The Role of Foxp3-Expressing Regulatory T Cells and T Helpers in Immunopathogenesis of Multidrug Resistant Pulmonary Tuberculosis

Subpopulation structure of regulatory T cells and T helpers of peripheral blood in patients with newly diagnosed pulmonary tuberculosis depending on the clinical form of disease and sensitivity of Mycobacterium tuberculosis to antituberculosis drugs has been analyzed in this work. It has been shown that the leading part in immune suppression at infiltrative, dissemination, and fibrosis-cavity pulmonary tuberculosis is played by natural regulatory CD4+CD25+Foxp3+-T lymphocytes. Thus we estimate increase of their number in blood by drug-resistance and drug-susceptible patients. It has been demonstrated that in patients with fibrocavernous and infiltrative form of the disease and drug-resistant pulmonary tuberculosis the number of CD4+CD25−Foxp3+-regulatory T cells was increasing. In patients with infiltrative pulmonary tuberculosis, including multidrug-resistant M. tuberculosis, an increased number of CD3+CD4+CD25− T helpers is determined by the pathogenic features of the development of the tuberculosis infection and is connected with the activation of Th1-dependent immune response. Reduction in the number of T-helpers in the blood of patients with dissemination and fibrosis-cavity pulmonary tuberculosis mediates inefficient implementation of cell-mediated protective immunity

Hot Electron and X-ray Production from Intense Laser Irradiation of Wavelength-Scale Polystyrene Spheres

Hot electron and x-ray production from solid targets coated with polystyrene-spheres which are irradiated with high-contrast, 100 fs, 400 nm light pulses at intensity up to 2×1017 W/cm2 have been studied. The peak hard x-ray signal from uncoated fused silica targets is an order of magnitude smaller than the signal from targets coated with submicron sized spheres. The temperature of the x-rays in the case of sphere-coated targets is twice as hot as that of uncoated glass. A sphere-size scan of the x-ray yield and observation of a peak in both the x-ray production and temperature at a sphere diameter of 0.26 μm, indicate that these results are consistent with Mie enhancements of the laser field at the sphere surface and multipass stochastic heating of the hot electrons in the oscillating laser field. These results also match well with particle-in-cell simulations of the interaction

Экспрессия провоспалительных и костимулирующих молекул на макрофагах in vitro у больных туберкулезом легких

