A new isolation and culture method for granulosa cells.

Granulosa cells are the cell population who have an increasing importance in the female genital system and reproduction. Thus, nowadays in vitro studies to address these cells are also gaining importance and attracts researcher's attention. The aim of our study is to develop a more feasible, low-cost granulosa cell isolation and culture method compared to methods defined so far. Granulosa cells were isolated from follicular fluids obtained from both healthy women donors (n = 19) and polycystic ovary syndrome (n = 15) applied to in vitro fertilization treatment process. Granulosa cells were isolated by using Lymphosep® separation fluid that was not used for this purpose before. The isolated cells were cultured in suitable culture dishes with a mixture of BIO-AMFTM-1 and DMEM/F12 in the first seeding and only complete DMEM/F12 in the following feeds. Complete medium contains only 5% fetal calf serum, 4% L-glutamine and 1% penicillin-streptomycin-amphotericin. The new methods we have developed in granulosa cell isolation and in vitro culture have been successful. Reduction in supplement types and amount; improved the proliferation rate of the granulosa cells in culture. Our new methods of isolation and cell culture for granulosa cells from healthy women, have been also successful in samples of polycystic ovarian patients. With these developed methods, granulosa cells, which belong to humans and have an important role in the ovary, could be isolated and subsequently be maintained to reproduce (proliferate) more easily and cheaper.

Convex Hemiepiphysiodesis: Posterior/anterior in-situ Versus Posterior-only With Pedicle Screw Instrumentation: An Experimental Simulation in Immature Pigs.

STUDY DESIGN: Experimental study. BACKGROUND: Convex growth arrest (CGA) has been commonly used in the treatment of long-sweeping congenital deformities of the immature spine. As there are major drawbacks about the anterior procedure in the conventional CGA method, a new modification has been documented that using only posterior spinal approach with pedicle screw instrumentation. The aim of the study was to compare posterior-only CGA using pedicle screws with combined anterior/posterior in-situ CGA for the findings in histologic, radiologic, and manual palpation examinations in an immature pig model. METHODS: Twelve 10-weeks old pigs were grouped into 2. In group 1, posterior-only, pedicle screw instrumented CGA was performed on the left side of L1-L4 vertebrae. In group 2, conventional combined posterior and anterior CGA was performed to the left side of L1-L4 vertebrae without instrumentation. All animals were killed twelve weeks after surgery. T11-L5 segments were en-bloc resected and radiologic, histologic, and manual palpation examinations were done. RESULTS: Marked scoliotic (12.2±2.5 and 9.2±1.3 in group 1 and 2, respectively) and kyphotic (11.2±1.0 degrees for the group 1 and 12±5.2 degrees for the group 2, respectively) deformities were noted in both groups, which were caused by hemiepiphysiodesis effect. Anterior and posterior parts of group 2 and posterior part of group 1 demonstrated fusion in histologic and radiologic analyzes. In anterior part of the group 1, marked narrowing on the disk spaces and thinning of growth plates were noted in radiologicg examination, chondrocyte degeneration, and newly-formed bone trabeculae in disk-space were noted in histological examination. In manual palpation, no motion was detected in group 1 and motion was detected in only one segment of one animal in group 2. CONCLUSIONS: Anterior growth of the vertebrae can be controlled by application of posterior transpedicular screws and rod. Such an effect can eliminate the need for anterior surgical intervention in convex hemiepiphysiodesis procedures. CLINICAL RELEVANCE: The instrumented CGA technique provides a satisfactory epiphysiodesis effect both anteriorly and posteriorly, as previously demonstrated by clinical studies.

Investigation of FF-MAS oxysterole's role in follicular development and its relation to hedgehog signal pathway.

