Neuroendocrine modulation by metamizole and indomethacin: investigating the impact on neuronal markers and GnRH release.

PURPOSE: Some evidence that non-steroidal anti-inflammatory drugs have neuroprotective effects indicates their potential for use in a new field. However, their effects on hormone secretion have yet to be adequately discovered. Therefore, we aimed to evaluate the effects of metamizole and indomethacin on neuronal markers as well as the GnRH expression in the GT1-7 cell line. METHODS: The effects of these drugs on proliferation were evaluated by MTT analysis. The effect of 10-50-250 µM concentrations of the drugs also on the expression of neuronal factors and markers, including NGF, nestin and ßIII Tubulin, and additionally GnRH, was determined by the RT-qPCR method. RESULTS: NGF and nestin mRNA expressions were increased in all concentrations of both metamizole and indomethacin. No changes were detected in ßIII Tubulin. While metamizole showed an increase in GnRH mRNA expression, there was no change at 10 and 50 µM concentrations of indomethacin, but a remarkable decrease was observed at 250 µM concentrations. CONCLUSIONS: The results of our study showing an increase in the expression of neuronal factors reveal that metamizole and indomethacin may have possible neuroprotective effects. Moreover, the effects on the GnRH expression appear to be different. Animal models are required to confirm these effects of NSAIDs on neurons.

An in vitro pilot study investigating placenta-derived mesenchymal stem cell coating on polypropylene mesh materials.

INTRODUCTION AND HYPOTHESIS: Polypropylene meshes (PM) used in pelvic organ prolapse surgery are being withdrawn from the market. Although concerns about the usage of PMs in stress incontinence surgery have been raised, it is still one of the best methods of curing stress urinary incontinence. With advancements in stem cell-based therapies, especially mesenchymal stem cells (MSCs), it is believed that coating the synthetic meshes with MSCs may minimize excessive tissue reactions ultimately leading to clinical problems such as pain, erosion or extrusion of the implanted material. In our study we tried to show the possibility of coating the PM with placenta-derived MSCs. METHODS: Mesenchymal stem cells obtained from six placentas were isolated, cultured, and identified. MSCs were then soaked in either fibronectin or collagen prior to co-culturing with strips of PMs. One group is used as a control, and hence was not pretreated before co-culturing. Specimens were fixed and stained with both Gram and hematoxylin and eosin and marked with Vybran Dil and DAPI. All preparations were examined under a light microscope. The IMAGEJ program was utilized to determine the surface area of meshes coated with MSCs. RESULTS: We clearly showed that PMs can be coated successfully with placenta-derived MSCs. The percentage of the coated area is significantly increased when meshes were pretreated with fibronectin or collagen (p<0.0001). CONCLUSIONS: Placenta-derived MSCs can successfully coat PMs. The immunomodulatory properties of MSCs, which may be of great advantage in preventing the side effects of meshes, should be tested by in vivo and hopefully human studies before clinical applications.

Effect of milk and whey on proliferation and differentiation of placental stromal cells.

Fetal bovine serum (FBS), which is widely used in cell culture media, has the potential to cause medical and ethical problems. Here, an experimental study using milk or whey proteins containing essential nutrients and growth factors is presented to limit the use of FBS in cell culture media produced for cell and tissue regeneration. Study groups were formed by culturing human placenta mesenchymal stem cells, known to have high proliferation and differentiation capacity, with milk or whey solution at increasing concentrations, alone or in combination with FBS. Osteogenic and adipogenic differentiation capacities of proliferating cells were observed in FBS, milk or whey groups. Milk, whey or FBS groups obtained in P3 and after differentiation were separately analyzed for protein mRNA expression by reverse transcriptase-polymerase chain reaction (RT-qPCR). Fibroblast Growth Factor 2 (FGF2), Octamer-binding Transcription Factor 4 (OCT4), Bone Morphogenetic Protein 6 (BMP6), and adipogenic differentiation marker Peroxisome Proliferator-Activated Receptor Gamma (PPARG) were analysed by RT-qPCR. Proliferation was more pronounced in FBS alone and in its combinations with milk-whey compared to the groups in which only milk and whey were used. OCT4 mRNA and FGF2 mRNA expression decreased in differentiated cells. BMP6 mRNA expression increased with osteogenic and adipogenic stimuli. As expected, PPRG expression also increased with adipogenic stimulation. With this experimental study, evidence has been obtained that milk or whey can provide nutritional support to the culture media of repair cells and preserve the functional capacity of the cells, with a slightly more limited capacity than FBS. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00585-z.