The aim of this study was to identify features of the expression of pro-inflammatory and co-stimulatory molecules on the surface of macrophages in vitro in patients with pulmonary tuberculosis, depending on the clinical form of the disease and sensitivity of the pathogen to anti-TB drugs.Materials and methods. 40 patients (36 men and 4 women) with pulmonary tuberculosis (TB) were examined: 18 patients (16 men and 2 women, average age (44.56 ± 8.10) years) with disseminated tuberculosis (DTB) and  22 patients (20 men and 2 women, average age (46.54 ± 5.24) years) with infiltrative tuberculosis (ITB). Of those, 30 patients secreted Mycobacterium tuberculosis (MBT) sensitive to the basic anti-TB drugs (ATBD), and 10 patients secreted MBT resistant to first-line anti-TB drugs. Venous blood was the study material. To isolate monocytes from the whole blood in order to transform them into macrophages, ficoll density gradient centrifugation with gradient density of 1.077 g/cm3 was used followed by immunomagnetic separation of CD14+ cells. Monocytes were cultured in a complete culture medium X-VIVO 10 with gentamicin and phenol red with the addition of the macrophage colony-stimulating factor (M-CSF) (5 ng/ml) at a concentration of 1×106 cells/ml with the following stimulators: interleukin (IL) 4 (10 ng/ml) and interferon (IFN) γ (100 ng/ml). Immunophenotyping of macrophages was performed using monoclonal antibodies to CD80, CD86, and HLA-DR on a Beckman Coulter CytoFLEX LX flow cytometer (Beckman Coulter, USA). The analysis of the obtained data was carried out using the CytExpert 2.0 software application. The results were analyzed using statistical methods.Results. The number of intact and cytokine-stimulated (IL-4 and IFNγ) CD80-positive macrophages in patients with ITB and drug-resistant TB (DR TB) exceeded their number not only in healthy donors, but also in patients with DTB and drug-sensitive TB (DS TB), respectively. In addition, an increase in CD86 expression on the surface of macrophages was registered in patients with ITB and DR TB after adding IFNγ (M1-activation inducer) to the suspension culture. In contrast, in patients with DTB and DS TB, the number of macrophages with expression of B7 family co-stimulating molecules decreased or remained within the normal values in the absence of a reaction to cytokines during cytokine induction. Deficiency of HLA-DR-positive macrophages was found in all TB patients. The minimal number of macrophages expressing HLA-DR was found in patients with DTB and DS TB after cell incubation with IL-4 (M2-activation inducer).Conclusion. Evaluation of the expression of B7 (CD80/86) and HLA-DR membrane molecules on macrophages in TB patients allows to conclude that anti-TB immune response is impaired at stages of antigen presentation (in all examined patients with TB) and co-stimulation (in DTB and DS TB). An increase in the expression of macrophage surface molecules CD80 (with M1- and M2-stimulation) and CD86 (with M1-stimulation) in patients with ITB and DR TB indicates an increase in cell reactivity in these forms of TB. In addition, deficit of expression of HLA-DR (a key marker of pro-inflammatory cell activation) on the surface of macrophages in TB can be considered as a general (independent of the clinical form of the disease and drug sensitivity of the pathogen) pathogenetic factor of immune imbalance in pulmonary tuberculosis.Цель работы – установить особенности экспрессии провоспалительных и костимулирующих молекул на макрофагах in vitro у больных туберкулезом легких в зависимости от клинической формы заболевания и чувствительности возбудителя к противотуберкулезным лекарственным средствам.Материалы и методы. Обследованы 40 пациентов (36 мужчин и 4 женщины): 18  пациентов с диссеминированным туберкулезом легких (ДТБ) (16 мужчин и 2 женщины,  средний возраст (44,56 ± 8,10) лет) и 22 пациента с инфильтративным туберкулезом легких (ИТБ) (20 мужчин и 2 женщины, средний возраст (46,54 ± 5,24) лет) c туберкулезом легких (ТБ). Из них было 30 пациентов, выделяющих Mycobacterium tuberculosis (MBT), чувствительные к основным противотуберкулезным средствам (ПТС), и 10 пациентов, выделяющих MBT, устойчивые к лекарственным  средствам основного ряда противотуберкулезной терапии. Группу сравнения составили 15 здоровых доноров с сопоставимыми характеристиками по полу и возрасту.Материалом исследования являлась венозная кровь. Для выделения моноцитов из цельной крови с целью их трансформации в макрофаги использовали метод центрифугирования в градиенте фиколла плотностью 1,077 г/см3 с последующей иммуномагнитной сепарацией CD14+ клеток. Моноциты культивировали в полной питательной среде X-VIVO 10 с добавлением колониестимулирующего фактора макрофагов (M-CSF) (5 нг/мл) в концентрации 1×106 клеток/мл со стимуляторами: интерлейкином (IL) 4 (10 нг/мл) и интерфероном (IFN) γ (100 нг/мл).  Иммунофенотипирование макрофагов проводили с использованием моноклональныхантител к CD80, CD86, HLA-DR на проточном цитометре Beckman Coulter CytoFLEX LX (Beckman Coulter, США). Анализ полученных данных осуществляли при помощи программного приложения CytExpert 2.0 (Beckman Coulter, США). Полученные результаты анализировали статистическими методами.Результаты. Количество интактных и стимулированных цитокинами (IL-4 и IFNγ) CD80- позитивных макрофагов у больных ИТБ и с лекарственно-устойчивым ТБ (ЛУ ТБ)  превышало их число не только у здоровых доноров, но и у больных ДТБ и с лекарственно-чувствительным ТБ (ЛЧ ТБ) соответственно. Кроме того, у больных ИТБ и ЛУ ТБ регистрировалось повышение экспрессии CD86 на макрофагах после добавления в суспензионную культуру IFNγ (индуктор М1-активации). У больных ДТБ и ЛЧ ТБ количество макрофагов с экспрессией костимулирующих молекул семейства В7 при индукции цитокинами, напротив, снижалось или сохранялось в пределах нормы в отсутствие реакции на цитокины. Дефицит HLA-DR-позитивных макрофагов обнаруживался у всех больных ТБ. Минимальное число макрофагов, экспрессирующих  HLADR, установлено у больных ДТБ и ЛЧ ТБ после инкубации клеток с IL-4 (индуктор  М2-активации).Заключение. Оценка экспрессии мембранных молекул B7 (CD80/86) и HLA-DR на макрофагах у больных ТБ позволяет сделать вывод о нарушениях  противотуберкулезного иммунного ответа на стадии презентации антигена (у всех обследованных больных ТБ) и костимуляции (при ДТБ и ЛЧ ТБ). Увеличение экспрессии макрофагами поверхностных молекул CD80 (при М1- и М2-стимуляции) и  CD86 (при М1-стимуляции) у больных ИТБ и ЛУ ТБ свидетельствует о повышении реактивности клеток при данных формах течения ТБ. Наряду с этим дефицит экспрессии на макрофагах HLA-DR (ключевого маркера провоспалительной активации клеток) при ТБ можно рассматривать как общий (не зависящий от клинической формы болезни и  лекарственной чувствительности возбудителя) патогенетический фактор иммунного  дисбаланса при туберкулезе легких.

