Heat Shock Protein HSP27 and the Status of the Glutathione System in Dexamethasone-Induced Apoptosis of Jurkat Tumor Cells.

We studied the effect of the HSP27 inhibitor, 5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl)-isoxazole, at a final concentration of 0.1 µM and/or the apoptosis inducer dexamethasone at a final concentration of 10 µM on the content of hydroxyl radical, reduced and oxidized glutathione, HSP27, activity of glutathione reductase, glutathione peroxidase, caspase-3, and the number of Annexin+ Jurkat tumor cells. The involvement of HSP27 in apoptosis of Jurkat tumor cells was demonstrated. Simultaneous exposure to the HSP27 inhibitor and dexamethasone resulted in an increase in the level of HSP27 against the background of developing oxidative stress (increase in the concentration of hydroxyl radicals and changes in the state of the glutathione system).

Human Mesenchymal Stem Cells as a Carrier for a Cell-Mediated Drug Delivery.

A number of preclinical and clinical studies have demonstrated the efficiency of mesenchymal stromal cells to serve as an excellent base for a cell-mediated drug delivery system. Cell-based targeted drug delivery has received much attention as a system to facilitate the uptake a nd transfer of active substances to specific organs and tissues with high efficiency. Human mesenchymal stem cells (MSCs) are attracting increased interest as a promising tool for cell-based therapy due to their high proliferative capacity, multi-potency, and anti-inflammatory and immunomodulatory properties. In particular, these cells are potentially suitable for use as encapsulated drug transporters to sites of inflammation. Here, we studied the in vitro effects of incorporating synthetic polymer microcapsules at various microcapsule-to-cell ratios on the morphology, ultrastructure, cytokine profile, and migration ability of human adipose-derived MSCs at various time points post-phagocytosis. The data show that under appropriate conditions, human MSCs can be efficiently loaded with synthesized microcapsules without damaging the cell's structural integrity with unexpressed cytokine secretion, retained motility, and ability to migrate through 8 µm pores. Thus, the strategy of using human MSCs as a delivery vehicle for transferring microcapsules, containing bioactive material, across the tissue-blood or tumor-blood barriers to facilitate the treatment of stroke, cancer, or inflammatory diseases may open a new therapeutic perspective.

Graphene Oxide Nanoparticels Interaction with Jurkat Cell Line in Cell-IQ System.

In recent years, materials based on graphene oxide (GO) have been actively studied for their use in biomedicine. The aim of our study was to investigate the increase in cell mass and viability of Jurkat tumor line T cells during 24 h of contact with GO nanoparticles in the Cell-IQ system of intravital observation. We used nanoparticles of different sizes coated with linear or branched polyethylene glycol (PEG) at concentrations of 5 and 25 µg/mL. It was shown for the first time that direct contact with GO nanoparticles reduced the growth in cell mass at the visualization points by more than twofold, regardless of nanoparticle size and concentration. Moreover, the number of live cells in the culture decreased by 5-9% after 24 h of monitoring. Thus, PEG-coated GO nanoparticles were found to suppress the proliferation and viability of Jurkat cell line T lymphocytes.

Surface characterization and biological assessment of corrosion-resistant a-C:H:SiOx PACVD coating for Ti-6Al-4V alloy.

The paper focuses on the SiOx-doped amorphous hydrocarbon (a-C:H:SiOx) coating on the titanium (Ti-6Al-4V) alloy substrate obtained by plasma-assisted chemical vapor deposition (PACVD) in a mixture of argon gas and polyphenylmethylsiloxane vapor using a bipolar substrate bias. It is shown that the a-C:H:SiOx coating deposition results in the formation of a negative surface potential important for application of this coating for medical implants. The a-C:H:SiOx coatings improve the corrosion resistance of Ti alloy to 0.5 M NaCl solution and phosphate-buffered saline. In particular, the corrosion current density of the a-C:H:SiOx-coated sample in a 0.5 M NaCl solution at 22 °C decreases from 1∙10-8 to 1.7∙10-10 A/cm2, that reduces the corrosion rate from 9∙10-5 to 15∙10-7 mm/year. The a-C:H:SiOx coating facilitates the surface endothelization of an implant located in the thoracic aorta of a mini pig, and reduces the risk of thrombosis and implant failure. This effect can be explained by the ability of the a-C:H:SiOx coating ability to reduce in vitro a 24-hour secretion of pro-inflammatory interleukins (IL-6, IL-12(p70), IL-15, and IL-17) and cytokines (IFN-g and TNF-a) by blood mononuclear cells (MNCs) and elevates the concentration of anti-inflammatory interleukin IL-1Ra. In vitro analysis shows no cytotoxicity of the a-C:H:SiOx coating for the human blood MNCs, suggesting a promising PACVD on Ti alloys for cardiovascular implants, including pumps for mechanical heart support systems.

