumors. We also demonstrated that high claudin-4 expression in BBC is associated with a more aggressive phenotype (lack of steroid receptors, HER2 overexpression, and high Ki-67). It is possible that claudins down- and upregulation may depend on different triggers and lead to various consequences in UBC and BBC. [ABSTRACT FROM AUTHOR]

The extent of tight junction (TJ) formation is one of many factors that regulate motility, invasion, and metastasis. Claudins are required for the formation and maintenance of TJs. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are highly expressed in the majority of ovarian cancers. We report here that CLDN3 and CLDN4 each serve to constrain the growth of human 2008 cancer xenografts and limit metastatic potential. Knockdown of CLDN3 increased in vivo growth rate by 2.3-fold and knockdown of CLDN4 by 3.7-fold in the absence of significant change in in vitro growth rate. Both types of tumors exhibited increase in birth rate as measured by Ki67 staining and decrease in death rate as reflected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Knockdown of either claudin did not alter expression of other TJ protein but did reduce TJ formation as measured by transepithelial resistance and paracellular flux of dextran, enhance migration and invasion in in vitro assays, and increase lung colonization following intravenous injection. Knockdown of CLDN3 and CLDN4 increased total lung metastatic burden by 1.7-fold and 2.4-fold, respectively. Loss of either CLDN3 or CLDN4 resulted in down-regulation of E-cadherin mRNA and protein, increased inhibitory phosphorylation of glycogen synthase kinase-3β (GSK-3β), and activation of β-catenin pathway signaling as evidenced by increases in nuclear β-catenin, the dephosphorylated form of the protein, and transcriptional activity of β-catenin/T-cell factor (TCF). We conclude that both CLDN3 and CLDN4 mediate interactions with other cells in vivo that restrain growth and metastatic potential by sustaining expression of E-cadherin and limiting β-catenin signaling.

Background: Environmental enteropathy (EE) is associated with stunting, impairment of responses to oral vaccines, and other adverse health consequences in young children throughout the developing world. EE is characterized by chronic low-grade intestinal inflammation and disrupted epithelial barrier integrity, partly resulting from dysregulation of tight junction proteins, observed in other enteropathies such as celiac disease. During EE, this dysregulation of tight junction expression amplifies translocation of pathogenic bacteria across the intestinal mucosa. Aims: The aim was to determine whether enteropathogen-mediated epithelial barrier failure can be ameliorated using contra-pathogenicity therapies. Methods: Intestinal epithelial barrier damage was assessed in Caco-2 cells incubated with three important enteropathogens identified in EE patients: Enteropathogenic Escherichia coli (EPEC), Citrobacter rodentium (C. rodentium), and Cryptosporidium parvum (C. parvum). Potential therapeutic molecules were tested to detect effects on transepithelial resistance (TER), bacterial translocation (BT), claudin-4 expression, and regulation of the inflammatory cytokine response. Results: All three enteropathogens compared to uninfected cells, reduced TER (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0007), reduced claudin-4 expression, and permitted BT (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0003) through the monolayer. Zinc, colostrum, epidermal growth factor, trefoil factor 3, resistin-like molecule-β, hydrocortisone, and the myosin light chain kinase inhibitor ML7 (Hexahydro-1-[(5-iodo-1-naphthalenyl)sulfonyl]-1H-1,4-diazepine hydrochloride); ML7) improved TER (up to 70%) and decreased BT (as much as 96%). Only zinc demonstrated modest antimicrobial activity. Conclusion: The enteropathogens impaired intestinal–epithelial barrier integrity with dysregulation of claudin-4 and increased bacterial translocation. Enteropathogen-mediated damage was reduced using contra-pathogenicity agents which mitigated the effects of pathogens without direct antimicrobial activity. [ABSTRACT FROM AUTHOR]

The involvement of claudins in urothelial carcinogenesis is controversial. In this study, we analyzed Claudin-4 immunoexpression in 50 cases of bladder urothelial carcinomas depending on the main prognostic parameters of the lesions represented by the tumor grade and tumor extension. Claudin-4 immunoexpression scores were significantly higher in high-grade urothelial carcinomas and in tumors with invasion in muscularis propria. The results obtained indicate the involvement of Claudin-4 in the progression of urothelial bladder carcinomas. [ABSTRACT FROM AUTHOR]

Oral vaccine has been become the most effective strategy in the fight against intestinal infections because of its economy, ease, and capability on inducing responses not only systemic but also local, especially in mucosal compartments. The major obstruction to potent vaccine development is antigen dispersion and tolerance. To overcome this hindrance, we aimed to target M cells, which are the main sentinel gateway for taking up luminal antigens and initiating specific mucosal responses. In this study, 12-mer peptide (CPE12) derived from the Cterminal of 30 amino acids of Clostridium perfringens (CPE30) was predicted to be a good binder using molecular docking and molecular dynamics simulations. To confirm the interaction between CPE12 and M cell receptor, the coding gene for CPE12 was cloned and expressed, CPE12 was then purified, and evaluated ex vivo afterwards. As a result, ex vivo assessment on murine M cells demonstrated that CPE12 had lower binding on murine M cells surface comparing to CPE30. These present results suggest that CPE12 could be a competent candidate peptide for oral vaccine development. [ABSTRACT FROM AUTHOR]

