HEPATIC SOD1 GENE EXPRESSION CHANGES IN THE NAFLD PATHOGENESIS IN OBESITY

Steatosis in the liver in obesity increases the work of mitochondria to utilize excess lipids. An overload of β-oxidation of fatty acids, the tricarboxylic acid cycle, and oxidative phosphorylation leads to a decrease in ATP and an increase in the formation of reactive oxygen species. Normally, mitochondria can efficiently remove elevated levels of reactive oxygen species using the cell's antioxidant system and metabolic adaptation to altered conditions. This study aimed to investigate the role of hepatic SOD expression in the pathogenesis of NAFLD in obesity. It was found that the level of SOD1 expression in the liver in obese patients with and without type 2 diabetes with a BMI > 40 kg/m2 was lower than in healthy donors. The copy number of mitochondrial DNA (mtDNA) in the liver in all obese patients was more than two times lower than in the control group. In the liver of obese patients without type 2 diabetes, the SOD1 protein level and the mtDNA copy number were interrelated and negatively correlated with the area of fatty inclusions. Thus, in obese patients, a decrease in antioxidant defense in the liver leads to the vulnerability of mitochondria, which, in turn, contributes to the progression of steatosis and insulin resistance

Significance of nutrient media choice for the long-term cultures of leukemic T-lymphoblasts

Correct choice of nutrient media for culturing different types of cells in various applications is one of the most important aspects of modern biotechnology, since chemical composition of the culture media largely contains the necessary metabolites to support certain cells’ growth lines outside the body. Jurkat line of human leukemic T-lymphoblast-like cells (hereinafter Jurkat T-cells) is actively used for in vitro modeling of intracellular signaling and activation of normal blood T-lymphocytes mediated by the T-cell receptor/CD3/ CD4 complex in toxicological studies of immune and secretory responses, to test medicinal substances and ions. Also, Jurkat T-cells are widely used for ex vivo testing in immunology, oncology, toxicology, orthopedics, and traumatology. The existing standards and numerous studies are mainly based on short-term in vitro cultivation of Jurkat T-cells in RPMI 1640 nutrient medium. Meanwhile, the issues of long-term maintenance of the growth of Jurkat T-cells culture are poorly presented in the research literature. This study aimed for studying the activity of Jurkat T-cells over 7 to 14 days of in vitro culture and comparing the relative value of RPMI 1640 and αMEM media for the behavior of immunocompetent tumor cells. Using flow cytometry, multiplex analysis, and phase contrast Cell-IQ microscopy, the proportions of living cells and those dying by apoptosis and necrosis, secretion of cytokines and chemokines, and the dynamics of cell biomass propagation were studied. It was found that the αMEM medium in the complete nutrient medium, as compared with RPMI 1640, is more appropriate to in vitro promotion of cell viability (increased proportion of viable cells by 13.5% at the day 14), their secretory ability for 23 из 27 tested biomolecules, shortened adaptation time (на 32%) in culture before growth initiation, 5-fold increase of the Jurkat Т-cell cellularity by the day 7. Potential significance of the chemical components of nutrient media and secreted biomolecules for these results is discussed. As based on the results obtained, we concluded on superior properties of αMEM medium for long-term in vitro cultures of Jurkat T-cells. Consequently, the in vitro testing of medical devices intended for long-term contact with the body, including those for cancer patients, using Jurkat T-cell leukemia line in RPMI 1640 medium, may lead to wrong predictions on their biocompatibility and potential antitumor activity

Клеточные реакции CD3+ CD4+ CD45RO+ Т-лимфоцитов на дексаметазон в норме и при ревматоидном артрите в системе in vitro

