The lecture analyzes the results of research on the role of humoral and cellular mediators, their interaction, as well as the imbalance of angiogenic factors in psoriatic arthritis. The information is presented with identification of the leading typical pathological processes: inflammation and microcirculation disorders, formed due to the activation of protein cascades and interaction of molecular proinflammatory mediators and angiogenic factors. It is known that the clinical phenotypes of psoriatic arthritis are diverse. A deeper understanding of the pathogenesis and changes in the predominant pathological process can become the basis for the development of a personalized treatment strategy based on the pathogenesis to minimize iatrogenic complications and economic costs, as well as for the introduction of modern diagnostic methods for verification, differentiation, and monitoring of psoriatic arthritis in order to timely correct drug treatment.

Aim of the study. To investigate the effect of recombinant galectin-3 on the cytokines secretion of various subpopulations of helper-lymphocytes (Th1, Th2, Th17, Treg) in culture in vitro. Material and methods. The material for the study was peripheral blood from healthy people (n=17), from which lymphocytes were isolated by gradient centrifugation. Lymphocytes were cultured for 72 hours in a CO2-incubator with recombinant galectin-3 and activating antibodies (antiCD3/antiCD28). The concentration of cytokines (IL-10, IL-13, IL-17A, IL-22, IFN, TNF, TGF1) in the supernatants of lymphocyte cultures was determined by enzyme immunoassay. Results. Recombinant galectin-3 in vitro enhanced the secretion of IL-17A, IL-22 by lymphocytes, acting at a dose of 0.5 g/ml; IL-13, TNF and IFN at doses of 0.5 g/ml and 1 /ml (more pronounced when acting at a dose of 0.5 g/ml) and inhibited the production of IL-17, IL-22 at a dose of 1 g/ml and IL-10, TGF1 when testing both concentrations. Conclusion: Galectin-3 has a dose-dependent modulating effect on the cytokine-producing function of healthy donors lymphocytes in vitro. Functional imbalance in blood lymphocytes under the action of recombinant galectin-3 is manifested by induction of pro-inflammatory (IFN, IL-17, IL-22, TNF) and anti-inflammatory (interleukin-13) cytokines secretion, against the background of oppression of the suppressor cytokines (IL-10 and TGF1) production. A detailed study of the immunotropic effects of galectin-3 in relation to individual lymphocytes subpopulations is relevant from the view point of development new approaches to the treatment of tumor and autoimmune diseases accompanied by excessive production of this lectin.

The objective : to evaluate the modulating effect of the Notch signaling pathway on differentiation of Th1 and Th2 lymphocytes in vitro in patients with infiltrative pulmonary tuberculosis. Subjects and Methods . 14 new patients with infiltrative pulmonary tuberculosis were enrolled in the study. Mononuclear leukocytes were isolated from blood by gradient centrifugation. Only Mycobacterium tuberculosis antigens in the form of the CFP10-ESAT6 protein or together with Y-secretase inhibitor DAPT (5 μМ/l or 10 μМ/l) were added to the incubation medium. Cells were cultured for 72 h in a complete nutrient medium with 5% CO 2 at 37°C. Counts of Th1 and Th2 lymphocytes were determined by flow cytometry by evaluating the expression of CD4 receptor and intracellular transcription factors T-bet and GATA-3. Results . Cell stimulation with the CFP10-ESAT6 protein was accompanied by increasing number of Th1 and Th2 lymphocytes only in patients with pulmonary tuberculosis resistant to isoniazid + rifampicin. Adding DAPT at the concentration of 10 μМ/L to the incubation medium in these patients led to the growing number of Th1 lymphocytes and decrease in Th2 lymphocytes. In pulmonary tuberculosis patients who were susceptible to isoniazid + rifampicin, only decrease in the number of Th2-lymphocytes was regeistered. In all groups of subjects, suppression of the Notch signaling pathway led increase in the Th1/Th2 index versus Th1/Th2 stimulated with CFP10-ESAT6 antigens.

