Long-term passages of human clonal mesenchymal stromal cells can alleviate the disease in the rat model of collagen-induced arthritis resembling early passages of different heterogeneous cells.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease of unknown cause. The interaction of immune system cells and the secretion of inflammatory cytokines with synovial cells leads to severe inflammation in the affected joints. Currently, medications, including non-steroidal anti-inflammatory drugs, glucocorticoids, and more recently, disease-modifying anti-rheumatic drugs, are used to reduce inflammation. However, long-term use of these drugs causes adverse effects or resistance in a considerable number of RA patients. Recent findings revealed the safety and efficacy of mesenchymal stromal cells (MSCs)-based therapies both in RA animal models and clinical trials. Here, the beneficial effects of bone marrow-derived heterogeneous MSCs (BM-hMSCs) and Wharton jelly-derived MSCs (WJ-MSCs) at early passages were compared to BM-derived clonal MSCs (BM-cMSCs) at high passage number on a rat model of collagen-induced arthritis. Results showed that systemic delivery of MSCs significantly reversed adverse changes in body weight, paw swelling, and arthritis score in all MSC-treated groups. Radiological images and histological evaluation demonstrated the therapeutic effects of MSCs. There was a decrease in serum level of anti-collagen type II immunoglobulin G and the inflammatory cytokines interleukin (IL)-1ß, IL-6, IL-17, and tumor necrosis factor-α in all MSC-treated groups. In contrast, an increase in inhibitory cytokines transforming growth factor-ß and IL-10 was seen. Notably, the long-term passages of BM-cMSCs could alleviate RA symptoms similar to the early passages of WJ-MSCs and BM-hMSCs. The importance of BM-cMSCs is the potential to establish cell banks with billions of cells derived from a single donor that could be a competitive cell-based therapy to treat RA.

Long-Term Inhibition of Notch in A-375 Melanoma Cells Enhances Tumor Growth Through the Enhancement of AXIN1, CSNK2A3, and CEBPA2 as Intermediate Genes in Wnt and Notch Pathways.

Notch suppression by gamma-secretase inhibitors is a valid approach against melanoma. However, most of studies have evaluated the short-term effect of DAPT on tumor cells or even cancer stem cells. In the present study, we surveyed the short-term and long-term effects of DAPT on the stem cell properties of A375 and NA8 as melanoma cell lines. The effects of DAPT were tested both in vitro and in vivo using xenograft models. In A375 with B-raf mutation, DAPT decreased the level of NOTCH1, NOTH2, and HES1 as downstream genes of the Notch pathway. This was accompanied by enhanced apoptosis after 24 h treatment, arrest in the G2-M phase, and impaired ability of colony and melanosphere formation at the short term. Moreover, tumor growth also reduced during 13 days of treatment. However, long-term treatment of DAPT promoted tumor growth in the xenograft model and enhanced the number and size of colonies and spheroids in vitro. The gene expression studies confirmed the up-regulation of Wnt and Notch downstream genes as well as AXIN1, CSNK2A3, and CEBPA2 following the removal of Notch inhibitor in vitro and in the xenograft model. Moreover, the Gompertz-based mathematical model determined a new drug resistance term in the present study. Our data supported that the long-term and not short-term inhibition of Notch by DAPT may enhance tumor growth and motility through up-regulation of AXIN1, CSNK2A3, and CEBPA2 genes in B-raf mutated A375 cells.

Dietary Vitamin E Is More Effective than Omega-3 and Omega-6 Fatty Acid for Improving The Kinematic Characteristics of Rat Sperm.

OBJECTIVE: Although key roles for dietary vitamin E (VITE) and fatty acid (FA) in fertility have been confirmed, limited data are available on the effects of VITE alone, or a constant level of VITE supplemented by dietary omega-6 and omega-3 FAs in combination on male reproduction. Consequently in this paper, the effects of VITE, sunflower oil, fish oil and their combination on rat sperm were investigated. MATERIALS AND METHODS: We divided 50 mature male Wistar rats into 5 groups (n=10) in a experimental completely randomized design for eight weeks: i. Control (CTR): standard diet; ii. Vitamin E diet (VITE): 2 times greater than recommendations; iii. Sunflower oil group (n-6) [gavaged with 0.5 ml/day/rat sunflower oil+VITE diet]; iv. Fish oil group (n-3): [gavaged with 0.5 ml/day/rat fish oil+VITE diet] and v. n-3+n-6 group [gavaged with 0.3 ml fish oil/day/rat+0.2 ml sunflower oil/day/rat+VITE diet]. The sperm parameters were measured by computer assisted semen analyzer (CASA). All data were analyzed with SPSS software. RESULTS: Feed intake decreased in groups which were administered sunflower oil compared with the other groups (P<0.05). The groups which received only VITE or fish oil+VITE had a significantly higher concentration of sperm compared with the n-6+n-3 and CTR group (P<0.05). VITE and n-3 showed significant improved progressive motility compared to the CTR group, whereas the n-6 and n-6+n-3 groups were in the middle (P<0.05). The highest sperm kinematic parameters were observed in the VITE only group. There was no strong correlation between sperm parameters and blood lipid profiles. CONCLUSION: Dietary VITE and fish oil+VITE can improve sperm quality. Our findings can be a focus for improvements in sperm quantity and motility in fertile animals using only dietary VITE.

