Syndecans, Exostosins and Sulfotransferases as Potential Synovial Inflammation Moderators in Patients with Hip Osteoarthritis.

The gradual deterioration of articular cartilage was thought to be the central event in osteoarthritis (OA), but recent studies demonstrated the importance of low-grade synovitis in the progression of OA. The Syndecan (SDC) family of membrane proteoglycans is known to be involved in the regulation of inflammation, but there is limited evidence considering the role of syndecans in OA synovitis. Our study aimed to investigate the hip OA synovial membrane expression patterns of SDC1, SDC2 and SDC4, as well as exostosins and sulfotransferases (enzymes involved in the polymerisation and modification of syndecans' heparan sulphate chains). Synovial membrane samples of patients with OA (24) were divided into two groups according to their Krenn synovitis score severity. The immunohistochemical expressions of SDC1, SDC2, SDC4, EXT1, EXT2, NDST1 and NDST2 in synovial intima and subintima were then analysed and compared with the control group (patients with femoral neck fracture). According to our study, the immunoexpression of SDC1, NDST1 and EXT2 is significantly increased in the intimal cells of OA synovial membrane in patients with lower histological synovitis scores and SDC4 in patients with higher synovitis scores, in comparison with non-OA controls. The difference in the expression of SDC2 among the OA and non-OA groups was insignificant. SDC1, SDC4, NDST1 and EXT2 seem to be involved as inflammation moderators in low-grade OA synovitis and, therefore, should be further investigated as potential markers of disease progression and therapeutic goals.

Unraveling the Impact of Dab1 Gene Silencing on the Expression of Autophagy Markers in Lung Development.

The purpose of this study was to evaluate the effects of Dab1 gene silencing on the immunoexpression of light chain 3 beta (Lc3b), glucose regulating protein 78 (Grp78), heat shock cognate 71 (Hsc70), mammalian target of rapamycin (mTOR) and lysosomal-associated membrane protein 2A (Lamp2a) in the lung tissue of developing yotari (Dab1-/-) and wild-type (wt) mice. The lung epithelium and mesenchyme of the embryos at gestational days E13.5 and E15.5 were examined using immunofluorescence and semi-quantitative methods. In the pulmonary mesenchyme and epithelium, Grp78 and Lc3b of moderate fluorescence reactivity was demonstrated in wt mice for both evaluated time points, while yotari mice exhibited only epithelial reactivity for the same markers. Mild punctate expression of Hsc70 was observed for both genotypes. A significant difference was present when analyzing mTOR expression, where wt mice showed strong perinuclear staining in the epithelium. According to our data, Dab1 gene silencing may result in autophagy abnormalities, which could then cause respiratory system pathologies via defective lung cell degradation by lysosome-dependent cell elimination.

Differential expression of HIF1A and its downstream target VEGFA in the main subtypes of renal cell carcinoma and their impact on patient survival.

Renal cell carcinoma (RCC) represents around 3% of all cancers, with the most frequent histological types being clear-cell RCC (ccRCC), followed by papillary (pRCC) and chromophobe (chRCC). Hypoxia-inducible factors (HIFs), which promote the expression of various target genes, including vascular endothelial growth factor (VEGF) and the high- affinity glucose transporter 1, have an important role in the pathogenesis of RCC. This study investigated the immunohistochemical expression of HIF-1α and VEGF-A, showing significantly higher HIF-1α nuclear expression in pRCC compared to ccRCC, while there was no significant difference in VEGF-A protein expression between the analyzed histological RCC subtypes. The quantitative reverse transcription polymerase chain reaction for HIF1A showed no statistical difference between histological types. Data from publicly available RNA sequencing databases were analyzed and showed that, compared to healthy kidney tissue, VEGFA was significantly up-regulated in ccRCC and significantly down-regulated in pRCC. The comparison between histological subtypes of RCC revealed that VEGFA was significantly up-regulated in ccRCC compared to both pRCC and chRCC. There was no statistically significant difference in survival time between HIF1A high- and low-expression groups of patients. As for VEGFA expression, pRCC patients with low expression had a significantly higher survival rate compared to patients with high VEGFA expression.