[Osteogenic and angiogenic properties of heparin as a system of biomolecule delivery for bone bioengineering: a brief critical review]. / Osteogennye i angiogennye svoistva geparina kak sistemy dostavki biomolekul pri bioinzhenerii kosti: kratkii kriticheskii obzor.

The review discusses the complex, ambiguous and individual effects of heparin and its derivatives on the bone and circulatory systems, in dependence of the dosage, the state of the cells and tissues of recipients. General data on the anticoagulant activity of heparin and its derivatives are presented; aspects of the effect of heparin on mesenchymal cells and tissues and its role in angiogenesis are considered in details. Particular attention is paid to the ability of heparin to bind osteogenic and angiogenic biomolecules: thus us especially important for the development of systems for their delivery and sustained controlled release. A schematic representation of the positive and side effects of heparin as a delivery system for biomolecules in tissue engineering is proposed.

Effects of Bariatric Surgeries on the Size of Myocardial Infarction and Ghrelin Level in Rats with Experimental Decompensated Type 2 Diabetes Mellitus.

The effects of bariatric surgeries (sleeve gastrectomy and ileal transposition) on the dynamics of changes in ghrelin level were studied in rats with severe decompensated type 2 diabetes mellitus under conditions of glucose challenge as well as on the size of myocardial infarction in these animals. Diabetes was modelled by high fat diet and a single administration of streptozotocin (25 mg/kg, intraperitoneally). Both bariatric surgeries significantly decreased glucose-induced ghrelin level in the blood of rats with type 2 diabetes mellitus, which attested to an increase in the tissue sensitivity to ghrelin. Sleeve gastrectomy resulted in a decrease in the size of myocardial infarction in diabetic rats, which was calculated as the ratio of the necrosis zone to the zone of the risk of myocardial infarction. Ileal transposition had no effect on this parameter. Our data can be used as the basis for optimization of treatment approaches when using bariatric surgery in the treatment of patients with severe forms of type 2 diabetes mellitus with a high risk of cardiovascular diseases.

[Secretion of niche signal molecules in conditions of osteogenic differentiation of multipotent mesenchymal stromal cells induced by textured calcium phosphate coating]. / Sekretsiia signal'nykh molekul krovetvornykh nish v usloviiakh osteogennoi differentsirovki mul'tipotentnykh mezenkhimnykh stromal'nykh kletok, indutsirovannoi rel'efnym kal'tsii-fosfatnym pokrytiem.

Secretion of 21 cytokines, chemokines and growth factors (LIF, SCF, SDF-1a, SCGF-b, M-CSF, MCP-3, MIF, MIG, TRAIL, GRO-a; IL-1a, IL-2ra, IL-3, IL-12(p40), IL-16, IL-18, HGF, TNF-b, b-NGF, IFN-a2, CTACK) has been studied in vitro in the culture of human adipose-derived multipotent mesenchymal stromal cells (hAMMSCs) in conditions of its osteogenic differentiation caused by 14-day contact with calcium phosphate (CP) surface with different roughness. Bilateral X-ray amorphous CP coatings were prepared on the samples of commercially pure titanium in the anodal regime using a micro-arc method. An aqueous solution prepared from 20 wt% phosphoric acid, 6 wt% dissolved hydrohyapatite nanopowder (particle diameter 10-30 nm with single agglomerates up to 100 nm), and 9 wt% dissolved calcium carbonate was used to obtain CP coating. hAMMSCs isolated from lipoaspirate were co-cultured after 4 passages with the CP-coated samples at final concentration of 1.5´105 viable karyocytes per 1.5 mL of standard nutrition medium (without osteogenic stimulators) for 14 days (a determination of [CD45,34,14,20], CD73, CD90 и CD105 cell immunophenotype; an analysis of secretory activity) and 21 days (alizarin red S staining of culture) with medium replacement every 3-4 days. Under conditions of in vitro contact with rough CP coating hAMMSCs differentiated into osteoblasts synthesizing the mineralized bone matrix; this was accompanied by 2-3-fold increasing ratio of [CD45,34,14,20]+ hemopoietic cells. The following humoral factors of hemopoietic niches acted as the signal molecules escalating in vitro the hemopoietic base in 14 days of differentiating three-dimensional culture of hAMMSCs: either leukemia inhibitory factor (LIF) and stem cell factor (SCF) cytokines under mean index of CP roughness Ra=2.4-2.6 mm or stromal derived factor-1 (SDF-1a, CXCL12 chemokine) under Ra=3.1-4.4 mm.

