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Immune relationships involved in a wide range of immunopathological conditions, including ankylosing spondylitis (AS), are formed due to the characteristics of the subset composition of follicular T helper cells (Tfh) and B lymphocytes. Expression of the HLA-B27 antigen can change the reactivity of cells of the immune system and, accordingly, their interaction and participation in the immunopathogenesis of AS. The aim of this study was to investigate the characteristics of the subset composition of Tfh and B cells in HLA-B27-positive and negative patients with AS. Material and methods. 66 patients (17 women and 49 men) aged 20–58 years with a diagnosis of AS were examined. Molecular genetic research on HLA-B27 expression was carried out using the quantitative PCR method with real-time detection. The subset composition of Tfh and B cells was studied using flow cytometry. Results. An increase in the amount of Tfh2 in the blood is observed in all patients with AS. The number of Tfh1 was reduced in HLA-B27-positive AS patients, but Tfh17 cell content was increased. Changes in the subset composition of B lymphocytes, which were found only in patients with an HLA-B27-positive form of the disease, manifest themselves primarily as an imbalance in the distribution of B cell memory. Only negative correlations of Tfh1 and Tfh17 content with “double-negative” B cell and plasmablast precursors percentage are detected in HLA-B27-negative AS patients. Tfh1 cell number correlate negatively with naïve and activated naïve B cell content in HLA-B27-positive disease, Tfh2 cell percentage – with memory B cell fraction number. CCR6+ Tfh and Tfh17 have positive regulatory effects on plasmablast precursors. Conclusions. The subset composition of Tfh characterizes the dominance in the immunopathogenesis of AS of the direction of the regulatory influence of follicular T helper cells on B lymphocytes regardless of the carriage of the HLA-B27 gene in AS patients. High levels of Tfh type 17 are also detected in HLA-B27-positive patients. The relationships between the subsets of Tfh and B cells in HLA-B27-negative AS patients characterize the presence of processes aimed at inhibiting B cells. The influence of Tfh1 is aimed at suppression of B-cell immunity in HLA-B27-positive AS while Tfh2 and Tfh17 stimulate B-cell mechanisms.

Introduction. Phosphodiesterases (PDEs) are a group of enzymes that hydrolyze the phosphodiester bond in cyclic nucleotides. PDE10A is mainly present in the medium-sized spiny neurons of the striatum. Functionally, PDE10A inhibition imitates the effect of D1-like agonists and D2-like dopamine receptor antagonists, and simultaneously modulating “direct” and “indirect” striato-thalamo-cortical brain pathway. To date, the effects of PDE10A inhibition have been characterized mainly, reproducing the inhibitory motor activity of D2-like dopamine receptor antagonists.The objective was to evaluate the stimulating motor activity of the effect of PDE10A inhibitors, as well as the possible development of tolerance to these effects when they are re-administered.Methods and materials. The hypodopaminergic state in male Wistar stock rats was modeled by administration of the VMAT2 inhibitor tetrabenazine (3 mg/kg). The effects of selective inhibitors PDE10A, MP-10 (0.3–5 mg/kg) and RO5545965 (0.1–0.9 mg/kg), on the motor activity of rats were evaluated with single and repeated administration (5 and 10 days).Results. Switching off PDE10A dose-dependently stimulated the motor activity of rats after administration of tetrabenazine. However, repeated administration of PDE10A inhibitors was accompanied by the development of tolerance to their paradoxical stimulating effect.Conclusion. The development of tolerance may limit the potential clinical use of PDE10A inhibitors to correct hypodopaminergic symptoms in patients with Parkinson’s disease. It is necessary to study the molecular mechanism of this phenomenon.

Aim. To study the functional activity of blood eosinophils in patients with Opisthorchis felineus (O. felineus) invasion.Material and methods. A total of 328 patients with O. felineus invasion (120 men and 208 women, mean age 40.3 years) and 34 practically healthy patients (14 men and 20 women, mean age 41.1 years) aged 18 to 70 years were examined. The main method for diagnosing opisthorchiasis was the determination of eggs or bodies of adult parasites in the duodenal contents and/or feces, which was used in all 328 patients with opisthorchiasis. All patients underwent a complete blood count and biochemical blood test, esophagogastroduodenoscopy and ultrasound examination of the abdominal organs. The study of the functional activity of blood eosinophils was carried out in 42 patients with opisthorchiasis and 34 healthy individuals from the control group by chemiluminescent analysis with the measurement of the intensity of production of reactive oxygen species in spontaneous and zymosan-induced reactions in lucigenin- and luminol-dependent processes.Results. The frequency of eosinophils in the complete blood count above 5% in the examined patients with opisthorchiasis was 19.5%. An increase in the content of eosinophils in the blood in patients with opisthorchiasis was associated with an increase in the frequency of complaints of pain in the right hypochondrium, pruritus, skin rashes, an increase in the proportion of leukopenia and neutropenia, an increase in the level of gamma-glutamyl transpeptidase in the blood, and an increase in the frequency of ultrasound signs of cholecystitis. At the same time, a pronounced increase in the functional activity of blood eosinophils, determined by the chemiluminescent method, was observed in patients with O. felineus invasion.Conclusions. Patients with O. felineus invasion with an increase in the content of eosinophils in the blood due to the likelihood of an aggressive course of the pathology deserve the close attention of practitioners with the need for a thorough comprehensive examination of patients, including immunological methods.

The aim of the work is to review current national and foreign specialized literature on various complications associated with the treatment of chronic limb-threatening ischemia. Atherosclerotic lesion of the lower limb arteries is the most common cause of tissue perfusion deficiency of the lower limbs and can lead to their loss. A radical method of treatment for chronic limb-threatening ischemia (CLTI) remains revascularization. Despite the modern possibilities of surgical treatment and many years of experience in such treatment, the percentage of surgical complications is still substantial. The level of providing qualitative care to patients with CLTI consists in the successful correction of the mentioned complications. That is why the analysis of this problem is an urgent issue of modern angiosurgery. Conclusions. Most complications after surgical treatment of atherosclerotic lower limb lesions could cause both a limb loss and a severe systemic disorder development in general. The presented data of the professional literature review of clinical and experimental studies justify the need for an early management of complications associated with surgical treatment of chronic limb-threatening ischemia. It would contribute not only to the limb preservation, but also prevent the occurrence of severe general somatic complications.