Connexin 37, 40, 43 and Pannexin 1 Expression in the Gastric Mucosa of Patients with Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. Although its pathogenesis is not fully understood, connexins (Cxs) and pannexins (Panx) could be involved in the process of fibrosis. We analyzed the protein expression of Cx37, Cx40, Cx43, and Panx1 in the gastric mucosa of patients with SSc and healthy volunteers, using immunofluorescence staining. Protein levels of Cx37 were slightly increased, while the levels of Cx40 were significantly decreased in the lamina propria of the gastric mucosa of SSc patients compared to the controls. The changes were proportional to SSc severity, with the most prominent changes found in patients with severe diffuse cutaneous SSc. No differences in Cx43 or Panx1 levels were found between the analyzed groups of samples. The lack of changes in Cx43 expression, which has been previously associated with fibrosis, could be due to the weak expression of Cx43 in the gastric mucosa in general. Further studies on full-thickness gastric biopsies containing muscle layers and animal SSc models are needed to fully elucidate the role of Cxs and Panxs in SSc-associated fibrosis.

The Effect of GLUT1 and HIF-1α Expressions on Glucose Uptake and Patient Survival in Non-Small-Cell Lung Carcinoma.

Lung cancer is the second-most-common cancer while being the leading cause of cancer deaths worldwide. It has been found that glucose transporter 1 (GLUT1) and hypoxia-inducible factor 1α (HIF-1α) are overexpressed in various malignancies and that they correlate with the maximum standard uptake values (SUVmax) on 18F-fluorodeoxyglucose-positron emission tomography/computed tomography (18F-FDG PET/CT) and poor prognosis. In this study, we aim to evaluate the relationship between the SUVmax, GLUT1, and HIF-1α expression with primary tumor size, histological type, lymph node metastases, and patient survival. Of the 48 patients with non-small-cell lung cancer, those with squamous cell carcinomas (SCCs) had significantly higher GLUT1 and HIF-1α immunohistochemical expressions in comparison to adenocarcinomas (ACs), while there was no statistically significant difference in FDG accumulation between them. No significant correlation was noted between either GLUT1 or HIF-1α protein expression and FDG uptake and overall survival. However, an analysis of tumor transcriptomics showed a significant difference in overall survival depending on mRNA expression; patients with SCC and high HIF-1α levels survived longer compared to those with low HIF-1α levels, while patients with AC and low GLUT1 levels had a higher average survival time than those with high GLUT1 levels. Further studies are needed to determine the prognostic value of the expression of these factors depending on the histologic type.

Disturbances in Switching between Canonical and Non-Canonical Wnt Signaling Characterize Developing and Postnatal Kidneys of Dab1-/- (yotari) Mice.

This study aims to determine the protein expression patterns of acetylated α-tubulin, inversin, dishevelled-1, Wnt5a/b, and ß-catenin in developing (E13.5 and E15.5) and early postnatal (P4 and P14) kidneys of Dab1-/- (yotari) mice, their role in regulating the Wnt signaling pathway, and the possible relation to congenital anomalies of kidney and urinary tract (CAKUT). The analysis of target protein co-expression, observed in the renal vesicles/immature glomeruli, ampullae/collecting ducts, convoluted tubules, metanephric mesenchyme of developing kidneys, but proximal convoluted tubules, distal convoluted tubules and glomeruli of postnatal kidneys, was performed using double immunofluorescence and semi-quantitative methods. The overall expression of acetylated α-tubulin and inversin during normal kidney development increases with higher expression in yotari mice as the kidney acquires mature morphology. An increase in ß-catenin and cytosolic DVL-1 levels, indicating a switch from non-canonical to canonical Wnt signaling, is found in the postnatal kidney of yotari mice. In contrast, healthy mouse kidney expresses inversin and Wnt5a/b in the postnatal period, thus activating non-canonical Wnt signaling. Target protein expression patterns in kidney development and the early postnatal period observed in this study could indicate that switching between canonical and non-canonical Wnt signaling is crucial for normal nephrogenesis, while the defective Dab1 gene product in yotari mice may promote CAKUT due to interfering with this process.

The Distribution of Innervation and Immune Cell Infiltration Is Different in Genital and Extragenital Variants of Lichen Sclerosus.

