Letrozole Decreased Testosterone-Induced Cell Proliferation and Prolactin Secretion also Increased Apoptosis in MMQ and GH3 Rat Prolactinoma Cell Lines.

Aromatase enzyme plays an essential role in estrogen-induced tumorigenesis. It is expressed in the normal pituitary and more significantly in prolactinoma tissues. The aim of this study was to investigate the effects of an aromatase inhibitor, letrozole, on MMQ and GH3 rat prolactinoma cell lines and evaluate the possible mechanism of action. MMQ and GH3 cells were characterized with demonstrating aromatase enzyme and estrogen receptor alpha expression by PCR and immunofluorescence staining. After dose optimization for testosterone (T) and letrozole (L), four groups were established: only the testosteron-treated group (T) to detect cell proliferation; only letrozole-treated group (L) to investigate apoptotic effects; testosterone and letrozole concomitant-treated group to demonstrate inhibition of testosterone induced cell proliferation with letrozole treatment s(T + L) and control group (C) with no treatment. The proliferation rate of cells was determined by WST-1. For the detection of apoptotic and necrotic cells, Annexin V and caspase-3 labeling was used. Prolactin and estrogen levels were measured with ELISA, and the mRNA expression of aromatase and Esr1 was also determined. Testosterone induced the proliferation of MMQ and GH3 cells and further increased prolactin and estradiol levels. Adding letrozole to testosterone resulted in decreased cellular proliferation and even induced apoptosis. Also, letrozole administration significantly decreased prolactin and estradiol levels. However, letrozole alone had no effects on proliferation and apoptosis. Gene expression of aromatase and Esr1 was also significantly decreased by letrozole treatment. This in vitro study demonstrated that treatment of testosterone proliferating cells with letrozole resulted in decreased prolactin levels and cell proliferation and induced apoptosis, and further loss of aromatase and Esr1 mRNA expression were observed. Although this is an in vivo study, the results showed unique and novel findings which may easily be adapted to clinical use for further verification.

Determination of antibiotic susceptibility of bacteria by flow cytometric method.

In this study, it was aimed to determine the antibiotic susceptibility of bacterial strains by using flow cytometry method by comparing them with current standardized methods. Eleven clinical isolates and 6 standard bacterial strains were included in the study. MIC values were determined by broth microdilution method (BMD), VITEK 2® automated system and flow cytometric method (FCM). FCM was performed with the Accuri C6 flow cytometer. For all strains except P. aeuruginosa ATCC 27853 [BMD-FCM:r = 0.557(p = 0.048); VITEK 2-FCM:r = 0.529(p = 0.063)], E. faecalis ATCC 29212 [BMD-FCM:r = 0.393(p = 0.295); BMD-VITEK 2:r = 0.393(p = 0.295)], and vancomycin-resistant E. faecium clinical isolate [BMD-FCM:r = 0.452(p = 0.063)] r values were in the range of 0.802-0.969 for BMD-FCM (p < 0.001), 0.655-0.941 for BMD-VITEK 2 (p < 0.005) and 0.667-0.953 for FCM-VITEK 2 (p < 0.005). Correlation values of antibiotic susceptibility test results between three methods for Gram-negative bacteria were found as follows; r = 0.927(p < 0.001) for BMD-FCM, r = 0.851(p < 0.001) for BMD-VITEK 2, r = 0.807(p < 0.001) for VITEK 2-FCM. Correlation values were found as follows for Gram positive bacteria; r = 0.848(p < 0.001) for BMD-FCM, r = 0.877(p < 0.001) for BMD-VITEK 2, r = 0.800(p < 0.001) for VITEK 2-FCM. When all bacteria included in the study were evaluated as a total; it was r = 0.911(p < 0.001) for BMD-FCM, r = 0.888(p < 0.001) for BMD-VITEK 2, r = 0.835(p < 0.001) for VITEK 2-FCM. The methicillin resistance of the clinical methicillin resistant S. aureus isolate could not be detected by FCM. It was determined that there was a high level of correlation between methods. FCM shortens the duration of antibiotic susceptibility tests by 12-14 h and gives results within the same day. However, it has not been standardized to be widely used in microbiology laboratories and experienced personnel are needed for its implementation.

