[The features of regulatory T-blood cells subpopulation and the level of CD25 expression in patients with Graves' disease in dynamics after radioactive iodine therapy].

BACKGROUND: The content of regulatory T cells (Treg) at different stages in formation of effector subpopulations and the level of CD25 expression on the membrane of their various fractions in Graves' disease can determine the long-term autoimmune process persistence and be the target of immunotropic therapy of the disease. AIM: To study the features of regulatory T-blood cells subpopulation and the level of CD25 expression in patients with Graves' disease in dynamics after radioactive iodine therapy (RIT) to identify the specific Treg subpopulations for potential immunotropic therapy targets of the disease. MATERIALS AND METHODS: A single-center, prospective, cohort, open, controlled study was conducted with the participation of women with laboratory-confirmed Graves' disease. The features of regulatory T-blood cells subpopulation and the level of expression (MFI) CD25 surface receptor were studied by flow cytometry using direct immunofluorescence using monoclonal antibodies. RESULTS: The study included 36 women with recurrent Graves' disease, middle age 46.34±14.32 years. In patients with Graves' disease before and during the entire period after RIT a low percentage of naive (CD45R0-CD62L+) and terminally differentiated (CD45R0-CD62L-) Treg was established relative to the control, and on 3 and 6 months after RIT a significant decrease of cells with this phenotype was revealed relative to the values detected in patients before and 1 month after RIT (p<0.001). Against the background of compensated hypothyroidism the most significant changes of expression CD25 receptor in patients with Graves' disease were found on 3 and 6 months after RIT: reduced levels of MFI CD25 on surface of naive and terminally differentiated Treg. CONCLUSION: A decrease in the level of naive Treg was found (apparently due to a violation of differentiation processes in thymus) and terminally differentiated Tregs (due to maturation and survival processes), which are supplemented by a reduced expression of the CD25 receptor on the surface of these cells and do not depend on hyperthyroidism compensation, the titer of TSH receptor antibodies, previous conservative therapy with thiamazole and RIT. The obtained new data reveal the role of naive and terminally differentiated Treg subpopulations in immunopathogenesis and help to outline further ways to develop approaches for immunotropic therapy.

Phenotype of Urine Sediment Cells in Patients with Bladder Cancer.

Phenotype of urine sediment cells were studied in patients with bladder cancer depending on the cancer stage and recurrence prognosis. In T1N0M0 stage, the number of lymphocytes decreased, in T2N0M0 stage, the most pronounced shift was an increase in the number of erythrocytes. Irrespectively of the disease stage, we observed increased number of innate immunity cells and cells that inhibit antitumor immunity in the composition of the leukocyte fraction of urine sediment cells. At T1N0M0 stage, the epithelial-endothelial fraction was characterized by increased content of cells expressing CD13 marker (responsible for tumor growth and metastasis) and reduced number of cells expressing CD15 marker (responsible for intercellular adhesion). In patients developing relapse of bladder cancer, the number of lymphocytes was decreased in urine sediment cells and the number of epithelial and endothelial cells expressing CD13 marker was increased.

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MicroRNAs epigenetically regulate physiological and pathological processes. Previously, we found that miR-204-5p is expressed at low levels in melanoma cells, and an increase in its level leads to a change in proliferation, migration, and invasion of these cancer cells. Now, using bioinformatics analysis, it has been shown that the target of miR-204-5p is FOXC1 transcription factor, which is implicated in carcinogenesis. Using the luciferase reporter assay, it was found that miR-204-5p suppresses expression of the FOXC1 gene by binding to its 3' non-coding region. Transfection of small interfering RNA (siRNA) targeting FOXC1 into melanoma cells caused a decrease in miR-204-5p levels, which is consistent with the generally accepted concept of feedback regulation of miRNA expression by target genes. According to the results of the MTT test and fluorescence microscopy, the proliferation level of melanoma cells under the influence of siRNA to FOXC1 decreased 72 h after transfection. Changes in the ratio of cells by cell cycle phase were analyzed using flow cytometry. Regulatory relationships between FOXC1 and miR-204-5p, and an inhibitory effect of FOXC1 knockdown on melanoma cell proliferation were revealed. Based on the results, it can be assumed that miR-204-5p regulates proliferation of melanoma cells by affecting FOXC1 expression.