Lichen sclerosus (LS) is a progressive skin disease that is characterized by chronic inflammation of either genital or extragenital skin, and it disproportionately affects women. We analyzed the distribution of nerve fibers, vanilloid receptors, cell proliferation, mast cells and macrophages in genital and extragenital LS samples, as well as in healthy skin, by using immunohistochemistry. The total amount of intraepidermal nerve fibers was lower in LS samples compared to healthy controls, while the total amount of subepidermal nerve fibers and calcitonin gene-related peptide (CGRP) positive fibers was higher in genital LS samples compared to both extragenital LS and healthy controls. Cell proliferation, macrophage and mast cell density were increased in LS samples compared to healthy controls. Genital LS had a higher macrophage density compared to the extragenital variant. Mast cell distribution significantly differed between genital and extragenital LS samples, even though their total mast cell densities were similar. These findings could explain the differences between pruritic symptoms of genital and extragenital LS and provide targets for the research of novel therapeutic strategies for LS management.

Fibrosis-Associated Signaling Molecules Are Differentially Expressed in Palmar Connective Tissues of Patients with Carpal Tunnel Syndrome and Dupuytren's Disease.

Carpal tunnel syndrome (CTS) and Dupuytren's disease (DD) are fibrotic conditions that affect the connective tissue of the hand and limit its functionality. The exact molecular mechanism underlying the fibrosis is unknown, and only some profibrotic factors have been investigated. In this cross-sectional study, we analyzed the expression of FGF signaling pathway molecules associated with fibrotic changes in the palmar fascia and the flexor retinaculum of 15 CTS patients and both clinically affected and unaffected palmar fascia of 15 DD patients, using immunofluorescence techniques. The expression of FGFR1, FGFR2, and CTGF in the blood vessel walls and surrounding connective tissue cells differed significantly between the analyzed groups, with changes in expression present even in clinically unremarkable tissues from DD patients. We also found altered expression of the analyzed factors, as well as TGF-ß1 and syndecan-1 in DD-associated sweat glands, possibly implicating their role in the pathophysiology of the disease. The increased expression of profibrotic factors in the clinically unaffected palmar fascia of DD patients may indicate that more extensive excision is needed during surgical treatment, while the profibrotic factors could be potential targets for developing pharmacological therapeutic strategies against DD-associated fibrosis.

Aberrations in FGFR1, FGFR2, and RIP5 Expression in Human Congenital Anomalies of the Kidney and Urinary Tract (CAKUT).

This study aimed to explore the spatio-temporal expression patterns of congenital anomalies of kidney and urinary tract (CAKUT) candidate genes, Fibroblast Growth Factor Receptor 1 (FGFR1), Fibroblast Growth Factor Receptor 2 (FGFR2) and Receptor-Interacting Protein Kinase 5 (RIP5), in human fetal kidney development (CTRL) and kidneys affected with CAKUT. Human fetal kidneys from the 22nd to 41st developmental week (duplex, hypoplastic, dysplastic, and controls) were stained with antibodies and analyzed by epifluorescence microscopy and RT-qPCR. The effect of CAKUT candidate genes on kidney nephrogenesis and function is confirmed by statistically significant variations in the spatio-temporal expression patterns of the investigated markers. The nuclear localization of FGFR1, elevated expression score of FGFR1 mRNA, the increased area percentage of FGFR1-positive cells in the kidney cortex, and the overall decrease in the expression after the peak at the 27th developmental week in dysplastic kidneys (DYS), suggest an altered expression pattern and protein function in response to CAKUT pathophysiology. The RT-qPCR analysis revealed a significantly higher FGFR2 mRNA expression score in the CAKUT kidneys compared to the CTRL. This increase could be due to the repair mechanism involving the downstream mediator, Extracellular Signal-Regulated Kinase 1/2 (ERK1/2). The expression of RIP5 during normal human kidney development was reduced temporarily, due to urine production and increased later since it undertakes additional functions in the maturation of the postnatal kidney and homeostasis, while the expression dynamics in CAKUT-affected kidneys exhibited a decrease in the percentage of RIP5-positive cells during the investigated developmental period. Our findings highlight the importance of FGFR1, FGFR2, and RIP5 as markers in normal and pathological kidney development.

Differences in Immunohistochemical and Ultrastructural Features between Podocytes and Parietal Epithelial Cells (PECs) Are Observed in Developing, Healthy Postnatal, and Pathologically Changed Human Kidneys.

