JAK/STAT signaling pathway affects CCR5 expression in human CD4+ T cells.

CCR5 serves as R5-tropic HIV co-receptor. Knocking out CCR5 in HIV patients, which has occurred <10 times, is believed important for cure. JAK/STAT inhibitors tofacitinib and ruxolitinib inhibit CCR5 expression in HIV+ viremic patients. We investigated the association of JAK/STAT signaling pathway with CCR5/CCR2 expression in human primary CD4+ T cells and confirmed its importance. Six of nine JAK/STAT inhibitors that reduced CCR5/CCR2 expression were identified. Inhibitor-treated CD4+ T cells were relatively resistant, specifically to R5-tropic HIV infection. Furthermore, single JAK2, STAT3, STAT5A, and STAT5B knockout and different combinations of JAK/STAT knockout significantly reduced CCR2/CCR5 expression of both RNA and protein levels, indicating that CCR5/CCR2 expression was positively regulated by JAK-STAT pathway in CD4+ T cells. Serum and glucocorticoid-regulated kinase 1 (SGK1) knockout affected CCR2/CCR5 gene expression, suggesting that SGK1 is involved in CCR2/CCR5 regulation. If cell surface CCR5 levels can be specifically and markedly down-regulated without adverse effects, that may have a major impact on the HIV cure agenda.

Co-Culture of Cryopreserved Healthy Sertoli Cells with Testicular Tissue of Non-Obstructive Azoospermia (NOA) Patients in Culture Media Containing Follicle-Stimulating Hormone (FSH)/Testosterone Has No Advantage in Germ Cell Maturation.

Different cell culture conditions and techniques have been used to mature spermatogenic cells to increase the success of in vitro fertilization. Sertoli cells (SCs) are essential in maintaining spermatogenesis and FSH stimulation exerts its effect through direct or indirect actions on SCs. The effectiveness of FSH and testosterone added to the co-culture has been demonstrated in other studies to provide microenvironment conditions of the testicular niche and to contribute to the maturation and meiotic progression of spermatogonial stem cells (SSCs). In the present study, we investigated whether co-culture of healthy SCs with the patient's testicular tissue in the medium supplemented with FSH/testosterone provides an advantage in the differentiation and maturation of germ cells in NOA cases (N = 34). In men with obstructive azoospermia (N = 12), healthy SCs from testicular biopsies were identified and purified, then cryopreserved. The characterization of healthy SCs was done by flow cytometry (FC) and immunohistochemistry using antibodies specific for GATA4 and vimentin. FITC-conjugated annexin V/PI staining and the MTT assay were performed to compare the viability and proliferation of SCs before and after freezing. In annexin V staining, no difference was found in percentages of live and apoptotic SCs, and MTT showed that cryopreservation did not inhibit SC proliferation compared to the pre-freezing state. Then, tissue samples from NOA patients were processed in two separate environments containing FSH/testosterone and FSH/testosterone plus co-culture with thawed healthy SCs for 7 days. FC was used to measure 7th-day levels of specific markers expressed in spermatogonia (VASA), meiotic cells (CREM), and post-meiotic cells (protamine-2 and acrosin). VASA and acrosin basal levels were found to be lower in infertile patients compared to the OA group (8.2% vs. 30.6% and 12.8% vs. 30.5%, respectively; p < 0.05). Compared to pre-treatment measurements, on the 7th day in the FSH/testosterone environment, CREM levels increased by 58.8% and acrosin levels increased by 195.5% (p < 0.05). Similarly, in medium co-culture with healthy SCs, by day 7, CREM and acrosin levels increased to 92.2% and 204.8%, respectively (p < 0.05). Although VASA and protamine levels increased in both groups, they did not reach a significant level. No significant difference was found between the day 7 increase rates of CREM, VASA, acrosin and protamine-2 in either FSH/testosterone-containing medium or in medium additionally co-cultured with healthy SCs (58.8% vs. 92.2%, 120.6% vs. 79.4%, 195.5% vs. 204.8%, and 232.3% vs. 198.4%, respectively; p > 0.05). Our results suggest that the presence of the patient's own SCs for maturation of germ cells in the culture medium supplemented with FSH and testosterone is sufficient, and co-culture with healthy SCs does not have an additional advantage. In addition, the freezing-thawing process would not impair the viability and proliferation of SCs.