The effect of mesenchymal stromal cells in the microenvironment on cancer development.

Inflammatory signals secreted from the tumor microenvironment are thought to promote tumor growth and survival. It has been reported that stromal cells in the tumor microenvironment have similar characteristics to tumor-associated cells. In addition miRNAs play critical roles in various diseases, including cancer. In this study, we aimed to investigate the effects of co-culture of cancer cells and stromal cells isolated from normal and malignant breast tissue on each other and the possible effects of miRNAs on these interactions. The characterized stromal cells were co-cultured with an MDA-MB-231 cancer cell line. The proliferation capacity of the experimental groups was evaluated using the WST-1 assay. The expression of breast cancer-specific miRNAs and related genes were assessed by real-time PCR. ELISA assay was performed to determine the concentration of some cytokines and chemokines. We found that the microenvironment plays an important role in the development of cancer, confirming the changes in the expression of oncogenic and tumor suppressor miRNA and their target genes after co-culture with malignant stromal cells. As a result of the studies, specific gene expressions of related signaling pathways were detected in correlation with miRNA changes and the effects of tumor microenvironment on tumorigenesis were revealed in detail. miRNAs have been shown to play an important role in cancer development in recent studies. The idea that these small molecules can be used in diagnosis and treatment is becoming stronger day by day. We believe that new treatment approaches involving the tumor microenvironment and using miRNAs as markers are promising.

Protective role of melatonin against testicular damage caused by polymicrobial sepsis in adult rats.

BACKGROUND: This study aimed to investigate the possible protective effects of melatonin (MEL) against the damage to testicular tissue in rats caused by polymicrobial sepsis as a result of cecal ligation and perforation (CLP). METHODS: In this study, 21 male Wistar albino rats were used. The rats were randomly divided into three groups (n=7): Sham Control (Group 1), CLP (Group 2), and CLP + MEL (Group 3). Sepsis was created using the CLP method. MEL was administered intraperitoneally in two equal doses of 10 mg/kg at 30 min before and 6 h after perforation. Tissue sections taken from paraffin blocks were stained with hematoxylin and eosin (H and E) and examined histopathologically under a light microscope. Intracellular H2O2 and apoptosis evaluations were carried out using the flow cytometric method. RESULTS: Sepsis caused a significant reduction in all sperm parameters. There was a significant decrease in sperm density, motility and cell numbers with normal morphology (p<0.05). Intracellular H2O2 level and apoptotic cell percentages increased in sperm cells in the CLP group. MEL treatment was found to significantly reduce sperm abnormalities, testicular damage, intracellular H2O2 levels, and apoptosis. CONCLUSION: This study showed that melatonin administration could be a potential treatment option to reduce acute testicular tissue damage due to sepsis.

Age-related changes in the immunomodulatory effects of human dental pulp derived mesenchymal stem cells on the CD4+ T cell subsets.

Mesenchymal stem cells (MSCs) are powerful immunomodulatory cells. The effects of the aging on these abilities of MSCs have not been adequately clarified. In this study, alterations in immunomodulatory abilities of MSCs caused by aging were investigated. For this, dental pulp (DP) MSCs and peripheral blood mononuclear cells (PBMCs) of elderly and young donors were co-cultured age-matched and cross. We detected that the effects of DP-MSCs on Th1 and Th2 cells and their specific cytokines IFN-γ and IL-4 are not affected by aging. However, we observed that young and elderly DP-MSCs have different effects on Th17 and Treg cells. Th17 frequencies of young and elderly PBMCs were significantly increased only by young DP-MSCs, in contrast, Treg frequencies were significantly increased by elderly DP-MSCs. IL-6, IL-17a and HGF levels of both young and elderly PBMCs showed a significant increase only by young DP-MSCs, but TGF-ß levels were significantly increased only by elderly DP-MSCs. The oral cavity is home to a rich microflora. The interactions of dental tissues with this microflora can lead them to acquire different epigenetic modifications. Aging can affect the microflora composition of the oral cavity and change this process in different directions. According to our findings, DP-MSCs are effective cells in the regulation of CD4+ T cells, and their effects on Th1 and Th2 cells were not affected by aging. However, pleiotropic molecules IL-6 and HGF expressions, which are important in dental and bone tissue regeneration, decreased significantly in elderly DP-MSCs. This situation may have indirectly made a difference in the modulation effects of young and elderly DP-MSCs on the Th17 and Treg cells.