The Hedgehog signaling pathway plays a crucial role in folliculogenesis; however, the association between FF-MAS oxysterol activity in folliculogenesis and the Hedgehog signaling pathway has not been revealed. The evaluation of FF-MAS activity in polycystic ovary syndrome (PCOS) with folliculogenesis disorder might provide a new approach to tackle follicular and oocyte maturation failure. The question is: does FF-MAS oxysterol affect granulosa cell (GC) proliferation? If so, is this effect facilitated through the Hedgehog pathway? To answer these questions, GCs were isolated from follicle fluids obtained from patients undergoing oocyte retrieval during in vitro fertilization (IVF) treatment. After the isolated GCs were incubated in different cell culture media, the levels of Hedgehog pathway components (SMO, Gli1) were measured by using immunohistochemical methods, cytoELISA, and qRT-PCR. Meanwhile, cell proliferation rates were determined. Significant increases (p < 0.001) in SMO and Gli1 expressions and cell proliferation were observed in the FF-MAS-treated subgroups of both PCOS and male factor participants compared to the FF-MAS deficient subgroup. Remarkably, FF-MAS positively affected the pathway components despite the pathway inhibitor cyclopamine. Although the increase in Hedgehog pathway components was slightly higher in the male factor group (MF), it was not statistically significant. In our study, we demonstrated for the first time the molecular effect of FF-MAS on human GCs in folliculogenesis. Since FF-MAS is already used in assisted reproductive techniques in animals and is known to be synthesized in the human body, it could be considered a new approach in human IVF treatments.

Taurine Protects Doxorubicin-Induced Hepatotoxicity via Its Membrane-Stabilizing Effect in Rats.

BACKGROUND: Doxorubicin (dox) is a chemotherapeutic agent widely used against various tumors. However, the clinical use of this agent is limited due to various organ toxicities. Taurine is an intracellular free ß-amino acid with antioxidant properties. The present study investigated the protective mechanism of taurine on dox-induced hepatotoxicity. METHODS: In total, 31 male Sprague-Dawley rats were used in the study. The control group received intraperitoneal (i.p.) 0.9% NaCl alone for 14 days; the taurine (Tau) group received i.p. taurine 150 mg/kg body weight/day for 14 days; the dox group received dox on days 12, 13, and 14 at a cumulative dose of 25 mg/kg body weight/3 days; and the tau+dox group received taurine and dox together at the same dose and through the same route. On day 15, biochemical evaluations were performed on blood samples taken from the left ventricle followed by histological examinations on liver samples. RESULTS: Dox was found to increase liver function enzymes and tissue protein carbonyl levels, causing congestion and tissue damage, thereby leading to dysfunction. Tau was found to histologically preserve the liver morphology without showing any corrective effect on oxidative stress parameters. These findings suggest that the membrane-stabilizing effect of taurine may be more effective than its radical scavenging activity in preventing dox-induced toxicity. CONCLUSION: Taurine can prevent doxorubicin-induced hepatotoxicity through non-antioxidant pathways.

Effects of aging and inflammation on catalase activity in human dental pulp.

OBJECTIVES: In this study, it was aimed to examine the catalase activity in clinically healthy and caries-related inflamed symptomatic human dental pulps of young and old individuals. DESIGN: Sixty pulp samples from young and old healthy donors, were collected depending on pulpal status. 48 samples were used for spectrophotometric analysis and 12 samples for immunohistochemistry. Healthy pulps were maintained from non-carries, extracted third molars. Reversible and irreversible pulpitis samples were obtained by pulp extirpation during endodontic treatment. Following homogenization catalase enzyme activity was determined by spectrophotometry. Additionally, two pulp tissue samples from each group were fixed and evaluated for catalase immunoreactivity. RESULTS: Catalase enzyme activity in old healthy pulp samples were significantly higher than healthy young samples (p ≤ 0.05). Reversible and irreversible pulpitis samples indicated significantly decreased activity compared to healthy samples in elderly group (p ≤ 0.05). Young reversible pulpitis samples showed significantly increased catalase activity when compared to irreversible pulpitis and the reversible pulpitis samples in elderly group (p ≤ 0.05). Immunohistochemical evaluation indicated that there was intense catalase immunoreactivity in young patients with reversible pulpitis compared with reversible pulpitis in elderly group. However, weak immunoreactivity was observed in young irreversible pulpitis and elderly reversible pulpitis samples. CONCLUSIONS: The pulp tissues presented different levels of catalase activities against pulpitis and aging.