The effect of thymoquinone and propranolol combination on epidermoid laryngeal carcinoma cell.

PURPOSE: We aimed to evaluate the effects of thymoquinone and propranolol on Hep-2 cells representing laryngeal Ca cell type in comparison with cisplatin. We also evaluated their combined effects. METHODS: Apoptotic effects were directly analyzed via mitochondrial membrane potential and caspase-3 assays. In addition, effects on apoptosis and cell cycle via Bcl-2, Bax, P53, and Cyclin D1 mRNA expressions and effects on angiogenesis via VEGFA mRNA expression were evaluated by RT-qPCR. RESULTS: According to our results, it was determined that the anticancer effects of thymoquinone on Hep-2 cells were higher than propranolol. Our JC-1 and caspase-3 results showed an effect close to cisplatin, especially for 50 µM thymoquinone. Significant differences were also obtained in Bcl-2, Bax, P53, and cyclin D1 results for similar concentrations compared to the control. No effect of thymoquinone was seen for VEGFA. Propranolol alone had no significant effect on JC-1 and Caspase-3. Propranolol had an effect on Bcl-2, Bax mRNA expressions compared to the control, only at 250 µM concentration. Propranolol and its combinations increased VEGFA mRNA expression-like cisplatin. CONCLUSION: Thymoquinone induced apoptosis and blocked the cell cycle in Hep-2 cells. The effects of propranolol, which was reported to have an antiangiogenesis effect in some studies, on apoptosis and cell cycle were limited except at high concentrations. For this cell line, why propranolol causes an increase in VEGFA expression should be evaluated extensively. Thymoquinone shows promise for cancer therapy, but studies need to be designed in vivo to evaluate the effects more reliably.

The effects of metamizole on hematopoietic progenitor cells: Suppression of hematopoiesis stimulation in vitro.

BACKGRAUND: There is evidence that the adverse effects of metamizole occur due to the effect of the drug on the hematopoietic stem/progenitor cells, and therefore, the disruption of hematopoiesis. Therefore, our study aimed to evaluate the effects of metamizole on hematopoietic stem/progenitor cells using cell culture techniques. MATERIAL AND METHODS: In our study, samples were taken from stem cell products of healthy allogeneic stem cell transplant donors. The colony-forming unit (CFU) assay was used for the cells obtained from these samples. In addition, the drug effects on cell proliferation were evaluated with the MTT. Furthermore, the cell colonies were labelled with immunofluorescent antibodies and the effects of metamizole on cell types formed in culture were evaluated. RESULTS: We determined that metamizole negatively affects the proliferation of cells, especially starting from 10 µM. As a result of the evaluation of colonization, we saw that the number of colonies decreased with increasing concentrations. Granulocyte-macrophage colonies were more affected at increasing concentrations than other colonies. As a result of the evaluations of our in vitro study, it was also shown as an important finding that the individual effects of the drug were highly variable. CONCLUSION: CFU method can be used as a suitable method to investigate the effects of drugs and toxic substances on hematopoiesis. We also think it may be suitable for pre-analysing hematopoietic side effects in new drug research. In addition, using stem cell samples in studies may contribute more easily to the in vitro simulation of hematopoietic differentiations (Fig. 7, Ref. 29). Text in PDF www.elis.sk Keywords: metamizole, hematopoietic progenitor cells, hematopoiesis, CFU assay, adverse effect.

Adrenergic receptor behaviors of mesenchymal stem cells obtained from different tissue sources and the effect of the receptor blockade on differentiation.