The objective: to evaluate the role of IL-23 in the development of Th17 lymphocytes in patients with different clinical and pathogenetic forms of pulmonary tuberculosis. Subjects and Methods. 165 pulmonary tuberculosis patients were examined. Venous blood was used for tests. Mononuclear leukocytes were isolated by centrifugation and monocytes were extracted and transformed into dendritic cells. The concentration of IL-23 in the supernatants of culture suspensions of dendritic cells was determined by ELISA. Immunophenotyping of Th17 lymphocytes (CD4+CD161+IL-17A+ cells) was performed by flow cytometry. Real-time PCR was used to determine the expression of the RORC2 transcription factor gene in lymphocytes. Results. In patients with infiltrative drug susceptible and drug resistant pulmonary tuberculosis against the background of normal production of IL-23 by dendritic cells, an increase in blood level of Th17 lymphocytes and the level of mRNA of the RORC2 transcription factor gene was registered. The course of disseminated pulmonary tuberculosis (regardless of drug susceptibility and resistance) is associated with pronounced decrease in the concentration of IL-23 in vitro and the absence of response from Th17 lymphocytes.

Data on the role of regulatory T lymphocytes (Treg) in the immunopathogenesis of tuberculosis are actively accumulating in the current literature. The overwhelming effect of Treg cells on the proliferation, functional activity of Th1 lymphocytes and antigen-presenting cells allows to consider this population as a possible target of modulation of the immune response in patients with tuberculosis. The Notch signaling pathway participates in the regulation of FoxP3 transcription factor expression and, therefore, is capable of supporting suppressor activity of Treg lymphocytes. A key role in the functioning of the Notch signaling cascade belongs to the enzyme γ-secretase that cleaves the intracellular domain of the receptor (Notch ICD), with the subsequent formation of a complex that regulates cell differentiation. The actively studied inhibitor of γ-secretase is DAPT – N-[N-(3.5-difluorophenacetyl)-L-alanyl]-S-phenylglycine tert-butyl ester). Mononuclear leukocytes isolated from the blood of patients with drug-sensitive and drug-resistant pulmonary tuberculosis by gradient centrifugation before the start of anti-tuberculosis therapy were used as the material for the study. The cells were cultured under conditions of stimulation with Mycobacterium tuberculosis antigens CFP10-ESAT6 or with the addition of γ-secretase inhibitor (DAPT) at doses of 5 μM/L and 10 μM/L in combination with CFP10-ESAT6 at 37 °C and 5% CO 2 for 72 h to the incubation medium. The number of Treg lymphocytes was assessed by flow cytofluorimetry by determining the expression of the CD4 surface receptor (FITC) and the intracellular transcription factor FoxP3 (PE). In intact cell cultures of pulmonary tuberculosis patients, the relative number of Treg lymphocytes was statistically significantly (p < 0.001) higher than that of healthy donors. Stimulation of cells with CFP10-ESAT6 antigens was accompanied by an increase in the proportion of CD4 + FoxP3 + cells in both groups of tuberculosis patients. Addition of γ-secretase inhibitor at a concentration of 5 μM/L to the incubation medium did not lead to statistically significant changes in the number of Treg lymphocytes. The increase in DAPT concentration up to 10 μM/L was accompanied by a decrease in the number of Treg lymphocytes in comparison with the corresponding indices upon stimulation with CFP10-ESAT6 antigens in all groups of the subjects. Regardless of cultivation conditions, the number of CD4 + FoxP3 + cells in patients with drug-resistant mycobacteria exceeded their number in patients with drug-sensitive pulmonary tuberculosis. Inhibition of the Notch signaling pathway by a γ-secretase inhibitor (DAPT) at a concentration of 10 μM/L contributed to a decrease in the number of Treg lymphocytes in patients with drug-sensitive and drug-resistant pulmonary tuberculosis. Reduction of Treg lymphocyte number by γ-secretase inhibitor confirms the importance of Notch signaling cascade as a potential target for correction of Treg lymphocytes immunosuppressive activity and pathogenetic therapy of tuberculosis.