Expression analysis of RNA-binding motif gene on Y chromosome (RBMY) protein isoforms in testis tissue and a testicular germ cell cancer-derived cell line (NT2).

BACKGROUND: RNA-binding motif gene on Y chromosome (RBMY), a germ cell-specific nuclear protein, is known as a key factor in spermatogenesis and disorders associated with this protein have been recognized to be related to male infertility. Although it was suggested that this protein could have different functions during germ cell development, no studies have been conducted to uncover the mechanism of this potential function yet. Here, we analyzed the expression pattern of RBMY protein isoforms in testis compared to NT2, a testicular germ cell cancer-derived cell line, to test probability of differential expression of RBMY protein isoforms at different spermatogenesis stages. METHODS: Full length and a segment of RBMY gene were cloned and expressed in E. coli. Anti-human RBMY antibody was produced in rabbit using the recombinant proteins as antigen. Western-blot and immunofluorescence were conducted for detection and comparison of RBMY protein isoforms. RESULTS: Selected segment of RBMY protein resulted in producing a mono-specific antibody. As results shows, only the longest isoform of RBMY was expressed at protein level in NT2 cell line, while three isoforms of this protein were detected in the whole testis lysate. CONCLUSION: The results imply that different alternative splicing may happen in testis cells and probably difference of RBMY function during spermatogenesis is due to the differential expression of RBMY protein isoforms. These results and further experiments on RBMY isoforms can help to obtain a better understanding of the function of this protein, which may increase our knowledge about spermatogenesis and causes of male infertility.

Relationship of leptin administration with production of reactive oxygen species, sperm DNA fragmentation, sperm parameters and hormone profile in the adult rat.

PURPOSE: Leptin, an adipose tissue-derived hormone, plays an important role in energy homeostasis and metabolism, and in the neuroendocrine and reproductive systems. The function of leptin in male reproduction is unclear; however, it is known to affect sex hormones, sperm motility and its parameters. Leptin induces mitochondrial superoxide production in aortic endothelia and may increase oxidative stress and abnormal sperm production in leptin-treated rats. This study aims to evaluate whether exogenous leptin affects sperm parameters, hormone profiles, and the production of reactive oxygen species (ROS) in adult rats. METHODS: A total of 65 Sprague-Dawley rats were divided into three treated groups and a control group. Treated rats received daily intraperitoneal injections of 5, 10 and 30 µg/kg of leptin administered for a duration of 7, 15, and 42 days. Control rats were given 0.1 mL of 0.9 % normal saline for the same period. One day after final drug administration, we evaluated serum specimens for follicle-stimulating hormone (FSH), leutinizing hormone (LH), free testosterone (FT), and total testosterone (TT) levels. Samples from the rat epididymis were also evaluated for sperm parameters and motility characteristics by a Computer-Aided Semen Analysis (CASA) system. Samples were treated with 2',7'-dichlorofluorescein-diacetate (DCFH-DA) and analyzed using flow cytometry and TUNEL to determine the impact of leptin administration on sperm DNA fragmentation. RESULTS: According to CASA, significant differences in all sperm parameters in leptin-treated rats and their age-matched controls were detected, except for TM, ALH and BCF. Serum FSH and LH levels were significantly higher in rats that received 10 and 30 µg/kg of leptin compared to those treated with 5 µg/kg of leptin in the same group and control rats (P < 0.05). ROS and sperm DNA fragmentation was significantly higher in rats injected with 10 and 30 µg/kg of leptin for 7 and 15 days compared with rats treated with 5 µg/kg of leptin and the control group (P < 0.05) for the same time period. However, at day 42 of treatment, ROS and sperm DNA fragmentation levels significantly decreased in all groups (P < 0.05). CONCLUSION: According to these results, leptin can possibly affect male infertility by ROS induction or hormone profile modulation.