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The article presents the data on assessment of functional features of neutrophils in 34 patients with systemic lupus erythematosus (SLE). Development of neutrophil extracellular traps (NETs) was evaluated in cell cultures incubated in vitro for 30 and 150 minutes (basal levels, NETBAS30 and NETBAS150, respectively), and in the presence of heat-inactivated S. аureus (strain ATCC 25923, 108 CFU/ml) (stimulated levels, NETST30 and NETST150, respectively). NET looks like thin free-lying extracellular fibrillar structures, 2-3 times exceeding the size of unchanged granulocyte. The result was expressed as percentage and relative amount of extracellular traps per 100 counted leukocytes. Phagocytic activity of neutrophils was evaluated as phagocytosis of S. аureus by counting the percentage of neutrophils that engulfed phagocytic index of microbial particles (PI); the average number of phagocytosed objects per neutrophil phagocytic number (PC). ROS-producing activity was determined in the reduction of Nitroblue Tetrazolium tested in spontaneous and stimulated S. аureus variants (NBTBAS and NBTST, respectively). The result was expressed as the percentage of formazan-positive cells per 100 white blood cells. Nitroxide-producing properties were determined using the Crow (1999) method in spontaneous and stimulated samples for the accumulation of the nitrated amino acid tyrosine (3-nitrothyrosine, 3-NTBAS, and 3-NTST, respectively). We revealed a decrease in ROS production, phagocytosis and NO-forming activity of neutrophils associated with increased netosis. Activation of the netosis was observed in cell cultures without stimulation, indicating the in vivo formation of networks in SLE. The NET increase is most pronounced in the patients with lupus nephritis (p < 0.05), and in remission of the disease (p < 0.05). We have revealed a correlation of NET formation parameters with duration and degree of SLE activity (rs = -0.6; p = 0.001, and rs = 0.39; p = 0.02, respectively); autoantibody titers (anti-dsDNA and ANA) (rs = 0.67; р = 0.047 and rs = 0.59; р = 0.034, respectively); prothrombin complex activity (rs = 0.6; p = 0.036), as well and urea and creatinine levels (rs = 0.47; p = 0.037 and rs = 0.39; p = 0.048, respectively). The parameters of NETs can be considered a promising biomarker for verifying the diagnosis of SLE, evaluation of clinical activity, disease severity, and predicting the development of complications.

В статье представлено изучение особенностей мембранного потенциала митохондрий (МПМ) гранулоцитов у детей с врожденными пороками развития легких (ВПРЛ). Выявлено увеличение в 5 раз доли клеток с пониженным МПМ гранулоцитов по сравнению с группой контроля. В периоды обострения заболевания число гранулоцитов со сниженным потенциалом увеличивалось в 8 раз, сопровождаясь повышением люминол- и люцигенин зависимой хемилюминесценции фагоцитов цельной крови спонтанной, достоверным снижением индексов стимуляции I-lum и I-luc на 30 и 25%. Отрицательная динамика индекса стимуляции указывает на истощение резервных возможностей фагоцитарной системы у детей исследуемой группы. Выявлены статистически значимые прямые корреляционные зависимости между уровнем гранулоцитов со сниженным МПМ и показателями фагоцитарной активности лейкоцитов., The article presents the study of the characteristics of the membrane potential of mitochondria (MPM) granulocytes in 72 children with congenital lung malformations (CLM). There was a 5-fold increase in the proportion of cells with reduced granulocyte MPM(ΔΨm) compared to the control group. During the periods of exacerbation of the disease, the number of granulocytes with reduced potential increased by 8 times accompanied by the growth of luminol-and lucigenin-dependent chemiluminescence of whole blood phagocytes with spontaneous, significant decrease in the I-lum and I-luc stimulation indices by 30 and 25%. Negative dynamics of the stimulation index indicates the depletion of reserve capacities of the phagocytic system in children of the study group. There are statistically significant direct correlations between the level of granulocytes with reduced MPM(ΔΨm) and indicators of phagocytic activity of leukocytes, Якутский медицинский журнал, Выпуск 4 (72) 2020, Pages 10-13

