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

Молекулярно-генетические факторы туберкулеза легких

The modulating influence of a causative agent on the character of an immune reaction at lung tuberculosis is analyzed. Peculiarities of immunopathological reactions depending on the etiological version of a disease and the spectrum of bacterial resistance to antituberculous preparations are discussed with the justification of possible approaches to their correction. Main fields of research activity at the Chair of Pathophysiology of the Siberian State Medical University (Tomsk) concerning immunopathology of the tuberculosis infection are presented.В лекции анализируется модулирующее влияние возбудителя на характер иммунного ответа при туберкулезе легких. Обсуждаются особенности иммунопатологических реакций в зависимости от этиологического варианта заболевания и спектра бактериорезистентности к противотуберкулезным препаратам с обоснованием возможных подходов к их коррекции. Представлены основные направления научно-исследовательской работы кафедры патофизиологии Сибирского государственного медицинского университета (г. Томск) в области иммунопатологии туберкулезной инфекции

Цитокиновый профиль крови в зависимости от полиморфизма генов цитокинов у больных хроническим гнойным средним отитом

The goal is to reveal features of changes in the ratio of cytokines IL-1β, IL-6, TNF-α and IL-10 in blood depending on polymorphism of genes IL1В, IL6, TNFА and IL10 at different clinical forms of chronic inflammation of the middle ear in the stage of exacerbation before treatment and on the 10th day of treatment.Materials and methods. A total of 299 patients (129 men and 170 women) with chronic purulent otitis media were examined: 146 with tubotympanic form and 153 with epitympanoantral form. The control group included 183 relatively healthy donors, comparable to those of 299 patients by sex and age. Human genomic DNA was isolated from the blood by a standard method and was analyzed with the use of the polymerase chain reaction (PCR) in real-time. The concentration of cytokines IL-1β, IL-6, TNF-α and IL-10 in blood was measured by solid-phase enzyme-linked assay before and after 10 days of treatment. Statistical processing of the obtained data was performed using software package STATISTICA 6.0 (StatSoft Inc., USA). The critical level of significance when testing statistical hypotheses was taken to be less than 0.05.Results. In patients with chronic purulent otitis media, an increase in the concentration of IL-1β, IL-6 and TNF-α in the blood varies depending on polymorphism of genes IL1В (-3953, -511, -31), IL6 (-174), TNFА (-308) and the clinical form of the disease. The concentration of IL-10 in the blood depends more on the polymorphism of the IL10 gene (-1082, -592) than on the clinical form of the disease. The combined increase of concentration of IL-10 and proinflammatory cytokines in the blood before the beginning and on the 10th day of combined treatment of patients with tubotympanic and epitympanoantral forms of chronic purulent otitis media, in carriers of high-producing alleles inflammatory cytokines of polymorphic variants of genes IL1B, IL6 and TNFA, indicates a genetic predisposition to hyperergic the course of inflammation.Цель – выявить особенности изменений соотношения цитокинов IL-1β, IL-6, TNF-α и IL-10 в крови в зависимости от полиморфизма генов IL1В, IL6, TNFА и IL10 при разных клинических формах хронического гнойного воспаления среднего уха в стадии обострения до и на 10-е сут лечения.Материалы и методы. Обследованы 299 пациентов (129 мужчин и 170 женщин) с хроническим гнойным средним отитом (ХГСО): 146 с туботимпанальной формой и 153 с эпитимпано-антральной формой. Группу контроля составили 183 относительно здоровых донора, сопоставимых с больными ХГСО по полу и возрасту. Геномную ДНК выделяли из крови стандартным методом и анализировали методом полимеразной цепной реакции (ПЦР) в режиме реального времени. Концентрацию цитокинов IL-1β,  IL-6, TNF-α и IL-10 в крови измеряли методом твердофазного иммуноферментного анализа до и через 10 сут лечения. Статистическую обработку полученных данных проводили с использованием пакета программ STATISTICA 6.0 (StatSoft Inc., США). Критический уровень значимости при проверке статистических гипотез принимался равным 0,05.Результаты. У больных ХГСО повышение концентрации IL-1β, IL-6 и TNF-α в крови варьирует в зависимости от носительства полиморфных вариантов их генов (IL1В в локусах -3953, -511, -31, IL6 в локусе -174 и TNFА в локусе -308) и клинической формы заболевания. Концентрация IL-10 в крови в большей степени зависит от полиморфизма гена IL10 (в локусах -1082, -592), чем от клинической формы заболевания. Сочетанное увеличение концентрации IL-10 и провоспалительных цитокинов до начала и на 10-е сут лечения у больных с туботимпанальной и эпитимпано-антральной формами ХГСО, являющихся носителями «высокопродуцирующих» аллелей полиморфных вариантов генов IL1В, IL6 и TNFА, свидетельствует о генетической предрасположенности к гиперергическому течению воспаления