The aim of the present study was to evaluate the subpopulation profile of T and B lymphocytes, and their relationships during therapy of the patients with Graves’ disease (GD) treated by means of radioactive iodine. We have examined 36 women with verified diagnosis of GD. The contents of thyroid hormones were determined by immunoradiometric analysis. The levels of thyroid-stimulating hormone receptor autoantibodies (rTSH) were evaluated by enzyme-linked immunosorbent assay. On the basis of comprehensive pre-therapeutic examination, all patients were exposed to the fixed-activity therapy with radioactive iodine-131 at a dose of 400 to 700 MBq administered orally in isotonic aqueous solution of sodium iodide. 56 practically healthy women were examined as a control group. The phenotype of T and B cells in whole blood was studied by flow cytometry using direct immunofluorescence. It was shown that the patients, prior to treatment with radioactive iodine, had high levels of cellular functional activity, as determined by expression of CD25 antigen on T cells and CD23-antigen on B lymphocytes. Higher functional activity of the cells responsive for adaptive immunity in the patients with GD manifests in the presence of increased levels of autoantibodies to rTSH. By means of correlation analysis, we found that the patients with GD examined before the therapy had the thyroid status may determine the functional stimulation of T and B cells, thus increasing the levels of autoimmune processes. One month after radioiodine therapy (RIT), the GD patients, along with transient hyperthyroidism with increased concentration of autoantibodies to rTSH, showed a reduction of activated T lymphocyte contents (including T helpers and cytotoxic T cells) to control values. However, the level of cytotoxic T lymphocytes in the blood remained low, and the content of Treg cells was significantly increased in the patients. Decreased contents of B cells activated memory B cell to the control levels were found in patients with GD over 1 month after RIT when studying the phenotype of blood B lymphocytes. In this case, increased levels of naive B lymphocytes and B2 cells were detected, as well as decreased numbers of activated B1 lymphocytes. The observed changes in the subpopulation composition of T and B cells, and in their phenotype developed against the background of complete absence of relationships between the studied parameters, thus suggesting loss of thyroid control of immune processes and cooperative cell interaction during the development of the immune response. Generally, the phenotypic changes of T and B lymphocyte subsets in the blood of patients with GD through 1 month after treatment with radioactive iodine may reflect a trend for decreased functional activity of adaptive cellular immunity which may also account for inhibition of autoimmune processes.

Introduction. Changes in the expression of the dopamine transporter (DAT) have been identified in patients with a number of neuropsychiatric disorders, but their significance for pathogenesis remains unclear. DAT knockout rats are a promising model of frontostriatal dysfunctions involved in adapting learning processes to current organism's needs, motivation, and experience.The objective was to evaluate the effect of DAT disruption on the interaction of classical and instrumental conditioning processes (associative learning).Methods and materials. Rats from a local colony were used: knockouts (n=31), heterozygotes (n=32), and wild type rats (n=24). DAT knockout (Slc6a3) was detected by PCR followed by restriction analysis. The rats were kept individually with limited access to food and unlimited access to water. In the operant chambers equipped with a MED-PC interface (MED Associates, USA), 3 experiments were performed: 1) classical conditioning; 2) autoshaping of operant responding; 3) reward learning based on secondary reinforcement. Statistical analysis was performed using SigmaPlot 12.5 (Systat Software Inc., USA) and SPSS Statistics 21 (IBM, USA).Results. We revealed that DAT disruption in rats did not affect the formation of conditioned reflex connections in classical conditioning (1) but was accompanied by impairments in the autoshaping of the operant response (2) and learning based on secondary reinforcement (3).Conclusion. The observed impairments of associative learning might be associated with a decreased incentive value of stimuli in hyperdopaminergic state.

Introduction. There is a discussion about which genotype of viral hepatitis C (HCV) is the most aggressive. Some authors consider that the 1st genotype is the most aggressive, others define the 3rd HCV genotype as a factor that determines the high activity of the pathological process. The solution of this issue is important for optimizing the tactics of patient management.Aim. To compare the clinical and laboratory characteristics and the incidence of liver fibrosis in patients with chronic viral hepatitis C of the first and third genotypes.Materials and methods. 297 patients with genotype 1 of HCV and 231 patients with genotype 3 of HCV were examined. The diagnosis of chronic viral hepatitis C was established according to the recommendations of the European Association for the Study of the Liver (2016, 2018). Liver fibrosis was studied by shear wave transient elastometry with METAVIR score.Results and discussion. The frequency of elevated ALT in the blood prevailed in patients with HCV genotype 3 compared with persons with HCV genotype 1 (90.5% vs. 82.8%, p = 0.02). ALT levels above 3 norms were registered in 29.0% of patients with genotype 3 and in 16.8% patients with HCV genotype 1 (p = 0.001). The frequency of liver fibrosis F2 according to METAVIR was 11.8% in patients with genotype 1 and 21.2% in patients with genotype 3 of HCV (p = 0.005); the frequency of liver fibrosis F3–F4 according to METAVIR was 20.5% in patients with genotype 1 and 32.5% in patients with genotype 3 of HCV (p = 0.003). The combination of high viral load and high inflammatory activity, which was associated with liver fibrosis F3–F4 according to METAVIR in both compared groups, was determined in 16.9% of patients with genotype 3 and only in 10.4% of patients with genotype 1 of HCV (p = 0.04).Conclusion. The obtained data allow us to consider that in the surveyed population the course of chronic viral hepatitis C with genotype 3 is clearly more aggressive than in persons with genotype 1 of HCV.