[The effect of different types of bariatric surgery on the metabolic and hormonal parameters in rats with decompensed form of type 2 diabetes mellitus.]

Currently, one of the approaches to correct metabolic disorders in the type 2 diabetes mellitus (DM2) with obesity are bariatric surgery (BS), including sleeve gastrectomy (SG), gastric bypass (GB) and ileal transposition (IT). However, their effectiveness and impact on the hypothalamic signaling and hormonal status in severe forms of DM2 without obesity remain little studied. The aim of the work was to study the effect of IT, SG and GB on the insulin, leptin, ghrelin and glucagon-like peptide-1 (GLP-1) levels in the blood and on the expression of the genes encoding the main components of the hypothalamic signaling systems in rats with decompensated form of DM2, which was induced by a high-fat diet (3 months) and a single low dose of streptozotocin (25 mg/kg, 2 months after the start of the diet). In diabetic rats, a significantly expressed hyperglycemia, an impaired glucose tolerance, a decrease in glucose-stimulated GLP-1 level, a slight decrease in the insulin and leptin levels and an slight increase in ghrelin level were detected. In the hypothalamus, the expression of the genes encoding GLP-1 receptor, orexigenic agouti-related peptide (AgRP), as well as phosphotyrosine phosphatase 1B and SOCS3, the negative regulators of the leptin and insulin pathways was increased. In diabetic rats, the IT reduced the glucose levels 120 minutes after glucose load, increased the basal and glucose-stimulated GLP-1 levels, normalized the gene expression for phosphotyrosine phosphatase 1B, SOCS3, AgRP and GLP-1 receptor, which indicates the restoration of the hypothalamic signaling responsible for the control of energy metabolism and insulin sensitivity. In the case of SG and GB, an improvement in the glucose tolerance was found, and in the case of SG, an increase in the basal and glucose-stimulated GLP-1 levels was shown. However, no significant effect on the expression of the hypothalamic genes in SG and GB was found. Thus, IT is the most effective of all studied BS in the treatment of severe forms of DM2 without obesity.

[Factors governing development of nonalcoholic fatty liver disease and insulin resistance in obesity]. / Faktory, sposobstvuiushchie razvitiiu nealkogol'noi zhirovoi bolezni pecheni i insulinorezistentnosti pri ozhirenii.

The factors promoting development of non-alcoholic fatty liver disease in patients with obesity and different state of carbohydrate metabolism have been studied. 43 patients were examined; these included 26 patients with abdominal obesity (BMI=52.9±7.9 kg/m2). The control group consisted of 17 conditionally healthy donors without obesity (BMI=18.9-24.9 kg/m2), seven of them formed a comparison group that was included to compare the results of study on the levels of tissue-specific expression of HSP70 mRNA. The study of mRNA expression was performed by real-time PCR. The concentration of IL-6 and TNF-a was measured in blood serum by the ELISA method. In patients with obesity with diabetes mellitus type 2 (DM2), a significant increase in the serum level of proinflammatory cytokines was found in comparison with the group of patients without DM2 and control. The results of histological examination of liver biopsy specimens in obese patients revealed the most pronounced changes in the group of DM2 patients. Regardless of the stage of nonalcoholic fatty liver disease in obese DM2 patients, an increase in the area of fatty inclusions (relative to the group without type 2 diabetes) was recorded. The study of the HSP70 gene expression in peripheral blood mononuclear cells allowed its significant increase relative to the comparison group. The relationship between the level of expression of the HSP70 gene in metabolically active tissues (visceral, subcutaneous adipose tissue and liver) established in all obese patients with the serum content of proinflammatory cytokines (IL-6 and TNF-a) may indicate suppression of HSP70 expression in these tissues, background of systemic and local inflammation in obesity.