In this study, it was aimed to analyze behavioral changes of adrenergic receptors (ARs) in first three passages and osteogenic/adipogenic differentiation of mesenchymal stem cells (MSCs) derived from placenta fetal membrane (FM) and bone marrow (BM). It was also aimed to evaluate effects of receptor blockade on differentiation. We obtained first three passages of MSCs from placenta and BM samples. For cell identification, the cells were analyzed by flow cytometry using CD34, CD45 and CD3, CD105 antibodies in each passage. The effects of propranolol and phenoxybenzamine at incremental doses were analyzed by MTT. In addition, cell cultures were separately maintained with the blockers or without after second passage. After each passage and differentiation, α1A, α1B, α2A, α2B, ß1, ß2, ß3 AR-mRNA expressions analyzed by RT-qPCR technique. BMP6 and PPARG mRNA expressions only after differentiation and passage 3 were analyzed. A microscopic examination was also performed. Our results showed that AR expression behaviors were different in MSCs obtained from different tissue sources. In particular, α1A-AR and α2A-AR were expressed with considerably high coefficients in differentiation under blocker effect in BM-derived MSCs. No such coefficients were observed in any group of placental MSCs. In addition, it was found that the blockers stimulated adipogenesis in BM-derived MSCs during osteogenic differentiation. MSCs exhibit protein expressions that vary according to source of tissue and differentiation. Given that MSCs from different sources are used for repair and modulation, our study makes implications of this variable expression intriguing in the clinical practice.

Biological behaviors of muscarinic receptors in mesenchymal stem cells derived from human placenta and bone marrow.

OBJECTIVES: Cells perform their functional activities by communicating with each other through endogenous substances and receptors. Post-translation, stem cells function properly in new host tissue by carrying specific cell surface receptors. We aimed to characterize muscarinic receptor subtypes in mesenchymal stem cells (MSCs) together with osteogenic and adipogenic differentiation markers. MATERIALS AND METHODS: mRNA levels of 5 muscarinic receptor subtypes (CHRM1 to 5), BMP-6, and PPARγ during osteogenic and adipogenic differentiation, under the effect of atropine blockade, were measured in MSCs obtained from human fetal membrane (FM) and bone marrow (BM). Additionally, the effect of atropine on differentiation in the 1st, 2nd, and 3rd passages of MSCs, obtained from human FM and BM, were analyzed by RT-qPCR. RESULTS: CHRM1 mRNA levels increased in the FM group, while decreasing in the BM group. We found significant decreases in CHRM3 and CHRM5 mRNA levels in FM and BM groups, respectively. Atropine had variable effects based on cell source and receptor type. BMP-6 mRNA levels in differentiated osteogenic cells increased significantly compared to undifferentiated cells in both FM and BM groups. In MSCs derived from both sources, PPARγ mRNA levels in differentiated adipogenic cells increased significantly. Atropine showed no effect on MSCs differentiation. CONCLUSION: These results indicate that expressions of muscarinic receptors in MSCs derived from BM and FM can vary and these cells keep the potential of osteogenic and adipogenic differentiation in vitro. Besides, atropine had no effect on adipogenic and osteogenic differentiation of MSCs.

Effects of two doses of anti-T lymphocyte globulin-Fresenius given after full-match sibling stem cell transplantation in acute myeloblastic leukemia patients who underwent myeloablative fludarabine/busulfan conditioning.

OBJECTIVE/BACKGROUND: Anti-T lymphocyte globulin Fresenius (rATG-F; ATG-Fresenius) and antithymocyte globulin (thymoglobulin), which are included in transplant protocols, are used to reduce the risk of chronic graft-versus-host disease (cGVHD) or suppress allograft rejection. Available clinical studies have been conducted in heterogenous patient populations and with different administration protocols including stem cell sources. Additionally, the pharmacokinetics of ATG is variable, and the clinically effective dose of rATG-F, in particular, is not exactly known. The aim of the study was to investigate the clinical outcomes of acute myeloid leukemia (AML) patients who underwent hemopoietic peripheral stem cell transplantation from full-matched sibling donors and given two different doses of r-ATG-F. METHODS: This was a single-center, retrospective chart review conducted between July 2005 and July 2016. Sixty-nine consecutive AML patients who underwent transplant with fludarabine- and busulfan-based conditioning were included in the study. Patients in Group 1 received 15 mg/kg body weight rATG-F to 2013 (n = 46), and Group 2 received 30 mg/kg of rATG-F dose begining in 2013 to reduce to cGVHD (n = 23). Cyclosporine and methotrexate were used to treat acute GVHD (aGVHD) prophylaxis. Outcome parameters were compared between the groups. RESULTS: Although the recommended dose r-ATG-F had led to a decrease in the cumulative incidence of cGVHD (27 [58.7%] vs. 8 [34.8%]; p = .03), it also increased the infection rate at 1 year (3 [6.5%] vs. 4 [17.4%]; p = .02). The two groups were similar in terms of engraftment time, aGVHD, relapse, nonrelapse mortality, and rATG-F-related toxicity. A Cox regression model revealed that aGVHD III-IV was associated with increased nonrelapse mortality at 1 year (hazard ratio = 18.2; 95% confidence interval, 1.667-199.255; p = <.02). No patients developed rATG-F-related severe adverse events (Common Terminology Criteria grade 4 or 5). CONCLUSION: Dose difference of rATG-F did not influence survival parameters; however, increasing the dose to 30 mg/kg seems to be effective for reducing cGVHD with an increase in infection rate requiring close monitoring of infections in AML patients who received myeloablative fludarabine/busulfan conditioning.