The aim of the study was to investigate the phenotype of effector T lymphocytes in patients with chronic viral hepatitis C (CVHC) before and after of treatment with direct antiviral drugs depending on the genotype of the virus. 50 patients with CVHC without signs of liver cirrhosis were examined. The diagnosis was made on the basis of epidemiological and clinical laboratory data as recommended by the European Association for the Study of the Liver when specific serological markers of CHCV and RNA of hepatitis C virus (HCV) were detected (EASL, 2016). The determination of HCV RNA was carried out by the method of quantitative polymerase chain reaction in real time. The degree of liver fibrosis in patients with CVHC was assessed using ultrasound elastography. Patients were treated for 3 months with direct antiviral drugs according to the recommendations of the European Association for the Study of the Liver (2016). The control group included 46 practically healthy individuals with severe chronic diseases of various organs and systems excluded during a routine examination, no health complaints, having normal clinical and biochemical blood tests in the absence of markers for viral hepatitis B and C, antibodies to opisthorchis and denying history of alcohol abuse. The study of the subpopulation composition of helper and cytotoxic T lymphocytes was carried out by direct immunofluorescence of whole peripheral blood. We obtained a 100% sustained virological response in patients with 1, 2 and genotypes of CHCV without signs of liver cirrhosis when using therapy with Sofosbuvir (400 mg) and Daclatasvir (60 mg) for 12 weeks. It was found that in CVHC patients were found characteristic features in the phenotypic composition of effector T lymphocytes before and after treatment with direct antiviral drugs in depending on the genotype of HCV. Patients with HCV genotypes 1 and 3 had an increase in the content of terminal differentiated effector (TEMRA) T helpers and effector memory (EM). Only patients with HCV genotype 2 had a decrease in the level of EM T-helper cells in the blood. A decrease in the relative number of T helpers of central memory (СM) was independent of the HCV genotype. The level of effector subpopulations of cytotoxic T lymphocytes in patients with CVHC was consistent with or exceeded control levels in depending on the genotype of HCV. The level of all investigated subpopulations of effector cytotoxic T lymphocytes in patients with HCV genotype 1 was equal to the control values. The number of naïve cytotoxic T cells and CM in peripheral blood in patients with HCV genotype 2 was increased. The content of naïve cytotoxic T lymphocytes, CM and TEMRA in patients with genotype 3 HCV in the blood was increased. The highest viral load was detected in patients with CVHC with genotype 1 HCV. Liver fibrosis was most pronounced in patients with CVHC infection with HCV genotypes 2 and 3. After 3 months of treatment with direct antiviral drugs the patients with CVHC had a reduced content of CM T helpers regardless of the HCV genotype. In addition, patients with HCV genotypes 1 and 3 had a decrease in the number of naïve T helpers and patients with HCV genotypes 2 and 3 had a normalization of the content of naïve cytotoxic T lymphocytes.

The aim of current study was to compare profiles of T cell subsets in the patients with ankylosing spondylitis (AS) who received different modes of genetically engineered biological therapy (GEBT). The research involved 58 patients aged 20 to 58 years diagnosed with AS and treated with anti-TNFα and antiIL-17 drugs, as well as those receiving common anti-inflammatory therapy. The AS diagnostics was based on the modified New York criteria. Disease activity was assessed by means of nomenclature approved by the Assessment of Spondyloarthritis International Society and Outcome Measures in Rheumatology. 45 healthy people aged 18 to 57 were included into the control group. Peripheral blood T cell subsets were analysed by multicolor flow cytometry. It was found that the T lymphocyte subpopulation profiles in AS patients showed significant differences depending on the therapy type. First, T lymphocyte counts were decreased in AS patients receiving traditional anti-inflammatory therapy, whereas relative numbers of T cells with high levels of effector potential and cytokine secretion were increased. Negative correlations between the levels of effector memory and pre-effector cytotoxic T cells and other laboratory and clinical indexes of inflammatory activity in AS may reflect lower efficiency of traditional therapy. Next, the levels of main T cell subsets in AS patients during antiIL-17 therapy fully corresponded to the control values. However, based on numerous correlations between immunological and clinical laboratory parameters, it was concluded that anti-IL-17 therapy had an inhibitory effect on the joint inflammation activity, while the state of T cell subsets was mainly dependent on standard anti-inflammatory therapy. The most pronounced changes in T cell subsets were found in AS patients during anti-TNFα therapy was associated with decreased effector potential of Th cells and cytotoxic T lymphocytes. At the same time, the lowest frequency of extraskeletal manifestations was found in AS patients treated with anti-TNFα drugs. Finally, the higher efficiency of GEBT, compared with conventional methods of therapy, is determined by the effects upon immune targets of AS pathogenesis which manifested, e.g., by changes in the T lymphocyte subpopulation profile. Moreover, usage of anti-TNFα versus anti-IL-17 inhibitors was associated with greater effect upon phenotypic profile of T cells.

The aim of the study was to investigate the phenotypic features of peripheral blood B-lymphocytes in patients with widespread purulent peritonitis (WPP) during postoperative treatment depending on the disease outcome. 52 patients with acute surgical diseases and injuries of the abdominal organs complicated by WPP were examined. Blood sampling was performed before surgery (preoperative period), as well as on day 7, 14 and 21 of the postoperative period. All patients with WPP were divided into two groups depending on the disease outcome during postoperative period: patients with favorable outcome (n = 34), patients with unfavorable outcome (n = 18). 68 healthy subjects were included into control group. Immunophenotyping of blood B-lymphocytes was assessed by flow cytometry and direct immunofluorescence of whole peripheral blood stained with monoclonal antibodies. It was found that patients with WPP in preoperative period contained lowered B1-cell subset compared to control group that was paralleled with low absolute B-lymphocyte count. Moreover, a higher number of activated (based on upregulated CD23 expression) peripheral blood B1-lymphocytes was observed in the patients with unfavorable than in patients with favorable WPP outcome. In addition, dynamics of changes in frequency of B-lymphocytes during postoperative period (day 7—21) varied profoundly depending on the disease outcome. In particular, patients with favorable WPP outcome during postoperative period were found to contain decreased percentage of the most B-cell subsets (including activated cells) examined, whereas patients with unfavorable disease outcome had virtually unaltered B-cell composition in the postoperative period. Moreover, count of total B-cells, naive B-cells and B2-cells negative or positive for CD23 expression was higher in patients with unfavorable outcome than in patients with favorable WPP outcome throughout entire postoperative period. Percentage of some other B-cell subsets in patients with unfavorable than with favorable outcome was also higher only at certain stages of postoperative treatment. It is assumed that such features in peripheral blood B-cell subset composition were closely linked to the disease outcome so that patients with unfavorable WPP outcome turned out to have disturbed B-cell maturation and migration in developing immune response that might be due to lowered total sensitivity of host body to postoperative antibiotic therapy.