[The role of single nucleotide polymorphisms in GIPR gene in the changes of secretion in hormones and adipokines in patients with obesity with type 2 diabetes]. / Rol' odnonukleotidnykh polimorfizmov gena GIPR v reguliatsii sekretsii gormonov i adipokinov pri ozhirenii, oslozhnennom sakharnym diabetom 2 tipa.

The relationship between the rs2302382, rs8111428 and Glu354Gln (rs1800437) polymorphisms in GIPR (glucosedependent insulinotropic polypeptide receptor) gene and plasma levels of mediators involved in the regulation of carbohydrate metabolism in obese patients with type 2 diabetes (before and after a test breakfast) was investigated. The contribution of polymorphic variants of rs2302382, rs8111428 in GIPR gene in the predisposition to type 2 diabetes in individuals belonging to the Slavic population of Russia was found. Polymorphisms rs2302382 and rs8111428 in the GIPR gene were characterized by the nonequilibrium cohesion. The decrease in the level of expression of the GIPR gene in adipose tissue of the small intestine mesentery in the carriers of the CC genotype rs2302382 and AA rs8111428 was associated with the increase in the plasma leptin level, whereas during normal expression, the plasma content of insulin, and GIP (in persons with the genotype of the polymorphism rs2302382 and AG polymorphism rs8111428), resistin and ghrelin (in individuals with the genotype of the polymorphism rs2302382) increased. We propose the stimulating effect of GIP on the secretion of resistin, leptin and ghrelin, with an increase in insulin production in obese patients with type 2 diabetes.

Cell-IQ visualization of motility, cell mass, and osteogenic differentiation of multipotent mesenchymal stromal cells cultured with relief calcium phosphate coating.

The Cell-IQ continuous surveillance system allowed us to establish the following changes in a 14- day culture in vitro: a twofold suppression of the directional migration of multipotent mesenchymal stromal cells of human adipose tissue (MMSC-AT) towards the samples with a microarc calcium phosphate (CP) coating from synthetic hydroxyapatite; a tenfold decrease in the cell mass on the interphase with the samples, which was accompanied by a slight reduction in the expression of membrane determinants of stromal stem cells; and an enhancement of their osteogenic differentiation (osteocalcin secretion and mineralized matrix formation) on the 21st day of the study. Calcium phosphate particles, but not the calcium and phosphorus ions, may trigger the phenotypic transformation of the MMSC-AT behavior in vitro.

[The influence of methylprednisolone on the ability of CD4+CD95+HLA-DR+ T-cells to produce proinflammatory medators in cultures of TCR-activated CD3+CD45RO+ T-lymphocytes from patients with rheumatoid arthritis]. / Vliianie metilprednizolona na sposobnost' CD4+CD95+HLA-DR+ T-kletok v kul'turakh TCR-aktivirovannykh CD3+CD45RO+ T-limfotsitov bol'nykh revmatoidnym artritom produtsirovat' provospalitel'nye mediatory.

The effect of different concentrations of the glucocorticoid (GC) methylprednisolone (MP) on CD4+CD95+HLA-DR+ T-cells and their ability to produce proinflammatory mediators in cultures of TCR-stimulated CD3+CD45RO+ T-lymphocytes in the in vitro system was investigated. T cells were obtained from healthy donors and patients with rheumatoid arthritis (RA).Under conditions of TCR-activation, MP increased the number of CD4+HLA-DR+CD95+ cells in CD3+CD45RO+ cultures obtained from RA patients and did not change their content in the control group. In general, MP decreased production of proinflammatory factors (IFN-, IL-2, IL-17, IL-21 and TNF-) by TCR-activated CD3+CD45RO+ cells from healthy donors and RA, consistent with the overall immunosuppressive mechanism of GC action. The correlation between CD4+CD45RO+HLA-DR+CD95+ T-cell contents and parameters reflecting production of proinflammatory mediators (IL-17, IL-21 and TNF-) in RA patients indicates maintenance of the pro-inflammatory potential of this T-cell population exposed to GC action. We suggest that relative resistance of CD4+CD45RO+CD95+HLA-DR+ T-cells of RA patients to the suppressor effect of GC leads to maintenance and even enhancement in the functional capacities of autoreactive cells in the pathogenesis of RA.