Production of angiogenesis mediators and the structure of the vascular wall in the heart in ischemic cardiomyopathy

Background. In the pathogenesis of ischemic cardiomyopathy (ICMP), angiopoiesis remains unexplored.The aim. To describe the vasculature of the heart and the imbalance of angiogenesis mediators in the coronary circulation in association with the number of endothelial progenitor cells (EPC) and desquamated endothelial cells (DEC) in the blood of patients with coronary heart disease (CHD), suffering and not suffering from ICMP.Methods. Fifty-two patients with CHD (30  patients with ICMP, 22  patients without  ICMP), 15  healthy donors were examined. The content of EPC (CD14+CD34+VEGFR2+) in the blood from the cubital vein and DEC (CD45–CD146+) in the blood from the coronary sinus and the cubital vein was determined by flow cytometry. The concentrations of VEGF-A (vascular endothelial growth factor A), PDGF (platelet-derived growth factor), and SDF-1 (stromal cell-derived factor 1) in blood plasma were recorded using immunofluorescence assay; the angiopoietin-2, MMP-9 (matrix metallopeptidase 9) were recorded using enzyme immunoassay. In myocardial biopsies the specific area of vessels and the expression of αSMA (smooth muscle alpha-actin) were determined by morphometric and immunohistochemical methods.Results. In the peripheral blood of patients with CHD, regardless of the presence of ICMP, the DEC content exceeded the physiological level, and the VEGF-A, PDGF, angiopoietin-2, and MMP-9 corresponded to the norm. In CHD patients without cardiomyopathy, there was an excess of SDF-1 and EPC in the blood from the cubital vein, and in ICMP, their physiological significance was noted. In the coronary blood flow in patients with CHD without cardiomyopathy, an increase in the concentration of PDGF was found, which was not determined in patients with ICMP, who had an increased content of DEC, angiopoietin-2 and MMP-9. The specific area of the vessels in the patients of the two groups was comparable; the expression of αSMA in ICMP was 6.2 times lower than in patients with CHD without cardiomyopathy.Conclusion. The development of ICMP is accompanied by impaired maturation of vessels in the myocardium, associated with the absence of a compensatory reaction of activation of cellular and humoral factors of angiogenesis

Макрофаги при бактериальных болезнях легких: фенотип и функции (обзор)

This literature review is devoted to the analysis of the role of macrophages in the immunopathogenesis of infectious lung diseases of bacterial etiology. The article summarizes information about the origin of macrophages, their phenotypic and functional heterogeneity. The mechanisms of impaired protective function of innate immunity are associated with the polarization of the program of maturation and activation of macrophages in the direction to tolerogenic or immunoregulatory cells with phenotype of M2. Alveolar macrophages perform a variety of functions (from pro-inflammatory to regenerative) in the development of inflammation in the respiratory organs. Their inherent plasticity suggests that the same macrophages can change their phenotype and function depending on the microenvironment in the inflammatory focus at different stages of the disease. Understanding the mechanisms that regulate macrophage plasticity will be an important step towards realizing the potential of personalized immunomodulatory therapy.Обзор литературы посвящен анализу роли макрофагов в иммунопатогенезе инфекционных заболеваний легких бактериальной этиологии. В статье обобщены сведения о происхождении макрофагов, их фенотипической и функциональной гетерогенности. Механизмы нарушений защитной функции врожденного иммунитета связаны с поляризацией программы созревания и активации макрофагов в направлении толерогенных или иммунорегуляторных клеток с фенотипом М2. Альвеолярные макрофаги выполняют разнообразные функции (от провоспалительной до регенераторной) при развитии воспаления в органах дыхания. Присущая им пластичность свидетельствует, что одни и те же макрофаги могут изменять свой фенотип и функции в зависимости от микроокружения в очаге воспаления на разных стадиях заболевания. Понимание механизмов, которые регулируют пластичность макрофагов, станет важным шагом на пути реализации потенциала персонифицированной иммуномодулирующей терапии