Effects of reprogrammed splenic CD8+ T-cells in vitro and in mice with spontaneous metastatic Lewis lung carcinoma.

BACKGROUND: Metastatic disease is a major and difficult-to-treat complication of lung cancer. Considering insufficient effectiveness of existing therapies and taking into account the current problem of lung cancer chemoresistance, it is necessary to continue the development of new treatments. METHODS: Previously, we have demonstrated the antitumor effects of reprogrammed CD8+ T-cells (rCD8+ T-cells) from the spleen in mice with orthotopic lung carcinoma. Reprogramming was conducted by inhibiting the MAPK/ERK signalling pathway through MEKi and the immune checkpoint PD-1/PD-L1. Concurrently, CD8+ T-cells were trained in Lewis lung carcinoma (LLC) cells. We suggested that rCD8+ T-cells isolated from the spleen might impede the development of metastatic disease. RESULTS: The present study has indicated that the reprogramming procedure enhances the survival and cytotoxicity of splenic CD8+ T-cells in LLC culture. In an LLC model of spontaneous metastasis, splenic rCD8 + T-cell therapy augmented the numbers of CD8+ T-cells and CD4+ T-cells in the lungs of mice. These changes can account for the partial reduction of tumors in the lungs and the mitigation of metastatic activity. CONCLUSIONS: Our proposed reprogramming method enhances the antitumor activity of CD8+ T-cells isolated from the spleen and could be valuable in formulating an approach to treating metastatic disease in patients with lung cancer.

The Role of Cancer and Somatic Stem Cells in the Anti-Inflammatory and Antitumor Effects of Aconitum baicalense Extract on Experimental Breast Cancer.

We studied the effects of the extract of the terrestrial part of Aconitum baicalense in BALB/c female mice at the early stages after the injection of N-methyl-N-nitrosourea (MNU). The extract reduced inflammatory activity and tumor growth in the mammary gland. The antitumor and anti-inflammatory effects of the extract are based on the inhibition of cancer stem cells, hematopoietic stem cells, and hematopoietic progenitor cells that promote inflammation. The extract of A. baicalense disrupted the recruitment of epithelial progenitor cells and angiogenesis precursors to the mammary gland preventing neovascularization and transformation of epithelial cells into tumor cells.

[Aminothiols in blood plasma at different subtypes of ischemic stroke]. / Aminotioly plazmy krovi pri razlichnykh podtipakh ishemicheskogo insul'ta.

OBJECTIVE: To evaluate the hemostasis of plasma aminothiols in different subtypes of ischemic stroke (IS). MATERIAL AND METHODS: The study included 177 patients, aged 62 (55-68) years, admitted in the first 8-24 hours since IS onset. The pathogenetic subtype of IS was clarified according to the results of clinical and instrumental examination by the Trial of ORG 10172 in Acute Stroke Treatment criteria. Determination of the total plasma aminothiols levels, their reduced forms and redox status was performed using the ultra-efficient Acquity H-Class UPLC liquid chromatograph (Waters, CSHA). RESULTS: Large-artery atherosclerosis was diagnosed in 24.3% patients, cardioembolic stroke in 20.3%, lacunar stroke in 55.4%. Significant differences in total levels of cysteine (Cys), glutathione (Gsh) and homocysteine (Hcy) were identified in patients with different IS subtypes. Patients with large-artery atherosclerosis and lacunar stroke showed the highest level of Hcy, patients with cardioembolic stroke had the lowest levels of Cys and Gsh. CONCLUSION: Total levels of plasma aminothiols are associated with different subtypes of IS.

Application of the capillary electrophoresis method for the study of plasma proteins homocysteinylation.