During human kidney development, cells of the proximal nephron gradually differentiate into podocytes and parietal epithelial cells (PECs). Podocytes are terminally differentiated cells that play a key role in both normal and pathological kidney function. Therefore, the potential of podocytes to regenerate or be replaced by other cell populations (PECs) is of great interest for the possible treatment of kidney diseases. In the present study, we analyzed the proliferation and differentiation capabilities of podocytes and PECs, changes in the expression pattern of nestin, and several early proteins including WNT4, Notch2, and Snail, as well as Ki-67, in tissues of developing, postnatal, and pathologically changed human kidneys by using immunohistochemistry and electron microscopy. Developing PECs showed a higher proliferation rate than podocytes, whereas nestin expression characterized only podocytes and pathologically changed kidneys. In the developing kidneys, WNT4 and Notch2 expression increased moderately in podocytes and strongly in PECs, whereas Snail increased only in PECs in the later fetal period. During human kidney development, WNT4, Notch2, and Snail are involved in early nephrogenesis control. In kidneys affected by congenital nephrotic syndrome of the Finnish type (CNF) and focal segmental glomerulosclerosis (FSGS), WNT4 decreased in both cell populations, whereas Notch2 decreased in FSGS. In contrast, Snail increased both in CNF and FSGS, whereas Notch2 increased only in CNF. Electron microscopy revealed cytoplasmic processes spanning the urinary space between the podocytes and PECs in developing and healthy postnatal kidneys, whereas the CNF and FSGS kidneys were characterized by numerous cellular bridges containing cells with strong expression of nestin and all analyzed proteins. Our results indicate that the mechanisms of gene control in nephrogenesis are reactivated under pathological conditions. These mechanisms could have a role in restoring glomerular integrity by potentially inducing the regeneration of podocytes from PECs.

Determining the immunohistochemical expression of GLUT1 in renal cell carcinoma using the HSCORE method.

The aim of the present study was to compare the immunohistochemical expression of glucose transporter 1 (GLUT1) between the most common histological types of renal cell carcinoma (RCC), and to determine whether a correlation between GLUT1 expression and nuclear grade or tumor size exists. A total of 19 RCC samples were selected for the study, consisting of 8 clear cell (cc)RCC and 11 non-ccRCC tissues. Immunohistochemistry for GLUT1 was performed on formalin-fixed and paraffin-embedded sections using GLUT1 antibodies. All data analyses were performed using the MedCalc software. There was a higher immunohistochemical expression of GLUT1 in the ccRCC group compared with the non-cc group, but there was no difference in GLUT1 expression between groups of RCCs with differing nuclear grades. No significant correlation between GLUT1 expression and tumor size was found. The higher immunohistochemical expression of GLUT1 in ccRCC may be a contributing factor to the clinical characteristics and behavior of that group of carcinomas. These results suggest that GLUT1 expression cannot be used as a prognostic factor for RCC, but it may be used as a predictive factor in the future.

Expression Pattern of 5-HT (Serotonin) Receptors during Normal Development of the Human Spinal Cord and Ganglia and in Fetus with Cervical Spina Bifida.

The expression of 5-HT (serotonin) receptors (sr) was analyzed in the spinal cord and ganglia of 15 human conceptuses (5-10-weeks), and in the 9-week fetus with spina bifida. We used immunohistochemical method to detect sr-positive, apoptotic (caspase-3) and proliferating (Ki-67) cells, double immunofluorescence for co-localization with protein gene peptide (pgp) 9.5 and GFAP, as well as semiquantification and statistical measurements. Following the neurulation process, moderate (sr1 and sr2) and mild (sr3) expression characterized neuroblasts in the spinal cord and ganglia. During further development, sr1 expression gradually increased in the motoneurons, autonomic and sensory neurons, while sr2 and sr3 increased strongly in floor and roof plates. In the ganglia, sr3 expression increased during limited developmental period, while sr1 and sr2 increased throughout the investigated period. Co-expression of sr/pgp 9.5 characterized developing neurons, while sr/GFAP co-localized in the roof plate. In the spinal cord and ganglia of malformed fetus, weaker sr1 and sr2 and stronger sr3 expression accompanied morphological abnormalities. Anomalous roof plate morphology showed an excess of apoptotic and proliferating cells and increased sr3 expression. Our results indicate a human-species specific sr expression pattern, and the importance of sr1 in neuronal differentiation, and sr2 and sr3 in the control of the roof plate morphogenesis in normal and disturbed development.