Gastric cancer is one of most challenging cancers and a significant cause of death worldwide. Gastric cancer is, associated with a high incidence and recurrence rate of metastatic disease and poor survival for those with metastatic disease. Claudin-4 is a transmembrane protein that plays an important role in tight junctions. Increasing experimental research has demonstrated that claudin-4 plays an important role in the progression of gastric cancer, including the occurrence of epithelial to mesenchymal transition, intestinal metaplasia, and gastric cancer. In addition, claudin-4 regulates cell proliferation, invasion, migration and apoptosis. Claudin-4 may represent a potential biomarker for gastric cancer patient prognosis and is useful in the classification of gastric cancer. Therefore, in this review, we summarize current information on claudin-4 and gastric cancer, describing the role of claudin-4 in gastric cancer progression and its application in clinical treatment to provide a basis for further research and promote the claudin-4 gene as a potential target to diagnose and treat gastric cancer. [ABSTRACT FROM AUTHOR]

Aims: Anaplastic carcinoma arising in a mucinous tumour of the ovary and rarely in the retroperitoneum is an uncommon neoplasm with three morphological patterns; rhabdoid, sarcomatoid and pleomorphic. We investigated expression of switch/sucrose non‐fermentable (SWI/SNF) chromatin remodelling complex components and claudin‐4 expression. Methods and results: Twenty‐two ovarian and three retroperitoneal mucinous tumours were investigated using antibodies against SMARCB1, SMARCA4, SMARCA2, ARID1A and claudin‐4. Loss of nuclear staining for any SWI/SNF protein was observed in the anaplastic component of nine of 25 (36%), with retained expression within the mucinous component of all tumours. Five (56%) showed loss of more than one protein, with dual loss of SMARCA4 and SMARCA2 in two, loss of SMARCA2 and ARID1A in two and loss of SMARCB1 and SMARCA2 in one. Retained expression of claudin‐4 was seen in 39% of the anaplastic carcinomas and within the mucinous component of all tumours. Rhabdoid morphology was associated with poor prognosis [stages III or IV disease (six of six, 100% versus four of 14, 29%; P = 0.0108] and death from disease (three of four, 75% versus one of 13, 8%; P = 0.0223). Although loss of a SWI/SNF protein was not significantly associated with death from disease (three of five, 60% versus one of 12, 8%; P = 0.0525), it showed a trend in correlation with poor prognosis and was often noted in tumours with rhabdoid morphology within this small cohort. Conclusions: Our report adds to the growing list of female genital tract malignancies with loss of SWI/SNF proteins, underlining their broad differential diagnosis and the importance of careful, context‐dependent interpretation of SWI/SNF protein loss. [ABSTRACT FROM AUTHOR]

Claudin-2-dependent pore function mediates paracellular cation permeability and can result in pathogenesis of many diseases. Although existing various types of claudins, including barrier-forming and pore-forming claudins, their heterodimeric interaction affecting barrier and pore functions has never been fully elucidated yet. Recently, Shashikanth and colleagues demonstrated that expression of claudin-4 was able to antagonize paracellular pore activity of claudin-2. This commentary will emphasize the mechanism underlying claudin-4-mediated claudin-2-dependent pore inhibition and discuss its potential therapeutic and prognostic applications. [ABSTRACT FROM AUTHOR]

Background: Triple-negative breast cancer is a heterogeneous subtype of breast cancer. Claudin is an epithelial tight junctional protein, and also it is a receptor for clostridium perfringens enterotoxin and shows impairment of expression in several cancers. The chief purpose of this study is to assess the claudin-4 expression in triple-negative breast cancer (TNBC) Iranian patients and evaluate its correlation with some clinicopathological factors. Materials and Methods: In this study, 81 TNBC patients were evaluated for the claudin-4 expression by immunohistochemistry. The slides' staining intensity was examined and scored from 0 to 3. Then, slides were reviewed to assess the percentage of cells with membrane and cytoplasmic staining; the obtaining scores were 1–4. Finally, added the resulting two numbers from two stages, and the final number was a maximum of 7. Final scores of 0–3 were considered the low expression, and 4–7 were considered the high expression. Finally, the collected data were analyzed using the Chi-square test. Results: Eighty-one women with breast cancer and a mean age of 49 ± 12 years participated in the study. In 80% of the patients, there was a high expression of claudin-4 marker, and 20% had low expression. The expression level of the marker was not significantly correlated with age, tumor size, lymph node involvement, tumor grade, disease stage, Ki-67, and metastasis. Conclusion: The present study confirmed the high frequency of claudin-4 antigen expression in TNBC patients, and no significant correlation was observed between the expression of antigen and demographic or clinicopathological factors.