IL-33 induces ADAMTS5 expression and cell migration in glioblastoma multiforme.

Glioblastoma multiforme (GBM), characterized by a high rate of proliferation and migration capacity, is an incurable brain tumor in adults. Interleukin-33 (IL-33), a member of the IL-1 cytokine superfamily, and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), a family of zinc dependent metalloproteinases, are known to have essential roles in GBM migration and invasion. Previous studies have separately revealed elevated expressions of IL-33 and ADAMTS5 in GBM; however, the interaction between IL-33 and ADAMTS5 in GBM remains unclear. Here, using publically available GlioVis and GEPIA programs, we showed that mRNA expressions of IL-33 and ADAMTS5 are significantly high in GBM cells, and a positive correlation between IL-33 and ADAMTS5 was also determined in these cells. In parallel with the mRNA data of IL-33 and ADAMTS5, by Western blot analysis, protein levels were found to be elevated in GBM tissues and increased gradually with the disease progression. Primary GBM cells and low-grade glioma cells were then treated with IL-33 to examine its stimulating effect on ADAMTS5 expression. Exposure to IL-33 raised ADAMTS5 protein levels in a dose-dependent manner. Finally, the wound-healing method was performed to confirm the impact of IL-33 on migration in primary GBM cells. IL-33 promoted migration of primary GBM cells three times higher than untreated GBM cells. Thus, the current study suggests for the first time that IL-33 might have a role in playing a part in GBM progression through induction of ADAMTS5 expression and promotion of migration in GBM cells.

Induction of apoptosis in human hormone-refractory prostate cancer cell lines by using resveratrol in combination with AT-101.

The aim of this study was to determine the appropriate doses of AT-101 and resveratrol combination in the in vitro hormone-refractory prostate cancer (PC) cell lines, in order to evaluate the cytotoxic and genotoxic effects of this combination on the proliferation of cancer cells, namely PC-3, DU-145 and LNCAP. Cytotoxicity in PC cell lines was analysed by using the XTT Cell Proliferation Assay. DNA damage was performed with the cell death assay. Apoptotic protein levels were performed by Roche Human Apoptosis Array. IC50 values were determined by XTT analysis. The strongest combined doses (100 µM resveratrol + 5µM AT-101) were found to have the strongest synergistic apoptotic and cytotoxic effects on DU-145 cells at 72 hr. While the combined use of resveratrol and AT-101 increased the expression of markers in apoptotic cell pathways on cells, a decrease in the expression of anti-apoptotic markers was detected (p Ë‚ 0.05). Combined applications of these compounds showed an important synergism in the hormone-refractory PC cell lines, and it was determined that after the post-translational modification, they were significantly effective on the apoptotic pathway. These results have revealed that the combination of resveratrol and AT-101 holds great expectation as a new chemotherapeutic application in the treatment of human prostate cancer.

Relationship between functional Nrf2 gene promoter polymorphism and sperm DNA damage in male infertility.

This study examines the association of the -617 C > A polymorphism in the Nrf2 gene (rs6721961) with male infertility in a Turkish population and determines its functional role in spermatogenesis in correlation with the impact of different levels of DNA damage on the genotypes. A total of 100 infertile men and 100 healthy fertile men were included in the study. Nrf2 genotyping was performed with the PCR-based restriction fragment length gene polymorphism (RFLP-PCR) analysis. According to our results, the Nrf2 CC, CA, and AA genotype distribution frequencies were 58.6%, 38.4%, and 3% in the control group, respectively, and 38%, 48%, and 14% in the infertile men, respectively. The AA genotype was significantly higher in the patient group. In smokers, a significant difference was found in progressive motility values between the genotypes (p = 0.001). Also, sperm progressive motility and concentration decreased significantly in those smokers with the AA genotype; smokers carrying this genotype were also 5.75 times more likely to have oligoasthenozoospermia than those with CC (p < 0.05). There was a significant relationship between the number of cases with high sperm-DNA damage when comparing the frequency of Nrf2 AA genotype carriers with the CC genotype 16.3% vs. 6.9%, respectively (p < 0.001). These results suggest the importance of the Nrf2 gene C > A (rs 6,721,961) polymorphism in the etiology of sperm DNA damage as a risk factor for male infertility. Smokers carrying the AA genotype are more likely to impair seminal parameters through antioxidant mechanisms.Abbreviations: Polymerase chain reaction (PCR)-based restriction fragment length gene polymorphism (RFLP-PCR); reactive oxygen species (ROS); deoxyribonucleic acid (DNA); catalases (CATs); superoxide dismutase (SOD); glutathione peroxidase (GPX); glutathione-S-transferase (GST); Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2); basic leucine zipper (bZIP); antioxidant response element (ARE); World Health Organization (WHO);normospermia(NS);asthenozoospermia(AS);oligozoospermia(OS);oligoasthenozoospermia (OAS); follicle stimulating hormone (FSH); ultraviolet (UV); low-melting-point agarose (LMA); normal-melting-point agarose (NMA); arbitrary units (AU); total comet score (TCS); A one-way analysis of variance (ANOVA); standard deviation (SD); N-acetyltransferase (NAT2); small non-coding RNAs (ncRNAs); microRNAs (miRNA).

