Kseniya V. Nevskaya

Aim. To perform a comparative assessment of subset composition and functional activity of peripheral blood lymphocytes in patients with tick-borne encephalitis (TBE) and ixodid tick-borne borreliosis (ITBB) in the acute phase of the disease.Materials and methods. The study involved 22 patients with febrile and meningeal TBE, 15 patients with ITBB with and without erythema, and 11 healthy controls. Subset composition of blood lymphocytes was determined by flow cytometry. The blast transformation assay was applied to assess lymphocyte proliferation. Cytokine-producing activity of cells was studied in 24-hour incubated mononuclear cell cultures. Cytokine concentrations (interleukin (IL)-2, IL-4, IL-10, interferon (IFN)γ) were determined in the supernatants by the enzyme-linked immunosorbent assay (ELISA).Results. Patients with TBE demonstrated an increase in the proportion of helper – inducer T-cells, a pronounced decrease in the proportion and absolute count of cytotoxic T cells, and low T lymphocyte count compared to the control values. The study in ITBB patients revealed an increase in the helper – inducer T-cell count and the proportion of NK-cells, a decrease in the cytotoxic T cell count, and the T lymphocyte count comparable to normal values. The most significant decrease in the levels of phytohemagglutinin-induced lymphocyte proliferation was found in patients with TBE. Patients of both groups showed a decrease in IL-2 secretion in the mononuclear cell culture, a rise in IL-4 and IL-10 production, and IFNγ production levels comparable to control values.Conclusion. The study of TBE patients revealed relative lymphocytopenia with changes in the subset composition of lymphocytes characterized by an increase in the proportion of helper – inducer T-cells and a decrease in the absolute cytotoxic T lymphocyte count. Patients with ITBB demonstrated an increase in the proportion of NK-cells and a more pronounced imbalance in the T-helper / cytotoxic T lymphocyte ratio. Changes in the functional phenotype of lymphocytes, regardless of the etiology of tick-borne infection, were characterized by reduced proliferative reserve, low IL-2 secretion, increased IL-4 and IL-10 production, and depressed reactivity of lymphocytes with respect to IFNγ secretion.

We propose an original strategy for metastasis prevention using a combination of three microRNAs that blocks the dedifferentiation of cancer cells in a metastatic niche owing to the downregulation of stemness genes. Transcriptome microarray analysis was applied to identify the effects of a mixture of microRNAs on the pattern of differentially expressed genes in human breast cancer cell lines. Treatment of differentiated CD44^- cancer cells with the microRNA mixture inhibited their ability to form mammospheres in vitro. The combination of these three microRNAs encapsulated into lipid nanoparticles prevented lung metastasis in a mouse model of spontaneous metastasis. The mixture of three microRNAs (miR-195-5p/miR-520a/miR-630) holds promise for the development of an antimetastatic therapeutic that blocks tumor cell dedifferentiation, which occurs at secondary tumor sites and determines the transition of micrometastases to macrometastases.

Background. A severe form of coronary heart disease (CHD) is ischemic cardiomyopathy (ICMP), the pathogenesis of which has not been fully studied. Disturbances in the mobilization of endothelial progenitor cells (EPC) due to an imbalance of angiogenesis mediators may exacerbate ischemia in ICMP. The aim — to establish the peculiarities of changes in the balance of early and late EPС and subpopulations of VEGFR2+ cells in the blood and bone marrow in relation to the content of angiogenesis mediators and the number of desquamated endothelial cells (DEK) in the blood of patients with CHD, suffering and not suffering from ICMP. Methods. A single-stage, clinical, controlled (case-control) study was conducted from March 2019 to June 2022. 52 patients with CHD who had a history of myocardial infarction were examined: 30 people suffering from ICMP and 22 people not suffering from ICMP, as well as 15 healthy donors. The content of VEGFR2+, VEGFR2+CD34+CD14+ (early EPC), VEGFR2+CD34+CD14– (late EPC), VEGFR2+CD34–CD14+, VEGFR2+CD34–CD14– cells in patients with CHD in the blood (before surgery) and bone marrow (sampling was performed at the beginning of coronary bypass) and in healthy individuals in the blood, as well as (in both groups) the content of CD45–CD146+ DEC in the blood was determined by flow cytometry. The concentration of SDF-1, VEGF-A, MCP-1, GM-CSF, G-CSF in blood plasma was measured by multiplex immunofluorescence analysis. Results. The development of CHD without cardiomyopathy was accompanied by an increase in the content of VEGFR2+CD34+CD14+ and VEGFR2+ cells (0.74 [0.46; 1.23]% and 10.00 [8.20; 11.60]%, respectively, versus 0.19 [0.13; 0.32]%, p 0.001 and 5.40 [4.30; 6.50]%, p = 0.005) and concentrations of SDF-1, MCP-1, GM-CSF (respectively 60.00 [50.00; 81.00] pg/ml; 223.0 [180.0; 297.0] pg/ml; 2.10 [1.45; 3.40] pg/ml versus 30.00 [5.00; 45.00] pg/ml, p = 0.041; 175.1 [140.0; 204.0] pg/ml, p = 0.046; 0.96 [0.46; 1.41] pg/ml, p = 0.038) in the blood relative to the norm. No such changes were observed in patients with ICMP. Regardless of the presence of ICMP, the content of VEGFR2+CD34+CD14–, VEGFR2+CD34–CD14+, VEGFR2+CD34–CD14– cells, VEGF-A, G-CSF in the blood of patients with CHD varied within physiological values, and the number of DEC exceeded the norm (7.26 [5.43; 17.94]×105/l, p = 0.039). The number of VEGFR2+ cells and their immunophenotypes in the bone marrow of patients with ICMP did not differ from the parameters in patients with CHD without cardiomyopathy. Prolonged bleeding from the venopuncture area was registered in one CHD patient without cardiomyopathy. Conclusion. The development of ICMP is associated with the absence of a compensatory response to atherogenesis in the form of increased mobilization of early EPC from the bone marrow due to the absence of a reaction associated with hyperproduction of SDF-1, MCP-1, GM-CSF, which is characteristic of CHD without cardiomyopathy. The content of EPС, VEGFR2+CD34–CD14+ and VEGFR2+CD34–CD14– cells, VEGF-A and G-CSF in the blood in СHD corresponds to physiological values, regardless of the presence of ICMP. The generation of EPC in the bone marrow in ICMP is not impaired.