Chlamydia trachomatis (Ct) causes the most prevalent bacterial sexually transmitted disease leading to ectopic pregnancy and infertility. Swine not only have many similarities to humans, but they are also susceptible to Ct. Despite these benefits and the ease of access to primary tissue from this food animal, in vitro research in swine has been underutilized. This study will provide basic understanding of the Ct host–pathogen interactions in porcine oviduct epithelial cells (pOECs)—the counterparts of human Fallopian tube epithelial cells. Using NanoString technology, flow cytometry, and confocal and transmission-electron microscopy, we studied the Ct developmental cycle in pOECs, the cellular immune response, and the expression and location of the tight junction protein claudin-4. We show that Ct productively completes its developmental cycle in pOECs and induces an immune response to Ct similar to human cells: Ct mainly induced the upregulation of interferon regulated genes and T-cell attracting chemokines. Furthermore, Ct infection induced an accumulation of claudin-4 in the Ct inclusion with a coinciding reduction of membrane-bound claudin-4. Downstream effects of the reduced membrane-bound claudin-4 expression could potentially include a reduction in tight-junction expression, impaired epithelial barrier function as well as increased susceptibility to co-infections. Thereby, this study justifies the investigation of the effect of Ct on tight junctions and the mucosal epithelial barrier function. Taken together, this study demonstrates that primary pOECs represent an excellent in vitro model for research into Ct pathogenesis, cell biology and immunity.

Background/Aims: Altered pressure natriuresis is an important mechanism of hypertension, but it remains elusive at the molecular level. We hypothesized that in the kidney, tight junctions (TJs) may have a role in pressure natriuresis because paracellular NaCl transport affects interstitial hydrostatic pressure. Methods: To assess the association of salt-sensitive hypertension with altered renal TJ protein expression, Dahl salt-sensitive (SS) and salt-resistant (SR) rats were put on an 8% NaCl-containing rodent diet for 4 weeks. Systolic blood pressure (SBP) and urine NaCl excretion were measured weekly, and kidneys were harvested for immunoblotting and quantitative PCR analysis at the end of the animal experiments. Results: SBP was significantly higher in SS rats than in SR rats during the first to fourth weeks of the animal experiments. During the first and second week, urinary NaCl excretion was significantly lower in SS rats as compared with SR rats. However, the difference between the two groups vanished at the third and fourth weeks. In the kidney, claudin-4 protein and mRNA were significantly increased in SS rats as compared with SR rats. On the other hand, occludin protein and mRNA were significantly decreased in SS rats as compared with SR rats. The expression of claudin-2, claudin-7, and claudin-8 did not vary significantly between the two groups. Conclusions: In SS rats, SS hypertension was associated with differential changes in renal TJ protein expression. Both upregulation of claudin-4 and downregulation of occludin might increase paracellular NaCl transport in the kidney, resulting in impaired pressure natriuresis in SS rats.

