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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.

The aim of the study was to evaluate the expression of scavenger receptors (CD163, CD204, CD206) on macrophages in patients with pulmonary tuberculosis, depending on the clinical form of the disease and sensitivity of the pathogen to anti-tuberculosis drugs.Materials and methods. 64 patients with pulmonary tuberculosis (TB) were examined: 26 patients with disseminated pulmonary tuberculosis (DTB) and 38 patients with infiltrative pulmonary tuberculosis (ITB). Of these, 42 patients secreted Mycobacterium tuberculosis (MBT) sensitive to basic antituberculosis drugs (ATBD), and 22 patients secreted MBT resistant to first-line anti-TB drugs. Material for the study was venous blood. To isolate monocytes from the whole blood in order to transform them into macrophages, Ficoll density gradient centrifugation with a density of 1.077 g / cm3 was used followed by immunomagnetic separation of CD14+ cells. Monocytes were cultured in the X-VIVO 10 medium with gentamicin and phenol red with the addition of macrophage colony-stimulating factor (M-CSF) (5 ng / ml) at a concentration of 1×106 cells / ml with stimulators: interleukin (IL)-4 (10 ng / ml) and interferon (IFN) γ (100 ng / ml). Immunophenotyping of macrophages was performed using monoclonal antibodies to CD163, CD204, and CD206 on the Beckman Coulter CytoFLEX LX Flow Cytometer. The analysis of the obtained data was carried out using the CytExpert 2.0 software. The results were analyzed using statistical methods.Results. Switching the phenotype of macrophages from the M1-like proinflammatory phenotype to M2-like antiinflammatory one contributes to the chronic course of pulmonary TB, dissemination, and persistence of infection. In the present study, we analyzed the features of the expression of CD163, CD204, and CD206 scavenger receptors on macrophages in patients with pulmonary TB. An increase in the number of macrophages carrying markers of the M2 subpopulation (CD163, CD204, and CD206) on their surface was noted, regardless of the clinical form of pulmonary TB and drug resistance of M. tuberculosis.Conclusion. Studying the mechanisms underlying M1 or M2 activation of macrophages is necessary for a deeper understanding of the immunopathogenesis of TB and the role of innate immunity cells in protecting the body from mycobacteria. The analysis of the expression of scavenger receptors CD163, CD204, and CD206 on macrophages allowed to conclude that, in pulmonary TB, especially in patients with drug resistant M. tuberculosis and infiltrative TB, regulatory mechanisms that suppress the activation of innate immunity are implemented together with polarization of macrophage differentiation towards the M2 phenotype. It may be the cause of immune deficiency induced by the pathogen.

We examined expression pattern of CD80 and HLA-DR pro-inflammatory molecules on the monocytes in patients with pulmonary tuberculosis (TB), depending on the clinical form of the disease and susceptibility of the pathogen to anti-tuberculosis drugs. The study involved forty-five patients with newly diagnosed pulmonary TB (25 men and 20 women aged 18 to 55 years, average age — 44.0±12.4 years). The control group included 15 healthy donors with similar socio-demographic characteristics as in TB patients. Venous blood was used as biomaterial for assays. Studies of the monocyte immunophenotype were carried out by flow cytometry of whole blood cells using Cytoflex flow cytometer (Beckman Coulter, USA) with specific monoclonal antibodies (eBioscience, USA). We determined the content of cells expressing surface markers of monocytes, i.e., CD14, CD45, CD80, and HLA-DR. The results of this study were evaluated using SPSS Statistics 17.0 standard software package and Microsoft Excel. In the course of the study, we have suggested a working hypothesis that the monocytes in TB patients, still being in circulation, can express activation markers during their migration to inflammation focus, especially CD80 and HLA-DR molecules. Analysis of the total CD14+ monocyte number showed its decrease in all forms and variants of clinical course of pulmonary tuberculosis compared with the control group. Assessment of pro-inflammatory markers expressed on CD14 positive monocytes, i.e., HLA-DR activation marker and CD80 co-stimulatory molecule, showed that the number of monocytes with HLA-DR expression in all TB patients was higher than in healthy donors. HLA- DR expression on CD14+ monocytes in the group of patients with infiltrative TB proved to be 15% higher than in patients with disseminated TB. The expression of CD80 on CD14+ monocytes in TB patients showed no differences between the groups and varied within the normal range. Hence, an imbalance within monocyte population in patients with pulmonary tuberculosis, regardless of its clinical form and drug sensitivity of the pathogen is developed, due to decrease in total number of CD14+ cells, along with increased relative number of monocytes expressing HLA-DR activation marker (pro-inflammatory phenotype). Meanwhile, expression of the CD80 co-stimulatory molecule on monocytes was within normal values.

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.

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.