The aim of the study was to analyze the influence of glucocorticoid (GC) dexamethasone (Dex) on changes in CD4+ T-cells expressing the surface molecule of activation (CD25, CD71, HLA-DR and CD95) and their ability to produce proinflammatory mediators in cultures of TCR-stimulated CD3+CD45RO+ T-lymphocytes obtained from healthy donors and patients with rheumatoid arthritis in vitro.Materials and methods. The study included 50 patients and 20 healthy donors. T-cell cultures (CD3+ CD45RO+) were obtained from mononuclear leukocytes of immunomagnetic separation (MACS® technology). As an activator of T-lymphocytes, antibiotic particles with biotinylated antibodies against CD2+, CD3+, CD28+, which simulate the process of costimulation of T cells by antigen-presenting cells, were used. The following concentrations of dexamethasone (2, 8, 16, 32, 64 mg) were used in the experiment. The change in the immunophenotype of T-lymphocytes was analyzed by flow cytofluoometry. The secretion of CD3+CD45RO+ T-cells of proinflammatory cytokines IL-2, IFNγ, TNFα, IL-17 and IL-21 was evaluated by enzyme-linked immunosorbent assay.Results. The general suppressor effect of Dex on CD3+CD45RO+ T-cell cultures mediated by a decrease in the number of CD4 + T cells expressing activation molecules (CD25) and proliferation (CD71), as well as inhibition of the production of inflammatory mediators: IFNγ, IL-2 and TNFα. It is shown that against the background of TCR activation Dex increases the number of CD4+CD95+HLA-DR+ cells in CD3+CD45RO+ cultures obtained from RA patients and does not change their content in the control. The correlations between the number of proinflammatory factors (IL-17, IL-21 and TNFα) in CD4+CD45RO+CD95+HLA-DR+ T cells in supernatants of cell cultures in RA patients indicate the presence of a pro-inflammatory potential of this population of T cells. We assume that the resistance of CD4+CD45RO+CD95+HLA-DR+ T cells in RA patients to the suppressor effect of GC generally leads to the preservation and enhancement of the functionality of autoreactive cells in the pathogenesis of RA. Целью исследования явился анализ влияния глюкокортикоида (ГК) дексаметазона (Dex) на изменение числа CD4+ Т-клеток, экспрессирующих поверхностные молекулы активации (CD25, CD71, HLA-DR и CD95), и их способности продуцировать провоспалительные медиаторы в культурах TCRстимулированных Т-лимфоцитов CD3+CD45RO+, полученных у здоровых доноров и больных ревматоидным артритом (РА), в системе in vitro. В исследование включены 50 больных и 20 условно здоровых доноров.Материал и методы. Культуры T-клеток (CD3+CD45RO+) получали из мононуклеарных лейкоцитов методом иммуномагнитной сепарации (технология MACS®). В качестве активатора Т-лимфоцитов использовали антибиотиновые частицы с биотинилированными антителами против CD2+, CD3+, CD28+ человека, имитирующие процесс костимуляции Т-клеток антиген-презентирующими клетками. В эксперименте использованы следующие концентрации дексаметазона – 2; 8; 16; 32; 64 мг. Методом проточной цитофлуориметрии проанализировано изменение иммунофенотипа Т-лимфоцитов; иммуноферментным анализом оценена секреция Т-клетками CD3+CD45RO+ провоспалительных цитокинов: IL-2, IFNγ, TNFα, IL-17 и IL-21.Результаты. Подтвержден общий супрессорный эффект Dex на культуры Т-клеток CD3+CD45RO+, опосредованный снижением числа Т-клеток CD4+, экспрессирующих молекулы активации (CD25) и пролиферации (CD71), а также угнетением продукции медиаторов воспаления: IL-2, IFNγ и TNFα. Показано, что на фоне TCR-активации Dex повышает число клеток CD4+CD95+HLA-DR+ в культурах СD3+CD45RO+, полученных от больных РА, и не изменяет их содержание в контроле. Корреляции между числом Т-клеток CD4+CD45RO+ CD95+HLA-DR+ с уровнем провоспалительных факторов (IL-17, IL-21 и TNFα) в супернатантах клеточных культур у больных РА свидетельствуют о наличии провоспалительного потенциала этой популяции Т-клеток. Предполагается, что резистентность Т-клеток CD4+CD45RO+CD95+HLA-DR+ больных РА к супрессорному действию ГК в целом приводит к сохранению и усилению функциональных возможностей аутореактивных клеток в патогенезе РА.