Ethnopharmacological Relevance: Reflux esophagitis (RE) is a common chronic inflammatory disease of the esophageal mucosa with a high prevalence and recurrence rate, for which a satisfactory therapeutic strategy is still lacking. Chinese medicine has its characteristics and advantages in treating RE, and the clinical application of Xuanfu Daizhe Tang (XDT) in treating RE has achieved sound therapeutic effects. However, there needs to be more research on its mechanism of action., Aim of the Study: The present work aimed to investigate the mechanism of XDT action in RE through the Signal Transducer and Activator of Transcription 1 (STAT1)/Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) pathway., Materials and Methods: The main active components of XDT were analyzed by ultra-performance liquid chromatography-mass spectrometer (UPLC-MS). The effect of XDT on RE was evaluated in a rat model of RE induced by "Cardioplasty + pyloric ligation + Roux-en-Y esophagojejunostomy". Each administration group was treated by gavage. The degree of damage to the esophageal mucosa was evaluated by visual observation, and the Potential of Hydrogen (PH) method and Hematoxylin-eosin staining (HE) staining were performed. Serum levels of Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Tumor Necrosis Factor alpha (TNF-α), and Inducible Nitric Oxide Synthase (iNOS) were measured by ELISA. Quantitative Real-time PCR (qPCR), Western Blot (WB), and Immunofluorescence (IF) methods were used to detect Claudin-4, Claudin-5, TREM-1, and p-STAT1 in esophageal tissues for studying the mechanism of action and signaling pathway of XDT. Immunohistochemistry (IHC) analysis was used to detect the expression of TREM-1 and CD68 in esophageal tissues. Flow Cytometry (FC) was used to detect the polarization of macrophages in the blood. After conducting preliminary experiments to verify our hypothesis, we performed molecular docking between the active component of XDT and STAT1 derived from rats and parallel experiments with STAT1 inhibitor. The selective increaser of STAT1 transcription (2-NP) group was used to validate the mechanism by which XDT acts., Results: XDT alleviated esophageal injury and attenuated histopathological changes in RE rats. XDT also inhibited the inflammatory response and decreased serum IL-1β, IL-6, TNF-α, and iNOS levels in RE rats. qPCR and WB results revealed that XDT inhibited the expression of Claudin-4, Claudin-5, TREM-1, and STAT1 in the esophageal mucosa of RE rats. IHC and FC results showed that XDT reduced TREM-1 levels in esophageal tissues and polarized macrophages toward M2. The molecular docking results showed that rat-derived STAT1 can strongly bind to Isochronogenic acid A in XDT. The parallel experimental results of STAT1 inhibitor showed that XDT has anti-inflammatory effects similar to STAT1 inhibitors. The 2-NP group confirmed that XDT exerts its therapeutic effect on reflux esophagitis through the STAT1/TREM-1 pathway, with STAT1 as the upstream protein., Conclusions: This study suggests that XDT may treat reflux esophagitis by modulating the STAT1/TREM-1 pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background/Aim: It was hypothesized that endometrial tight junction morphology and expression of tight junction proteins i.e., claudin-4 and occludin in the uterus, are affected by testosterone. Therefore, the effects of testosterone on these parameters in the uterus during receptivity period were investigated. Materials and Methods: Ovariectomized adult female rats were given testosterone (1 mg/kg/day) alone or in combination with flutamide or finasteride between days 6 to 8 of sex-steroid replacement treatment, which was considered the period of uterine receptivity. Ultramorphology of tight junctions was visualized by transmission electron microscopy while distribution and expression of claudin-4 and occludin were examined by immunofluorescence and real-time polymerase chain reaction respectively. Results: Administration of testosterone caused loss of tight junction complexity and down-regulated expression of claudin-4 and occludin in the uterus. Conclusion: Decreased endometrial tight junction complexity and expression of claudin-4 and occludin in the uterus during receptivity period by testosterone may interfere with embryo attachment and subsequent implantation. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the utility of claudin‐4 as a pan‐carcinoma marker in cell‐blocks of effusion specimens and compare results with Ber‐Ep4 staining. Methods: Effusion cell‐blocks (n = 284) were stained for claudin‐4 and results compared with Ber‐Ep4. Cases included 172 metastatic malignancies (137 adenocarcinomas, 20 small cell lung tumours, eight metastatic melanoma, four squamous cell carcinoma, three urothelial cell carcinoma), 49 benign reactive cases and 63 mesotheliomas. Results: All 49 benign effusions were negative. Only 1/63 (1.6%) mesotheliomas was positive for claudin‐4. Claudin‐4 staining was positive in 131/137 (95.6%) adenocarcinoma cases. Cases negative for claudin‐4 included single cases of metastases from breast, colon, stomach, prostate, kidney and ovary. Claudin‐4 outperformed Ber‐Ep4. Sensitivity (95.6% vs 85.4%), specificity (99.1% vs 86.6%), negative predictive value (94.9% vs 82.9%) and positive predictive value (99.2% vs 88.6%) were all higher for claudin‐4 compared with Ber‐Ep4, respectively. Only two cases were claudin‐4−/Ber‐Ep4+. Significantly (P < .0064) more cases of metastatic adenocarcinoma stained positive for claudin‐4 (131/137; 95.6%) than Ber‐Ep4 (117/137; 86.2%). Claudin‐4 staining was present in 15/20 (75%) of neuroendocrine carcinomas, 3/4 (75%) squamous cell carcinoma and 3/3 (100%) urothelial cell carcinoma. All eight cases of melanoma were negative for both claudin‐4 and Ber‐Ep4. Conclusions: Claudin‐4 staining is a useful addition to IHC panels for effusions specimens with superior performance to Ber‐Ep4. The utility of claudin‐4 as a pan‐carcinoma marker in cell blocks of effusion specimens was investigated and compared with Ber‐Ep4 staining results. Claudin‐4 was found to be superior to Ber‐Ep4, staining 95.6% (131/137) metastatic adenocarcinomas, 1.6% (1/63) mesotheliomas and no reactive cases (0/49). [ABSTRACT FROM AUTHOR]