The aim was to examine functional activity of peripheral blood neutrophilic granulocytes in patients with Opisthorchis felineus invasion and skin syndrome. Materials and methods. A total of 92 patients with chronic opisthorchiasis were examined, of which 38 patients (20 males and 18 females, average age 35.7±3.9 years) and 54 patients (28 males and 26 females, average age 36.5±4.1 years) with/without skin syndrome, as well as 32 apparently healthy subjects (17 males and 15 females, average age 41.5 years). Detection of adult parasite eggs or bodies in the duodenal contents and/or feces was used as the major diagnostic method of opisthorchiasis used in all 92 opisthorchiasis patients. Liver fibrosis was assessed by elastometry method according to the METAVIR scale in all 92 opisthorchiasis patients. Functional activity of peripheral blood neutrophilic granulocytes was examined in all 92 opisthorchiasis patients and 32 healthy individuals from the control group by chemiluminescent analysis measuring intensity of reactive oxygen species production in spontaneous and zymosan-induced reactions in lucigenin and luminol-dependent processes. Results. Opisthorchiasis patients with vs. without skin syndrome suffered at higher rate from pain in the right hypochondrium, dyspepsia, astheno-vegetative syndrome, skin itching, blood eosinophilia, hyperbilirubinemia, cholestatic syndrome, cholecystitis, hepatomegaly, and had elevated serum immunoglobulin E level. Liver fibrosis F3—F4 according to METAVIR was determined in 18.4% and 11.1% of opisthorchiasis patients with vs. without skin syndrome (OR = 1.78; CI 0.57—5.57; p = 0.05). Both luminol- and lucigenin-dependent processes in opisthorchiasis patients with vs. without skin syndrome revealed significantly decreased functional activity of neutrophilic granulocytes which was evidenced by significantly decreased maximum intensity of reactive oxygen species production and the area under the chemiluminescence curve both in spontaneous and zymosan-induced reaction. Conclusion. The results suggest that the presence of skin syndrome in patients with opisthorchiasis is accompanied by more prominent clinical and laboratory manifestations, decreased functional activity of peripheral blood neutrophils, thereby allowing to consider it as a marker of intense pathology.

The aim is to examine activity of peripheral blood monocytes from patients with opisthorchiasis coupled to intensity of liver fibrosis. Materials and Methods. A total of 74 patients with chronic opisthorchiasis (39 males and 35 females, average age 42.3 years) and 32 apparently healthy subjects (17 males and 15 females, average age 41.5 years) aged 24 to 60 years were enrolled. Opisthorchiasis was mainly diagnosed by parasitological assays to detect adult parasite eggs or bodies in duodenal contents and/or feces in all 74 patients. Liver fibrosis was assessed by elastometry according to the METAVIR scale. Functional activity of peripheral blood monocytes was performed in all patients with opisthorchiasis and healthy individuals from the control group by chemiluminescent analysis to measure intensity of reactive oxygen species production in lucigenin- and luminol-dependent spontaneous and zymosan-induced reactions. Results. Liver fibrosis F2 and F3—F4 stage according to METAVIR scale was found in 20.3% and 17.6% of patients with opisthorchiasis, respectively. While analyzing total pool of reactive oxygen species in the luminol-dependent process in patients with opisthorchiasis, a significantly decreased monocyte functional activity was observed as compared to healthy subjects that was evidenced by significantly decreased maximum intensity of produced reactive oxygen species as well as area under the chemiluminescence curve both in spontaneous and zymosan-induced reaction. Such parameters in liver fibrosis F3—F4 compared to F0—F1 in zymosan-induced response were lowered. Monocyte functional activity in spontaneous luminol-dependent reaction did not differ significantly depending on liver fibrosis intensity in patients with opisthorchiasis. The phagocytosis activation index in patients with opisthorchiasis with liver fibrosis F3—F4 compared to F0—F1 and F2 stage was lower. Similar changes were observed in the lucigenin-dependent reaction. Conclusion. The data obtained undoubtedly provide promising evidence to interpret the mechanisms behind liver fibrosis in patients with opisthorchiasis and create new opportunities for development of diagnostic and therapeutic technologies.

Aim. To study the relationship between the levels of synthesis of reactive oxygen species (ROS) by platelets and neutrophils in patients with coronary heart disease (CHD) before and after coronary artery bypass grafting (CABG), depending on sensitivity to acetylsalicylic acid (ASA).Material and methods. The study included 95 patients with coronary artery disease who are indicated for CABG surgery. The control group consisted of 30 healthy donors. The antiplatelet therapy was stopped for at least 5 days before CABG. In the postoperative period, from the first day, all patients were received 100 mg of an enteric form of acetylsalicylic acid (ASA). Resistance to ASA was determined at the level of platelet aggregation with arachidonic acid ≥20% by optical agregometry at least at one observation point: before CABG, on 1-3 day and on 8-10 day after surgery. We evaluated the spontaneous and induced lucigenin-dependent chemiluminescence (CL) of platelets (ADP induction) and neutrophils (zymosan induction) by the exit time to maximum intensity (Tmax), maximum intensity (Imax) and area (S) under the CL curve.Results. 70.5% sensitive (sASA) and 29.5% resistant (rASA) to ASA patients were revealed. Prior to CABG, in sASA patients, the Imax of spontaneous and zymosan-induced neutrophil CL and CL platelet activity was increased relative to control values. Tmax of spontaneous platelet CL, Imax and S under the ADP-induced platelet CL curve were lower in sASA patients, if to compare with rASA patients. On the 1st and 8-10th day after CABG in sASA patients, the CL indicators of neutrophil and platelet activity also remained elevated compared to control values. On the 1st day after CABG decreased levels of S under the spontaneous CL curve of neutrophils in rASA patients was established compared with sASA patients, and increased levels of Imax and S under the curve of induced neutrophil CL were detected in comparison with the control range. In rASA patients, the values of Tmax of spontaneous platelet CL decreased in relation to the values detected in the control group and sASA patients. On the 8–10th day after CABG, most indicators of spontaneous and zymosan-induced CL neutrophils in rASA patients were also increased compared to control values. In rASA patients a positive correlation of Imax-induced CL was found (r=0.83) on the 1st day after CABG and negative correlations of Tmax of spontaneous CL (r=- 0.75) and S under the curve induced CL (r=-0.70) on the 8-10th day were detected between platelets and neutrophils.Conclusion. In sASA patients with coronary heart disease before and after CABG, a high level of synthesis of superoxide radical by neutrophils and platelets was detected. The relationship between the levels of the synthesis of superoxide radical by neutrophils and platelets was found only after CABG in rASA patients. Increased synthesis of superoxide radical due to metabolic and regulatory relationships in neutrophils and platelets stimulates pro-inflammatory processes in coronary artery disease and determines the sensitivity of platelets to ASA.