Aim. To identify disturbances of differentiation and subpopulation composition of VEGFR2+ cells in the blood and bone marrow associated with the features of the cytokine profile in the blood and bone marrow in patients with coronary artery disease (CAD) with and without ischemic cardiomyopathy (ICM).Materials and methods. The study included 74 patients with СAD with and without ICM (30 and 44 people, respectively) and 18 healthy donors. In all patients with СAD, peripheral blood sampling was performed immediately before coronary artery bypass grafting, and bone marrow samples were taken during the surgery via a sternal incision. In the healthy donors, only peripheral blood sampling was performed. In the bone marrow and blood samples, the number of VEGFR2+ cells (CD14+VEGFR2+ cells) and their immunophenotypes CD14++CD16-VEGFR2+, CD14++CD16+VEGFR2+, CD14+CD16++VEGFR2+, and CD14+CD16-VEGFR2+ was determined by flow cytometry. Using enzyme-linked immunosorbent assay, the levels of VЕGF-А, TNFα, M-CSF, and IL-13, as well as the content of MCP-1 (only in the blood) and the M-CSF / IL-13 ratio (only in the bone marrow) were determined.Results. The content of CD14+VEGFR2+ cells in the blood of CAD patients with and without ICM was higher than normal values due to the greater number of CD14++CD16-VEGFR2+, CD14++CD16+VEGFR2+, and CD14+CD16++VEGFR2+. In the bone marrow of the patients with ICM, the content of CD14++CD16-VEGFR2+, CD14+CD16++VEGFR2+, and CD14+CD16-VEGFR2+ was lower than in patients with CAD without ICM, and the number of CD14++CD16+VEGFR2+ cells corresponded to that in the controls. Regardless of the presence of ICM in CAD, a high concentration of TNFα and normal levels of VEGF-A and IL-13 were observed in the blood. In CAD without ICM, an excess of MCP-1 and deficiency of M-CSF were revealed in the blood. In the bone marrow, the levels of VEGF-A, TNFα, M-CSF, and IL-13 were comparable between the groups of patients against the background of a decrease in the M-CSF / IL-13 ratio in the patients with ICM.Conclusion. Unlike CAD without cardiomyopathy, in ICM, no excess of VEGFR2+ cells and MCP-1 in the blood is observed, which hinders active migration of CD14+CD16++VEGFR2+ cells from the myeloid tissue, and a decrease in the M-CSF / IL-13 ratio in the bone marrow disrupts differentiation of other forms of VEGFR2+ cells, preventing vascular repair.