Basaloid squamous cell carcinomas (BSCCs) in oral lesions are extremely rare, and the histology is not well understood. Histologically, they are often similar to conventional squamous cell carcinoma (SCC). The present study was designed with an aim to distinguish BSCC from SCC using claudin-4, occludin, SRY-box 2 (SOX2) and proliferating cell nuclear antigen (PCNA) immunoreactivities and staining patterns. Three BSCCs (with abundant, with moderate, and without squamous components) specimens and 20 SCC specimens were selected for comparison of their immunoreactivity. These specimens were stained with claudin-4, occludin, SOX2 and PCNA. In addition to histological analysis, the expression of claudin-4, occludin and PCNA was determined in oral cancer HSC2 and HSC3 cells with or without SOX2 overexpression, and cell proliferation was determined by XTT assay. Claudin-4 had strong and occludin had weak immunoreactivity as detected in the membrane of squamous components of BSCC but not in cancer cells. No obvious detection of squamous components and cancer cells were observed in SCC. SOX2 and PCNA immunoreactivities in SCC had dot-like staining patterns in the nuclei of partial and marginal cancer cells. In contrast, in BSCCs, SOX2 and PCNA had diffuse staining patterns in almost all cancer cells. SOX2 overexpression had little effect on the expression levels of claudin-4, occludin and PCNA. It also had little effect on the cell proliferation of HSC2 and HSC3 cells. Differences in immunoreactivity and staining pattern may be valuable to distinguish between BSCC and SCC in diagnosis. [ABSTRACT FROM AUTHOR]

The expression pattern of tight junction proteins (TJPs) varies among organs and tumor types. In this study, we examined the immunoreactivity of claudin (CLDN)-1, -4, and -7, and JAM-A in salivary gland tumors (SGTs) by histological types and cell types to estimate their usefulness as differential diagnostic markers. Immunoreactivity of CLDN1 was higher in ductal epithelium cells of SGTs than in non-tumor tissues. Conversely, immunoreactivity of CLDN1 was significantly decreased in basal/myoepithelium cells of SGTs compared with that in non-tumor tissues. There was no significant difference between the immunoreactivity of CLDN1 in benign tumors and that in malignant tumors. Immunoreactivity of CLDN4, CLDN7, and JAM-A in ductal epithelium cells was higher in many SGTs than in non-tumor tissues. There was a difference depending on the histological type of SGT in immunoreactivity of CLDN4, CLDN7, and JAM-A in basaloid/myoepithelial cells. It was possible to classify SGTs by a hierarchical clustering using immunoreactivity of TJPs. The results suggest that an immunohistochemical marker panel including these TJPs may be useful for differential diagnosis of SGTs and that CLDN1 is associated with tumorigenesis of SGTs. [ABSTRACT FROM AUTHOR]

Background: In Egypt, bladder cancer occupies the second rankamong reported cancers in men. Claudins are tight junctions that have a critical role in tumor pathogenesis, invasion, progression, and metastasis and currentlyare a focus of interest for targeting therapies., Objectives: We aimed to evaluatethe immunohistochemical expression of Claudin-1 and Claudin-4 in urinary bladder urothelial carcinoma and investigate the relationshipbetweenthe expressed Claudins with differentclinicopathological parameters., Methods: Claudin-1 and Claudin-4 immunohistochemical expression was studied in 62 cases of urinary bladder urothelial carcinomas. The cases were classified into two categories; low and high Claudin-1 and Claudin-4 expression., Results: High Claudin-1 expression was detected in67.7% of the studied urothelial carcinomas while 32.3% showed low expression. Claudin-1 expression was reduced significantly with high tumor grade, non-papillary tumors, muscle invasion, schistosomal infestation, and perineural invasion (p-value < 0.05). Claudin-4 high expression was detected in 82.3% of our cases while low expression was detected in 17.7%. Claudin-4 reduced expression was significantly associated with non-papillary tumors, muscle invasion, advanced T stages, and associated lympho-vascular emboli (P-value < 0.05)., Conclusion: According to the results ofthe present study, the reduced expressions of Claudin-1 and Claudin-4 provide clues concerning the progression of urothelial carcinoma. Consequently, it is thought that Claudin-1 and Claudin-4 could help to differentiatelow-grade from high-grade and muscle-invasive from non-muscle-invasive urothelial carcinomas. In addition, it can be introduced as a possible therapeutic target.

