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.

The article presents the data on the level of reactive oxygen species, irreversible and reversible oxidative modification of proteins, the state of glutathione system components, activity of catalase, and implementation of apoptosis in intact blood lymphocytes. By modulating oxidative stress in vitro, we studied the role of hydrogen peroxide in the final concentration of 0.5 mM in apoptosis implementation. We also evaluated the contribution of the glutathione system components and irreversible and reversible oxidative modification of proteins to molecular mechanisms of apoptosis in blood lymphocytes. We showed that apoptotic death in blood lymphocytes under experimental oxidative stress was characterized by activation of protein glutathionylation and carbonylation. The obtained findings prove that protein molecules are potential redox-dependent targets for regulating cell metabolism in blood lymphocytes.

HBL-100 breast epithelial cells were cultured with a blocker (N - ethylmaleimide) and protector (1,4-dithioerythritol) of SH groups. The study assessed changes in redox potential of glutathione and thioredoxin systems, intensity of oxidative modification of proteins, ROS production, and cell proliferation. The roles of thioredoxin system and protein oxidative modification in HBL-100 cell proliferation under redox status modulation were established. The role of carbonylated thioredoxin in arrest of the cell cycle in S-phase was demonstrated, which could be used for targeted therapy of the diseases accompanied by oxidative stress and disturbed redox status.

DOI: http://dx.doi.org/10.15690/vramn1189 Ю.В. Колобовникова1*, О.И. Уразова1, 2, О.А. Васильева1, Е.В. Романова1, А.А. Комар3, Л.С. Литвинова3, В.С. Полетика1, С.П. Чумакова1, В.В. Новицкий1, 2 1 Сибирский государственный медицинский университет, Томск, Российская Федерация 2 Томский государственный университет систем управления и радиоэлектроники, Томск, Российская Федерация 3 Балтийский федеральный университет им. И. Канта, Калининград, Российская Федерация

Представлены данные о иммуномодулирующем, противовоспалительном и нейро-протективном действии антибиотика рифампицина, значении этих эффектов при химиотерапии рифампицином туберкулеза. Рифампицин повышает экспрессию ядерного рецептора pregnane X receptor, нарушает функции Toll-подобных рецепторов, препятствует активации ядерного фактора транскрипции NF-kB и металлопротеиназ в легких, увеличивает синтез оксида азота и тормозит образование простагландина Е2. Нейропротективное действие рифампицина обусловлено торможением продукции β-амилоида и агрегации α-синуклеина в головном мозге.

We analyzed in vitro secretion of growth factors (TGFβ1, VEGF, and EGF) by peripheral blood eosinophil granulocytes in patients with gastric and colon cancer according to the presence of tumor-associated tissue eosinophilia. In gastrointestinal cancer with tissue eosinophilia, an increase in basal in vitro secretion of TGFβ1 and VEGF (in gastric cancer) was detected, while basal eosinophil-derived EGF production in gastric and colon cancer did not differ significantly from that in the comparison group (without tissue eosinophilia). Additionally, it was shown that in patients with gastric cancer and colon cancer, regardless of the presence of tissue eosinophilia, the levels of basal and r-IL-5-induced secretion of TGFβ1, VEGF, and EGF by eosinophil granulocytes in vitro were comparable. An absence of the secretory response of eosinophils to cytokine stimulation (by r-IL-5) indicates a decrease in the functional reactivity of the cells.

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.

We analyzed the expression of galectin-1 and galectin-3 in tumor tissue in stomach and colorectal cancer with and without tissue eosinophilia. Low expression of galectin-3 was detected in all patients with malignant gastrointestinal tumors irrespective of the presence of eosinophilia. Low expression of galectin-1 was detected only in patients with gastrointestinal cancer associated with eosinophilia. Association of galectin-1 expression with eosinophilic infiltration of the tumor tissue in stomach and colorectal cancer was detected.