Changes in the Subpopulation Composition and Phagocytic Activity of Monocytes in Patients with Kidney Cancer under the Influence of Metabolites In Vitro.

We studied changes in the subpopulation composition and phagocytic activity of monocytes in patients with kidney cancer under the influence of metabolites of the tumor microenvironment in vitro (lactate, ADP, and glutamate). Incubation with metabolites caused similar shifts in the monocyte subsets in kidney cancer patients (an increase in the relative content of classical CD14++CD16- monocytes and a decrease in the content of the fraction of intermediate CD14++CD16+ monocytes), but different changes in their phagocytic activity. The regulatory effect of metabolites on cells is realized by various mechanisms: receptor, metabolic, and epigenomic. The reactions of monocytes to metabolites in vitro confirm the existence of a distant metabolic effect of the tumor on blood cells that should be taken into account when developing new immunotherapeutic methods.

[T-lymphocytes phenotypic composition of peripheral blood in patients with Graves' disease undergoing conservative therapy with thiamazole].

BACKGROUND: Effective control of autoimmune inflammation in Graves' disease determines necessity to study the T helper (Th) and cytotoxic T-lymphocytes dysfunction, as well as the level of regulatory T-cells (Treg) activation in patients with Graves' disease on thyrostatic medication, which will clarify the immunomodulatory effects of long-term thiamazole treatment serve as targets for more specific therapies. AIM: To study the phenotypic composition of T-lymphocytes in the peripheral blood of patients with Graves' disease to assess the direction of immune response depending on thimazole-induced euthyroidism duration. MATERIALS AND METHODS: A single-center, cohort, continuous, open-label, controlled trial was conducted to assess the phenotypic composition of T-lymphocytes in peripheral blood in women with Graves' disease on long-term thiamazole treatment. The phenotypic composition of T-lymphocytes was determined by flow cytometry using direct immunofluorescence with conjugated FITC monoclonal antibodies depending on the duration of thimazole-induced euthyroidism of long-term thiamazole treatment. RESULTS: The study included 135 women with Graves' disease, mean age 43.09±12.81 years, 120 (88.91%) with a relapse of the disease and 15 (11.09%) with newly diagnosed hyperthyroidism. An increase of activated CD3+CD4+CD25+ was found in patients with Graves' disease with a duration of thimazole-induced euthyroidism 5-8 months and 9-12 months, respectively, Me=0.94 (0.48-1.45), p=0.020) and Me=0.95 (0.41-1.80), p=0.025), in control group - Me=0.12 (0.03-0.68). Compared to the control an increase of CD4+CD25+CD127Low (Treg) was found in patients with a duration of thimazole-induced euthyroidism 5-8 and 9-12 months. The content of Treg in peripheral blood in Graves' disease patients with a duration of thimazole-induced euthyroidism more than 12 months decreases, but remains elevated relative to the control. CONCLUSION: In patients with Graves' disease with a duration of thimazole-induced euthyroidism 5-8 months and 9-12 months the level of Treg has been increased. The increase of activated Th (CD3+CD4+CD25+) persists independently of thimazole-induced euthyroidism. In patients with Graves' disease with a duration of thimazole-induced euthyroidism for more than 12 months, there is a compensatory increase in regulatory T-lymphocyte, and the total number of T-helpers is restored to the control.

[Functional activity of blood neutrophilic granulocytes in patients with opisthorchiasis, depending on the severity of liver fibrosis].