The aim of the work is to establish general regularities and features of differentiation of blood monocytes into 4 subpopulations in diseases associated with circulatory and respiratory hypoxia.Materials and methods. 18 patients with ischemic heart disease (IHD), 12 patients with ischemic cardiomyopathy (ICMP), 14 patients with chronic obstructive pulmonary disease (COPD), 15 patients with newly diagnosed infiltrative pulmonary tuberculosis (PTB) and 12 healthy donors were examined. In whole blood, we determined the relative number of different subpopulations of monocytes by flow cytometry. The results were analyzed by statistical methods.Results. It is shown that an increase in the number of classical (80.56 [77.60; 83.55]%) and the deficit of intermediate (10.38 [9.36; 11.26]%), non-classical (6.03 [5.24; 6.77]%) and transitional (2.14 [1.41; 3.92] %) monocytes in the blood is determined in patients with COPD when compared with the group of healthy donors (p < 0.05). In groups of patients with PTB and IHD, an increase in the number of intermediate monocytes (26.24 respectively [22.38; 42.88] % and 25.27 [15.78; 31.39]%) and the lack of transitional cells (1.77 [1.36; 3.74]% and 2.68 [2.63; 4.0]%) at the normal content of classical and non-classical forms of monocytes (p < 0.05) is detected. In patients with ICMP, a decrease in the number of non-classical monocytes (up to 5.05 [4.08; 6.58]%) is combined with the normal cell content of other subpopulations (p < 0.05). The interrelation between the number of classical and intermediate monocytes in patients with COPD (r = –0.63; p < 0.05), PTB (r = –0.72; p < 0.01), IHD (r = –0.59; p < 0.05), ICMP (r = –0.58; p < 0.05) was established.Conclusion. In COPD associated with generalized hypoxia, an increase in the number of classical monocytes is combined with a deficiency of their other subpopulations in the blood. In PTB and IHD, antigenic stimulation of the immune system mediates accelerated differentiation of monocytes from classical to intermediate forms with a decrease in the number of transitional cells regardless of the etiology of the disease (infectious or non-infectious) and the type of hypoxia (respiratory or circulatory).

In the present time, incidence of pulmonary tuberculosis (TB) becomes broader, due to spreading resistance of Mycobacterium tuberculosis (MBT) to anti-tuberculosis drugs and infection with highly virulent strains of M. tuberculosis. The MBT antigens can cause dysfunction of the receptors and modulate the cytokine secreting function of immunocompetent cells. Polymorphic genes of pro-inflammatory cytokines involved in the mechanisms of defense responses of innate immunity, determine the degree of resistance to individual mycobacterial infection, as well as severity and duration of the disease in cases of clinical manifestations. The aim of the study was to investigate the connections between allelic polymorphisms of IL2, IFNG and TNFA genes and changes in secretion of the corresponding pro-inflammatory cytokines IL-2, IFNγ, and TNFα in vitro in patients with the newly diagnosed pulmonary tuberculosis (TB), depending on the clinical form of the disease.A total of 334 patients (220 men and 114 women) aged 23 to 50 years with newly diagnosed infiltrative and disseminated TB were enrolled into the study. The control group consisted of 183 healthy donors (130 men and 53 women) of corresponding age. The material of the research included DNA extracted from the whole blood and supernatants of culture suspensions of mononuclear leukocytes isolated from venous blood in healthy volunteers and patients with TB. The evaluation of cytokines secretion was performed by measuring their concentration in the blood mononuclear cell culture supernatants. using enzyme-linked immunosorbent assay (ELISA). To study polymorphic regions of cytokine genes, a polymerase chain reaction (PCR) was applied. Analysis of the obtained data was carried out by means of the program Statistica for Windows Version 6.0 (StatSoft Inc., USA).It was found that the imbalance of secretion of pro-inflammatory cytokines in TB patients was associated with the polymorphic variants of genes of these cytokines. It was found that the hypo-secretion of IL-2 is determined by the carriage of the G allele and genotype GG (T-330G) of the IL2 gene in both the control group and in patients with TB, regardless of the clinical form. In patients with DTB carriers of the homozygous genotype TT (T-330G) of the IL2gene, increased protein secretion was established. The maximum secretion of TNFб was recorded in patients with the AA genotype (G-308A) of the TNFA gene in the control group and in ITB patients; the minimum concentration of TNFα was associated with the carrier of the homozygous GG genotype (G-308A) of the TNFA gene in all the examined groups. In patients with ITB and DTB, an increase in IFNγ secretion by mononuclear blood leukocytes is not associated with the carrier of polymorphism +874A/T of the IFNG gene.Reduced secretion of IL-2 and TNFα in TB patients is associated with polymorphisms of their genes – (T-330G) of IL2 gene and (G-308A) of TNFA gene, respectively. The polymorphism (+874A/T) of the IFNG gene does not have a modulatory effect on the secretion of IFNγ in patients with TB, regardless of clinical form of the disease.

