New approaches concerning the testis of Astyanax lacustris (Characidae): immunohistochemical studies.

Astyanax lacustris, locally known as lambari-do-rabo-amarelo, is a study model for Neotropical fish. Testis of A. lacustris shows deep morphophysiological changes throughout the annual reproductive cycle. This work analyzed the distribution of claudin-1, actin, and cytokeratin as elements of the cytoskeleton in germinal epithelium and interstitium; the distribution of type I collagen, fibronectin, and laminin as extracellular matrix compounds; and the localization of androgen receptor in the testis of this species. Claudin-1, cytokeratin, and actin were present in the Sertoli cells and modified Sertoli cells, and actin was also detected in peritubular myoid cells. Type I collagen were in the interstitial tissue, laminin in the basement membrane of germinal epithelium and endothelium, but fibronectin was additionally detected in the germinal epithelium compartment. The labeling of androgen receptor was higher in peritubular myoid cells and undifferentiated spermatogonia, and weaker labeling was detected in type B spermatogonia. Therefore, the present work highlights new aspects of the biology of the testis of A. lacustris, and contribute to amplify the understanding of this organ.

The abundance and localization of claudin-1 and -5 in the adult tomcats (Felis catus) testis, tubules rectus, rete testis, efferent ductules, and epididymis.

Tight junctions (TJ) are the anatomical component of blood-testis (BTB) and blood-epididymis (BEB) barriers and contain many proteins, including claudins. The presence of claudins in domestic cat testis and epididymis has not been previously described. This study aimed to determine whether claudin-1 and claudin-5 participate in the structure of BTB and BEB and whether their amounts differ between the testis and epididymal segments of adult cats, using Western blotting (WB) and immunohistochemistry. WB results demonstrated that claudin-1 was significantly lower in the testis than in all epididymal segments and higher in the corpus epididymis than in the cauda, while claudin-5 in the testis was significantly lower than in the caput and corpus. Claudin-1 was absent at the Sertoli-Sertoli junctions, while claudin-5 was detected at the level of the BTB during stages I and VIII. Both claudins were observed in the pachytene spermatocytes and the developing acrosome of the round and elongating spermatids. Claudin-5 was also detected in the cytoplasm of some spermatogonia, Sertoli cells, and late spermatid acrosome. In the epididymal segments, both claudins were localized to the area of the tight junctions and along the entire length of the lateral plasma membranes of adjacent principal cells and between principal and basal cells. These results may indicate that in the domestic cat, claudin-1 and -5 participate as both tight junction proteins and adhesion molecules in the BEB's structure, claudin 5 is a component of the BTB, and both proteins may be involved in postmeiotic germ cell development, especially acrosome development.

Tissue Content and Pattern of Expression of Claudin-3 and Occludin in Normal and Neoplastic Tissues in Patients with Colorectal Cancer.

BACKGROUND: Metastasis is the worst prognostic variable of patients with colorectal cancer (CRC). For the development of metastases, it is necessary that cancer cells detach from the primary tumor, migrate into the angiolymphatic system, and invade the tissue where they will develop. The breakdown of the tight junctions (TJs) plays an important role in colorectal metastatic tumors. Claudin-3 and occludin are the main component proteins of TJs. AIM: To analyze the expression and tissue content of claudin-3 and occludin in normal and neoplastic tissues of patients with metastatic CRC. METHODS: Fifty-seven consecutive patients with stage III and IV CRC were included. Fragments of neoplastic tissue were collected from the tumor margins, and samples of the normal tissue were collected from the same patient in a standardized distance of 10 cm from the cranial margin of the tumor. Immunohistochemistry technique was used to identify the tissue staining of claudin-3 and occludin. To measure the content of both proteins in cellular membranes of normal and cancer cells, a validated immunoscore was used. RESULTS: Claudin-3 and occludin in normal tissues are in the apical and lateral membranes of cells, while in the neoplastic, in cytoplasm. The mean of the tissue content of claudin-3 in the normal tissue was 2.57 ± 0.16, while in the neoplastic tissue was 1.03 ± 0.13. The contents of occludin were 2.77 ± 0.1 in normal tissue, while in the neoplastic were 1.08 ± 0.14. CONCLUSION: There is a reduction in the content of the claudin-3 and occludin in the cell membranes of the neoplastic tissue in patients with CRC.