Investigation of CD133 and CD24 as candidate azoospermia markers and their relationship with spermatogenesis defects.

Spermatogenesis is initiated when spermatogonial stem cells (SSCs) in the mature testes enter mitosis and trigger differentiation. Thus, spermatogenesis and the ability to maintain a continuous source of spermatogonia relies on the ability to differentiate SSCs. Many studies around the world have been performed to investigate the etiology of male infertility and recent studies have focused on the presence and identification of biomarkers. CD133 and CD24 are stem cell markers locating in the testis and spermatogonia. The aim of this study was to investigate the relationship of the CD133 and CD24 genes with spermatogenesis defects and examine them as a candidate a useful biomarker for azoospermia men. The association of CD133 and CD24 with spermatogenesis defects was investigated in patients with obstructive (O) and non-obstructive azoospermia (NOA). NOA cases were histopathologically classified into Hypospermatogenesis (HS), Maturation Arrest (MA), and Sertoli Cell Only Syndrome (SCO) groups. A qRT-PCR analysis of these genes was performed and protein expression levels were measured by Western blot analysis. CD133 expression in NOA group was found to be significantly different from OA and this was confirmed by immunohistochemistry and immunocytochemical assays. The qRT-PCR analysis revealed that gene expression of CD133 and CD24 had fold changes of 0.80 ±â€¯0.34 and 1.59 ±â€¯0.31 compared to controls, respectively in the HS group (p > 0.05) and 0.04 ±â€¯0.01 and 0.54 ±â€¯0.08 in the MA group (p < 0.05). In the SCO group, CD24 showed a 1.55 ±â€¯0.35-fold increase (p > 0.05). CD133 gene expression was not detected at the transcriptional level in the SCO group. Western blot analysis of CD133 protein expression revealed 1.83, 4.11, and 11.4-fold decreases in the HS, MA and SCO groups, respectively, compared to controls (p < 0.05). CD24 showed fold changes of 1.18, 0.38, (p < 0.05), and 0.89 in the HS, MA, and SCO groups, respectively. Immunohistochemical analysis of CD133 revealed moderate, partial staining in the HS group, compared to substantial, wide-spread staining in the OA group. No staining was detected in either the MA or SCO groups. The localization of CD133 in healthy sperm was determined to be prominent in the tail and partly expressed in the head by confocal laser scanning microscopy analysis. It was also found that the expression of CD133 protein was high in healthy commercially-sourced Sertoli cells as well as in the Sertoli cells of OA individuals. Data from this study show that CD133 exhibits different profiles in infertile patient groups and thus may be considered as a candidate biomarker. CD24 can be associated with blockage of germ cell maturation in the MA group. Curative protocols for spermatogenesis defects may be possible with the use of these markers and thus their identification is extremely valuable in terms of human reproductive health.

ADAMTS1 and ADAMTS5 metalloproteases produced by Sertoli cells: a potential diagnostic marker in azoospermia.