Redox proteins (thioredoxin, glutaredoxin) are key macromolecules capable of modulating intracellular processes. This determines research choices in the field of redox-dependent cell proliferation management. The study of the molecular mechanisms of the onset, development and progression of malignant neoplasms underlies the search for tumor-associated markers and potential targets for personalized antitumor therapy. Purpose. To establish the role of the “thioredoxin – thioredoxin-reductase” system in the impaired proliferation of mammary adenocarcinoma cells under the action of the cyclin-dependent protein kinase roskovitin blocker. Materials and methods. The study was carried out using the culture of mammary adenocarcinoma cells of the MCF-7 line incubated in the presence and absence of roskovitin at a final concentration of 20 μM for 18 h. The intracellular content of thioredoxin and protein regulators of proliferation (cyclin E and cyclin-dependent protein kinase 2) were determined by Western blotting technique, the expression level of thioredoxin mRNA was determined by real-time polymerase chain reaction and the activity of thioredoxin-reductase was measured by a spectrophotometric method. Results. It was established that the decrease in proliferative activity of MCF-7 tumor cells incubated in the presence of roskovitin was accompanied by a decrease in the content of cyclin E and cyclin-dependent kinase on the background of a decrease in the expression level of thioredoxin mRNA and an increase in the activity of thioredoxin-reductase. Conclusion. The involvement of the components of the thioredoxin system (thioredoxin, thioredoxinreductase) in disrupting the proliferation of MCF-7 tumor cells was detected under the action of the cyclindependent protein kinases of roskovitin.

A real-time PCR with hybridization and fluorescent detection was used to analyze the distribution of p53 G215C, p21A1026G, and G369C gene polymorphisms in patients with stomach cancer and healthy subjects. It was found that allele C, genotypes of CC and GC of G215C p53, and G369C p21 polymorphisms and allele A and AA and GA genotypes of A1026G p21 polymorphism are significantly associated with the risk of stomach cancer development.

The complex histologic, immunogictochemic, electronic-microscopic estimation biopsiа mucous membrane of bronchial in 87 patients with heavy sensitive and therapeutic resistant bronchial asthma. It is established, that weighting of a bronchial asthma is characterised by high indicators of functional activity of corpulent cages and ultrastructural signs of strengthening secretions processes in bocalotia mucous membrane of bronchial. Therapeutic resistant bronchial asthma of a phenotype «brittle» differs development of the Th2-dependent immune answer with eosinohyils an inflammation, ultrastructural a dyskinesia resnitch epitels. A therapeutic resistant bronchial asthma of a phenotype «the asthma with the fixed bronchial obstruction» neutrophil inflammation, is accompanied a mucous membrane of bronchial tubes with a thickening basals membranes, height decrease epitels a layer with a degeneration epithelium and strengthening of an expression of receptors to the transforming factor of growth β 1 .