The aim of the present study was to analyze the relationships between expression of activation and adhesion receptors on peripheral blood neutrophils, and intracellular activity of some neutrophil enzymes in patients with kidney cancer (KC). Patients and methods: the KC patients (n = 72) (T3N0M0, clear-cell type) were examined prior to surgical treatment at the Krasnoyarsk Regional Oncology Center. The diagnosis was verified histologically for all KC patients. The phenotype of blood neutrophils was studied using flow cytometry. The surface receptor expression levels of the neutrophils were evaluated by mean fluorescence intensity. NAD and NADP-dependent dehydrogenases activities in purified peripheral blood neutrophils were measured by bioluminescent method. Results: we have found that the phenotypic alterations in circulating KC patients’ neutrophils appeared along with inhibition of main intracellular metabolic processes and were closely linked with them. The features of the phenotypic imbalance in the neutrophils from KC patients were associated with a decrease in blood cells expressing adhesive (CD11b and CD62L) and functional (CD64 and HLA-DR) receptors. Moreover, the patient’s neutrophils expressed CD11b, CD16 and HLA-DR on their cell surface more intensively, than neutrophilic leukocytes from control group. These phenotypic changes in KC patients’ blood neutrophils occurred in parallel with pronounced decrease in immature cells numbers. The metabolic changes of neutrophil cytoplasmic compartment in KC patients were determined by a decrease in Glu6PDH activity (a key and initializing enzyme of the pentose phosphate cycle) and NADH-LDH (anaerobic glycolysis). Mitochondrial metabolism in neutrophils of KC patients was characterized by multidirectional changes in the activity of NAD- and NADP-dependent glutamate dehydrogenases (decreased activity of NAD-dependent and increased activity of NADP-dependent) and a decrease in NADH-MDH activity. The established features in mitochondrial enzymes activities suggest some disturbances of NAD-dependent processes that could lead to down-regulation of aerobic energy processes. We guess that the decreased activity of plastic and energy processes in blood neutrophils of KC patients could affect the receptor expression levels. By means of correlation analysis, we have found that the relationships in KC patients were determined by negative effects of NADHGDH and NADH-LDH activities upon expression of activation and adhesion receptors in blood neutrophils. Of these enzymes, only glutathione reductase activity in neutrophils from KC patients was positively linked with the CD23 and HLA-DR expression. Thus, an increase in activity of energy processes (e.g., coupling the tricarboxylic acid cycle to amino acid metabolism) in blood neutrophils from the patients with kidney cancer could stimulate expression levels of activation and adhesion receptors and potentially increase antitumor activity of neutrophils.

Geometrical parameters of the “wheelset—rail track” system are mutually related. Currently, a very topical issue is the question of a possible adjustment of the regulatory framework in the “wheelset—rail track” system in order to take into account the actual state of the track, turnouts and wheel park on the railway network.Any changes made in order to reduce the maintenance of wheelsets or tracks are possible only when the safety of train traffic is unconditionally ensured.The most difficult problem in determining the possibility of changing the standard for the minimum thickness of the flange of car wheels is the problem of passing the turnouts. In the event of an incorrect setting of the standard for the minimum thickness of the flange of the wheels, the passage of carriage along the turnout can be accompanied by unfavorable and even dangerous phenomena. The most dangerous of these are wheel strikes against retracted point rail, ramps on the crosspiece at large angles, and strikes against counter rails. These phenomena cause both disruptions in the geometry of the rail track of the turnout switches and crosses, and fractures of the elements of the turnouts. They should be excluded.Calculations and modeling based on the results of the study of statistical distributions of dimensions obtained by mass measurements on the railway network are the basis for the assignment of norms and tolerances in the “wheelset—rail track” system. The results obtained in the calculations should be verified by dynamic-strength tests directly on the track.The article discusses the results of dynamic and strength tests to determine the effect of cars with different geometric parameters of wheelsets on turnouts with pre-formed average network and maximum permissible dimensions of the rail track parameters on the turnout and in the cross node. The minimum thickness of the wheel flange in the tests was 21.5 mm. The carriages of the test train during the test trips were loaded to the maximum permissible size and in an empty state. The tests were carried out at the Test Center of the JSC “VNIIZhT”.

