Native High-Density Lipoprotein and Melatonin Improve Platelet Response Induced by Glycated Lipoproteins.

Activated platelets and glycated lipoproteins are responsible for atherothrombosis in diabetics. Melatonin and native high-density lipoproteins are crucial in the preservation of pro/oxidant-antioxidant balance. The aim of the present study was to investigate the in vitro effects of native high-density lipoproteins and melatonin on altering the platelet response induced by glycated lipoproteins. Low-density lipoproteins and high-density lipoproteins were purified from plasma by ultracentrifugation and were glycated with glucose for three weeks. After incubation with or without melatonin/or native highdensity lipoproteins, low-density lipoproteins, glycated low-density lipoproteins/glycated high-density lipoproteins were added to ADP-induced platelets. Oxidative parameters, caspase-3/9 and nitric oxide levels were measured spectrophotometrically; CD62-P/ annexin-V expression was determined by flow cytometry. In glycated low-density lipoprotein/glycated high-density lipoprotein-treated groups, platelet malondialdehyde/ protein carbonyl, P-selectin, annexin-V, caspase-3/9 levels were increased (ranging from P < 0.001 to P < 0.01); glutathione and nitric oxide levels were reduced (ranging from P < 0.001 to P < 0.01). In glycated low-density lipoprotein/glycated high-density lipoprotein-treated groups, melatonin treatment reduced malondialdehyde, protein carbonyl, CD62-P, annexin-V and caspase-3/9 (P < 0.001, P < 0.01) levels and elevated nitric oxide (only glycated low-density lipoproteins). In glycated low-density lipoprotein/glycated high-density lipoprotein-treated groups, native high-density lipoprotein treatment reduced malondialdehyde, protein carbonyl, annexin-V, caspase-3/9 levels (P < 0.001, P < 0.01) and increased glutathione; nitric oxide levels (only with gly-HDL). Both melatonin and high-density lipoproteins should be regarded as novel promising mechanism-based potential therapeutic targets to prevent atherothrombosis in diabetics.

Dual effects of testosterone in Behcet's disease: implications for a role in disease pathogenesis.

Behcet's disease (BD) exhibits more severe disease course and higher mortality among male patients. However, underlying mechanisms of gender differences in clinical manifestations and disease severity are unclear. The aim of this study was to determine whether testosterone (T) has any role on BD pathogenesis. We studied peripheral blood mononuclear cells (PBMC) and neutrophils of BD patients and controls. Functional assay of neutrophils, cytokine measurements of culture supernatants and gene expressions on both cells were analyzed before and after T incubation. Neutrophils were significantly activated after incubation with T in only BD patients. Incubation with T caused significantly elevated interleukin (IL)-12 and IL-2 in BD. Gene expression of IL-10 was significantly downregulated after incubation with T in BD, especially in male patients. The same difference was observed in IL-10 levels in culture supernatant after T. Baseline TLR4 expression was significantly higher in BD patients compared to healthy donors (HC). Toll-like receptor (TLR) 4 expression on PBMC was significantly elevated in female BD patients. ERAP1 expressions of all patients and controls were decreased under the T effect but it differed significantly between BD vs HC. Baseline IL23R expression was higher in BD males compared with females but the difference disappeared after T. When BD patients were analyzed separately, baseline C-C motif chemokine receptor1 (CCR1), STAT4, TLR4 and KLRC4 expressions were lower in males. Despite immunosuppressive behavior in healthy subjects, T causes neutrophil hyperactivation and TH1 type immune alterations in BD patients. Our results suggest that T may have a role in BD pathogenesis by altering the expression level of IL-10, TLR4, ERAP1, CCR1.

In vitro effects of nitric oxide donors on apoptosis and oxidative/nitrative protein modifications in ADP-activated platelets.

Nitric oxide (NO) is an important physiological signaling molecule. However, when produced in excessive amounts, NO can also have toxic effects. The aim of this study is to investigate the effects of exogenous- and endogenous-derived NO on oxidative modifications of proteins and apoptosis in activated platelets. Washed platelets were incubated with L-arginine or nitroso-glutathione (GSNO) in the presence of adenosine diphosphate (ADP). After incubation, caspase-3 activity, phosphatidylserine (PS) externalization and the potential of mitochondrial membrane as markers of apoptosis were measured. In addition, the alterations in protein carbonylation (PCO) and nitrotyrosine (NT) formation as markers of protein oxidation were examined. Platelet activation with ADP (20 µM) significantly increased PCO and NT levels and apoptotic events. After incubation with L-arginine, platelet NO production increased significantly. This L-arginine-induced increase caused decreases in formerly increased PCO and NT levels associated with ADP-induced platelet activation. Stimulation of NO production with L-arginine protected platelets from apoptosis. GSNO caused an increase in protein NT levels. Despite this change, GSNO was effective in inhibition of P-selectin expression, platelet aggregation, protein carbonylation and apoptosis. The results suggest that L-arginine and GSNO-mediated NO leads to the inhibition of key apoptotic processes including caspase-3 activation, PS exposure and low mitochondrial membrane potential in washed platelets. The inhibitory effect of platelet clearance of L-arginine and GSNO may be a novel useful therapeutic property in clinical application.

Influence of platelet γ-glutamyltransferase on oxidative stress and apoptosis in the presence of holo-transferrin.

