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.

Anticancer Effects of Spiperone in C57BL/6 Mice with Emphysema and Lung Carcinoma.

The antitumor and antimetastatic activity of dopamine D2 receptor antagonists spiperone was studied in C57BL/6 mice in a model of combined pathology (emphysema and lung cancer). Emphysema was induced by administration of LPS and cigarette smoke extract. Lung cancer was induced by injection of Lewis lung carcinoma cells into the lung. It has been shown that under conditions of combined lung pathology, spiperone prevents inflammatory infiltration and emphysematous expansion of the lungs and reduces the size of the primary tumor node, the number of metastases, and the area of the lungs affected by metastases. Spiperone reduces the number of cancer stem cells (CSCs) in the lungs and blood of mice with combined pathology. CSCs isolated from the lungs and blood of mice with combined pathology treated with spiperone had a significantly lower potential to form a tumorosphere in vitro than CSCs from untreated mice with emphysema and lung carcinoma. Thus, blockade of dopamine D2 receptors is a promising approach for correcting combined lung pathology and can be used in the development of a method for treating lung cancer in patients with emphysema.

[MicroRNA Biogenesis in Cell Senescence Induced by Chronic Endoplasmic Reticulum Stress].

MicroRNAs are small noncoding RNAs that regulate gene expression; stabilize the cell phenotype; and play an important role in cell differentiation, development, and apoptosis. A canonical microRNA biogenesis pathway includes several posttranscriptional steps of processing and transport and ends with cytoplasmic cleavage of pre-miRNA by type III ribonuclease DICER to form a mature duplex, which is included in RISC. MicroRNA biogenesis and role in cell stress are still poorly understood. Using flow cytometry and high-throughput analysis of gene expression, we have shown that chronic endoplasmic reticulum (ER) stress, which is associated with improper protein folding in the ER, induce a cellular senescence phenotype in fibroblast-like FRSN cells. While acute ER stress can reduce miRNA biogenesis, chronic stress does not cause a significant drop in global microRNA expression and is accompanied by only a slight decrease in DICER1 mRNA expression. Heterogeneity with respect to lysosomal ß-galactosidase activity was found to increase in the cell population exposed to ER stress. We do not exclude induced cell heterogeneity regarding expression of components of the microRNA biogenesis pathway.

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.

Effect of Platinum Nanoparticles on the Structure of Protein Molecules and Regulation of the Tone of Brain Vessels in Experimental Animals.

The effects of platinum nanoparticles on the morphological structures of the solid phase of blood serum and the tone of cerebral microvessels as indicators of the dynamics of homeostasis were studied on outbred albino male rats (n=40) weighing 300-350 g. Platinum nanoparticles were injected to experimental animals in 1 ml of physiological saline. For systemic BP measurements and blood sampling, the femoral artery was isolated and catheterized. The study of solid phase structures of blood serum was conducted by the method of cuneiform dehydration. The results showed that injection of platinum nanoparticles significantly affected the body of experimental animals.

A Marker of Cerebral Ischemia in Solid State Structures of Blood Serum.

Reversible cerebral ischemia of medium severity was reproduced in male Wistar rats by bilateral occlusion of the common carotid arteries. Solid-phase structures (anisomorphons) were obtained by marginal dehydration of the serum. Small focal isotropic defects in the serum anisomorphon texture were found in 100% cases during occlusion of the carotid arteries. Similar signs were detected in all patients with chronic cerebral ischemia, which proved specificity of this morphological marker of the disease.

Wavelet Analysis of Cerebral Microcirculation in Hypovolemic Shock.

Hypovolemic shock leads to severe cerebral ischemia and increases spectral amplitudes of cerebral blood flow oscillations in respiratory frequency range.

[REGRESSION OF ATHEROSCLECROTIC LESIONS: MEDICAMENTAL AND ALIMENTARY FACTORS].

The article reports results of clinical studies aimed to elucidate the influence of medicines on the size and density of atherosclerotic plaques in the walls of coronary and cerebral arteries. The phenomenon of regression of atherosclerotic lesions in the survivors of Leningrad siege during a long period of starvation is analyzed. The influence of inhibitors of angiotensin-converting enzyme on apoptosis of smooth muscle and foam cells of atherosclerotic plaques in the sanological mechanisms of atherosclerosis is discussed. The concept of natural regression of atherosclerosis is formulated and the necessity of development of the methods for is pharmacological activation are formulated.

Blood serum structures integrated assessment of pathophysiological changes in experimental cerebral ischemia.

The purpose: The purpose. To reveal solid-phase structures of blood serum during and after experimental cerebral ischemia. Methods: Integrated estimation pathophysiological changes at brain ischemia received on solid-phase structures of blood serum (BS) at 27 rats-male Vistar in weight of 300-390 g. Reversible brain ischemia of moderate severity was reproduced imposing surgical clips on both common carotids for 15 minutes then clips removed and provided bloodstream restoration on carotids. Blood took away in Eppendorf`s test tube in volume on 1 ml before imposing clips, directly ahead of their removal and in 30 minutes after bloodstream restoration. By the Method cuneiform dehydration received BS facia (dry a film) which structures analyzed by means of stereomicroscope MZ12 firm «Leica¼. Results of researches fixed on structural parameters BS before imposing clips, after removal clips and in 30 minutes after bloodstream restoration. Results: Before ischemia the integrated picture facias BS rats had the harmonious system organisation: the radial or partially-radial arrangement of the cracks, accurately generated konkrecias that testified to normal physiological condition of experimental animals. During the period occlusions carotids there were markers of a ischemia, activation of a capillary blood-groove, inflammatory reaction and stress. Partially these signs remained at reperfusion. Conclusion: The conducted researches of structures BS on experimental animals have allowed to reveal marker structures BS similar revealed at inspection of people with an ischemia of a brain.