Lactobacilli are widely used in clinical practice as probiotics, biologically active additives and probiotic products for functional nutrition. Some probiotics can be considered as bacterial vaccines due to induction of immune response, accompanied by production of specific antibodies. The aim of the present study was to evaluate the state of cellular and humoral immunity in women by using probiotic strains of lactobacilli. The study included 31 healthy women aged 25-45 years. As a source of probiotic lactobacterial complex, we used the “Provag” preparation (RU 77.99.11.003.E.003746.02.11 of 11.02.2011, 1 capsule contains 109 Lactobacillus gasseri 57C, Lactobacillus fermentum 57A и Lactobacillus plantarum 57B). The drug was used for 30 days, at a rate of one capsule per day. The immune system was examined twice: before administering the drug and after 30 days of treatment. The study of blood lymphocyte populations and subpopulations was performed by flow cytometry using direct immunofluorescence technique. The concentration of IgA, IgM, IgG in blood serum was determined using enzyme immunoassay. To determine specific antibodies, we used passive hemagglutination reaction with erythrocyte diagnosticum. The complex of probiotic lactobacilli Lactobacillus gasseri, Lactobacillus fermentum and Lactobacillus plantarum corresponding to the “Provag” preparation was used as a source of antigen. It has been revealed that the number of T and B lymphocytes in peripheral blood increased after 30 days of treatment with the probiotic preparation “Provag” in healthy women. Elevated contents of T cells was due to the T helper cell fraction. Increased levels of T helpers and B lymphocytes were associated with stimulation of humoral immunity, as evidenced by increasing concentration of IgA and IgG in blood serum. By means of passive hemagglutination reaction, we have found that 90% of healthy women showed increased concentrations of specific IgA in blood after 30 days of treatment with “Provag” preparation.

The aim of this work was to reveal the interrelations between the number of T regulatory cells (Tregs) in patients with kidney cancer (KC) and phenotype of peripheral blood monocytes and their capacities to produce ROS. Patients with KC (T3N0M0, clear cell type) were examined prior to surgical treatment. Tregs phenotype and blood monocytes were identified by flow cytometry. ROS production of purified monocytes was carried out through the determination of lucigenin- and luminol-dependent spontaneous and zymosan-induced chemiluminescence activity. It has been found that the relative number of Tregs within total lymphocyte subset in KC patients was increased if compared to control values (in KC patients — Me = 6.3%). Then the patients were divided into two groups according to the median of Tregs number (less and more than 6.3%). The most pronounced changes in the phenotype of monocytes and their chemiluminescent activity were found in KC patients with the Tregs count of less than 6.3%. Our findings suggest that low frequency of Tregs in the periphery was associated with increased relative numbers of “intermediate” and “non-classical” (“pro-inflammatory”) monocytes as it was shown on the samples from patients with KC with a low level of Tregs. According to our data, both groups of KC patients had low levels of HLA-DR expression when comparing to control group. Furthermore, both groups of patients had decreased rates of HLA-DR and CD64 co-expressing cells. Changes in the phenotype of monocytes in patients with KC were closely linked with imbalance in ROS production. Thus, the monocytes spontaneous superoxide radical (primary ROS) synthesis in KC patients with a low Treg numbers were characterized by redused NADPH-oxidase activation time and increased level of its activity if compared to patients with a high Treg rates in peripheral blood. Next, the activation index for lucigenin-dependent chemiluminescence in KC patients was reduced, as well as it was independent of circulating Tregs rates and was determined apparently by the insufficiency of metabolic reserves. Similarly, spontaneous secondary ROS production by the monocytes in KC patients was lower then in healthy controls and was also independent of circulating Tregs rates. Finally, the induced secondary ROS synthesis and activation index for their synthesis in monocytes were reduced only in patients with KC with a low number of Tregs in the blood. In general, the characteristics of the chemiluminescent reaction of monocytes in patients with KC determined the imbalance in peripheral blood monocytes primary and secondary ROS production. Monocytes in patients with KC with a low number of Tregs in the blood were characterized by more pro-inflammatory activity due to the rapid activation and intensity of the synthesis of primary ROS.

Aim. To study the production of reactive oxygen species (ROS) by platelets in patients with coronary artery disease (CAD) before and after coronary artery bypass grafting (CABG), depending on their sensitivity to acetylsalicylic acid (ASA) as a part of ASA monotherapy and dual antiplatelet therapy (DAPT) (ASA+clopidogrel).Material and methods. The study included 104 patients with CAD (ASA monotherapy, 64 patients; DAPT, 40 patients). From day 1 after CABG, they took 100 mg a day of enteric-coated ASA. In the DAPT group, clopidogrel was prescribed for 2-3 days after CABG. All measurements were performed before surgery, on the 1st day and days 8-10 after surgery. Control group consisted of 36 healthy donors. Resistance to ASA was determined at a level of optical platelet aggregation with arachidonic acid >20% at least at one observation point. The spontaneous and ADP-induced chemiluminescence (CL) of platelets with luminol and lucigenin was assessed according to the following parameters: time to maximum intensity (Tmax), maximum intensity (Imax), area (S) under the CL curve, and the ratio of ADP-induced CL S to spontaneous CL S.Results. Throughout the study, 71 patients with CAD were sensitive to ASA (sASA) (ASA monotherapy, 46 patients; DAPT, 25 patients), three patients — resistant (rASA) (ASA monotherapy, 1; DAPT, 2). Sensitivity of other 30 patients (ASA monotherapy, 17; DAPT, 13) changed in different follow-up periods. Compared to the control group, sASA patients had increased values of platelet CL parameters throughout the study, while in the rASA group (ASA monotherapy), Tmax was higher before CABG, and in the rASA group (ASA therapy+clopidogrel), Imax and S were higher on the first day after CABG, while Imax — on days 8-10 after CABG. Compared to sASA, the values of S and Imax before CABG, Imax after CABG, as well as Imax and S on the days 8-10 after CABG in rASA (ASA monotherapy) were significantly lower, while in rASA (ASA therapy+clopidogrel), only the Tmax values were lower on the 8-10 days after CABG.Conclusion. In patients with CAD, depending on the sensitivity to ASA and antiplatelet therapy after CABG, the metabolic activity of platelets in terms of ROS production differs. In sASA patients, ROS synthesis is higher than in healthy individuals, while, in rASA patients (ASA monotherapy), platelets produce ROS levels lower than in sASA. CABG surgery and the addition of clopidogrel to ASA therapy leads to increased ROS production in rASA patients in the postoperative period.

Investigation of the activity of supra- and supra (e)-dependent dehydrogenases of the Staphylococcus epidermidis strains isolated from the pharynx mucous membrane of healthy subjects showed that the level of the substrate exchange between the tricarbonic acid cycle and the amino acid metabolism reactions, as well as the intensity of the glycolysis terminal reactions and the glutathione reductase activity depended on the bacteria susceptibility to cefotaxime.