Особенности клеточного иммунитета и регенерации при алкогольном фиброзе печени

Purpose. The subpopulation composition of peripheral blood lymphocytes was evaluated in patients with alcoholic liver fibrosis (ALF).Materials and methods. The study included 62 patients with ALF; 15 patients abusing alcohol without liver fibrosis and 20 conditionally healthy donors. In samples of lysed peripheral blood, the number of cells bearing surface markers was determined by flow cytometry. In patients with ALF at terminal stages of fibrosis, significant lymphopenia was recorded with a change in the composition of the main subpopulations of lymphocytes relative to the values of conditionally healthy donors and the comparison group.Results. We identified in the blood of ALF patients with terminal (III–IV) stage (relative to control and comparison group) of the relative number of naive (TN) and central memory T-lymphocytes (TCM) associated with an increase in the number of effector cells (TEM and TEMRA) allows us to suggest in this category of patients the direct differentiation of TN and TCM lymphocytes to effector (TEM and TEMRA), which can aggravate the course of the tissue-destructive process due to the high biocidal activity of the latter. Elevated levels of hematopoietic (CD34 and CD133) cells in the peripheral blood at the initial and moderate stages. (I–II) fibrosis (relative to control and comparison group) may be due to persistent inflammation in the liver parenchyma and an increasing imbalance between the processes of its damage and reparative capabilities. Whereas the decrease in their number at the terminal station fibrosis may indicate an increasing decompensation and depletion of the regenerative potential of the organism in the final stages of the degenerative process.Conclusions. In general, the obtained data demonstrate new aspects of the immune regulation of the processes of fibrogenesis in chronic alcoholism.Цель. Оценка субпопуляционного состава лимфоцитов периферической крови у больных алкогольным фиброзом печени (АФП).Материалы и методы. В исследование были включены 62 больных АФП; 15 пациентов, злоупотребляющих алкоголем, без фиброза печени и 20 условно здоровых доноров. В образцах лизированной периферической крови методом проточной цитометрии определяли число клеток, несущих поверхностные маркеры.Результаты. У больных АФП на терминальных стадиях (ст.) фиброза регистрировалась значительная лимфопения с изменением состава основных субпопуляций лимфоцитов относительно значений условно здоровых доноров и группы сравнения. Выявленное нами в крови больных АФП с терминальными (III–IV) ст. заболевания снижение (относительно контроля и группы сравнения) относительного числа наивных (TN) и Т-лимфоцитов центральной памяти (ТСМ), ассоциированное с ростом количества эффекторных клеток (TEM и TEMRA), позволяет нам предположить у этой категории больных факт прямой дифференцировки лимфоцитов TN и ТСМ в эффекторные (TEM и TEMRA), что может усугублять течение тканедеструктивного процесса за счет высокой биоцидной активности последних. Повышенный уровень гемопоэтических (CD34 и CD133) клеток в периферической крови на начальных и умеренных (I–II) ст. фиброза (относительно контроля и группы сравнения) может быть обусловлен персистирующим воспалением в паренхиме печени и нарастающим дисбалансом между процессами ее повреждения и репаративными возможностями. Тогда как снижение их количества на терминальных ст. фиброза может свидетельствовать о нарастающей декомпенсации и истощении регенераторного потенциала организма на финальных этапах дегенеративного процесса.Заключение. В целом полученные данные демонстрируют новые аспекты иммунной регуляции процессов фиброгенеза при хроническом алкоголизм

EFFECTS OF γс-CYTOKINES (IL-2, IL-7 AND IL-15) UPON IN VITRO MATURATION AND DIFFERENTIATION OF CD4⁺CD45RО⁺/CD8⁺CD45RО⁺ T CELLS

Effect of γс-cytokines (IL-2, IL-7 и IL-15) upon maturation and differentiation of cytotoxic and helper CD45RО+ Tcell population was studied in the homeostatic in vitro culture conditions. We have found that IL-2, IL-7 and IL-15 mediate maturation and differentiation of CD8 T cells central memory, replenishing the population of cells that exhibit effector functions. Action of IL-2 on CD4+ central memory T cells is accociated with generation of effector memory cells by reducing the number of immature effectors (CD62L–CD27+), whereas IL-7 promotes the formation of immature (CD62L–CD27+) effectors. IL-2, IL-7 and IL-15 initiate formation of terminally-differentiated cells in a subpopulation of CD8+CD45RO+ lymphocytes, providing a stable immunological memory to a pathogen reinfestation

Cellular reactions of CD3+ CD4+ CD45RO+ T-lymphocytes on dexamethason in in normal patients and in patients with with rheumatoid arthritis in vitro