Purpose: Purpose. This article describes the use of capillary electrophoresis with UV detection to determine the ratio of protein-bound homocysteine and cysteine concentrations in human plasma. Methods: Plasma samples were reduced with dithiothreitol and derivatized by thiocarbonyldiimidazole before being filtered again for purification of proteins. The pre-concentration of analytes was carried out directly in the capillary (48.5 cm in length and an inner diameter of 50 mkm) by NaOH post-injection. The eletrophoretic separation of analytes was carried out using 0.2 M ammonium acetate with 25 mM hexadecyltrimethylammonium bromide. Results: Limit of quantitation for homocysteine was 0.8 mkM, reproducible ratio of cysteine/homocysteine <5%, full analysis time 15 min. Conclusion: The ratio of bound cysteine to homocysteine is characterized by the same regularity as the ratio of their total content. It has a fairly high degree of correlation with the level of bound homocysteine and it is characterized by less variability than the level of total homocysteine. This has the advantage of use the bound cysteine/homocysteine ratio for assessing the risk of cardiovascular disease complications.

S-Adenosylhomocysteine Assay in the Urine by Capillary Electrophoresis.

We present a simple and effective method for measuring urine S-adenosylhomocysteine by capillary electrophoresis without using modifiers. The detection threshold of the method is 0.1 µM S-adenosylhomocysteine, the time of analysis 13 min, reproducibility at physiological concentrations within 4%.

[Methylation in the Regulation of the Expression of Chromosome 3 and microRNA Genes in Clear-Cell Renal Cell Carcinomas].

The methylation of CpG islands in promoter regions, together with the interaction of miRNAs with the mRNAs of their target genes on the posttranscriptional level, are complex epigenetic mechanisms that perform the delicate and dynamic regulation of genes and signal transduction pathways in the cell. This review summarizes the results obtained by the authors, as well as the literature data, on the roles of methylation in regulating the protein-coding genes of chromosome 3 and a number of miRNA genes in clear-cell renal cell carcinomas. The results are based on the use of genomic NotI-microarrays (which allow the identification of both methylation and deletions in genes containing CpG islands) and on some other approaches. The application of NotI-microarray technology to the analysis of the chromosome-3 short arm, a region of frequent deletions in tumors, gave us the opportunity to identify many novel genes associated with kidney cancer pathogenesis. The relationship between alterations in the expression leyels and methylation of chromosome 3 genes, kidney cancer progression, and metastasis was shown. New microRNAs involved in kidney cancer pathogenesis were identified as well. The functions of microRNA genes methylated in kidney cancer were discussed.

Upregulation of RHOA and NKIRAS1 genes in lung tumors is associated with loss of their methylation as well as with methylation of regulatory miRNA genes.

Methylation of CpG-islands in promoter regions as well as interaction of miRNAs with messenger RNAs of target genes are related to multilayer mechanisms regulating gene expression. The goal of this study was to assess a possibility for miRNA gene methylation to influence indirectly activation of their target genes in lung tumors. By using a unified collection of samples of non-small cell lung cancer, it was demonstrated that elevated levels of mRNA for RHOA and NKIRAS1 genes were significantly (Spearman rank correlation, P < 10(-11)) associated both with loss of methylation in their CpG-islands and methylation in a number of miRNA genes, which, according to the miRWalk database, were predicted to possess regulatory functions. Novel potential regulatory miRNAs for RHOA (miR-9-1/-3, -34b/c, -129-2, -125b-1, -375, -1258) and NKIRAS1 (miR-34b/c, -129-2, -125b-1, -193a, -124a-1/-2/-3, -212, -132) genes in lung cancer were identified.

Effect of cerebral ischemia on redox status of plasma aminothiols.

We measured the content of reduced aminothiols (cysteine, homocysteine, and glutathione) after mechanical occlusion of the middle cerebral artery in rats. During acute period of ischemia (3 h), these parameters decreased by ~10 times. In 7 days, the content of reduced aminothiols in some animals remained low, but in the others surpassed the initial levels. These results indicate significant impairment of the redox status of the circulating aminothiol pool apparently caused by oxidative stress of the peripheral vascular endothelial occurring as a response to cerebral ischemia. Thus, the total amount of homocysteine is considered a risk factor for stroke, while its reduced fraction can reflect the post stroke state.