The aim of the study was to investigate the regulatory effect of monocytes and their subpopulations on the population composition of granulocyte leukocytes and the state of their respiratory burst in widespread purulent peritonitis (WPP). The study involved 24 patients aged 30-65 with acute surgical diseases and injuries of abdominal organs complicated by WPP. As a control 25 relatively healthy people of the same age range were examined. A study of the population composition of monocytes and granulocyte leukocytes in blood was performed using a two-platform technology on the hematological analyzer Sysmex XE-5000 (Sysmex Inc., USA) and FC-500 flow cytometer (Beckman Coulter, USA) using the Cytodiff antibody kit (Beckman Coulter, USA). A study of the monocytes number expressing HLA-DR- and CD64-receptor was performed by flow cytometry using direct immunofluorescence of whole peripheral blood. The respiratory burst state of neutrophilic granulocytes was studied by chemiluminescence analysis on a 36-channel chemiluminescence analyzer BLM-3607 (MedBioTech, Russia). As indicators of chemiluminescence were used luminol and lucigenin. The enhancement of chemiluminescence induced by zymosan was evaluated by the ratio of the area of the induced chemiluminescence to the spontaneous area and was defined as the activation index. It has been established that the immune-inflammatory process in WPP is characterized by a decrease in the number of classical monocytes in the peripheral blood and an increase in the content of non-classical monocytes. In WPP in peripheral blood the level of monocytes expressing HLA-DR receptors decreases. The change in the ratio of monocytes subpopulations characterizes the increase in the role of the proinflammatory fraction in the WPP pathogenesis. Changes in the population composition of granulocytes in the blood in patients with WPP also characterize the development of an acute inflammatory process. In this case, there is a decrease in the number of basophils in the peripheral blood, which, apparently, is determined by the presence of an allergic component in WPP and, accordingly, their migration to the inflammation area. In patients with WPP activation of a respiratory burst of granulocytes of blood was detected, the intensity of which is determined by the synthesis of primary and secondary active oxygen species. The results of the correlation analysis made it possible to establish that in WPP the regulatory role of non-classical monocytes increases aimed at stimulating the inflammatory processes (an increase in the number of mature and immature forms of neutrophils and stimulation of the activity of a respiratory explosion of granulocytes). The revealed features of the regulatory effect of monocytes on the population composition and the intensity of the respiratory burst of granulocytes can be used in the development of immunotherapeutic methods aimed at reducing the activity of the inflammatory process in WPP.