Background: Claudin-4, a tight junction associated protein expressed in epithelial cells, is purported as a highly specific and sensitive marker for epithelial malignancies. Our aim was to assess the sensitivity, specificity and real-time utility of claudin-4 immunocytochemistry (ICC) in the diagnostic work-up of suspected malignant effusions., Methods: Claudin-4 (3E2C1 clone) ICC was performed prospectively in effusion cell blocks where other ICC markers were being performed as part of reporting over 3 months. Based on claudin-4 staining in unequivocal malignant and reactive effusions, the sensitivity and specificity was calculated. In cases signed out as inconclusive encompassing atypia of undetermined significance (AUS) and suspicious for malignancy (SFM) and negative for malignancy, change in diagnostic category based on addition of claudin-4 ICC was assessed., Results: Study included 107 effusions. Claudin-4 stained 100% of metastatic adenocarcinomas including those with primaries in lung, breast, ovary, female genital tract, gastrointestinal tract and pancreatic-biliary tract, and was negative in all reactive mesothelial and mesothelioma effusions with sensitivity of 100% (48/48) and specificity of 95% (20/21) for adenocarcinoma. Claudin-4 upgraded the diagnostic category to positive in 70% (16/23) of SFM, 20% (1/5) of AUS, and in 50% (5/10) of negative effusions. Among cases with confirmed serosal involvement status on follow-up, claudin-4 showed sensitivity, specificity, positive predictive value and negative predictive values of 85% (11/13), 100% (3/3), 100% (10/10) and 75% (3/4), respectively, for metastatic adenocarcinoma., Conclusion: Claudin-4 as a single marker is sensitive and specific for adenocarcinoma and is a valuable addition to the ICC armamentarium., (© 2023 Wiley Periodicals LLC.)

Background: The dysregulation between pro-inflammatory and anti-inflammatory responses during sepsis is a crucial factor in driving sepsis progression. Acute lung injury (ALI) resulting from excessive production and accumulation of inflammatory mediators in the lungs contributes to impaired lung barrier function. The activation of the NF-κB signaling pathway during inflammation leads to the transcriptional activation of multiple inflammatory genes. Given the plausible impact of NF-κB signaling suppression in mitigating lung injury, substantive evidence demonstrates beta-sitosterol (BS)'s proficient ability to block NF-κB activation. Therefore, the aim of the present investigation was to delve into the impacts of BS in the context of sepsis-induced acute lung injury, employing both a mouse model and a model involving lung epithelial cells., Methods: Sepsis-induced lung injury was simulated in mice through cecum ligation and puncture (CLP). To emulate injury in murine lung epithelial (MLE-12) cells, an experiment involving lipopolysaccharide (LPS) was administered. Evaluation of alterations in lung tissue permeability encompassed techniques such as lung wet/dry (W/D) mass ratio, Evans blue staining, and quantification of total protein concentration in bronchoalveolar lavage fluid (BALF). Lung tissue histopathological shifts were ascertained via hematoxylin and eosin (HE) staining. Additionally, the concentrations of inflammatory cytokines IL-6 and TNF-α were quantified in every lung tissue and cell group by implementing enzyme-linked immunosorbent assay (ELISA). Protein quantification for signal biomarkers was carried out using Western blotting and immunofluorescence methodologies. In tandem, the assessment of MLE-12 cell permeability was conducted by evaluating fluorescein isothiocyanate (FITC)-dextran extravasation., Results: BS mitigated lung tissue pathologies, reduced inflammatory factors, and lowered tissue and cell permeability. BS inhibited NF-κB signaling and increased claudin-4 and claudin-5 expression, enhancing septic lung epithelial cell permeability., Conclusions: Through suppressing the NF-κB signaling cascade, BS effectively curtails the levels of inflammatory mediators. Simultaneously, it orchestrates the modulation of claudin-4 and claudin-5 expression, culminating in the augmentation of lung epithelial cell barrier competence, thus improving sepsis-induced lung injury.

Mesenchymal stem cell-derived exosomes and long non-coding RNAs (lncRNAs) have been identified to play a role in acute lung injury (ALI). In this study, we investigated whether exosomal lncRNAs could regulate ALI and the underlying mechanisms. Bone marrow mesenchymal stem cells (BM-MSCs) were pretreated with hypoxia or normoxia, and exosomes were subsequently extracted from normoxic BM-MSCs (Nor-exos) and hypoxic BM-MSCs (Hypo-exos). A rat model of ALI was established via an airway perfusion of lipopolysaccharide (LPS). Exosomes were administered via the tail vein to evaluate the in vivo effect of exosomes in ALI. LPS-exposed RLE-6TN cells were incubated with exosomes to explore their in vitro effect in ALI. A luciferase reporter assay was used to evaluate the interaction between lncRNA XIST and miR-455-3p, as well as miR-455-3p and Claudin-4. We found that the exosomes attenuated LPS-induced ALI and Hypo-Exos exerted a greater therapeutic effect compared with Nor-exos both in vitro and in vivo. Moreover, an abundance of lncRNA XIST was observed in Hypo-exos compared with Nor-exos. Mechanistically, LncRNA XIST functioned as a miR-455-3p sponge and targeted Claudin-4 in ALI. Our results provide novel insight into the role of exosomal lncRNA XIST for the treatment of ALI. Thus, hypoxic pretreatment may represent an effective method for improving the therapeutic effects of exosomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2023 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