AIM: To study the activity of neutrophilic granulocytes in patients with opisthorchiasis, depending on the severity of liver fibrosis. MATERIALS AND METHODS: A total of 74 patients with chronic opisthorchiasis (39 men and 35 women, average age 42.3 years) and 32 practically healthy patients (17 men and 15 women, average age 41.5 years) aged 24 to 60 years were examined. Diagnosis of opisthorchiasis was carried out by two methods: coprooscopy and identification of eggs or bodies of adult parasites in duodenal content. Liver fibrosis was determined by the method of elastometry according to the METAVIR scale in all 74 patients with opisthorchiasis. The study of the functional activity of neutrophils in the blood was performed to all 74 patients with opisthorchiasis and 32 healthy individuals from the control group by chemiluminescent analysis with measurement of the reactive oxygen species (ROS) production intensity in a spontaneous and zymosan-induced reaction in lucigenin and luminol-dependent processes. RESULTS: Liver fibrosis F2 by METAVIR was registered in 20.3% of the examined individuals, liver fibrosis F3F4 by METAVIR was detected in 17.6% of patients with opisthorchiasis. In patients with opisthorchiasis with liver fibrosis F3F4 by METAVIR, a significant decrease in the functional activity of neutrophilic granulocytes was registered in comparison with individuals with liver fibrosis F0F1 by METAVIR, as evidenced by a significant decrease in the maximum intensity of ROS production (Imax) and the area under the curve (S) chemiluminescence in lucigenin and luminol-dependent processes both in the spontaneous and zymosan-induced reaction. CONCLUSION: These results provide new information to explain the mechanisms of liver fibrosis in patients opisthorchiasis and create opportunities for the development of diagnostics and preventive technologies.

Chemiluminescent Analysis of Reactive Oxygen Species Synthesis by Platelets from Patients with Coronary Heart Disease.

A novel chemiluminescent method was developed to evaluate ROS generation by platelets. This method allows measuring activities of NADPH oxidase (NOX2) and enzymes synthesizing secondary ROS (superoxide dismutase, catalase, etc.) in resting and ADP-activated platelets (inductor of platelet aggregation and ROS generation) using a small number of cells. The method was tested in the examination of patients with coronary heart disease. It was found that platelets from patients with coronary heart disease were characterized by NOX2 activation, while cell metabolism is tuned for a long-term intensive production of superoxide anion radical. The enzymes synthesizing secondary ROS were also activated, but cell metabolism could not maintain their enhanced activity for a long time.

The Spatial Metabolic Heterogeneity of Solid Type of Enrlich Carcinoma.

In this work, we investigated the NAD(P)-dependent dehydrogenase activity in different fields of the solid form of Ehrlich carcinoma. It was shown that there is a metabolic distinction between different fields of the solid tumor. In this way, there is a significant difference between the ascites and solid type of Ehrlich carcinoma. In the central area of the tested sample of tumor tissue, there is a high intensity of energetic metabolism and biosynthetic processes as opposed to the peripheral areas. Previously, we proposed the concept of nonspecific metabolic reaction of the cell in organism upon the influence of negative factors. In accordance with this concept, our results indicate the development of adaptation in the tumor cells in the central area of the tested sample.

[Possible Genetic Predictors of Cardiovascular Complications After Coronary Artery Bypass Surgery].