PECULIARITIES OF CD3/CD28-INDUCED SECRETION OF INTERLEUKIN-2 AND SUBPOPULATION COMPOSITION OF T-LYMPHOCYTES IN PATIENTS WITH PULMONARY TUBERCULOSIS

The article presents the results of the research of CD3/CD28-induced production of interleukin-2 by blood lymphocytes in vitro as well as subpopulation composition of peripheral blood T-lymphocytes in patients with drug-sensitive and drug-resistant pulmonary tuberculosis. We detected the decrease in the total amount of CD3-positive cells and suppression of IL-2- secretion during CD3/CD28-induction of lymphocytes, which was most expressed in a disseminated form of drug-resistant TB. We also demonstrated the reduction in the percentage value of CD3+CD28+IL-2+ and CD3+CD28+IL-2- cells in patients with pulmonary TB against the background of the increase in the percentage value of CD3+CD28-IL-2-. Besides, it was also shown that the detected changes are unidirectional, don't depend on a clinical form of TB and are maximally manifested in patients with a drug resistant variant of the TB process

Cytokines and HIF-1α as dysregulation factors of migration and differentiation of monocyte progenitor cells of endotheliocytes in the pathogenesis of ischemic cardiomyopathy

Background. Angiogenic endothelial dysfunction and progenitor endothelial cells (EPCs) in ischemic cardiomyopathy (ICMP) have not been studied enough.The aim. To establish the nature of changes in the cytokine profile and HIF-1α in blood and bone marrow associated with impaired differentiation of monocytic progenitor cells of endotheliocytes (CD14+VEGFR2+) in the bone marrow and their migration into the blood in patients with coronary heart disease (CHD), suffering and not suffering from ICMP.Materials and methods. A single-stage, single-centre, observational case-control study was conducted involving 74 patients with CHD, suffering and not suffering from ICMP (30 and 44 people, respectively), and 25 healthy donors. In patients with CHD, bone marrow was obtained during coronary bypass surgery, peripheral blood – before surgery. Healthy donors were taken peripheral blood. The number of CD14+VEGFR2+ in bone marrow and blood was determined by flow cytometry; the concentration of IL-6, TNF-α, M-CSF, GM-CSF, MCP-1 and HIF-1α – by the method of enzyme immunoassay.Results. A high content of CD14+VEGFR2+ cells in the blood of patients with CHD without cardiomyopathy was established relative to patients with ICMP against the background of a comparable number of these cells in myeloid tissue. Regardless of the presence of ICMP in the blood, patients with CHD showed an excess of TNF-α, a normal concentration of IL-6, GM-CSF, HIF-1α and a deficiency of M-CSF, and in the bone marrow supernatant, the concentration of IL-6 and TNF-α exceeded that in the blood plasma (the level of GM-CSF – only in patients without cardiomyopathy). With ICMP, the normal concentration of MCP-1 was determined in the blood plasma, and with CHD without cardiomyopathy, its elevated content was determined.Conclusion. The formation of ICMP is accompanied by insufficient activation of EPCs migration with the CD14+VEGFR2+ phenotype in blood without disruption of their differentiation in the bone marrow, which associated with the absence of an increase in the concentration of MCP-1 in blood plasma and not associated with the plasma content of M-CSF, GM-CSF, HIF-1α, IL-6 and TNF-α

Экспериментальное обоснование эффективности «массажа» ретинальных вен для лечения окклюзии вен сетчатки