A leading role of phagocytes in prevention of M. tuberculosis infection is well established. Various clinical variants of tubercular inflammatory process necessitate further studies of functional and metabolic features of blood phagocytes in the patients with different forms of lung tuberculosis, being the main goal of this study. We have observed a total of 124 persons including 25 healthy subjects, and 99 patients with tuberculosis who manifested with different types of tubercular inflammatory process, i.e., 31 patients had a limited specific process (tuberculoma); in 44 patients, an infiltrative lung tuberculosis was diagnosed, and 24 patients had fibro-cavernous tuberculosis of lungs. We studied activation markers of neutrophils and monocytes (phagotest, burst-test, CD11b+, CD11c+, HLA-DR-Ag), as well as main indicators of cellular immunity (CD45+CD3+, CD45+CD19+, CD45+CD3- CD16+56+). Statistical evaluation was carried out in the «Microsoft Office Excel 2007» and «Statistica for Windows v. 6.1» environment. A considerable decrease in proportion of superoxide anion-producing monocytes was found in 10% of the patients with fibro-cavernous tuberculosis as compared to the patients with tuberculoma and infiltrative tuberculosis. Similarly, the fibro-cavernous tuberculosis was characterized by higher expression of adhesion markers, e.g., CD11b, by 49%, and CD11c, by 73.5%, when compared with the two other groups of patients. A considerable decrease of absorbing granulocyte function was found in the patients with active forms of tuberculosis, as compared with limited clinical forms (tuberculoma). Fibro-cavernous tuberculosis was associated with increased absolute numbers of granulocyte that produce both superoxide anion, and express surface CD11b+ and CD11c+. We have revealed a relative decrease in lymphocyte quantities in the patients from tuberculoma which corresponded to increased granulocyte quantities of granulocytes and monocytes in the patients’ blood. The conducted study allowed us to make a conclusion that each clinical form of tuberculosis id characterized by a specific immunological pattern. In the patients with tuberculoma, we have revealed a decrease of phagocytic, functional and metabolic activities of monocytes is noted, along with increased quantities of CD11b+ and CD11c+ adhesion molecules on granulocytes, increased numbers of T-lymphocytes, and decreased amounts of NK-cells. Infiltrative tuberculosis is characterized by increased contents and higher HLA-DR expression of the monocytes, with enhanced expression of CD11b+ and CD11c+ adhesion molecules on the granulocytes, and decreased number of T-lymphocytes. In the fibro-cavernous tuberculosis we observed leukocytosis, monocytosis, granulocytosis. The main functional feature of this clinical form is an increased amount of CD 11b+ and CD 11с+-bearing leukocytes, higher functional and metabolic activity of granulocytes, as well as expansion of CD11b+ expressing cell population and increased numbers of B-cells in peripheral blood.

To reveal the specific features of hematopoiesis in patients with head and neck squamous cell carcinoma (HNSCC), the authors analyzed the indicators of 41 bone marrow aspirates in relation to the clinical and morphological characteristics of the tumor and micrometastases detected in bone marrow. Puncture samples were morphologically and molecularly examined at the Laboratory of Hematopoiesis Immunology, N.N. Blokhin Russian Cancer Research Center. The found changes in the bone marrow cell composition in HNSCC patients showed themselves as an increase in the relative count of segmented neutrophils and a decrease in the level of immature granulocytes with no alternations in the neutrophil maturation index and in the percentage of granulocyte lineage cells. Bone marrow hypocellularity is a characteristic feature of high-grade HNSCC. The significantly enhanced leuko-erythroblastic ratio was due to a reduction in red blood cells: the erythroid cell maturation index rose, by increasing the percentage of bone marrow oxyphilic normoblasts in patients with locally advanced HNSCC. A highly sensitive method for detecting mRNA of the specific squamous cell carcinoma protein E48 was perfected using reverse-transcription polymerase chain reaction and Southern blotting, followed by an enzyme immunoassay that could recognize bone marrow micrometastases in 35 % of the patients with HNSCC. The patients with found bone marrow micrometastases showed a significant decrease in the proportion of polychromatophilic normoblasts and a significant increase in granulocytes in the presence of normal values of lymphocytes and other cellular elements of myelograms. It was established that in the presence of regional lymph node involvement, bone marrow micrometastases were significantly more frequently in the patients with HNSCC and tumor progression over a period of up to 9 months.

Aim. To study the effects of nitroglycerin on proliferative and migratory activity of mononuclear cells and endothelial progenitor cells in patients with chronic heart failure. Materials and methods. Proliferative potential of peripheral blood mononuclear cells in donors and patients were studied in MTT-test in the presence of 50 and 100 mg/ml nitroglycerin. Proliferation and migration of endothelial progenitor cells in patients were evaluated by changes in the cell impedance in real-time in the presence of various doses of nitroglycerin. Results. It is shown that nitroglycerin reduces in vitro both spontaneous and Concanavalin A stimulated proliferative activity of mononuclear cells in patients with chronic heart failure. Peripheral blood mononuclear cells in patients with chronic heart failure, obtained after procedure of mobilization by granulocyte colony-stimulating factor respond to nitroglycerin dose of 50 mg/ml with the increased proliferative activity, and with the suppressed proliferative activity to nitroglycerin dose of 100 mg/ml. Mononuclear cells of healthy donors respond to 50 mg/ml of nitroglycerin with the activation of proliferative capacity, but at a dose of 100 mg/ml spontaneous proliferative activity of mononuclear cells inhibit. It is also shown that the presence of nitroglycerin in medium dose in stimulating test leads to the inhibition of mononuclear cells proliferation from healthy donors. It is shown the stimulating effect of nitroglycerin on proliferative and migration potential of endothelial progenitor cells. Conclusion. Multidirectional and dose-dependent effects of nitroglycerin to the various types of human cells in vitro were observed.