3D-technology of the formation and maintenance of single dormant microspheres from 2000 human somatic cells and their reactivation in vitro.

We developed an original reproducible 3D-technology for preparation of single dormant microspheres consisting of 2000 somatic cells. The dynamics of microsphere assembly from mesenchymal and epithelial cells of retinal pigment epithelium was traced using time-lapse microscopy: formation of a loose aggregate over 24 h followed by its gradual consolidation and formation of a compact viable microsphere with a diameter of 100-150 µ by day 7. The cell number in the formed microspheres remains unchanged. Reactivation observed upon fusion of epithelial and/or mesenchymal microspheres results in the formation of a united compact microtissue. The fusion dynamics reproduces spherogenesis irrespective of the initial amount of co-cultured microspheres. Reactivation via two-step induced angiogenesis opens new prospects for production of vascularized microspheres and microtissues.

[Total aminothioles of rat plasma under intraperitoneal and subcutaneous introduction of homocysteine].

In the presented work a variation of total aminothiols (cysteine, glutathione, cysteinylglycine and homocysteine) in blood plasma have been shown at modelling hyperhomocysteinemia by daily intraperitoneal (0.6 mkmol/g body weight) and subcutaneous (0.12 mkmol/g body weight) introduction of homocysteine. During two weeks of the intraperitoneal introduction a significant concentration growth (from -40 to 180 mkM) of cysteine was observed. We also observed a moderate change of concentration levels for glutathione (from 10-15 to 30 mkM) and cysteinylglycine (from 1,5 to 4,5 mkM). The homocysteine level has decreased from 300 to 200-250 mkM at second week of experiments. Experimental results with subcutaneous introduction were similar. In this case a stable homocysteine level (-70 mkM) and increase of cysteine level (to 60 mkM) was observed at second week. These data reflect dose-depended processes of organism adaptation to hyperhomocysteinemia, i.e. reinforced capability for homocysteine metabolism and at the same time retention low glutathione level which correlates with hyperhomocysteinemia degree and duration.

[Induction of the endoplasmic reticulum stress in conditions of acid-base imbalance in human cells of T-lymphoblastic leukemia].

Endoplasmic reticulum stress - typical molecular pathophysiological process that underlies many cardiovascular, endocrine and other diseases. Violations of the protein conformational maturation processes in the endoplasmic reticulum can cause proteotoxic stress. Compensatory mechanisms are activated in response to ER stress include increased expression of enzymes involved in the formation of disulfide bonds in proteins. The sulfhydryl groups oxidation in the electron transport chain (PDI-ERO1-O2) is associated with reactive oxygen species (ROS) generation. Increased activity of oxidoreductase ERO1 could be one of the mechanisms of oxidative stress - however, a direct relationship of ER stress with the overproduction of ROS remains a subject of debate. In this study we have shown that induced by dithiothreitol (DTT) violation of the redox balance with low ROS production leads to the endoplasmic reticulum stress in Jurkat cells. ER-stress in these cells is not associated with increased ROS production, DTT treatment leads to induction of apoptosis. Modulation of intracellular levels of ROS can influence the apoptosis-inducing effects of ER-stress. Given the possible involvement of ROS in the generation of disulfide bonds, the role of ROS in ER stress may be a modulation of disulfide proteome including client proteins.

[A simple method for quantification of modified low-density lipoproteins].

A simple method for quantification of modified low-density lipoproteins (mLDL) in the blood serum containing 8.9% polyvinylpyrrolidone solution 12600 +/- 2700 has been developed. The results show that 10 min incubation of serum in a buffer containing 8.9% PVP leads to complete aggregation mLDL. Atherogenicity of aggregated mLDLs is experimentally confirmed by two independent tests (mLDLs bind and activate the complement system of a guinea pig (pro-inflammatory effect) and cause platelet hyperaggregation (thrombogenic effect)). The proposed method is simple and involves only two steps: mixing the serum with a solution of PVP and recording the turbidity. The method allows to register the presence of mLDL directly in serum without its prior fractionation.