Imbalance of morphofunctional responses of Jurkat T lymphoblasts at short-term culturing with relief zinc- or copper-containing calcium phosphate coating on titanium.

Morphofunctional response of Jurkat T cells that were cultured for 24 h on substrates prepared from commercially pure titanium with relief microarc bilateral calcium phosphate coating containing copper or zinc was studied. Changes in the concentration of essential trace elements contained in this coating can cause significant imbalance of molecular processes of differentiation, secretion, apoptosis, and necrosis and reduce tumor cell survival.

Comparative Evaluation of ß-Catenin and E-Cadherin Expression in Liquid Aspiration Biopsy Specimens of Thyroid Nodules.

We compared the results of gene molecular and immunocytochemical studies of ß-catenin and E-cadherin in different variants of nodular thyroid disease (nodular colloid goiter, follicular thyroid adenocarcinoma, papillary thyroid cancer) and revealed changes of the function of the E-cadherin/ß-catenin complex leading to switching from adhesion function of ß-catenin in nodular colloid goiter to predominantly transcriptional activity in papillary carcinoma. The results confirm the important role of disturbances in E-cadherin-ß-catenin interactions in the mechanisms of malignant transformation of follicular epithelium.

MORPHOFUNCTIONAL CHANGES OF JURKAT LINE T-LYMPHOBLASTS AT SHORT-TERM CONTACT WITH RELIEF CALCIUM PHOSPHATE SURFACE.

Human leukemic T-lymphoblastoid cells (hereinafter Jurkat T-cells) were used to model T-lymphocytes morphofunctional reaction to 24-h in vitro contact with relief (roughness index Ra = 2.2--2.7 mm) pure titanium substrates (12_12_1 mm3) covered by calcium phosphate (CP) bilateral coating that was prepared by micro-arc method. Jurkat T-cell culture on plastic surface of well plate (2D control of culture growth), as well as the cells that contacted for 24 h with oxide (TiO2) micro-arc coating on pure titanium substrate (3D control) served as comparison tests. 27--98 % of immortalized cells in 2D control culture had CD3+CD4+CD71+CD45RA+ immunophenotype and secreted IL-2, IL-4, IL-8, IL-10 and TNFa, but not IL-1b and IL-6. Other markers of cell activation, differentiation, maturation and death (CD8, CD16, CD56, CD25, CD95) were found at 0­2.5% of the cell population. Microtextured CP surface elevated statistically IL-8 outcome to 183 and 160 % of corresponding control values in 2D- and 3D-cultures of Jurkat T-cells. CD4/CD8 ratio fell to 9 : 1 (at 13 : 1 and 82 : 1 in 2D- and 3D-controls, respectively) both by CD4+ depletion and by CD8+ cell percent raise. Total amount of cells (TAC) in Jurkat T-cell culture after 24-h contact with CP coating was decreased to 88 % 2D control level (P < 0.04) that could favor to a suppression of cell division. TAC reduction in Jurkat T-cell culture was accompanied by accelerated IL-8 spontaneous secretion (r = ­0.97, P < 0.00009). For all this, IL-8 induced apoptosis (r = 0.94, P < 0.0001) in low concentrations (pg/ml). The obtained result is the feature of Jurkat T-cells reaction on CP but not TiO2 coatings, and it may be used in case of materials selection for endoprosthesis replacement and fracture osteosynthesis in patients suffering by hematological and bone malignancies.

[PHENOTYPIC CHARACTERIZATION AND FUNCTIONAL FEATURES OF MEMORY T- AND B-CELLS].

In this review, we systematized the data that characterize phenotypic properties and functional features of T- and B-lymphocytes of immune memory. We examin the organization of T-cells of immune memory in a population, and their selective distribution in the organism according to their effector potential.