Введение. При клинической манифестации туберкулеза легких альвеолярные макрофаги накапливают микобактерии и перестают выполнять свои эффекторные функции. Это связано с конверсией их провоспалительного фенотипа М1 в противовоспалительный М2, что способствует хронизации инфекции. Научная гипотеза исследования предполагает влияние цитокинового статуса организма на поляризацию моноцитов в крови в процессе их миграции к очагу воспаления, определяя дифференцировку и пути активации макрофагов в тканях. Цель исследования - оценка иммунофенотипа моноцитов крови и исследование in vitro уровня секреции иммунорегуляторных цитокинов мононуклеарными лейкоцитами периферической крови у больных с различными клиническими формами туберкулеза легких. Методика. Обследовано 65 пациентов с впервые выявленным туберкулезом легких. Материалом исследования служили венозная кровь и мононуклеарные лейкоциты периферической крови. Исследование иммунофенотипа моноцитов проводили методом проточной цитометрии (цитофлуориметр Cytoflex, Becman Coulter, США) в цельной крови с использованием моноклональных антител («eВioscience», США). Обработку полученных данных проводили с помощью программы «CytExpert 2.0». Определяли количество клеток экспрессирующих поверхностные маркеры: CD14, CD163, CD204 и HLA-DR. Содержание цитокинов (IL-2, IL-10, TGFβ) в супернатантах клеточных культур оценивали с помощью твердофазного иммуноферментного анализа (ELISA). Результаты. Полученные результаты позволяют предположить, что при общем снижении численности циркулирующих CD14-позитивных моноцитов крови у больных туберкулезом легких, независимо от его клинической формы сохраняется высокая экспрессия маркеров активации клеток как по провоспалительному фенотипу М1 (HLA-DR-позитивные моноциты), так и противовоспалительному фенотипу М2 (CD163-позитивные моноциты). При диссеминированной форме заболевания повышается количество противовоспалительных CD204-позитивных моноцитов, предшественников М2-макрофагов, что свидетельствует о доминировании супрессорного типа иммунного ответа. Анализ цитокинового статуса in vitro показал, что течение болезни сопровождается угнетением эффекторных иммунных реакций и повышением уровня противовоспалительных цитокинов. Выявленные изменения в равной степени могут быть как причиной, так и следствием дефицита секреции IL-2. Показано также, что уровень секреции медиаторов с супрессорными эффектами (IL-10, TGFβ) меняется в зависимости от клинической формы заболевания и чувствительности возбудителя к противотуберкулезным препаратам: гиперсекреция IL-10 отмечается у больных с инфильтративным лекарственно-чувствительным, а TGFβ - при диссеминированном лекарственно-устойчивом туберкулезе легких. Заключение. Особенности дифференциации моноцитов крови у больных туберкулезом легких позволили прийти к заключению, что предшественники макрофагов - моноциты, уже в кровотоке начинают экспрессировать маркеры, характерные для разных по функциям М1- и М2-макрофагов, c поляризацией в направлении М2-иммунофенотипа. Следовательно, при развитии туберкулеза легких реализуются механизмы цитокиновой регуляции, подавляющие активацию врожденного иммунитета, что, возможно, является причиной хронизации воспалительного процесса в легких и формирования иммунодефицита индуцированного Mycobacterium tuberculosis. In clinical manifestation of pulmonary tuberculosis, alveolar macrophages perform as a reservoir where they accumulate mycobacteria and lose their effector functions due to the pathological conversion of macrophage pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, which provides transition to chronic infection. The study hypothesis suggested that the cytokine status, as evaluated by leukocyte secretion of cytokines in vitro, influences the monocyte polarization in the blood during their migration to the inflammatory focus, thereby determining differentiation and pathways of macrophage activation in tissues. The aim of this work was to assess the immunophenotype of blood monocytes and the in vitro secretion of immunoregulatory cytokines by mononuclear peripheral blood leukocytes from patients with different clinical forms of pulmonary tuberculosis taking into account the pathogen sensitivity to major anti-tuberculosis drugs. Methods. 65 patients with newly diagnosed pulmonary tuberculosis were evaluated. The study material was venous blood and peripheral blood mononuclear leukocytes. Monocyte immunophenotype was determined in whole blood by flow cytometry on a Cytoflex flow cytometer (Becman Coulter, USA) with monoclonal antibodies (eBioscience, USA). Results were processed with a CytExpert 2.0 software. The number of cells expressing surface markers, CD14, CD163, CD204 and HLA-DR, was determined. Content of cytokines (IL-2, IL-10, TGFβ) in supernatants of cell cultures was measured by enzyme-linked immunosorbent assay (ELISA). Results of the study were processed with a SPSS v.11.0 standard software package. Results. The study results suggested that with an overall decrease in the number of circulating CD14-positive blood monocytes in patients with pulmonary tuberculosis regardless of its clinical form, high expression of cell activation markers remained both for the pro-inflammatory M1 phenotype (HLA-DR-positive monocytes) and the anti-inflammatory M2 phenotype (CD163-positive monocytes). In disseminated tuberculosis, the number of anti-inflammatory CD204-positive monocytes, M2 macrophage precursors, increases indicating predomination of the immunosuppressive response. In vitro analysis of the cytokine status showed that tuberculosis progression is accompanied by inhibition of effector immune responses and increases in anti-inflammatory cytokine concentrations in vitro. These changes may be equally either a cause or a consequence of deficient IL-2 secretion. We also found that the secretion of mediators with suppressor effects (IL-10, TGFβ) varied depending on both the clinical form of tuberculosis and the pathogen sensitivity to anti-TB drugs; IL-10 hypersecretion was observed in patients with drug-sensitive, infiltrative tuberculosis whereas TGFβ hypersecretion was observed in disseminated, drug-resistant tuberculosis. Conclusion. Features of blood monocyte differentiation in patients with pulmonary tuberculosis allowed us to conclude that monocytes, the macrophage precursors, start expressing markers for different functions of M1 and M2 macrophages with polarization toward the M2 immunophenotype already in the bloodstream. Therefore, in the development of pulmonary tuberculosis, cytokine regulation mechanisms become involved in suppressing the activation of innate immunity, which possibly causes chronic inflammation in the lungs and formation of Mtb-induced immunodeficiency.