Organ damage mitigation with the Baskent Sickle Cell Medical Care Development Program (BASCARE).

The Eastern Mediterranean is among the regions where sickle cell disease (SCD) is common. The morbidity and mortality of this disease can be postponed to adulthood through therapies implemented in childhood. The present study focuses on the organ damage-reducing effects of the Baskent Sickle Cell Medical Care Development Program (BASCARE), which was developed by a team who lives in this region and has approximately 25 years of experience. The deliverables of the program included the development of an electronic health recording system (PRANA) and electronic vaccination system; the use of low citrate infusion in routine prophylactic automatic erythrocyte exchange (ARCE) programs including pregnant women; the use of leukocyte-filtered and irradiated blood for transfusion; the use of magnetic resonance imaging methods (T2) for the management of transfusion-related hemosiderosis; and the implementation of an allogeneic hematopoietic stem cell transplantation protocol for adult patients. The sample was composed of 376 study subjects and 249 control subjects. The hospital's Data Management System and the central population operating system were used for data collection. BASCARE enabled better analysis and interpretation of complication and mortality data. Vaccination rates against influenza and pneumococcal disease improved (21.5% vs 50.8% and 21.5% vs 49.2%, respectively). Effective and safe ARCE with low citrate infusion were maintained in 352 subjects (1003 procedures). Maternal and fetal mortality was prevented in 35 consecutive pregnant patients with ARCE. Chelating therapy rates reduced from 6.7% to 5%. Successful outcomes could be obtained in all 13 adult patients who underwent allogeneic peripheral stem cell transplantation from a fully matched, related donor. No patients died by day 100 or after the first year. Cure could be achieved without graft loss, grades III to IV acute graft versus host disease, extensive chronic graft versus host disease, or other major complications. The BASCARE program significantly improved patient care and thereby prolonged the life span of SCD patients (42 ± 13 years vs 29 ±â€Š7 years, P < .001). We may recommend using such individualized programs in centers that provide health care for patients with SCD, in accordance with holistic approach due to the benign nature but malignant course of the disease.

Dipyrone ameliorates behavioural changes induced by unpredictable chronic mild stress: gender differences.

PURPOSE: Antidepressant effects of analgesics have been investigate in both clinical and experimental studies. The purpose of this study was to investigate if the analgesic-antipyretic drug, dipyrone, also had antidepressant-like effects. METHODS: Depression-like effects were investigated in an unpredictable chronic mild stress (UCMS) model in both male and female mice. Cage changes, light-dark cycle reversal, cage tilting, wet floor, empty cage, foreign material on the floor and predator sounds were used to induce light stress at different times for six weeks. Dipyrone was administered intraperitoneally beginning from the third week. Splash, rota-rod (RR) and forced swimming (FST) tests were performed at the seventh week as behavioural tests to evaluate the antidepressant-like effects of dipyrone. Coat state score (CSS) and weights of animals were recorded at seventh weeks. Results were analyzed using one or two-way ANOVA followed by the Bonferonni post hoc test. RESULTS: Weight of UCMS-exposed mice did not change compared with controls; however, significant changes were observed in CSS in both sexes of stressed mice (p.