Comparative Analyses of Immunosuppressive Characteristics of Bone-Marrow, Wharton's Jelly, and Adipose Tissue-Derived Human Mesenchymal Stem Cells.

OBJECTIVE: Mesenchymal stem cells (MSCs), which possess immunosuppressive characteristics on induced T-cells, were shown to be applicable in prevention and treatment of graft-versus-host disease. However, knowledge of effective cell sources is still limited. In this study, MSCs from different human tissues, i.e. bone marrow (BM), Wharton's jelly (WJ), and adipose tissue, were isolated, and the immune suppression of stimulated T cells was analyzed comparatively. MATERIALS AND METHODS: MSCs were co-cultured with phytohemagglutinin-induced T-cells with co-culture techniques with and without cell-to-cell contact. After co-culture for 24 and 96 h, the proliferation rate of T cells was estimated by carboxyfluorescein succinimidyl ester and apoptosis by annexin V/PI methods. Both T cells and MSCs were analyzed with respect to gene expressions by real-time polymerase chain reaction and their specific protein levels by ELISA. RESULTS: The results showed that all three MSC lines significantly suppressed T-cell proliferation; BM-MSCs were most effective. Similarly, T-cell apoptosis was induced most strongly by BM-MSCs in indirect culture. In T cells, the genes in NFkB and tumor necrosis factor pathways were silenced and the caspase pathway was induced after co-culture. These results were confirmed with the measurement of protein levels, like transforming growth factor ß1, IL-6, interferon-γ, interleukin (IL)-2, and tumor necrosis factor-α. Additionally, IL-17A was detected in high levels in WJ-MSC co-cultures. We showed that IL-17A-producing Tregs are the key mediators in the treatment of graft-versus-host disease. CONCLUSION: BM-MSCs, which have been used in clinical applications for a while, showed the greatest immunosuppressive effect compared to other MSCs. However, a promising cell source could also be WJ, which is also effective in suppression with fewer ethical concerns. We described the molecular mechanism of WJ-MSCs in allogenic transplants for the first time.

Evaluation of Effect of Topical Tacrolimus Treatment on Herpetic Stromal Keratitis in a Rat Model.

OBJECTIVES: To investigate the effectiveness of topical tacrolimus treatment on herpetic stromal keratitis (HSK) in a rat model. METHODS: The development of HSK was monitored for 14 days after the inoculation of rats with herpes simplex type 1 virus. Rats that developed HSK were divided into four groups as follows: (1) topical antiviral treatment (control), (2) topical antiviral and 1% prednisolone acetate, (3) topical antiviral and 0.03% tacrolimus ointment, and (4) topical antiviral plus 0.1% tacrolimus ointment. After 14 days of treatment, the severity levels of HSK were scored and compared with the levels before the treatment. The expression of CD3, CD4, and CD8 was evaluated by flow cytometry. The development of the disease was evaluated clinically and histologically. RESULTS: Significant improvement in vascularization was observed in the groups with the drug treatment in addition to the antiviral agent (P<0.05), but there was no obvious difference within groups 2, 3, and 4 in the vascularization severity. The regression of corneal edema was 8.05%±6% in group 1, 25.17%±14.55% in group 2 (P=0.01), 36.40%±21.69% in group 3 (P=0.03), and 46.39%±14.96% in group 4 (P=0.00). A significant decrease in the number of inflammatory cells in the groups with the drug treatment was evaluated by immunohistochemical staining and confirmed by flow cytometry analysis. CONCLUSIONS: Topical tacrolimus treatment caused a significant decrease in corneal vascularization accompanied by a lower number of inflammatory cells in the experimental HSK corneal edema model. Therefore, topical tacrolimus has the potential to be used in the treatment of HSK.