Background: Monocytes and macrophages play an important role in atherogenesis and myocardial remodeling. Impaired differentiation of monocyte subpopulations may contribute to ischemic cardiomyopathy (ICMP). The aim of the present research was to study the features of the humoral cytokine-dependent regulation of differentiation of classical, intermediate, non-classical and transitional monocytes in bone marrow (BM) of CHD patients with or without ICMP. Materials and Methods: Forty-five patients with coronary heart disease (CHD), with and without ICMP (19 and 26 male patients, respectively), were examined. Subpopulations of classical (CD14++CD16-), intermediate (CD14++CD16+), non-classical (CD14+CD16+), and transitional (CD14+CD16-) monocytes in bone marrow (BM) samples were quantified by flow cytometry. Concentrations of IL-1β, IL-13, TNF-α, IFN-γ, and M-CSF in BM supernatants and blood plasma were evaluated by ELISA. Results: Concentrations of all cytokines in the blood and IL-1β, IL-13, TNF-α, М-CSF in BM supernatants—as well as the capacity of М-CSF to activate, and IL-13 to inhibit, differentiation of classical monocytes from intermediate forms—were not dependent on the clinical form of CHD. Monocytopoiesis in ICMP was characterized by elevated BM concentration of IFN-γ, low М-CSF/IL-13 ratio, and a decreased percentage of classical and intermediate monocytes, accompanied by an increased number of transitional cells in BM, as compared to patients without ICMP. Conclusion: Excess of IFN-γ and low M-CSF/IL-13 ratio in BM were associated with inhibition of differentiation of mature monocyte forms and development of ICMP.

The effects of a fluroquinolone levofloxacin on apoptosis of peripheral blood lymphocytes from patients with infiltrative pulmonary tuberculosis were studied in vitro. It was found that levofloxacin stimulated apoptotic cell death in tuberculosis. Addition of levofloxacin to cell suspension from patients with drug-susceptible form of tuberculosis led to an increase in the number of CD95(+) and AnnV(+) lymphocytes. In patients with drug-resistant form of tuberculosis, only the number of apoptotic lymphocytes, but not the count of CD95(+) cells increased under these conditions.

The study of subpopulation structure of IFNγ-producing T cells in patients with pulmonary tuberculosis revealed a decrease in the number of CD3+ IFNγ+ cells against the background of significantly increased IFNγ secretion in vitro irrespective of the clinical form of the disease and drug sensitivity of M. tuberculosis, most strongly expressed in case of the disseminated tuberculosis. In patients with infiltrative drug-sensitive and drug-resistant pulmonary tuberculosis, increased number of Th1/Th17 lymphocytes (CD4+ IFNγ+IL-17A+) and, conversely, decreased number of blood γδT cells was detected.

The effects levofloxacin (fluoroquinolone) and vaccinal BCG strain on cytokine production by blood mononuclear leukocytes was studied in patients with infiltrative pulmonary tuberculosis. Combined treatment with levofloxacin and vaccinal BCG strain suppressed the production of TNFα in drug-resistant pulmonary tuberculosis and production of IL-12 and IFNγ in drug-sensitive tuberculosis.

ЦИТОКИН-СЕКРЕТОРНАЯ АКТИВНОСТЬ T-ЛИМФОЦИТОВ-ХЕЛПЕРОВ 17 И T-ЛИМФОЦИТОВ-ХЕЛПЕРОВ 1/ T-ЛИМФОЦИТОВ-ХЕЛПЕРОВ 17 ПРИ ТУБЕРКУЛЕЗЕ ЛЕГКИХТ. Е. Кононова, О

Homeostasis of subpopulations Th17- and Treg-lymphocytes plays an important role in a holistic and coordinated process of eradication of pathogens and preventing the spread of infection in the body. Study of molecular mechanisms controlling the balance of these cells in the formation of immune deviation in the pathogenesis of infection are particularly relevant. The article presents the results of a study of mRNA expression of transcription factors Th17- and Treg-lymphocytes--RORC2 and FoxP3, respectively, as well as the presence of these cells in peripheral blood in infectious disease (based on an example of infection caused by Mycobacterium tuberculosis). It was established that during the infiltrative (regardless of drug susceptibility testing) and disseminated drug-susceptible pulmonary tuberculosis accompanied by Th17-polarized differentiation of T-lymphocytes, as evidenced by the increased number of CD4+CD161+IL17A+ cells in the blood in association with increased mRNA expression of the transcription factor RORC2 in lymphocytes. In disseminated drug-resistant pulmonary tuberculosis T-lymphocyte differentiation is carried out mainly in the direction of immunosuppressive Treg-cells, as evidenced by the increase in their number in the blood in association with elevated levels of mRNA expression of the transcription factor FoxP3 in lymphocytes.