Claudin-1 decrease impacts epidermal barrier function in atopic dermatitis lesions dose-dependently.

The transmembrane protein claudin-1 is a major component of epidermal tight junctions (TJs), which create a dynamic paracellular barrier in the epidermis. Claudin-1 downregulation has been linked to atopic dermatitis (AD) pathogenesis but variable levels of claudin-1 have also been observed in healthy skin. To elucidate the impact of different levels of claudin-1 in healthy and diseased skin we determined claudin-1 levels in AD patients and controls and correlated them to TJ and skin barrier function. We observed a strikingly broad range of claudin-1 levels with stable TJ and overall skin barrier function in healthy and non-lesional skin. However, a significant decrease in TJ barrier function was detected in lesional AD skin where claudin-1 levels were further reduced. Investigations on reconstructed human epidermis expressing different levels of claudin-1 revealed that claudin-1 levels correlated with inside-out and outside-in barrier function, with a higher coherence for smaller molecular tracers. Claudin-1 decrease induced keratinocyte-autonomous IL-1ß expression and fostered inflammatory epidermal responses to non-pathogenic Staphylococci. In conclusion, claudin-1 decrease beyond a threshold level results in TJ and epidermal barrier function impairment and induces inflammation in human epidermis. Increasing claudin-1 levels might improve barrier function and decrease inflammation and therefore be a target for AD treatment.

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

BACKGROUND: 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. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Immunoreactivity patterns of tight junction proteins are useful for differential diagnosis of human salivary gland tumors.

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.

Vitamin A and Retinoic Acid Exhibit Protective Effects on Necrotizing Enterocolitis by Regulating Intestinal Flora and Enhancing the Intestinal Epithelial Barrier.

BACKGROUND: Exaggerated inflammation that characterizes necrotizing enterocolitis (NEC) is caused by the invasion of pathogens through an immature intestinal barrier. Vitamin A (VA) and retinoic acid (RA) play important roles in the growth of epithelial tissue and in modulating immune function. OBJECTIVE: To investigate the roles of VA and RA in the development of NEC. METHODS: Levels of serum retinol in patients and in a NEC mouse model were detected with high-performance liquid chromatography. Bacterial communities of NEC mice treated with VA or PBS were detected by high-throughput sequencing. In vitro and in vivo, levels of inflammatory factors were measured by ELISA and RT-PCR, and expression levels of claudin-1, occludin, and ZO-1 were detected by Western blotting. Transepithelial electrical resistance (TEER) was measured in Caco-2 cell monolayers. RESULTS: The level of VA in the NEC patients was lower than in the control patients. In the NEC mice that were treated with VA versus PBS, the proportion of Escherichia-Shigella was lower, while the abundance of Bacteroides was markedly higher. Both in vivo and in vitro, the levels of inflammatory factors were significantly reduced, while the expression levels of claudin-1, occludin, and ZO-1 were increased, after the VA and RA treatments. Meanwhile, TEER was increased and lipopolysaccharide-induced damage was reduced in Caco-2 cell monolayers after RA treatment. CONCLUSIONS: These results suggest that VA may regulate intestinal flora, alleviate inflammatory reactions, and enhance the intestinal epithelial barrier in NEC. Thus, VA may be an effective drug for providing protection against NEC in newborns.

Perineurial-like Cells and EMA Expression in the Suprachoroidal Region of the Human Eye.