Human bone marrow mesenchymal cells express NG2: possible increase in discriminative ability of flow cytometry during mesenchymal stromal cell identification.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) exhibit non-specific hematopoietic cell and/or stromal cell markers (e.g. CD73, CD105 and CD166) that have been used to identify MSC by flow cytometry. Because a neural glial antigen, NG2 (a progenitor cell marker in the central nervous system), is expressed by several tissue cells originating in the mesenchyme but not hematopoietic cells, it might be useful for isolating and identifying MSC. We investigated NG2 expression on culture-expanded MSC by flow cytometry. METHODS: Human bone marrow (BM) samples taken from 12 donors were cultured for MSC to be used in up to nine serial passages. Using flow cytometry, the neural glial antigen NG2 and commonly used MSC markers CD73, CD105 and CD166, were analyzed on the surface of culture-expanded MSC. The multipotential differentiation of the MSC was examined by adipogenic and osteogenic induction. RESULTS: The percentage of cells positive for NG2 was similar to the percentages of cells positive for CD73, CD105 and CD166 in all passages of BM samples. The mean fluorescent intensities of NG2 did not change with culture passage. The MSC was successfully differentiated into adipogenic and osteogenic lines. The cells showed no karyotypic abnormalities. CONCLUSIONS: NG2 seems to be a promising marker for investigating the biology of MSC.

Flow cytometric evaluation of circulating endothelial cells: a new protocol for identifying endothelial cells at several stages of differentiation.

Several factors may influence the analysis of endothelial cells (ECs) by flow cytometry: separation of mononuclear cell, washing and centrifugation steps, panel of monoclonal antibodies, and the lack of standardization of gating technique. Therefore, the reliable quantification of ECs remains a technical challenge. The purpose of this study is to define a new flow cytometric protocol to characterize and quantitate ECs. In previous investigations, increased numbers of circulating ECs have been found in sickle cell disease. The patients with sickle cell disease might provide useful material for the study. We performed flow cytometry on whole blood from 20 normal controls and 31 patients with sickle cell disease (20 patients with steady-state disease and 11 patients with vaso-occlusive crises) using a lyse/no-wash procedure, specific and nonspecific antibody combinations (CD146, CD144, CD34, and CD117), and broad gating. This protocol produced much higher values for the number of circulating ECs (a mean of 2,396.55 +/- 658.37 ECs/mL in controls vs 6,709.60 +/- 1,772.32 ECs/mL in the steady-state group, or 18,213.50 +/- 8,451 in the vaso-occlusive crises group, P < 0.001 for both), and also showed variable EC size and granularity, which may reflect activated, or early release ECs. This novel protocol performed comparably in terms of reproducibility, reliability, and dilution linearity with a previously described protocol. This approach has significant advantages for the characterization and quantitation ECs compatible with the pathophysiology. Using the specific antibodies, CD146 and CD144, together may give more informative EC data than the general approach used.

The apoptosis of blood polymorphonuclear leukocytes in sickle cell disease.

BACKGROUND: The apoptosis of human polymorphonuclear leukocytes (PMNs) in patients with sickle cell disease (SCD) is not well understood. The goal of this study was to examine the apoptosis of PMNs in patients with SCD and in controls. METHODS: Flow cytometric quantitation of PMN apoptosis was performed in 17 patients during and after sickle cell vasoocclusive crisis and in 17 healthy volunteers. Plasma nitric oxide concentrations were also measured in patients with SCD. RESULTS: The mean of annexin-V and annexin-V/PI staining (early and late apoptotic cells) increased to a greater degree in patients with SCD than in healthy controls for patients with SCD during and after vasoocclusive crisis. The mean of PI staining showing dead cells was higher only in patients after SCD crisis than in healthy controls. In the SCD groups during and after vasoocclusive crisis, there was no difference between PMN apoptosis levels. Furthermore, plasma nitric oxide concentrations were not correlated with PMN apoptosis. CONCLUSIONS: There was an evidence that the alteration of blood PMN apoptosis could contribute to the pathogenetic mechanisms of vasoocclusion in patients with SCD. This can be attributed to the effects of numerous inflammatory mediators rather than simply the effects of nitric oxide.