In patients with infiltrative pulmonary tuberculosis, the increase in IL-6 secretion, the decrease in TGFβ production (in case of drug resistance of the causative agent), and unchanged level of IL-1β secretion by mononuclear leukocytes in vitro were associated with increased number of CD4+CD161+IL-17A+ Th17 lymphocytes in the peripheral blood. In patients with disseminated drug-resistant pulmonary tuberculosis, TGFβ hyperproduction promoted differentiation of CD4+CD25+FoxP3+ Treg lymphocytes with immunosuppressive activity.

The peripheral blood count of γδТ cells was investigated in patients with new-onset pulmonary tuberculosis depending on the clinical form of the disease, on Mycobacterium tuberculosis susceptibility to antituberculosis drugs, and on the response to intradermal tuberculin administration (Mantoux test). The count of γδТ cells was shown to decrease in all clinical forms of tuberculosis infection; however, the most significant increase in their count was noted in disseminated and fibrocavernous pulmonary tuberculosis, as well as in the presence of multidrug resistance in Mycobacterium tuberculosis (in infiltrative and fibrocavernous forms of the disease) and a negative response to the Mantoux test in patients with infiltrative pulmonary tuberculosis. The demonstrated decrease in the count of circulating γδТ cells in the blood of patients with pulmonary tuberculosis promotes a reduction in the protective immune response to Mycobacterium tuberculosis.

Increased content of CD4+CD161+IL-17A+ Th17 lymphocytes in the peripheral blood was found in patients with pulmonary tuberculosis irrespective of the clinical form (infiltrative, disseminated) and variant of the disease (drug-sensitive, drug-resistant). The elevated content of Th17 cells in pulmonary tuberculosis is associated with hypersecretion of Th17-associated cytokines IL-17A and IL-22 in vitro that was most pronounced (in case of IL-17A) in patients with disseminated pulmonary tuberculosis.

The peripheral blood count of γδТ cells was investigated in patients with new-onset pulmonary tuberculosis depending on the clinical form of the disease, on Mycobacterium tuberculosis susceptibility to antituberculosis drugs, and on the response to intradermal tuberculin administration (Mantoux test). The count of γδТ cells was shown to decrease in all clinical forms of tuberculosis infection; however, the most significant increase in their count was noted in disseminated and fibrocavernous pulmonary tuberculosis, as well as in the presence of multidrug resistance in Mycobacterium tuberculosis (in infiltrative and fibrocavernous forms of the disease) and a negative response to the Mantoux test in patients with infiltrative pulmonary tuberculosis. The demonstrated decrease in the count of circulating γδТ cells in the blood of patients with pulmonary tuberculosis promotes a reduction in the protective immune response to Mycobacterium tuberculosis.

The work analyzed subpopulation structure of peripheral blood T-regulatory cells (T-reg) and production of cytokines with immunosuppressor activity (IL-10, TGF-b)  in vitro  in patients with disseminated pulmonary tuberculosis, depending on the sensitivity of  Mycobacterium tuberculosis  to anti-tuberculosis drugs. It has been shown that Trn – natural T-regulatory lymphocytes (CD4 + CD25 + Foxр3 + ) and TGF-b cytokine, formed by them, play the leading role in the formation of immunosuppression under disseminated pulmonary tuberculosis. Besides, subpopulation imbalance of Treg-cells in patients with disseminated pulmonary tuberculosis, in case at multi-drug-resistance and drug-susceptible, is shown by the increased content of T-reg-cells with CD4 + CD25 + Foxр3 + phenotypein combination with the deficiency of CD4 + CD25 + Foxр3 –  T-reg lymphocytes in blood. The increase in the amount of CD4 + CD25 + Foxр3 +  T-reg-cells in blood of patients with disseminated multi-drug resistance pulmonary tuberculosis is associated with the increase in basal and BCG-induced production of TGF-b in vitro .