Doxycycline alleviates acute traumatic brain injury by suppressing neuroinflammation and apoptosis in a mouse model.

Traumatic brain injury (TBI) is one of the significant causes of death among young people worldwide. Doxycycline (DOX), an antibiotic with anti-inflammatory effects, has not been used as a therapeutic agent to modify the inflammatory response after the traumatic brain injury. In this study, intraperitoneal administration of DOX reduced significantly the acute inflammatory markers like IL-6 and CD3, microglial migration to the damaged area marked with Iba-1, and neuronal apoptosis assessed with TUNEL assay at 72 h after the trauma. The low dose, 10 mg/kg of DOX had a dominant anti-inflammatory effect; while the high dose, 100 mg/kg of DOX, was more effective in decreasing neuronal apoptosis. In early hours after the head trauma, use of a low dose (10 mg/kg) of DOX for decreasing the acute form of inflammation followed by a high dose (100 mg/kg) for the anti-apoptotic effects particularly in severe head traumas, would be a promising approach to alleviate the brain injury.

Papaverine provides neuroprotection by suppressing neuroinflammation and apoptosis in the traumatic brain injury via RAGE- NF-B pathway.

The receptor for advanced glycation end products (RAGE)- Nuclear Factor kappa B (NF-κB) signal pathway may represent a new target for the treatment of traumatic brain injury (TBI). The aim of the study is to investigate effects of papaverine on secondary signaling mechanisms through this pathway in mice TBI model.Immunohistochemically, while the number of RAGE and NF- κB positive cells, apoptotic cells increased, the number of NeuN positive cells reduced in TBI.Papaverine reduced the number of RAGE positive cells on glia and the number of NF- κB positive cells on both neuron and glia. At the same time, it decreased the number of microglia labeled with P2RY12 increased due to TBI. It also increased the NeuN positive cells and mitigated the brain edema. Results of this study showed that papaverine reduced TBI- induced neuroinflammation and apoptosis, also provided neuroprotection via the RAGE- NF-κB signal path, which is one of the possible mechanisms in TBI.

The evaluation of the distribution of CD133, CXCR1 and the tumor associated macrophages in different molecular subtypes of breast cancer.

Breast cancer has different molecular subtypes, which determine the prognosis and response to the treatment. CD133 is a marker for cancer stem cells in tumor microenvironment with diagnostic/therapeutic importance. The tumor associated macrophages (TAMs) interact with the cancer stem cells through the CXCR1 receptor. In this study, we wanted to investigate the expression of these markers in patients with different molecular subtypes, in order to detect pathophysiological mechanisms and new molecular targets for the prospective targeted therapies. In this study we hypothesized a difference in expression of these antigens among different subtypes. We investigated expression of antigens in breast cancer patients with luminal A (LA), luminal B (LB), HER2 overexpressing (HER2OE), triple negative (TN) subtypes (n=70) and control patients (n=10) without cancer diagnosis. We applied indirect immunohistochemistry and evaluated immunostaining. CD133 expression was at the periphery and CXCR1 expression was at the central area of the tumor. The cytoplasmic CXCR1, CD133 expressions and nuclear CD133 expression, which is prominent in the TN subtype, were observed in patients. There was a statistically significant difference between the groups for CD133 (p=0.004), CXCR1 (p=0.002) H-Score values and M2 macrophages/whole TAM ratios (p=0.022). Between the CD133 and CXCR1 H-scores, there was a weak positive correlation (r=0.249, p=0.035). This study showed the compartment specific expression of the CD133 and CXCR1 antigens in neoplastic cells. The use of CD133 as a stem cell marker may be limited to TN subtype, due to its heterogeneous expression.

In Vitro Cytotoxicity of Calcium Silicate-Based Endodontic Cement as Root-End Filling Materials.