In this study, our aim was to detect protein levels of A Disintegrin and Metalloproteinase with Thrombospondin Motifs 1 and 5 (ADAMTS1 and ADAMTS5) proteases and to examine the effect of in vitro FSH supplementation on protease production in cultured Sertoli cells. The expression of metalloproteases, ADAMTS1, and ADAMTS5 were investigated in Sertoli cell cultures as well as in ejaculate of azoospermic men which then were compared with ejaculates of the fertile control group. A total of 15 azoospermic men, diagnosed as obstructive (OA, n = 5) and nonobstructive (NOA, n = 10) azoospermia were included in the study. ADAMTS1, ADAMTS5 and FSH receptors (FSHR) were found to be expressed 2.56, 2.10, and 2.66-fold less in Sertoli cells of NOA patients, than those of OA (p < 0.05). After rFSH was added onto Sertoli cell cultures of NOA patients, their expression did not increase significantly and did not reach to levels of control group. Evaluation of ejaculates revealed that the expression of ADAMTS1 and ADAMTS5 were insignificantly 1.03 and 1.1-fold higher in OA group (p > 0.05), respectively; however, in the NOA group, their expression were 1.70 and 1.96-fold lower, respectively, when compared with the fertile control group (p < 0.05) which was statistically significant. As a conclusion, the present study has revealed that insufficiency of ADAMTS1 and ADAMTS5 expression in Sertoli cells may have an important role in the etiology of male infertility. As expected due to low FSHR expression, rFSH response is impaired in NOA patients with relatively low ADAMTS expression response; therefore, such patients might hardly benefit from rFSH treatment. Further studies with larger cohorts may reveal ADAMTSs' potential use as a predictive marker for positive sperm retrieval in azoospermic patients who are scheduled to undergo testicular sperm extraction. Abbreviations: ADAM: A Disintegrin and Metalloproteinase; ADAMTS1 and ADAMTS5: A Disintegrin and Metalloproteinase with 10 Thrombospondin Motifs 1 and 5; ADAMTS: A Disintegrin and Metalloproteinase with Thrombospondin; ABP: androgen binding protein; CAMs: cell adhesion molecules; ECM: extracellular matrix; FSH: follicle stimulating hormone; FSHR: FSH receptors; HRP: horseradish peroxidase; MMP: matrix metalloproteinases; MP: metalloproteinases; NOA: nonobstructive azoospermia; OA: obstructive azoospermia; TIMP-1: tissue inhibitor of metalloproteinase-1.

In-vitro Antioxidant, Cytotoxic, Cholinesterase Inhibitory Activities and Anti-Genotoxic Effects of Hypericum retusum Aucher Flowers, Fruits and Seeds Methanol Extracts in Human Mononuclear Leukocytes.

The present study investigates the antioxidant, anticancer, anticholinesterase, anti-genotoxic activities and phenolic contents of flower, fruit and seed methanol extracts of Hypericum retusum AUCHER. The amounts of protocatechuic acid, catechin, caffeic acid and syringic acid in methanol extracts were determined by HPLC. Total phenolic content of H. retusum seed extract was found more than fruit and flower extracts. The DPPH free radical scavenging activity of flower and seed methanol extracts showed close activity versus BHT as control. Among three extracts of H. retusum only flower methanol extract was exhibited considerable cytotoxic activities against to HeLa and NRK-52E cell lines. Moreover, seed methanol extract showed both acetyl and butyrl-cholinesterase inhibitory activity. The highest anti-genotoxic effects were seen 25 and 50 µg/mL concentrations. In this study, the extracts showed a strong antioxidant and anti-genotoxic effect. The seed extract was more efficient- than extracts of fruit and flowers. Our results suggest that the antioxidant and anti-genotoxic effects of extracts depend on their phenolic contents. Further studies should evaluate the in-vitro and in-vivo the benefits of H. retusum seed methanol extracts.

Effect of insulin on the mRNA expression of procollagen N-proteinases in chondrosarcoma OUMS-27 cells.