PURPOSE: to investigate associations of single nucleotide polymorphisms (SNPs) rs2046934, rs1126643, rs5918, rs6065, rs4244285; rs4986893 with cardiovascular complications (CVC) in patients after CABG. MATERIALS AND METHODS: We enrolled in this study 130 patients with stable functional class II-IV angina. After CABG 69 patients received ASA (100 mg of enteric form), 61 patients received dual antiplatelet therapy (ASA 100 mg + clopidogrel 75 mg). Mean follow up period was 10.9±5.2 months. The primary composite endpoint included all-cause mortality, myocardial infarction (MI), and ischemic stroke. The control group comprised 185 healthy volunteers. Platelet function was assessed using light transmission aggregometry with ADP (5µM) and arachidonic acid (1 mM). The following single nucleotide polymorphisms (SNPs) were identified by real-time PCR: rs2046934 (H1/H2) on P2RY12 [encoding platelet ADP receptor] (n=100); rs1126643 (C807T, Phe224Phe) on ITGA2 [encoding collagen receptor] (n=87); rs5918 (176T>C, Leu33Pro) on ITGB3 [encoding fibrinogen receptor) (n=91); rs6065 (Thr145Met) on GP1BA [encoding platelet receptor for Von Willebrand factor] (n=114); rs4244285 (*2) (n=84), and rs4986893 (*3) (n=83) on СYP2C19 [encoding cytochrome P450 activity]. RESULTS: The prevalence of rs5918, rs6065, rs4244285, rs4986893, rs2046934 did not differ significantly between patients and healthy volunteers. The mutant allele (T) of ITGA2 was detected more often in healthy volunteers: 67.2 % vs 51.7 % (р=0.021). Before and after CABG there was no significant difference in platelet aggregation between carries and non-carries of the mutant ITGA2 allele. Number of CVCs registered during follow-up was 12: 3 strokes, 6 MIs, 3 deaths. Patients with composite mutant alleles of ITGB3+СYP2C19*2 or СYP2C19*2+ITGA2, and with the mutant allele (*2) of СYP2C19 met end points more often than patients with other gene combinations (wild type homozygotes, presence of one mutant allele of ITGB3 or ITGA2, the composite of mutant alleles of ITGB3+ITGA2 or ITGB3+ITGA2+ СYP2C19*2) (hazard ratio 4.0, 95 % confidence interval 2,19-7.29, р=0.008). Carriers of ITGB3 mutant allele showed higher AA-induced platelet reactivity on the 1st-3rd day after CABG. Amplitude of platelet aggregation in carriers of mutant allele was 27.5 % vs 12.7 % in wild type (p=0.016). No differences in platelet reactivity among carriers of other mentioned mutant alleles and wild types were observed. CONCLUSION: Carriage of the combination of mutant alleles ITGB3+СYP2C19*2 or СYP2C19*2+ITGA2 or СYP2C19*2 is possible predictor of CVC in patients after CABG.

[Neutrophil respiratory burst and the state of hemostasis in patients with ischemic and hemorrhagic stroke]. / Osobennosti respiratornogo vzryva neitrofilov i sostoianie gemostaza u patsientov s ishemicheskim i gemorragicheskim insul'tom.

AIM: To study the relationship between the state of the respiratory burst of neutrophils and the indices of hemostasis in patients with ischemic (IS) and hemorrhagic (HS) strokes. MATERIAL AND METHODS: Thirty-three patients with IS and 31 patients with HS were examined in the first 24 hours after an acute cerebrovascular accident (ACA). The diagnosis of ASA was confirmed by neuroimaging methods: computed tomography and magnetic resonance imaging. The determination of the respiratory burst state of blood neutrophils was carried out using chemiluminescence analysis. Indicators of vascular-platelet and coagulation hemostasis were determined in all patients. RESULTS AND CONCLUSION: There were changes in the kinetics and activity of neutrophil respiratory burst in stroke patients. Patients with HS had higher (compared to those of patients with IS) level of spontaneous and induced synthesis of secondary ROS but with a decrease in metabolic resources for their synthesis. Activation of spontaneous and ADP-induced platelet aggregation was detected only in HS. Patients with HS had a higher level of fibrinolytic activity as compared to the patients with IS. There were positive correlations between the kinetic parameters of blood coagulation and the synthesis of primary ROS by neutrophils in patients with HS, whereas the intensity of all ROS synthesis negatively correlated with activated partial thromboplastin time. Therefore, in patients with HS the synergistic relationship between the respiratory burst of neutrophils (pro-inflammatory activity) and coagulation hemostasis was more pronounced compared to patients with IS.

The metabolic changes in tumor-associated macrophages during cancer grow in mice with Ehrlich ascites carcinoma.

In this work, we investigated the activity of the key NAD(P)-dependent dehydrogenases associated with macrophage tumors in mice with Ehrlich ascites carcinoma. It was shown that cancer grow is associated with the development of conditions in macrophages leading to a decrease in the substrate flow intensity in the tricarboxylic acid cycle, deceleration of oxidative deamination of L-glutamate, NADP regeneration, and a decrease in the antioxidant defense efficiency. There results are consistent with our recent concept on the nonspecific metabolic reaction of cells to extreme exposures.

[The alterations of indices of collagen-induced aggregation of thrombocytes in qualified sportsmen in the dynamics of annual macro-cycle.]