Background: M cells and intestinal tight junction proteins (TJPs) are important components of intestinal barrier which is compromised by high fat diet (HFD). Objective: To determine the effect of HFD on M cells and TJPs of intestinal villi of rats. Materials and Methods: Four weeks old, twenty male Wistar rats were divided into control (n = 10) and HFD (n = 10) groups. After 6 weeks, rats were sacrificed, ileum segments and feces were collected. Immunohistochemical staining and fecal calprotectin (FCP) level were measured. Data were presented as mean ± SD. Ap value of < 0.05 was considered as significant. Results: As compared to the control, M cell expression in the villi was significantly higher in HFD group (1.625 ± 0.518 vs 2.3 ± 0.458, p = 0.045). Claudin-3 was highly expressed in the villi of both groups. As compared to the control, claudin-4 expression in the villi was significantly lower in HFD group (1.975 ± 0.670 vs 1.55 ± 0.597, p = 0.015). As compared to the control, occludin expression in the villi was reduced but not significantly in HFD group (1.5 ± 0.592 vs 1.45 ± 0.497, p = 0.589). As compared to the control, FCP level was found significantly higher in HFD group (40.172 ± 4.0852 vs 49.058 ± 1.174, p = 0.016). Conclusion: HFD consumption for 6 weeks caused higher expression of M cell, no significant changes of claudin-3 expression, reduced claudin-4 and occludin expression in the villi of male Wistar rats which were associated with intestinal inflammation. [ABSTRACT FROM AUTHOR]

Aims: Dedifferentiated endometrial carcinomas (DDECs)/undifferentiated endometrial carcinomas (UECs) are aggressive endometrial cancers with frequent genomic inactivation of core components of switch/sucrose non‐fermentable (SWI/SNF) complex proteins. Claudin‐4, an epithelial intercellular tight junction protein, was recently found to be expressed in SWI/SNF‐deficient undifferentiated carcinomas but not in SWI/SNF‐deficient sarcomas. The aim of this study was to examine claudin‐4 expression in UECs/DDECs and other high‐grade uterine carcinomas. Methods and results: We examined claudin‐4 expression by immunohistochemistry (clone 3E2C1) on tissue microarrays that contained 44 UECs/DDECs (24 SWI/SNF‐deficient), 50 carcinosarcomas, 164 grade 3 endometrioid carcinomas, 57 serous carcinomas, and 20 clear cell carcinomas. Tumours with <5% claudin‐4 expression were considered to be negative. Nearly all SWI/SNF‐deficient, and most SWI/SNF‐proficient, UECs/DDECs showed a complete absence of claudin‐4 expression in the undifferentiated component, whereas the differentiated component in DDECs showed consistent and diffuse claudin‐4 expression. Only one SWI/SNF‐deficient DDEC showed focal expression of claudin‐4 in the undifferentiated component, as compared with diffuse expression in the corresponding differentiated component. Claudin‐4 expression was consistently absent in the sarcomatous component of carcinosarcoma, and it was absent in 24% of grade 3 endometrioid carcinomas and serous carcinomas. Conclusion: Claudin‐4 expression can be absent or very focal in a subset of high‐grade endometrial carcinomas, and is almost always absent in the undifferentiated components of SWI/SNF‐deficient UECs/DDECs, despite the apparent epithelial origin in the case of DDECs. Therefore, claudin‐4 expression cannot be used to infer mesenchymal or epithelial tumour origin in the endometrium. The consistent loss or down‐regulation of claudin‐4, a tight junction protein, in SWI/SNF‐deficient UECs/DDECs further supports the undifferentiated nature of these tumours. [ABSTRACT FROM AUTHOR]

A qualitative study is presented, where the main question was whether food-derived hemorphins, i.e., originating from digested alimentary hemoglobin, could pass the intestinal barrier and/or the blood--brain barrier (BBB). Once absorbed, hemorphins are opioid receptor (OR) ligands that may interact with peripheral and central OR and have effects on food intake and energy balance regulation. LLVV-YPWT (LLVV-H4), LVV-H4, VV-H4, VV-YPWTQRF (VV-H7), and VV-H7 hemorphins that were previously identified in the 120 min digest resulting from the simulated gastrointestinal digestion of hemoglobin have been synthesized to be tested in in vitro models of passage of IB and BBB. LC-MS/ MS analyses yielded that all hemorphins, except the LLVV-H4 sequence, were able to cross intact the human intestinal epithelium model with Caco-2 cells within 5-60 min when applied at 5 mM. Moreover, all hemorphins crossed intact the human BBB model with brain-like endothelial cells (BLEC) within 30 min when applied at 100 μM. Fragments of these hemorphins were also detected, especially the YPWT common tetrapeptide that retains OR-binding capacity. A cAMP assay performed in Caco-2 cells indicates that tested hemorphins behave as OR agonists in these cells by reducing cAMP production. We further provide preliminary results regarding the effects of hemorphins on tight junction proteins, specifically here the claudin-4 that is involved in paracellular permeability. All hemorphins at 100 μM, except the LLVV-H4 peptide, significantly decreased claudin-4 mRNA levels in the Caco-2 intestinal model. This in vitro study is a first step toward demonstrating food-derived hemorphins bioavailability which is in line with the growing body of evidence supporting physiological functions for food-derived peptides. [ABSTRACT FROM AUTHOR]