The aim of the study was to analyze the influence of glucocorticoid (GC) dexamethasone (Dex) on changes in CD4+ T-cells expressing the surface molecule of activation (CD25, CD71, HLA-DR and CD95) and their ability to produce proinflammatory mediators in cultures of TCR-stimulated CD3+CD45RO+ T-lymphocytes obtained from healthy donors and patients with rheumatoid arthritis in vitro.Materials and methods. The study included 50 patients and 20 healthy donors. T-cell cultures (CD3+ CD45RO+) were obtained from mononuclear leukocytes of immunomagnetic separation (MACS® technology). As an activator of T-lymphocytes, antibiotic particles with biotinylated antibodies against CD2+, CD3+, CD28+, which simulate the process of costimulation of T cells by antigen-presenting cells, were used. The following concentrations of dexamethasone (2, 8, 16, 32, 64 mg) were used in the experiment. The change in the immunophenotype of T-lymphocytes was analyzed by flow cytofluoometry. The secretion of CD3+CD45RO+ T-cells of proinflammatory cytokines IL-2, IFNγ, TNFα, IL-17 and IL-21 was evaluated by enzyme-linked immunosorbent assay.Results. The general suppressor effect of Dex on CD3+CD45RO+ T-cell cultures mediated by a decrease in the number of CD4 + T cells expressing activation molecules (CD25) and proliferation (CD71), as well as inhibition of the production of inflammatory mediators: IFNγ, IL-2 and TNFα. It is shown that against the background of TCR activation Dex increases the number of CD4+CD95+HLA-DR+ cells in CD3+CD45RO+ cultures obtained from RA patients and does not change their content in the control. The correlations between the number of proinflammatory factors (IL-17, IL-21 and TNFα) in CD4+CD45RO+CD95+HLA-DR+ T cells in supernatants of cell cultures in RA patients indicate the presence of a pro-inflammatory potential of this population of T cells. We assume that the resistance of CD4+CD45RO+CD95+HLA-DR+ T cells in RA patients to the suppressor effect of GC generally leads to the preservation and enhancement of the functionality of autoreactive cells in the pathogenesis of RA

CYTOKINE PROFILE IN PREGNANT WOMEN WITH METABOLIC DISORDERS

Serum concentrations of cytokines (IL-2, IL-6, IL-8, IL-10, GM-CSF, IFNγ, TNFα) were determined in blood serum of pregnant women with different types of metabolic disorders, i.e., gestational diabetes, obesity, and a combination of those disorders. The study was performed by means of flow fluorimetry, using a multiplex test system. Changes of cytokine profiles in pregnant women with overweight or obesity, and pregnant women with gestational diabetes showed some similarities. In pregnant women with overweight or obesity we have found an increase in blood concentrations of IL-2, IL-6, IL-10, IFNγ, and TNFa. Meanwhile, an increase in blood levels of IL-2, IL-6, IL-10 and TNFα was revealed in pregnant women with gestational diabetes. The changes in cytokine profile were most pronounced in cases of pregnancy complicated with gestational diabetes and obesity. In these women, increased IL-6, TNFα, GM-CSF and IFNg concentrations were revealed in blood, along with low contents of IL-2 and IL-10. Potential causes and consequences of suggested subclinical inflammation in pregnant women are discussed. We conclude that pregnant women with metabolic disorders may develop a subclinical inflammation at the early stages of obesity, or metabolic disturbances

Comparative Analysis of Biological Properties of Large-Scale Expanded Adult Neural Crest-Derived Stem Cells Isolated from Human Hair Follicle and Skin Dermis

Introduction. The adult neural crest-derived stem cells (NCSCs) have significant perspectives for use in regenerative medicine. The most attractive sources for adult NCSC isolation are the hair follicles (HF) and skin dermis (SD) because of easy access and minimally invasive biopsy. The aim of this study was to compare the biological properties of HF- and SD-derived NCSCs after their large-scale expansion. Methods. The conventional explant method was used to obtain HF NCSCs. For the isolation of SD NCSCs, a new combined technique consisting of preplating and subsequent culturing in 3D blood plasma-derived fibrin hydrogel was applied. The studied cells were characterized by flow cytometry, ICC, qPCR, Bio-Plex multiplex assay, and directed multilineage differentiation assays. Results. We have obtained both adult SD and HF NCSCs from each skin sample (n=5). Adult SD and HF NCSCs were positive for key neural crest markers: SOX10, P75 (CD271), NESTIN, SOX2, and CD349. SD NCSCs showed a higher growth rate during the large-scale expansion compared to HF NCSCs (p<0.01). Final population of SD NCSCs also contained more clonogenic cells (p<0.01) and SOX10+, CD271+, CD105+, CD140a+, CD146+, CD349+ cells (p<0.01). Both HF and SD NCSCs had similar gene expression profiling and produced growth factors, but some quantitative differences were detected. Adult HF and SD NCSCs were able to undergo directed differentiation into neurons, Schwann cells, adipocytes, and osteoblasts. Conclusion. The HF and SD are suitable sources for large-scale manufacturing of adult NCSCs with similar biological properties. We demonstrated that the NCSC population from SD was homogenous and displayed significantly higher growth rate than HF NCSCs. Moreover, SD NCSC isolation is cheaper, easier, and minimally time-consuming method