Опухолеассоциированная тканевая эозинофилия определяется как эозинофильная инфильтрация стромы опухоли. Эта реакция, как полагают, играет важную роль в канцерогенезе. Считается, что эозинофилы обладают противоопухолевой активностью за счет высвобождения цитотоксических белков. Цель исследования - изучение молекулярно-генетических механизмов формирования опухолеассоциированной тканевой эозинофилии у больных раком желудка и толстой кишки. Методика. Материалом исследования служили образцы опухолевой ткани пациентов с диагнозом рак желудка или толстой кишки . Подсчитывали количество эозинофилов, инфильтрирующих опухолевую ткань, а также оценивали экспрессию эотаксина-1 (ключевого фактора хемотаксиса эозинофилов) и его рецептора CCR3 в опухолевой ткани методом иммуногистохимии. Было изучено распределение полиморфных вариантов генов CCL11 (А384G) , CCR3 (T51С) , IL5 (С703Т) и IL5R (G80А) . Геномную ДНК выделяли из образцов опухолевой ткани. Генотипирование образцов ДНК по полиморфизмам генов осуществляли путем ПДРФ-анализа продуктов ПЦР-амплификации специфических участков генома с последующей визуализацией в ультрафиолетовом свете. Результаты. Показана более высокая экспрессия эотаксина-1 и CCR3 в ткани опухоли при раке желудка и толстой кишки, ассоциированном с эозинофилией. Установлена ассоциация эозинофильной инфильтрации опухолевой ткани с носительством генотипа СС и аллеля С полиморфизма T51С гена CCR3 и генотипа СС и аллеля С полиморфизма С703Т гена IL5 . Заключение. Формирование опухолеассоциированной тканевой эозинофилии при раке желудка и толстой кишки опосредованно действием эотаксина-1 и его рецептора. Результаты молекулярно-генетического исследования указывают на генетически детерминированный характер эозинофилии при раке желудка и толстой кишки. Tumor-associated tissue eosinophilia is defined as eosinophilic stromal infiltration of a tumor. Tumor-associated tissue eosinophilia is believed to play a significant role in carcinogenesis. It is believed that eosinophils have antitumor activity due to the release of cytotoxic proteins. The aim of this study was to investigate the molecular-genetic mechanisms of formation of tumor-associated tissue eosinophilia in patients with gastric cancer and colon cancer. Methods. Tumor tissue samples from patients with gastric cancer and colon cancer were reviewed. We counted the number of infiltrating eosinophils in neoplastic lesion tissue and we evaluated the expression of eotaxin-1 (a key factor of chemotaxis of eosinophils) and its receptor CCR3 in tumor tissue by immunohistochemical staining. The distribution of polymorphic variants of genes CCL11 (А384G), CCR3 (Т51С), IL5 (С703Т) and of the IL5R (G80А) was studied. DNA was isolated from tumor tissue samples. Genotyping of polymorphisms was carried out by polymerase chain reaction (PCR), followed by digestion with the appropriate restriction enzyme (PCR-RFLP) and agarose-gel electrophoresis. Results. It is shown that higher expression eotaxin-1 and CCR3 is determined in the tumor tissue of gastric cancer and colon cancer associated with eosinophilia. It is established that eosinophilic infiltration of the tumor tissue is associated with the carriers of CC genotype and C allele of the gene CCR3 (Т51С) and CC genotype and C allele of gene IL5 (С703Т) . Conclusion. The formation of tumor-associated tissue eosinophilia in gastric cancer and colon cancer is mediated through the action eotaxin-1 and its receptor. The results of molecular-genetic studies indicate a genetically determined character of eosinophilia in gastric cancer and colon cancer.

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

We compared the results of gene molecular and immunocytochemical studies of β-catenin and E-cadherin in different variants of nodular thyroid disease (nodular colloid goiter, follicular thyroid adenocarcinoma, papillary thyroid cancer) and revealed changes of the function of the E-cadherin/β-catenin complex leading to switching from adhesion function of β-catenin in nodular colloid goiter to predominantly transcriptional activity in papillary carcinoma. The results confirm the important role of disturbances in E-cadherin–β-catenin interactions in the mechanisms of malignant transformation of follicular epithelium.

MCF-7 breast cancer cells and HBL-100 breast epithelial cells were cultured with N-ethylmaleimide, a blocker of SH groups. Changes in redox potential of the glutathione system, activities of glutathione reductase, glutathione peroxidase, and intensity of apoptotic cell death were evaluated. The results indicate that incubation with N-ethylmaleimide led to glutathione system imbalance, reduced tumor cell redox potential, and induced their programmed death, which seemed useful for prospective target therapy of tumor diseases.

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.

The parameters characterizing antituberculous immune response at the stage of cytokinedependent activation of T cells were analyzed in 90 patients with pulmonary tuberculosis before etiotropic therapy. Immunophenotyping of T cells for IL-12Rβ 2 , WSX-1, and gp130 surface markers and T-bet intracellular transcription factor was carried out after specific IL-12/IL-27 induction of cells in vitro . An increase in the counts of CD3 + T-bet + , CD3 + IL-12Rβ 2 + , and CD3 + WSX-1 + gp130 + cells and the level of T cells with high expression of WSX-1 inhibitory molecule (CD3 + WSX-1 hi+ gp130 − and CD3 + WSX-1 hi+ gp130 + ) was detected. The type of disorders in the inductive stage of antituberculous immune response did not depend on the clinical form of disease.

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.