The aim of the investigation was to determine the characteristics of the immune response regulation for pulmonary tuberculosis (TB) and to analyze the role of regulatory T cells in the immunopathogenesis of TB with eosinophilia in the blood, depending on the clinical form of the disease and sensitivity of Micobacterium tuberculosis to anti-TB drugs.Materials and methods. 157 patients who were initially diagnosed with infiltrative and disseminated TB were examined. The material of the study was venous blood and culture of mononuclear leukocytes isolated from venous blood. The content of interleukin (IL) 4, IL-10 and transforming factor beta (TGFβ) in culture suspensions of mononuclear leukocytes in vitro and IL-5 in the blood was determined by enzyme-linked immunosorbent assay (ELISA) test. The expression of surface molecules CD4, CD20, CD25 and intracellular transcription factor Foxp3 by lymphocytes of the blood was evaluated by flow cytometry. The obtained results were analyzed by statistical methods.Results. It is shown that excessive generation of regulatory T cells in patients with TB is associated with eosinophilia of the blood and imbalance of immune response regulation mechanisms. In TB with eosinophilia, an increase in the number of Foxp3-positive regulatory T cells in the blood is combined with in vitro hypersecretion of anti-inflammatory cytokines TGFβ, IL-10, IL-4 and an increase in the content of CD20+ B lymphocytes and IL-5 in the blood. These changes are most pronounced in the disseminated form of TB in combination with drug resistance.Conclusion. Characteristics of immunoregulation at TB with blood eosinophilia are associated with activation of immunosuppression mechanisms and polarization of immune response towards Th2-dependent pathway.

The article provides an overview of scientific research to explain the mechanisms of inefficient implementation of antigen-specific immune response in patients with tuberculosis infection depending on the clinical form (infiltrative and disseminated pulmonary tuberculosis) and the course (drug-sensitive and drug-resistant pulmonary tuberculosis) of the disease. The mechanisms of immune imbalance in patients with pulmonary tuberculosis are associated with abnormal co-stimulation of a signal required for the activation of T-lymphocytes and immunosuppressive influence of regulatory T-cells both when they interact with dendritic cells at the inductive stage of the immune response, and in the process of differentiation and proliferation of effector cells with formation of suppressive mode of immune-regulation.

In present work, some immunogenetic aspects of pulmonary tuberculosis were studied, using modern techniques from molecular genetics and immunology. It is shown that carriage of Т allele and homozygous TT genotype in +874А/Т IFNγ gene polymorphism comprise a immunogenetic factor which correlated with a protective effect, regarding a susceptibility to pulmonary tuberculosis. Predisposition for tuberculosis infection is associated with A allele of this gene, as well as with АА and АТ genotypes of +874A/T IFNγ gene. Clinical evolution of pulmonary tuberculosis is accompanied by increase in IFNγ production.

Abstract. The work concerned some parameters of proliferation and apoptosis in peripheral blood lymphocytes in patients with primary pulmonary tuberculosis (Tbc), as dependent on clinical form of the disease and sensitivity of M. tuberculosis to specific medical drugs. It was shown that, in patients with infiltrative and disseminated Tbc, as well as in cases of multi-drug resistance of M. tuberculosis, an increased apoptosis of lymphocytes is observed, accompanied by inhibition of their proliferative activity. The numbers of apoptotic and proliferating lymphocytes remain within normal ranges in patients with fibrous/cavernous form of the disease, and in cases of drug-sensitive M. tuberculosis.

Abstract. The study dealt with subpopulation analysis of peripheral blood T-regulatory cells (Treg), and in vitro testing of immunosuppressive cytokine (IL-10, TGF-β) production in the patients with fibrous/cavernous pulmonary tuberculosis, depending on results of intracutaneous tuberculin tests (Mantoux test). We have shown that Trn, natural T-regulatory lymphocytes (CD4+CD25+FoxP3+), and TGF-β cytokine, produced by these cells, play a leading role for immune suppression developing in fibrosis-cavernous pulmonary tuberculosis. Moreover, an imbalance of Treg cell subsets has been revealed in patients with fibrous/ cavernous pulmonary tuberculosis, with either positive, or negative Mantoux reaction, as shown by increased content of Treg cells with CD4+CD25+FoxP3+ phenotype, along with higher amounts of CD4+CD25-FoxP3+ subpopulations, and deficiency of CD4+CD25+FoxP3- Treg lymphocytes in blood samples (in cases of positive Mantoux tests). Increased amounts of CD4+CD25+FoxP3+ Treg cells in peripheral blood of patients with fibrous/cavernous pulmonary tuberculosis is associated with increase in basal and BCG-induced TGF-β production, and decreased in vitro IL-10 secretory potential. (Med. Immunol., 2011, vol. 13, N 2-3, pp 267-272)

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.

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.