[The influence of testosterone and ß-estradiol on T-lymphocytes activation associated with IL-2 production and expression of CD25 (IL-2Rα) molecules].

We have shown that testosterone and ß-estradiol in vitro have effects on naïve (CD45RA+) and priming (CD45RO+) T-lymphocytes, which associated with the production of IL-2 and CD25/IL-2Rα expression. Testosterone can inhibit naïve and priming T-cell function activity in our study. Moreover it was shown that naïve lymphocytes are more sensitive to testosterone than primed. We have found dose dependent suppressive ß-estradiol effect on IL-2 production by activated CD45RA+ and CD45RO+ lymphocytes which leads to uniform decreasing CD25-positive T-cells number.

[Dose-response effect of steroid hormones on the Gfi1 and U2afil4 gene expression in T lymphocytes at different stages of differentiation].

Alternative splicing of Ptprc gene is a key event in memory T cell differentiation. This gene encodes transmembrane tyrosine phosphatase CD45. One of potential mechanisms of alternative splicing regulation is based on antagonistic effects of auxiliary splicing factor U2AF26 and transcription factor Gfi1. These two proteins regulate antigen-dependent T cell activation. We have shown that steroid hormones have different effects on U2af1l4 and Gfi1 transcription regulation in dissimilar differentiation stage cell culture, subjected to antigen-independent stimulation. Low concentrations of glucocorticoid (Dex) and female sex hormone (Est) can activate expression of U2af1l4 in re-stimulated cells that probably induce terminal receptor CD45 isoforms formation mechanism, whereas high doses of hormones inhibit the process. In the same conditions Dex in a wide range of concentrations (10(-5)-10(-7) M) and Est (10(-6) and 10(-7) M) activate U2af1l4 gene expression that probably leads to "surrogate memory T cells" formation. Dose dependent testosterone (Test) effect is opposite to Est and Dex effect on priming (CD45RO+) and naive (CD45RA+) lymphocytes. The role of steroid hormones in memory T cell differentiation in antigen-independent stimulation conditions is of great interest for the understanding of chronic hormonal and immune disbalance mechanisms.

[The impact of surgical correction of obesity (laparoscopic gastro-bypass surgery) under metabolic syndrome on the biochemical blood indicators].

In nowadays the wide propagation of surgical treatment of metabolic syndrome using the laparoscopic gastro-bypass surgery can be explained hy severe consequences of clinical manifestation of metabolic syndrome. The risk of the mentioned method has to be clear-cut assessed. The comprehensive analysis of changes in indicators of metabolism has to be applied to patients with metabolic syndrome before and after laparoscopic gastro-bypass surgery. The study was carried out on the sampling of 41 patients, 16 patients before and 25 after laparoscopic gastro-bypass surgery. In patients with metabolic syndrome after laparoscopic gastro-bypass surgery the normalization of concentration of glucose and glycated hemoglobin was established. The level of triglycerides, low density lipoproteins and reactive protein reliably decreased as compared with indicators of in non-operated patients. After laparoscopic gastro-bypass surgery, the positive dynamics of activity of enzymes of blood serum was noted.

[Cytokine-mediated apoptosis of granulocytes eosinophilicus in eosinophilia].

The influence of recombinant forms of cytokincs (IL-5, IL-3 and eotaxin) on programmed destruction of eosinophils obtained from patients with diseases associated with high eosinophilia of blood (malignant diseases of system of blood, opisthorchosis) was studied in vitro. It was shown that all examined categories of patients irrespective of their nosology demonstrated a low level of spontaneous apoptosis of eosinophils. Cultivation of cosinophils isolated from peripheral blood of the patients with invasion O. felineus in vitro with r-IL-5, r-IL-3 and r-eotaxin decreased the number of eosinophilic cells undergoing apoptosis in comparison with spontaneous one. At the same time, incubation of eosinophils obtained from the patients with malignant diseases of system of blood, associated with cosinophilic syndrome, with r-IL-5, r-IL-3 and r-eotaxin allowed to ascertain the absence of sensitivity of eosinophilic cells to the antiapoptotic effect of these cytocines.