Purpose: Despite its widespread use, the positron emission tomography (PET) radiotracer 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) has been shown in clinical settings to be ineffective for improving early diagnosis of pancreatic ductal adenocarcinoma (PDAC). A promising biomarker for PDAC detection is the tight junction protein claudin-4. The purpose of this study was to evaluate a new single-photon emission computed tomography (SPECT) imaging agent, [111In]anti-claudin-4 mAb, with regard to its ability to allow visualisation of claudin-4 in a xenograft and a genetically engineered mouse model of PDAC.Procedures: The ability of [111In]anti-claudin-4 mAb to selectively target claudin-4 was assessed using two human xenograft tumour models with differential claudin-4 status in mice. [111In]anti-claudin-4 mAb was also used to detect PDAC development in genetically engineered KPC mice. The PDAC status of these mice was confirmed with [18F]FDG-PET, magnetic resonance imaging (MRI), histology, and immunofluorescence microscopy.Results: High uptake of [111In]anti-claudin-4 mAb was observed in PDAC xenografts in mice, reaching 16.9 ± 4.5 % of injected dose per gram (% ID/g) at 72 h post-injection. This uptake was mediated specifically by the expression of claudin-4. Uptake of [111In]anti-claudin-4 mAb also enabled clear visualisation of spontaneous PDAC formation in KPC mice.Conclusions: [111In]anti-claudin-4 mAb allows non-invasive detection of claudin-4 upregulation during development of PDAC and could potentially be used to aid in the early detection and characterisation of this malignancy. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome (PRRS) is an important swine disease caused by infection of porcine reproductive and respiratory syndrome virus (PRRSV), which leads to huge loss in swine industry. How to effectively control PRRS is challenging. Long non-coding RNA (lncRNA) are key regulator of viral infections and anti-virus immunological responses, therefore, further understanding of lncRNAs will aid to identification of novel regulators of viral infections and better design of prevention and control strategies to viral infection related diseases and immune disorders. We demonstrated that PRRSV infection upregulated the expression of lncRNA LOC103222771 in Marc-145 cells and porcine alveolar macrophage cells (PAMs) and that LOC103222771 is mainly located in cytoplasm. Knockdown of LOC103222771 could inhibit the PRRSV infection in Marc-145 cells. RNA-seq analysis and subsequent validation revealed increased expression of Claudin-4 (CLDN4) in Marc-145 when LOC103222771 was specifically downregulated，suggesting that LOC103222771 might be an upstream regulator of CLDN4, an important component of tight junctions for establishment of the paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells. We and others showed that Downregulation of CLDN4 could boost the infection of PRRSV. Collectively, LOC103222771/CLDN4 signal axis might be a novel mechanism of PRRSV pathogenesis, implying a potential therapeutic target against PRRSV infection. • The role of LOC103222771 and CLDN4 in PRRSV infection in Marc-145 cells was studied. • PRRSV infection upregulates the LOC103222771 expression in Marc-145 cells and PAMs. • LOC103222771 RNA is mainly localized in cytoplasm in Marc-145 cells. • LOC103222771 RNA was found to downregulate CLDN4 to facilitate viral entry. • The mechanism by which LOC103222771 inhibits CLDN4 expression is discussed. [ABSTRACT FROM AUTHOR]

We previously reported that quercetin increases tight junction (TJ) barrier integrity in intestinal Caco-2 cells. This study was aimed at determining the molecular mechanism underlying the quercetin-mediated effect with a particular focus on the transcriptional regulation of claudin-4. Quercetin aglycone, but not its glycosides, enhanced TJ barrier integrity of Caco-2 cells, indicated by transepithelial electrical resistance. Immunoblot analysis, qPCR, and luciferase promoter assay revealed that quercetin transcriptionally increased the claudin-4 expression. Narrowing the claudin-4 promoter sequences from 1866 bp to 200 bp did not affect quercetin-mediated promoter activation. Site-directed mutation of SP1, AP-1 and GATA binding sites within the 200 bp sequence partially suppressed the effects of quercetin on claudin-4 promoter activity. Taken together, quercetin enhances the TJ barrier integrity in intestinal Caco-2 cells, with the transcription factors, SP1, AP-1, and GATA, involved in the quercetin-mediated transcriptional regulation of claudin-4.