The physical load provokes reliable alterations in the blood coagulation system, specifically increasing of blood coagulability, including increasing of thrombus formation. However, the long-term alterations in the blood coagulation system in sportsmen are still unexplored. The article demonstrates that in sportsmen parameters за aggregation capacity of thrombocytes have significant long-term alterations during annual training competition macro-cycle. The maximal alterations have to do with value of lag-phase and area under the curve of aggregation of thrombocytes. The lag-phase no the curve of aggregation of thrombocytes under stimulation with collagen is significantly increased in termination of competition period; it decreases up to control values during resting time in transition period and significantly falls down below control group indices during preparatory period. The area under the curve of aggregation of thrombocytes under stimulation with collagen is reliably decreased as to control values at the termination of competition period. It exceeds the control level during transition period and it has no reliable differences against control values during preparatory period. The alterations of functional activities of thrombocytes significantly effect functioning of immune system that modulates course of processes of adaptation to sport activities in general.

[Types of immune response in advanced suppurative peritonitis]. / Tipy immunnogo reagirovaniya pri rasprostranennom gnoinom peritonite (s kommentariem).

AIM: to assess types of immune response in patients with advanced suppurative peritonitis and course of disease. MATERIAL AND METHODS: We examined 79 patients with acute surgical abdominal diseases and injuries complicated by advanced suppurative peritonitis. Blood immunological parameters were estimated using flowing cytometry and enzyme immunoassay. RESULTS: It was concluded that functional parameters of immune system are very various in patients with advanced suppurative peritonitis. Cluster analysis defined 4 immune types which are determined by different state of congenital and acquired immunity. Immunodeficient and unreactive immune types are unfavorable. Immune types with activation of congenital and acquired immunity are the most favourable. This stratification personifies diagnosis and treatment of immune disorders in patients with advanced suppurative peritonitis.

Antitumor effect of arabinogalactan and platinum complex.

The article presents the results of investigation of antitumor properties of platinum-arabinogalactan complex. We showed the ability of the complex to inhibit the growth of Ehrlich ascites tumor cells. It is found that the distribution of the platinum-arabinogalactan complex is not specific only for tumor cells in mice. The complex was found in all tissues and organs examined (ascites cells, embryonic cells, kidney, and liver). The mechanism of action of the arabinogalactan-platinum complex may be similar to cisplatin as the complex is able to accumulate in tumor cells.

[Platelet Metabolism in Clopidogrel Resistant and Sensitive Patients With Ischemic Heart Disease].

OBJECTIVE: to investigate relationship between activity of platelet nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) dependent dehydrogenases in patients with ischemic heart disease (IHD) after coronary artery bypass grafting (CABG) and their sensitivity to clopidogrel. MATERIAL AND METHODS: We studied 51 male patients with chronic IHD before and after CABG. Control group comprised 35 healthy men. In IHD patients spontaneous and induced platelet aggregation was measured by aggregometry before CABG (in 2 weeks after cessation of antiplatelet drugs), after CABG during therapy with clopidogrel (75 mg/day), and in 6 months after surgery. Patients were classified as clopidogrel sensitive or resistant using cutoff of 50% inhibition of initial ADP (5 M) induced platelet aggregation after 2 weeks of treatment with clopidogrel. Activity of platelet NAD and NADP dependent dehydrogenases was measured by bioluminescent method. RESULTS: There were 41 clopidogrel sensitive and 10 clopidogrel resistant patients. Irrespective of sensitivity to clopidogrel platelet metabolism in IHD patients was characterized by reduced activity of anaerobic energetics, glutathione-dependent antioxidant system, reactions that transport products of lipid catabolism on glycolysis, and NADPH-dependent transfer of substrates from citric acid cycle to amino acid metabolism. Metabolic features of platelets characteristic of clopidogrel resistance were low activity of the key reaction of pentose phosphate cycle reflecting state of plastic metabolism, and substrate flow in the tricarbonic acid cycle - determinant of intensity of aerobic energy. Clopidogrel-sensitive patients had increased platelets NADP-malate dehydrogenase activity (the key reaction of lipid anabolism).

[MORPHOLOGICAL PECULIARITIES OF THE LIVER IN COMMON VOLES INHABITING THE TERRITORY OF BORODINO COAL DEPOSITS AND RECULTIVATION AREAS].