Objective: evaluate the effectiveness of treatment of retinal vein occlusion with the use of “massage” of retinal veins on the experimental model of photoinduced retinal venous occlusive disease.Materials and methods. Studies were carried out on 30 rabbits of the Chinchilla breed (30 eyes) weighing 1.5–2.0 kg. In the first stage of the experiment, for all animals the photoinduced thrombosis were modeled by laser irradiation of its retinal veins for 0.3–0.4 seconds at a density of 200 J/cm2 and a wavelength of 662 nm after the administration of “Photoditazine” at a dose of 2.5 mg/kg weight. In the second stage of the experiment, the animals were divided into the main group and the comparison group, depending on the method of treatment: 15 animals of the main group with “massage” of the retinal vein in the form of 5–7 movements with a silicone tip of the scanner Tano, 15 animals of the comparison group with epiretinal administration of recombinant prourokinase in a dose 500 international units (IU).Results. The formation of a parietal thrombus in the simulation of photoinduced the central retinal vein occlusion in the experiment leads to a slowing of perfusion in the affected vessel by (8.0 ± 2.5) s (according to fluorescence angiography), an increase in the thickness of the central parts of the retina by (70.2 ± 9.7) μm (according to optical coherence tomography), to the appearance of crimp and uneven lumen of the retina veins, extensive plasma and hemorrhages (according to the data of ophthalmoscopy). On the model of retinal veins thrombosis, a new method of treating retinal vein occlusion was tested, which showed significant efficacy. It has been established that the use of “massage” of the affected retinal vein reduces the time of venous perfusion by 1.3 times and 1.45 times accelerates the resorption of the edema of the central parts of the retina compared to enzymatic thrombolysis.Conclusions. “Massage” of the retinal veins is an effective method of treating retinal vein occlusion and is not accompanied by damage to chorioretinal structures during manipulation.Цель работы: на экспериментальной модели фотоиндуцированного тромбоза центральной вены сетчатки и ее ветвей оценить эффективность лечения окклюзии вен сетчатки с применением «массажа» ретинальных вен.Материалы и методы. Исследование проведено на 30 кроликах породы шиншилла (30 глаз) массой 1,5–2,0 кг. На первом этапе эксперимента всем животным моделировали фотоиндуцированный тромбоз при помощи лазерного облучения ретинальных вен в течение 0,3–0,4 с при плотности воздействия 200 Дж/см2 и длине волны 662 нм после введения фотодитазина в дозе 2,5 мг/кг массы. На втором этапе эксперимента животных разделяли на основную группу и группу сравнения в зависимости от метода лечения: 15 животным основной группы выполняли «массаж» пораженной ретинальной вены в виде 5–7 движений силиконовым наконечником скраппера Tano, 15 животным группы сравнения эпиретинально вводили рекомбинантную проурокиназу в дозе 500 международных единиц (МЕ).Результаты. Формирование пристеночного тромба при моделировании фотоиндуцированной окклюзии ретинальных вен в эксперименте приводит к замедлению перфузии в пораженном сосуде на (8,0 ± 2,5) с (по данным флуоресцентной ангиографии), увеличению толщины центральных отделов сетчатки на (70,2 ± 9,7) мкм (по данным оптической когерентной томографии), появлению извитости и неравномерности просвета вен сетчатки, обширных плазмо- и геморрагий (по данным офтальмоскопии). На модели тромбоза ретинальных вен апробирован новый метод лечения окклюзии вен сетчатки, показавший значительную эффективность. Установлено, что применение «массажа» пораженной ретинальной вены сокращает время венозной перфузии и в 1,45 раза ускоряет резорбцию отека центральных отделов сетчатки по сравнению с ферментным тромболизисом.Выводы. «Массаж» ретинальных вен является эффективным методом лечения окклюзии вен сетчатки и не сопровождается повреждением хориоретинальных структур во время манипуляции

FUNCTIONAL POLYMORPHISM OF THE PRO-INFLAMMATORY CYTOKINE GENES IN PULMONARY TUBERCULOSIS

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

Expression of CD80 and HLA-DR molecules on blood monocytes in patients with pulmonary tuberculosis

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

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