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.

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.

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%.

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.

[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.

[Endotoxin Is a Companent in Pathogenesis of Chronic Viral Diseases].

The level of endotoxin and indicators of activity of antiendotoxin immunity (antibody concentration to glycolipid Re-chemotype and general antigen of enterobacteria) were estimated in serum of 174 patients with persistent viral infections (viruses: herpes simplex, hepatitis C, human immunodeficiency). The presence of markers of systemic inflammatory response syndrome (interleukin IL-1ß) and acquired immunodeficiency (CD4+) in HIV-infected patients were also determined. Persistent viral infections are accompanied by endotoxin aggression intestinal origin (caused by them), which is able to induce the development of systemic inflammatory response syndrome. In HIV-infected patients with this syndrome is cyclical, when the phase of hyperactivity replaced immunodeficiency. Schematically, this process can be represented as the following sequence of events: HIV-mediated damage to the intestinal barrier--the development of endotoxin aggression--induction ofsystemic inflammatory response syndrome--the depletion of the immune system, which is transient and is related to the duration of activity of the virus replication cycle, i.e., with damage to enterocytes. Using antiendotoxin component (means of reducing levels of endotoxin in the blood) in the scheme of treatment of persistent viral infections can serve as an element of a successful prevention of complications.

[Endotoxin theory of atherosclerosis].

The involvement of bacterial lipopolysaccharides in the induction of atherogenesis is postulated. The excess of bacterial lipopolysaccharides (endotoxin aggression) is observed in the general blood circulation of volunteers with the presence of atherosclerotic risk factors (obesity, diabetes, stress, pathology of haemostasis, kidney, intestines, etc.). The results allow to be optimistic about the prospects for preventing the development and progression of atherosclerotic pathology.

[Changes of endotoxin concentration in blood serum in patients with uncomplicated acute myocardial Q-infarction].

The effectiveness of acute myocardial infarction (AMI) treatment does not correspond to high material costs for the study of its pathogenesis and development of new drugs. This circumstance gives the grounds to assume existence of nowadays unknown mechanisms of emergence and development of this disease. High probability of participation of endotoxin (ET) in the pathogenesis of AMI was theoretically proved by us for more than a quarter of the century ago, but it's clinical evidence to date is not found yet. As a result of the study a significant increase of endotoxin (ET) concentration in the blood serum of patients with AMI increasing from 1 to 14 day of the disease has been found. In women the concentration of ET was higher than in men. It allows to qualify the EA as a factor probably influencing the known difference in AMI tolerance in men and women. The source of ET were Bacteroides (most often--67.8% of patients), Klebsiella, Pseudomonas, Proteus, Escherichia coli. One or two bacteria more often took part in the development of EA. In 9.1% of patients the etiology of EA could not be verified, what indicates the presence of other sources of EA, not evaluated in this study. In 25% of patients with AMI serologic evidence of systemic candidiasis, caused by candida Albicans, has been found, what is able to enhance the biological effects of ET.

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.

[Molecular logic of the endoplasmic reticulum stress signal pathways: the system of unfolded protein response].

Endoplasmic reticulum (ER) is the central organelle of a eukaryotic cell, it provides the synthesis and maturation of the majority of secretory and transmembrane proteins. The intensity of ER-related protein synthesis and ER loading varies in different cell types and depends on the cell microenvironment, physiological state of the cells, the stage of cellular differentiation. Quality control of transmembrane and secretory proteins in ER is a high precision process. The proteins with non-native conformations which are difficult or energetically disadvantageous to refold undergo ubiquitin-dependent proteolytic degradation. Homeostatic control of protein maturation in ER is mediated by a system of interconnected signaling pathways represented sensors located in the lumen of the ER, and effectors, that transmit information to other cell compartments. This system of intracellular signaling pathways play an important role in the endoplasmic reticulum stress and initiates a complex cellular response to the proteins with non-native conformations (Unfolded Protein Response, UPR). However, if homeostasis is not restored, cell death is triggered via apoptosis, which is a supracellular level adaptation mechanism that protects the tissues and the whole organism from dysfunctional and potentially immunogenic unfolded proteins. Malfunctions of the UPR, as well as ER-associated protein degradation (ERAD) process contribute to the development of many diseases: cardiovascular, neurodegenerative, endocrine diseases, autoimmune. The list of factors and mechanisms involved in ER stress is constantly updated, which is a result of significant attention to ER stress as a typical pathophysiological process that forms the basis of many diseases.

[A method of rapid determination of cholesterol in immune complexes].

A method of rapid determination of cholesterol in immune complexes (CIC). In the proposed method precipitate immune complexes containing multiple modified low density lipoproteins from human serum are prepared by treatment with a buffer containing 8.3 PEC 3350, and 3.3% PVP 12600, in a ratio of 1:1.2, incubated for 10 min at room temperature. The precipitate containing the CIC is separated by centrifugation at 3100 g for 5 min at 23 degrees C, dissolved in a buffer without PEG and PVP, the cholesterol is determined using an enzymatic kit and at a level of CIC more than 8.3 mg/dl ascertain higher level. The method improves the accuracy of the quantitative determination of CIC, conduct extensive screening tests to detect atherosclerosis as the pre-clinical stage and monitor the effectiveness of the therapy.