The aim of this study was to evaluate the cytotoxicity of three types of calcium silicate-based endodontic cement after different incubation periods with human periodontal ligament fibroblasts. Human periodontal ligament fibroblasts were cultured from extracted third molars and seeded in 96-well plates. MTA, calcium enriched mixture (CEM) cement, and Biodentine were prepared and added to culture insert plates which were immediately placed into 96-well plates containing cultured cells. After incubation periods of 24, 48, and 72 hours, cell viability was determined with WST-1 assay. Data were analysed statistically by ANOVA with repeated measures and Bonferroni tests. There was no significant difference in cell viability amongst the test materials after each incubation period (P > 0.05). MTA and CEM presented more than 90% cell viability after 24 and 48 hours of incubation and showed statistically significant decrease in cell viability after 72 hours of incubation (P < 0.05). Biodentine showed significantly less cell viability (73%) after 24 hours of incubation, whereas more than 90% cell viability was seen after 48 and 72 hours of incubation (P < 0.05). Despite the significant changes in cell viability over time, materials presented similar cytotoxicity profile. Biodentine and CEM can be considered as alternative materials for root-end surgery procedures.

Apoptosis in chronic tonsillitis and tonsillar hypertrophy.

OBJECTIVE: Chronic tonsillitis is the persistent inflammation of the tonsillar tissue that occurs due to recurrent, acute or subclinical infection. The recurrent and chronic inflammation of palatine tonsils sometimes results in hypertrophy. Apoptosis provides an important balance between lymphocytes in tonsillar lymphoid tissue. The aim of this study is to investigate the apoptosis in tonsillar diseases. METHODS: 43 patients with chronic tonsilitis and tonsillar hypertrophy underwent tonsillectomy. The specimens were examined immunohistochemically for apoptosis. Tonsils were assembled into groups according to their size. Specimens were compared for their apoptotic cell count. RESULTS: The apoptosis difference between the tonsil size groups is not statistically significant (p>0.05). However, when the study group was divided into two at age 6, the difference was not statistically significant for patients at and below 6 years of age; but, the difference was statistically significant for patients above 6 years of age (p<0.05). The comparison of apoptosis in microcompartments of tonsil tissue (intrafollicular, interfollicular, subepithelial and intraepithelial) between tonsil size stages and between chronic tonsillitis and tonsillar hypertrophy groups revealed no statistical significance (p>0.05). There was a statistically significant positive correlation between intrafollicular and interfollicular, interfollicular and intraepithelial & subepithelial and intraepithelial areas (p<0.05). CONCLUSIONS: In the light of these findings, it was concluded that apoptosis played a role in the tonsillar hypertrophy and atrophy. Apoptosis functioned to balance lymphocyte proliferation in tonsil tissue. The association of apoptosis with tonsillar hypertrophy seemed to be age-dependent.

The effects of Gemcitabine and Vinorelbine on inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) distribution of MCF-7 breast cancer cells.

Gemcitabine, which induces S-phase arrest, and Vinorelbine, which arrests microtubule organization, are two agents that have demonstrate preferred anti-tumor activity. Nitric oxide acts in diverse functions including anti-tumor and anti-pathogenic activities. In this study, we aimed to examine the distribution of immunoreactivities of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in cells of the MCF-7 breast cancer cell line in response to treatment with Gemcitabine (G), Vinorelbine (V) and combination of Gemcitabine and Vinorelbine (G+V). The distributions of iNOS and eNOS were determined by using indirect immunoperoxidase or immunofluorescence methods and ELISA. Cells incubated with G, V and G+V for 24, 48 and 72h were immunolabelled with anti-eNOS and anti-iNOS primary antibodies. Apoptosis was determined by TUNEL assay. A significant increase of eNOS immunolabelling on MCF-7 cells treated with G and G+V was observed. Apoptotic cells were also detected in G, V and G+V treated MCF-7 cells. The immunolabelling of iNOS was detected in all groups but this immunoreactivity was not different among the groups. In conclusion, while G treatment, induced S-phase arrest, triggered the NOS pathway after treatment of MCF-7 cells, V treatment, arrested microtubule organization and did not change the NOS pathway. Detection of increased eNOS immunolabelling and apoptosis after G treatment of MCF-7 cells could be important to the treatment of human breast cancer.