Our study was aimed at investigating dynamic phenotype pattern of peripheral blood NK cells in patients with widespread purulent peritonitis (WPP) during postoperative period depending on disease outcome. A total of 48 patients aged 30–63 with acute surgical diseases and abdominal injuries complicated by WPP were examined. Blood sampling was performed before surgery (preoperative period) as well as on day 7, 14 and 21 during postoperative period. 40 apparently healthy age-matched subjects were included in control group. Peripheral blood NK cell phenotyping was performed by using flow cytometry with directly immunofluorescently tagged antibodies. Mean fluorescence intensity was measured to estimate expression levels of NK cell surface receptors was measured. It was found that in patients with a favorable WPP outcome during preoperative period the percentage of mature NK cells was decreased that was restored by the end of the postoperative period (21 days post-surgery) due to elevated mature, cytotoxic and cytokine-producing NK cell subsets. In addition, percentage of CD11b-positive NK cell subsets was increased upon favorable outcome by the end of postoperative period as well as frequency of CD57-positive NK cells relative to the preoperative period. However, frequency of mature NK cells with unfavorable WPP outcome vs. control vs. favorable outcome was decreased during preoperative and entire postoperative period. Moreover, amount of cytotoxic NK cells was elevated during examination period upon unfavorable WPP outcome. Further, percentage of mature CD11b-positive NK cells in this patient cohort was decreased during preoperative period and post-surgery. Percentage of CD57-positive NK cells was decreased during entire postoperative period in patients with unfavorable vs. favorable outcome vs. control group. At the same time, patients with unfavorable outcome of this infectious-inflammatory disease were shown to display upregulated expression of CD28 and CD57 markers on NK cells. such features identified in phenotype of peripheral blood NK cells in patients with unfavorable WPP outcome reflect abnormal mechanisms in NK cell maturation and migration, which, in turn, determines disturbance in events regulating acute inflammatory reaction in WPP.

Introduction. The urgency of the problem of opistorchiasis is dictated by the long clinical course of the disease with the possibility of formation of hepatobiliary pathology, including cancer. Aim. To study the peculiarities of the production of active oxygen forms of blood phagocytes in patients with opistorchiasis. Material and methods. A total of 42 patients with chronic opisthorchiasis (22 men and 20 women) and 35 apparently healthy patients (18 men and 17 women) between the ages of 24 and 45 were examined. Opistorchiasis was diagnosed by three methods: microscopic examination of duodenal bile, coproovoscopy and serological method. The control group was composed of healthy individuals who underwent routine medical examinations, which included biochemical blood tests, identification of markers for viral hepatitis and antibodies to opistorchs. The culture of neutrophils and blood monocytes was isolated by fractionation of cells on the ficoll with subsequent cleaning from adhering cells. Functional activity of neutrophils and blood monocytes was estimated by chemiluminescence method on production of active oxygen species: basic and after induction with zymosan. Results. In patients with opistorchiasis in comparison with healthy people, in neutrophil granulocytes, the maximum intensity of active oxygen species production decreased and the time to reach the maximum of active oxygen species production was increased both in spontaneous and zymosan-induced reactions in luminoland lucigenin-dependent processes. In monocytes during spontaneous reaction study it was discovered that the maximum intensity of oxygen production in luminoland lucigenindependent processes decreased and the time of reaching the maximum of oxygen production in luminol-dependent process increased in opistorchiasis patients in comparison with healthy people. When studying the zymosan-induced reaction in monocytes, a decrease in the maximum intensity of active oxygen species production in both reactions and an increase in the time to reach the maximum output of active oxygen species in the luminol-dependent process was registered in patients with opistorchiasis in comparison with healthy people. Conclusion. The data obtained indicate low functional activity of blood phagocytes in patients with chronic opistorchiasis in comparison with healthy people.

The aim of the study was to investigate an interdependence between the phenotype of dendritic cells (DC) differentiated from monocytes and the number of pro-inflammatory monocytes in peripheral blood of patients with kidney cancer (KC). The study involved 28 patients at the age of 40-55 years suffering with KC (Т3N0М0, clear cell type) before surgical treatment. The diagnosis was verified histologically. 31 healthy agematched persons were examined as a control group. Mononuclear cells were isolated from heparinized venous blood by centrifugation in a Histopaque®-1077 density gradient followed by plastic adsorption in RPMI 1640 medium supplied with 10% autologous serum. Immature DCs (iDCs) were generated from blood monocytes by culturing for 5 days with GM-CSF and IFNα. Activation of DCs (mDCs) was induced by incubation with the tumor cell lysate and TNFα, followed by incubation for 48 hours. A tumor fragment was used to prepare the lysate of autologous tumor cells. Phenotyping of blood monocytes and DC at various maturation stages was performed by flow cytometry. The numbers of CD14+CD16+ monocytes in peripheral blood of KC patients were decreased (up to 42% of the total monocyte level) against the control ranges. In this regard, the analysis of the dependence between the phenotype of DCs differentiated from monocytes and the number of pro-inflammatory blood monocytes was carried out by comparing the groups with a high content of pro-inflammatory monocytes in the blood in KC patients (> 42%, near-control range) and low content (resp., < 42%). We have found that the contents of tolerogenic iDC in cell culture are increased in KC patients with low amounts of pro-inflammatory monocytes in blood (< 42%). A relatively increased expression of antigen-presenting and co-stimulatory molecules proved to be the specific feature of iDC phenotype in patients with high contents (> 42%) of proinflammatory monocytes in blood. The phenotype of dendritic cells in KC patients with different content of proinflammatory monocytes during maturation/activation showed more differences. In the patients with low levels of pro-inflammatory monocytes, the cell pool of in vitro maturing DCs was characterized by low level of CD86 and HLA-DR receptor expression, thus reflecting a weak co-stimulating and antigen-presenting activity. In the patients with high levels of pro-inflammatory monocytes in blood, the in vitro activated DCs showed higher level of functional activity using the above markers. The revealed differences in the DC phenotype and interrelations with amounts of blood monocyte subpopulations in KC patients may presume the programmed cell differentiation mechanisms depending on the microenvironment, under pathogenic conditions (i.e., in presence of malignant tumor growth).