Chondrosarcoma is one of the most common bone tumors, and at present, there is no non-invasive treatment option for this cancer. The chondrosarcoma OUMS-27 cell line produces proteoglycan and type II, IX, and XI collagens, which constitutes cartilage tissue. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) proteases are a group of secreted proteases, which include the procollagen N-proteinases ADAMTS-2, -3 and -14. These procollagen N-proteinases perform a role in the processing of procollagens to collagen and the maturation of type I collagen. The present study aimed to improve the understanding of the causes of metastasis, local invasion and resistance to chemo- and radiotherapy in chondrosarcoma, as well as the effect of insulin on cancer cells. The present study was designed to reveal the effects of insulin on procollagen N-proteinases in chondrosarcoma OUMS-27 cells. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) alone or in DMEM containing 10 µg/ml insulin. The medium was changed every other day for 11 days. The cells were harvested on days 1, 3, 7 and 11, and total RNA isolation was performed immediately following harvesting. The expression levels of ADAMTS2, ADAMTS3 and ADAMTS14 mRNA were estimated by reverse transcription-quantitative polymerase chain reaction using appropriate primers. ADAMTS2 mRNA expression was found to be decreased on day 7 (P=0.028) and increased at day 11 compared with the control group (P=0.016). The increase in mRNA concentration at day 11 was significantly different compared to the concentrations on days 3 (P=0.047) and 7 (P=0.008). The expression of ADAMTS3 mRNA decreased immediately subsequent to insulin induction on day 1 compared with the control group (P=0.008). The most evident decrease in mRNA concentration was seen at day 7 subsequent to insulin induction (P=0.008). The present results demonstrated that ADAMTS2 and ADAMTS3 may perform a role in the invasion and metastasis of tumors, and may also possess proteolytic activity that results in the breakdown of the extracellular matrix (ECM). Insulin itself can modulate the biosynthesis of ECM macromolecules that are altered in diabetes through various pathways.

Analysis of the correlation between sperm DNA integrity and conventional semen parameters in infertile men.

OBJECTIVE: A male factor is responsible in approximately 30-40% of couples receiving infertility treatment. Routinely, such couples undergo semen analysis including parameters such as sperm count, motility and morphology. Generally, the analysis of sperm DNA damage, shown to have a significant clinical importance by many studies, is recognized as an advanced test that is not included in routine infertility tests. Intracytoplasmic sperm injection method, commonly employed in the current infertility treatment protocols, lowers the fertilization rate, however, fertilization can occur even with a damaged DNA which is known to pose a risk in the subsequent pregnancy period. The relation between sperm morphology and the degree of sperm DNA damage has not yet been understood clearly. In this study, we aimed to investigate the association between routine semen analysis and sperm DNA integrity assay, another advanced but costly method. MATERIAL AND METHODS: The degree of DNA damage was compared with the results of semen analysis, based on the WHO criteria, in 399 male patients who received comet assay for sperm DNA integrity. The statistical correlation analyses were performed with Windows SPPS statistical package program. RESULTS: Accordingly, the sperm DNA damage was found to be correlated with all 3 parameters (sperm count, forward motility, and morphology) examined by the semen analysis (p<0.001). Total sperm DNA Damage Count was 226, 216, and 210 arbitrary units in patients with a sperm count <15 mil/mL, forward moving motility <32%, and normal morphology <4%, respectively. The difference with the normal individuals was statistically significant (p<0.001). CONCLUSION: In light of the comet assay results, higher degree of sperm DNA damage is associated with significant impairment of all seminal parameters.

Increased DNA damage and oxidative stress among silver jewelry workers.

Silver has long been valued as a precious metal, and it is used to make ornaments, jewelry, high-value tableware, utensils, and currency coins. Human exposures to silver and silver compounds can occur oral, dermal, or by inhalation. In this study, we investigated genotoxic and oxidative effects of silver exposure among silver jewelry workers. DNA damage in peripheral mononuclear leukocytes was measured by using the comet assay. Serum total antioxidative status (TAS), total oxidative status (TOS), total thiol contents, and ceruloplasmin levels were measured by using colorimetric methods among silver jewelry workers. Moreover, oxidative stress index (OSI) was calculated. Results were compared with non-exposed healthy subjects. The mean values of mononuclear leukocyte DNA damage were significantly higher than control subjects (p < 0.001). Serum TOS, OSI, and ceruloplasmin levels were also found to be higher in silver particles exposed group than those of non-exposed group (p < 0.001, p < 0.001, p < 0.01, respectively). However, serum TAS levels and total thiol contents of silver exposed group were found significantly lower (p < 0.05, p < 0.001, respectively). Exposure to silver particles among silver jewelry workers caused oxidative stress and accumulation of severe DNA damage.