Aim. To evaluate the nature of changes in the expression of markers of endothelial progenitor cells (VEGFR2, CD34, CD14) and endothelial cells (CD146) in association with the expression of the leukocyte common antigen CD45 in the culture of blood mononuclear cells in the presence of M-CSF in patients with coronary heart disease (CHD) and healthy donors.Materials and methods. The study included 12 patients with CHD with class III–V angina pectoris and 10 healthy donors, from whom 30 ml of venous blood was taken on an empty stomach in the morning and stabilized with heparin. Blood mononuclear cells were isolated by Ficoll density gradient centrifugation (1.077 g / cm³) and subject to immunomagnetic separation using CD14-MicroBeads and CD34-MicroBead Kit (Miltenyi Biotec B.V. & Co. KG, Germany). The resulting CD14⁺ and CD34⁺ culture of mononuclear cells was incubated for 6 days in a complete nutrient medium with and without M-CSF 50 ng / ml (Cloud-Clone Corp., USA) with complete replacement of the medium and repeated application of M-CSF on day 3. After 6 days, the proportions of CD45⁺, CD14⁺, CD34⁺, VEGFR2⁺, and CD146⁺ cells in the culture were assessed by flow cytometry using CD14-FITC, CD34-PE, VEGFR2-Alexa Fluor 647; CD45-FITC and CD146-PerCP antibodies (BD Biosciences, USA).Results. It was shown that in healthy donors, the proportion of CD146⁺ cells in the co-culture of blood mononuclear cells with M-CSF exceeded their number in the sample without it, with comparable expression rates of CD45, CD14, and VEGFR2 markers between the control and stimulated cultures. In CHD patients, the number of CD146+ and VEGFR2⁺ cells did not change when M-CSF was added to the mononuclear cell culture; however, the proportion of CD14⁺ cells increased and the proportion of CD45⁺ cells decreased compared to the control sample. The number of CD34⁺ cells was comparable both between control and stimulated samples, and between the groups of examined individuals. At the same time, in patients with CHD, an increased proportion of VEGFR2⁺ cells was found in the control and stimulated samples compared to healthy individuals, while an increased proportion of CD14⁺ cells was detected only in the stimulated culture.Conclusion. The development of CHD disrupts the response of blood mononuclear cells to the effect of M-CSF, increasing the number of CD14⁺ and reducing the proportion of CD45⁺ cells in the culture in the absence of stimulating effects on the expression of endothelial cell marker CD146. At the same time, M-CSF does not affect the expression of CD34 and VEGFR2 in endothelial progenitor cells both in patients with CHD and in healthy individuals.

Highlights Dysregulation of angiogenesis may be the pathogenetic factor of ischemic cardiomyopathy (ICMP). Aim. To determine the content of growth factors and desquamated endothelial cells (DEC) in the blood from the coronary sinus and ulnar vein in association with the number of progenitor endothelial cells (PEC) in the blood from the ulnar vein in patients with coronary heart disease (CHD), suffering and not suffering from ICMP.Methods. The study included 30 patients with ICMР and 22 patients with CHD, and 18 healthy donors. The content of DEC (CD45^–CD146⁺) was determined in blood from the cubital vein (peripheral) and coronary sinus, and the content of DEC (CD14⁺CD34⁺VEGFR2⁺) was determined in peripheral blood by flow cytometry (antibodies “BD Biosciences”, USA). The concentrations of VEGF-A, VEGF-B, PDGF, SDF-1, SCF, FGF-1, TGF-β1 in blood plasma from both locations were evaluated by multiplex analysis (set “Cloud-Clone Corp.”, USA).Results. The content of DEC in peripheral blood was elevated in patients with CHD of both groups, and in patients with ICMP in sinus blood was higher than in peripheral. At the same time, in patients with CHD without cardiomyopathy, an excess of PEC and SDF-1 in the blood from the ulnar vein was established in combination with an increase in the concentration of PDGF and a decrease in the content of VEGF-B in the blood from the coronary sinus relative to the parameters of systemic blood flow. In patients with ICMP, these changes were not detected, but there was an increase in the concentration of TGF-β1 in sinus blood compared with peripheral blood. Regardless of the presence of ICMP, the concentration of SCF, FGF-1, VEGF-A in the blood from the ulnar vein corresponded to the norm and that in sinus blood; the content of VEGF-A in the coronary bloodstream exceeded its systemic level.Conclusion. In patients with ICMP, desquamation of the coronary vascular endothelium is enhanced against the background of violations of its repair processes due to insufficient (relative to CHD without cardiomyopathy) mobilization of PEC from the bone marrow due to the absence of an excess of SDF-1 in the blood and their insufficient homing into the myocardium due to weak PDGF production in the heart.