The suprachoroidal region of the eye comprises vascular channels, melanocytes, and thin fibroblasts with elongated cytoplasm that are positioned directly adjacent to the densely collagenous sclera. Morphological similarities between these suprachoroidal fibroblasts and arachnoid cells and perineurial cells have been recognized, but whether these fibroblasts have a perineurial cell-like immunophenotype is not known. To further examine the relationship of these three cell types, we investigated the comparative expression of epithelial membrane antigen (EMA), the tight junction protein claudin-1, glucose transporter-1 (Glut-1), and CD34 in suprachoroidal fibroblasts, arachnoid of the optic nerve sheath, and perineurium of ciliary nerves in eight human eye specimens. Granular, diffuse, and cytoplasmic EMA expression was seen in suprachoroidal fibroblasts, but this was not contiguous with the similar pattern of EMA expression in adjacent perineurium and arachnoid. CD34 expression in suprachoroidal fibroblasts was also seen, similar to arachnoid and perineurium. Claudin-1 and Glut-1 were not consistently expressed in suprachoroidal fibroblasts, distinguishing them from perineurial cells in particular and suggesting that these fibroblasts do not arise directly from adjacent arachnoid or perineurium. Nonetheless, the overlapping morphology and protein expression suggest phenotypic similarities in these cells that protect and support adjacent retina, optic nerve, and peripheral nerve.

Rhubarb Supplementation Promotes Intestinal Mucosal Innate Immune Homeostasis through Modulating Intestinal Epithelial Microbiota in Goat Kids.

The abuse and misuse of antibiotics in livestock production pose a potential health risk globally. Rhubarb can serve as a potential alternative to antibiotics, and several studies have looked into its anticancer, antitumor, and anti-inflammatory properties. The aim of this study was to test the effects of rhubarb supplementation to the diet of young ruminants on innate immune function and epithelial microbiota in the small intestine. Goat kids were fed with a control diet supplemented with or without rhubarb (1.25% DM) and were slaughtered at days 50 and 60 of age. Results showed that the supplementation of rhubarb increased ileal villus height (P = 0.036), increased jejujal and ileal anti-inflammatory IL-10 production (P < 0.05), increased jejunal and ileal Claudin-1 expression at both mRNA and protein levels (P < 0.05), and decreased ileal pro-inflammatory IL-1ß production (P < 0.05). These changes in innate immune function were accompanied by shifts in ileal epithelial bacterial ecosystem in favor of Blautia, Clostridium, Lactobacillus, and Pseudomonas, and with a decline in the relative abundance of Staphylococcus (P < 0.001) when rhubarb was supplemented. Additionally, age also affected (P < 0.05) crypt depth, cytokine production, Claudin-1 expression and relative abundances of specific genera in epithelial bacteria. Collectively, the supplementation of rhubarb could enhance host mucosal innate immune homeostasis by modulating intestinal epithelial microbiota during the early stages of animal development.

Comparison of suction blistering and tape stripping for analysis of epidermal genes, proteins and lipids.

Analysis of epidermal genes, proteins and lipids is important in the research and diagnosis of skin diseases. Although punch biopsy is the first-choice technique for the skin sampling, it is unnecessarily invasive for obtaining a sample just for the epidermal analysis. Here we compare two less invasive methods, suction blistering (SB) and tape stripping (TS), for the analysis of selected epidermal genes (quantitative real-time reverse transcription PCR, qRT-PCR), proteins (western blotting, WB), and lipids in ten healthy volunteers. TS provided significantly less material than SB and no viable epidermal layers could be obtained according to the reflectance confocal microscopy. Consistently, only the SC protein filaggrin and housekeeping GAPDH together with FLG and RPL13A mRNA were detected by TS. In the SB samples, WB and qRT-PCR could easily detect all the selected proteins (claudin-1, occludin, filaggrin, laminin and GAPDH) and genes (CLDN1, OCLN, FLG, LAMA3 and RPL13A), respectively. A single SB sample further provided enough of material for immunohistochemistry and lipid analyses, which was not feasible with the TS samples. Immunohistochemistry of the SB samples showed intact epidermal structure and a characteristic expression of claudin-1. Infrared spectroscopy showed well-ordered lipids with both orthorhombic and hexagonal packing and high-performance thin layer chromatography confirmed all lipid classes (including ceramide subclasses) in correct proportions. Taken together, SB represents a reliable sampling technique that can be utilized for multipurpose epidermal analyses in various studies.