The aim of the study was to investigate the phenotype of blood T-lymphocytes in the dynamics of the postoperative period in patients with widespread purulent peritonitis (WPP) depending on the outcome of the disease. A total of 36 patients aged 30–65 with acute surgical diseases and abdominal injuries complicated by WPP years were examined. Blood sampling was performed before the operation (preoperative period), and also on the 7th, 14th and 21st day of the postoperative period. 40 relatively healthy people of the same age range were examined as a control. Study of the phenotype of blood T-lymphocytes was carried out by the method of 5-color f low cytometry using direct immunof luorescence of whole peripheral blood. Mean levels of fluorescence were used to evaluate the expression levels of surface receptors. It was established that in the preoperative period in patients with WPP regardless of the outcome of the disease against the background of a decrease in the absolute amount of total lymphocytes in the blood the content of T cells increases. The T-lymphocytes of patients with WPP are significantly more intense expressing the CD28 and CD62L receptors than the cells of healthy people. In the preoperative period and within two weeks of postoperative treatment with a favorable outcome of the WPP an increased amount of CD62L+T-lymphocytes is detected in comparison with the indices of patients with an unfavorable outcome of the disease. Other features of the phenotypic composition of T-lymphocytes in patients with a favorable outcome of WPP is an increase in T-regulatory activity which manifests itself both in the preoperative period and within two weeks of postoperative treatment. With a favorable outcome of WPP an increase in the number of activated cytotoxic T-lymphocytes is observed on the 14th day of treatment which with an unfavorable outcome is observed only on the 21st day of treatment. It is assumed that T-cell suppression and activation of cytotoxic T cells are the factors determining a decrease in the intensity of inflammatory processes in WPP and thereby increasing the level of the favorable outcome of the disease. In case of an unfavorable outcome of the disease, postoperative therapy has a weaker or delayed effect on the dynamics of the studied parameters than with a favorable outcome. With a favorable outcome of WPP an increase in the CD3+CD57+cell count is observed already from the second week of treatment and is more pronounced whereas in the case of an adverse outcome of WPP an increase in the level of these cells is observed only at the third week of treatment. However, the level of expression of CD57 on T-lymphocytes is more pronounced throughout the course of the examination with an unfavorable outcome of WPP.

The aim of the study was to investigate a dependence of respiratory burst state in neutrophils on activities of their intracellular enzymes in patients with acute destructive pancreatitis (ADP) of different severity. The study included 50 patients with ADP of moderate (17 cases) and severe degree (33 cases). A group of 47 healthy people was examined as controls. The respiratory burst state was examined in neutrophilic granulocytes by means of chemiluminescence assays. A study of NAD(P)-dependent dehydrogenases activity in blood neutrophils was performed using bioluminescent analysis. We have revealed that a decrease in spontaneous and induced synthesis of superoxide radical by neutrophils was detected in ADP patients, independently of the disease severity. Kinetics of primary ROS synthesis was also impaired in patients with severe ADP. In patients with moderate disorder, the level of secondary ROS synthesis by neutrophils proved to be increased, whereas, in cases of severe disease, a disturbed kinetics of secondary ROS synthesis by neutrophils was detected at a resting state, showing increased synthetic level upon additional induction by zymosan. Metabolism of neutrophils in patients with ADP is characterized by activation of plastic processes (due to the products of the pentose phosphate cycle) and aerobic energy (increased substrate flow intensity in the cycle of tricarboxylic acids). However, NADPH neutrophilic pool in patients with moderate disorder could be additionally supported by enzymatic malic enzyme reactions and NADP-dependent glutamate dehydrogenase. Activation of peroxidation events in patients with severe ADP is revealed, which needs NADPH compensation. The state of energy processes in blood neutrophils in patients with ADP is characterized by lacking changes in glycolytic activity, and increased intensity of substrate flux along tricarboxylic acids cycle. Activity of aerobic processes in patients with moderate disease is maintained by the products of amino acid metabolism (via glutamate dehydrogenase), whereas, in severe ADP it may be provided by products of lipid catabolism. Using correlation analysis, a dependence of respiratory burst of neutrophils on the state of their metabolism was studied. We have found that intensity and kinetics of respiratory burst in the neutrophils of controls depends only on the activity of NADP-dependent dehydrogenases. The changes in cellular metabolic activity in the patients with moderate ADP led to disturbances of their regulatory effect upon the state of neutrophil respiratory burst. In patients with severe disorder, the degree a neutrophil respiratory burst is stimulated by reductive amination of α-ketoglutarate, being, however, inhibited by intracellular peroxidation processes.

Fungi are among the most diverse and ecologically important kingdoms in life. However, the distributional ranges of fungi remain largely unknown as do the ecological mechanisms that shape their distributions1,2. To provide an integrated view of the spatial and seasonal dynamics of fungi, we implemented a globally distributed standardized aerial sampling of fungal spores3. The vast majority of operational taxonomic units were detected within only one climatic zone, and the spatiotemporal patterns of species richness and community composition were mostly explained by annual mean air temperature. Tropical regions hosted the highest fungal diversity except for lichenized, ericoid mycorrhizal and ectomycorrhizal fungi, which reached their peak diversity in temperate regions. The sensitivity in climatic responses was associated with phylogenetic relatedness, suggesting that large-scale distributions of some fungal groups are partially constrained by their ancestral niche. There was a strong phylogenetic signal in seasonal sensitivity, suggesting that some groups of fungi have retained their ancestral trait of sporulating for only a short period. Overall, our results show that the hyperdiverse kingdom of fungi follows globally highly predictable spatial and temporal dynamics, with seasonality in both species richness and community composition increasing with latitude. Our study reports patterns resembling those described for other major groups of organisms, thus making a major contribution to the long-standing debate on whether organisms with a microbial lifestyle follow the global biodiversity paradigms known for macroorganisms4,5.