We have studied functional parameters of neutrophilic granulocytes in patients with polypous rhinosinusitis by means of luminol- or lucigeninependent chemiluminescence induced by zymosan and Staphylococcus aureus. Luminol-enhanced chemiluminescence registered the entire pool of reactive oxygen species (ROS), reflecting total activity of myeloperoxidase and NADPH oxidase etc., whereas lucigenindependent chemiluminescence detected production of superoxide radical anion (·О2 -), as well as NADPH oxidase activivity. Usage of the in vitro stimulation tests allows both modeling of disease conditions, and to suggest origins of granulocyte stimulation, their mobilization potential and integration of intracellular processes. We found the increased rate of ROS formation, both in spontaneous and induced chemiluminescent reaction in luminol-dependent process. We have studied neutrophilic granulocyte response to different stimuli and found an increased luminol-dependent ROS formation in polypous rhinosinusitis in response to bacterial induction.

In spite of similar clinical patterns, there are numerous differences in immunopathogenesis of intrinsiс and extrinsiс forms of atopic dermatitis. Patients with both forms have decreased levels of CD8+CD25+ lymphocytes, decreased telomere DNA length of CD4+T-cells, decreased migration inhibition index in delayedtype hypersensitivity, decreased levels of CD16+NK-cells, decreased Fc-dependent monocyte and granulocyte phagocytosis, increased hydrogen peroxide production by neutrophils, increased levels of CD19+B-cells, as well as high IgA and IgG immunoglobulin levels. Extrinsiс form of atopic dermatitis are characterized by more severe clinical course, and by involvement of both immediate hypersensitivity (hyperproduction of IgE and decreased T-cells with intracellular IFNγ production), like as delayed-type hypersensitivity (decreased migration index along with decreased migration inhibition index). CD8+ play a large role in extrinsiс form of atopic dermatitis that may be traced as decreased telomere length of CD8+T- cells, decreased levels of CD8+CD45RO+ cells, increased levels of CD8+CD45RA+ naive cells and increased levels of CD28+ costimulatory molecules on CD8+cells. Increased levels of CD4+CD25+bright cells and strongly alterations of innate immunity determined of decreased H2O2 production by monocytes are shown in extrinsiс form. Hence, the severity index of atopic dermatitis is more expressed in extrinsic form of bronchial asthma and it is, probably, determined by more exaggerated immunological alterations.

Acute respiratory distress syndrome (ARDS) was reproduced in a rat model, by means of intratracheal instillation of granulocyte lysates (a method protected by Russian patent). Expression of HSP-70 in lung cells was determined by immunohistochemical technique at each ARDS stage. A significant increase of HSP-70 expression by all cell types was revealed during exudative stage, being more intensive in alveolocytes type 1, and less expressed in endothelium. During proliferative stage of the disorder, a decreased HSP-70 expression was noted in all cell populations. At these terms, it proved to be high in neutrophils and alveveolocytes type 1, whereas lower expression was registered in endothelium. At fibrotic stage, HSP-70 synthesis remained at high levels in neutrophils, macrophages, fibroblasts and alveolocytes type 1. Endothelium and alveolocytes type 2 exhibited a recurrent increase at fibrotic stage of ARDS, however it did not reach the values typical to the initial stage of the syndrome.