Neuroprotective effects of intravitreally transplanted adipose tissue and bone marrow-derived mesenchymal stem cells in an experimental ocular hypertension model.

BACKGROUND AIMS: The purpose of this study was to investigate the neuroprotective effects of bone marrow bone marrow-derived and adipose tissue-derived mesenchymal stromal cells (MSCs) that were intravitreally transplanted in an experimental ocular hypertension (OHT) model. METHODS: An OHT rat model was generated by means of intracameral injection of hyaluronic acid into the anterior chamber. MSCs labeled with green fluorescence protein were transplanted intravitreally 1 week after OHT induction. At the end of the second and fourth weeks, retinal ganglion cells were visualized with the use of a flat-mount retina method and were evaluated by means of immunofluorescence staining against green fluorescence protein, vimentin, CD105, and cytokines (interleukin [IL]-1Ra, prostaglandin E2 receptor, IL-6, transforming growth factor-ß1, interferon-γ and tumor necrosis factor-α). RESULTS: The retinal ganglion cell numbers per area were significantly improved in stem cell-treated OHT groups compared with that in the non-treated OHT group (P < 0.05). The results of immunohistochemical analyses indicated that a limited number of stem cells had integrated into the ganglion cell layer and the inner nuclear layer. The number of cells expressing proinflammatory cytokines (interferon-γ and tumor necrosis factor-α) decreased in the MSC-transferred group compared with that in the OHT group after 4 weeks (P < 0.01). On the other hand, IL-1Ra and prostaglandin E2 receptor expressions were increased in the rat bone marrow-derived MSC group but were more significant in the rat adipose tissue-derived MSC group (P < 0.01). CONCLUSIONS: After intravitreal transplantation, MSCs showed a neuroprotective effect in the rat OHT model. Therefore, MSCs promise an alternative therapy approach for functional recovery in the treatment of glaucoma.

Neurogenic differentiation capacity of subacromial bursal tissue-derived stem cells.

In this study, analysis and comprehensive comparison of neurogenic differentiation capacity of human bursal tissue-derived-stem cells (hBT-SCs) was aimed with human bone marrow derived mesenchymal stem cells (hBM-MSCs). hBT-SCs was isolated from subacromial bursa tissue (n = 3) by collagen type-II digestion. The expression of stem cell markers, differentiation capacity and telomerase activity were determined for both cell lines. The expression levels of neurogenic cell markers were compared consecutively. With respect to the surface marker profile, both cells display similar pluripotency phenotypes. Both cells successfully differentiated into osteo- and adipogenic cell lines. The immune staining of mesenchymal, stem cell and neurogenic markers gave positive reaction. The gene expression level for Tubb3, Nestin, Gfap, Map2, Nf-h, and Nf-l was higher in hBT-SCs than hBM-MSCs. The high level of neurotrophic factors, like Tenascin C, NGF, BDNF, VEGF, and CNTF might indicate their regeneration and maintenance capacity in damaged neural tissue. Besides they are alternative source for human mesenchymal stem cells, hBT-SCs assess the possibility to use in clinical studies.

A unique Golgi apparatus distribution may be a marker for osteogenic differentiation of hDP-MSCs.

Stem cell markers are utilized to isolate or identify stem cells. So far, many stem-cell-specific markers have been described, although some of them turned out to be not as specific as it was originally proposed. In this study, we sought to search for a specific stem cell marker that would be phenotypically helpful, characteristically specific, economically affordable and easy to use. Because organelles are one of the major characteristics of eukaryotic cells, we asked the question of whether organelle characteristics might be a useful tool for stem cell characterization. We studied distribution and characteristics of the endoplasmic reticulum, the mitochondria and the Golgi apparatus in human dental-pulp-derived mesenchymal stem cells before and during osteogenic differentiation. Although it was not possible to find a useful macromolecular marker for stem cell characterization, we found that during osteogenic differentiation, the stem cells changed their Golgi characteristics and displayed a unique in vivo pattern. We analysed these unique Golgi structures and proposed a potential osteogenic differentiation marker for human dental-pulp-derived mesenchymal stem cells. This pattern may be used in the evaluation of osteogenic differentiation.