The role of cell fusion in physiological and reparative regeneration of the cerebral cortex.

The prefrontal (cognitive) cerebral cortex of rats was studied by morphological and physiological methods 56 days after stroke induced by photothrombosis. The cognitive capacity impaired after the intervention was completely restored by this time. The count of fused cells (dikaryons) increased significantly in experimental and sham-operated (control) animals in comparison with the early period (7 days) after surgery. Normalization of the dikaryon and mononuclear cell structure was observed after 56 days. Presumably, cell fusion promotes their morphological restoration and regeneration of the lost functional capacity. Fusion is regarded as a manifestation of physiological and reparative regeneration of the cortex.

[Experience with the multiple use of dialyzers]. / Ob opyte mnogokratnogo ispol'zovaniia dializatorov.

Biocompatibility assessed by values of dialysis leukopenia, complement system activation, that of blood elements was compared in the first and second use of capillary dialyzers equipped with various membranes. In the dialyzers re-use biocompatibility improved. Electron microscopy of hollow fiber sections suggested the role of protein layer safety on the membrane surface in biocompatibility under successive employment of the dialyzers. Treatment of the latter with acetic and peracetic acids mixture proved advantageous against other methods. Clinical effect due to dialyzer re-use changed positively.

[Thrombocyte aggregation and the release of ATP from their dense granules in immobilized rats]. / Aggregatsiia trombotsitov i osvobozhdenie ATF iz ikh plotnykh granul u immobilizirovannykh krys.

In August rats, 24-h immobilization stress (IS) decreased the rate and amplitude of platelet aggregation induced by collagen (2-4 microgram/ml) or ADR (0.25-5.0 microM) in platelet-rich plasma, but had no effect on ATP release reaction. Platelet aggregability unchanged in groups deprived of food and water for 24 h or subjected to 6-h IS. Only after 24-h IS were the effects of collagen and low concentrations of ADP potentiated by preincubation of platelets with epinephrine (1 microM). The IS-induced decrease in platelet aggregation to 2 micrograms/ml collagen correlated significantly with the thymus involution, and both effects were most pronounced in rats displaying low activity scores in a hole-board apparatus. The data strongly implicate behavioral variables in the stress-induced changes in platelet functions.

[Thermo-induced structural reorganizations of platelet membranes as affected by aggregation inducers and inhibitors]. / Termoindutsirovannye strukturnye perestroiki membran trombotsitov pod deistviem induktora i ingibitorov agregatsii.

Thermo-induced structural reorganizations in thrombocytes' membranes in the presence of aggregation inductor ADP and aggregation inhibitors--aspirin or alpha-tocopherol at the temperature range of 0-40 degrees C above zero, have been studied by mean of spin probe. It has been shown that in this temperature range alpha-tocopherol decrease structural flexibility of phospholipid fatty acid residua and increase the parameter of order S of 5-doxilstearin probe. ADP and aspirin increase structural flexibility of fatty acid residua of this cells. The temperature plots' study of 2A'11 parameter in EPR, proportional to parameter of order S made possible to find out essential difference between influences of these substances to thrombocyte membrane. The aggregation inductor ADP results in removal of the curve bent-point in Arrhenius coordinates to the lower temperature region (17-13 degrees C) when aspirin and alpha-tocopherol induce either low temperature removal of the bent-point and formation of the supplementary bent in higher temperature region (28 degrees C for alpha-tocopherol, 20 degrees C for aspirin). Such differently directed changes of parameter of order under the aggregation inhibitors' influence is probably connected with different mechanisms of their antiaggregation activity.