BACKGROUND: The problem of food allergy remains actual in pediatric practice. AIM: to establish the prevalence, clinical features and risk factors of food allergy in infants predisposed to allergy diseases in a cohort prospective study with a follow-up period of 12 months. MATERIALS AND METHODS: A prospective cohort study in children whose parents suffer from allergic diseases (n=151) was initiated. Prospective observation included: clinical examination at birth and at the age of 3, 9, 12 months, interviewing parents, assessment of allergen-specific IgE to food allergens in blood serum at the age of 12 months. Statistical analysis was performed using STATISTICA 13.3. RESULTS: 141 children completed the prospective follow-up. Suspected food allergy (reactions associated with the use of food products) was registered in 48.9% of predisposed children. The symptoms of food allergy include skin lesions and gastrointestinal symptoms. Oral allergic syndrome and respiratory symptoms were recorded only in some cases. Prevalence of suspected food allergy progressively increase by 12 months, mainly due to skin symptoms, while the prevalence of gastrointestinal symptoms, on the contrary, decreased by this age. Mixed feeding was recognized as a risk factor for suspected food allergy in predisposed children. The prevalence of IgE ― mediated food allergy, was 13.9%. The use of antibiotics in the first year of life was shown as risk factor for IgE-mediated food allergy in predisposed children. Multivariate logistic regression showed that pets (cats) owners had lower risk of food allergy. CONCLUSION: Primary allergy prevention and adherence to the principles of rational antibiotic therapy is necessary in children predisposed to allergic diseases.

Testing a number of N-[omega-(purin-6-yl)aminoalkanoyl] derivatives of 7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine in a panel of nine tumor cell lines has shown that the studied compounds exhibit high cytotoxic activity, especially against 4T1 murine mammary carcinoma, COLO201 human colorectal adenocarcinoma, SNU-1 human gastric carcinoma, and HepG2 human hepatocellular carcinoma cells. Synthesis and study of structural analogs of these compounds made it possible to find that the presence of both a difluorobenzoxazine fragment and a purine residue bound via a linker of a certain length is crucial for the manifestation of the cytotoxic activity of this group of compounds. The study of the effect of the most promising compound on the cell cycle of the human tumor cell lines, the most sensitive and least sensitive to cytotoxic action (MDA-MB-231 breast adenocarcinoma and COLO201 colorectal adenocarcinoma, respectively), allows us to conclude that this compound is an inhibitor of DNA biosynthesis. The found group of purine conjugates may be of interest in the design of new antitumor agents.

According to the latest concepts, for micrometastasis to develop into macrometastasis, differentiated cancer cells must revert to a dedifferentiated state. Activation of stemness genes plays a key role in this transition. Suppression of stemness gene expression using microRNAs can become the basis for the development of effective anti-metastatic drugs. This article provides an overview of the existing methods for assessing the effect of microRNAs on stemness genes and cancer cell dedifferentiation.

The aim of the study was mammosphere assay optimization for quantifcation of IL6-induced stemness in differentiated (СD44^– ) T47D breast cancer cells.Material and Methods. The effect of three commonly used cell-detaching methods (TrypLE, accutase, cell scrapper) at various confuence (40–50 % and 70–80 %) on cell viability, phenotypic profle and mammosphere formation was tested. The cell viability was examined using AnnexinV/propidium iodide assay. The phenotypic profle was analyzed by fow cytometry with fuorescent markers CD24 and CD44.Results. Detachment of the cells using scrapper led to substantial increase in early apoptotic and late apoptotic cells in comparison with TrypLE and accutase. Dissociation with TrypLE reduced the percentage of detected CD44⁺ positive cells, whereas accutase saved the surface marker. The number of mammosphere and their diameter did not differ between groups. Incubation of differentiated (CD44^– CD24⁺) T47D cells with IL-6 for 24 hours resulted in an appearance of CD44⁺CD24⁺ and CD44⁺CD24^–/low subpopulation. Furthermore, the differentiated cells after 24 hours of IL6 exposure formed 3 times more mammospheres compared to the control.Conclusion. Usage of cells with confuence of no more than 80 % and accutase for detachment of cells is recommended for mammosphere assay. Incubation of CD44^– CD24⁺ T47D cells with IL6 for 24 hours is suffcient for stimulation of stemness plasticity.