The aim of this work was to study the morphological changes of liver in common voles (Microtus arvalis Pallas) inhabiting the territories of brown coal deposition in Borodino coal opencast (Krasnoyarsk region) and on reclaimed dumps 10 and 20 years after its production. Trapping of the voles (10 animals in each group) living under natural conditions on each territory, was conducted for 30 days. Histological examination of the liver in all animals demonstrated degenerative changes and necrosis of hepatocytes, expressed to a various degree. Morphometric study has shown that the greatest changes in the structure of hepatic stroma and parenchyma took place in voles that lived in the dumps of coal, reclaimed 10 years before. It was found that in the animals of this group, the thickness of hepatocyte plates was increased 1.3 times, while the specific volume of necrotic hepatocytes was twice as much as this parameter in the animals that lived on intact territory.

Evaluation of NAD(P)-Dependent Dehydrogenase Activities in Neutrophilic Granulocytes by the Bioluminescent Method.

Bioluminescent method for measurements of the neutrophilic NAD(P)-dependent dehydrogenases (lactate dehydrogenase, NAD-dependent malate dehydrogenase, NADP-dependent decarboxylating malate dehydrogenase, NAD-dependent isocitrate dehydrogenase, and glucose- 6-phosphate dehydrogenase) is developed. The sensitivity of the method allows minimization of the volume of biological material for measurements to 104 neutrophils per analysis. The method is tried in patients with diffuse purulent peritonitis. Low levels of NADPH synthesis enzymes and high levels of enzymes determining the substrate flow by the Krebs cycle found in these patients can lead to attenuation of functional activity of cells.

[An effect of valdoxan on the hemostasis in patients with acute coronary syndrome in the combination with anxiety and depressive disorders].

OBJECTIVE: To explore the dynamics of hemostasis in patients with acute coronary syndrome (ACS) in the combination with anxiety and depressive disorders (ADD) during therapy with valdoxan (agomelatine). MATERIAL AND METHODS: We examined 196 male and female patients (mean age 64.2 ± 0.8 years) in the first 24 hours of ACS. The patients were divided into three groups: patients with ACS without ADD (n=88), patients with ACS and ADD without valdoxan therapy (n=58) and patients with ACS and ADD treated with valdoxan (n=50). Coagulation and vascular-platelet hemostasis were studied in the first 24 hours of hospitalization and in the 10th day of hospitalization. RESULTS: Patients with ACS revealed high levels of fibrinogen, soluble fibrin-monomer complexes, D-dimer at all stages of the study. In patients with ADD, these parameters were significantly higher compared to patients without ADD. The levels of D-dimer in the 10th day were similar in patients with ADD treated with valdoxan and in patients with ACS without ADD. In both groups of patients with ADD, the levels of spontaneous aggregation in the first and 10th days of hospitalization were not significantly higher than in the control group and in patients with ACS without ADD. All patients with ACS had high values of platelet aggregation stimulated by adenosine diphosphate in dose 0.1 uM. In patients with ACS and ADD treated with valdoxan, a significant reduction of these parameters was noted in the 10th day of treatment compared to the first day of hospitalization. CONCLUSION: The high procoagulant activity of the hemostatic system was observed during all stages of the study in the patients with ACS and ADD who received a combined antithrombotic therapy without valdoxan.

[Role of T- and B-cell immunity in the pathogenesis of cancer].

Adaptive immunity plays a crucial role in the system of anti-tumor defense of the organism. The theory of immune surveillance defines the presence of permanent monitoring of the organism for the detection of malignant transformed cells and their elimination or suppression of the growth. However in some cases tumor cells overcome immune surveillance that leads to the development of cancer.

[The State of Hemostasis and Immune System in Patients With Acute Coronary Syndrome Combined With Anxiety-Depressive Disorder].

Anxiety-depressive disorders (DD) have a negative impact on the course and prognosis of patients with ischemic heart disease, but the mechanisms of their correlation are not studied enough. The purpose of this study was to elucidate special characteristics of hemostasis and immune system in patients with acute coronary syndrome (ACS) combined with ADD. We included into the study 60 patients during first 24 hours of ACS. ADD was identified in 27 patients (45%). Parameters of plasma and vascular-platelet hemostasis and phenotype.