Several studies have documented that formation of oxidant mediators may induce apoptosis in nucleated and anucleated cells by modulating intracellular signalling pathways. Reactive oxygen species (ROS) play a very important role in the platelet function. γ-Glutamyltransferase (GGT), a novel source of cellular production of oxidants in the presence of iron and reduced glutathione (GSH), is also found on platelets. The role of platelet-bound GGT in platelet apoptosis and oxidative stress is unknown. The aim of our study was to determine the effects of platelet GGT activity on oxidative stress and apoptotic events in vitro via determination of lipid peroxidation (LPO), protein oxidation, GSH, catalase, caspase-3 activation and phosphatidylserine (PS) exposure in the presence of holo-transferrin (Tf). Stimulation of platelet GGT activity with GSH and glycylglycine (GlyGly) increased caspase-3 activation and PS exposure. A significant increase in lipid and protein oxidation and decrease in GSH and catalase levels was also observed in platelets with stimulation of GGT activity in the presence of Tf. Inhibition of GGT activity effectively reduced all the markers. These results suggest that generation of ROS by the GGT/GSH/Tf system can modify the platelets' redox environment and induce apoptosis in in vitro conditions.

The investigation of effects of quercetin and its combination with Cisplatin on malignant mesothelioma cells in vitro.

Malignant Mesothelioma (MM) is an aggressive and lethal tumour of the serosal surfaces with poor prognosis. In this study, we have investigated the antiproliferative effect of Quercetin (QU) and its combination with Cisplatin (CIS) on SPC212 and SPC111 cell lines. Our experiments showed that QU significantly reduced the proliferation of cell lines, altered the cell cycle distribution, and increased the level of Caspase 9 (C9) and Caspase 3 (C3) in concentration and time-dependent manner. Additionally, the combination of QU + CIS was found more effective when compared with individual treatment of agents.

Oxidized-LDL and Fe3+/ascorbic acid-induced oxidative modifications and phosphatidylserine exposure in human platelets are reduced by melatonin.

Low-density lipoprotein (LDL) modifications and platelet activation are major risk factors for cardiovascular diseases. When platelets are exposed to oxidative stress, they become activated. Oxidized LDL (ox-LDL) and metal-catalysed oxidation systems such as Fe3+/ascorbic acid increase free radical production. We wanted to verify whether melatonin has a protective effect against oxidative modifications and phosphatidylserine externalization in platelets induced by ox-LDL and Fe3+/ascorbic acid. For in vitro effects of melatonin on platelets, ADP-activated platelets were incubated with ox-LDL or Fe3+/ascorbic acid for 1 h at 37 degrees C with or without melatonin. Then platelet malondialdehyde, protein carbonyl and glutathione levels were measured. Platelet phosphatidylserine exposure was measured with annexin-V using flow cytometry. Malondialdehyde, protein carbonyl and phosphatidylserine levels of platelets treated with Fe3+/ascorbic acid significantly increased compared to the control group. Glutathione contents of Fe3+/ascorbic acid-treated platelets significantly decreased. Melatonin pre-treatment of Fe3+/ascorbic acid-treated platelets caused a mar ked reduction in malondialdehyde and phosphatidylserine levels and a marked increase in glutathione levels. Melatonin also caused non-significant reduction in protein carbonyl contents of Fe3+/ascorbic acid-treated platelets. Malondialdehyde, protein carbonyl and phosphatidylserine levels of platelets treated with ox-LDL also significantly increased compared to the control group. Platelet glutathione levels non-significantly decreased with ox-LDL. With addition of melatonin, malondialdehyde, protein carbonyl and phosphatidylserine levels of platelets treated with ox-LDL significantly decreased. These data suggest that melatonin may protect platelets from iron overload-induced and ox-LDL-induced oxidative modifications and also from the triggering signals of apoptosis activation, possibly due to its scavenger effect on toxic free radicals.

Separation of mesenchymal stem cells with magnetic nanosorbents carrying CD105 and CD73 antibodies in flow-through and batch systems.

The aim of this study is to develop magnetically loaded nanosorbents carrying specific monoclonal antibodies (namely CD105 and CD73) for separation of mesenchymal stem cells from cell suspensions. Super-paramagnetic magnetite (Fe3O4) nanoparticles were produced and then coated with a polymer layer containing carboxylic acid functional groups (average diameter: 153 nm and polydispersity index: 0.229). In order to obtain the nanosorbents, the monoclonal antibodies were immobilized via these functional groups with quite high coupling efficiencies up to 80%. These nanosorbents and also a commercially available one (i.e., microbeads carrying CD105 antibodies from Miltenyi Biotec., Germany) were used for separation of CD105+ and CD73+ mesenchymal stem cells from model cell suspension composed of peripheral blood (97.6%), human bone marrow cells (1.2%) and fibroblastic cells (1.2%). The initial concentrations of the CD105+ and CD73+ cells in this suspension were measured as 5.86% and 6.56%, respectively. A flow-through separation system and a very simple homemade batch separator unit were used. We were able to increase the concentration of CD105+ cells up to about 86% in the flow-through separation system with the nanosorbents produced in this study, which was even significantly better than the commercial one. The separation efficiencies were also very high, especially for the CD73+ cells (reached to about 64%) with the very simple and inexpensive homemade batch unit.