Introduction. Despite the rapid development of high-throughput sequencing for Ig genes, the main method is still Sanger sequencing from single B lymphocytes. Using this method, a number of monoclonal antibodies have been generated for the treatment and prevention of COVID-19.

Memory B cells or plasma cells usually act as a source of Ig genes. Another attractive object for sequencing Ig genes and the subsequent generation of antigen-specific monoclonal antibodies are circulating plasmablasts, which combine such properties as high mRNA expression and expression of the surface B cell receptor.

The aim of the study is to optimize the method for sequencing Ig genes from single circulating plasmablasts. The study was carried out using the example of plasmablasts formed during acute SARS-CoV-2 infection.

Material and methods. Blood was collected from a patient with a severe form of COVID-19 on day 10 from the onset of the first symptoms. Plasmablasts were isolated from the mononuclear fraction of blood cells using a flow cell sorter by the CD19⁺CD27⁺⁺CD38⁺⁺CD3^–CD16^–CD14^– phenotype. IgG secretion was determined by enzyme-linked immunosorbent assay, and antibody-secreting cells were determined by ELISpot. For PCR, the cells were distributed one cell per tube. After cell lysis, cDNA synthesis was performed using the Invitrogen SuperScript III kit. The sample was then divided in half and semi-nested PCR was performed separately to amplify the variable genes of the heavy (VH) and light (VL) chains. The resulting PCR products were sequenced according to Sanger. Nucleotide sequences were analyzed using the Igblast online tool with the IMGT domain definition system and UGENE (version 50.0). PCR products obtained from one sample were cloned into the expression vectors AbVec-hIgG1-799 and AbVec-hIgKappa-798 for subsequent IgG production. HEK293 cells were co-transfected with plasmids encoding the Ig light and heavy chains.

Results. In preliminary experiments, it was found that the isolation of B cells using a standard immunomagnetic separation kit resulted in a loss of the plasmablast population. Plasmablasts were then isolated by flow cytometric sorting. The percentage of plasmablasts in total B lymphocytes for the patient studied was 15 %. Antibody-secreting cells accounted for 38 % of the plasmablast population. Fifteen paired sequences of the VL and VH genes were obtained. 14 unique nucleotide sequences of the VL genes and 10 unique nucleotide sequences of the VH genes were identified. The obtained sequences differed from the germline configuration of VH and VL within 0 to 25 % (median 9 %) and from 0 to 11 % (median 7 %), respectively. The CDR3 regions of the VH genes differed significantly from each other, while the CDR3 regions of the VL genes were quite similar. Based on the determined nucleotide sequences, two IgG monoclones were obtained, which was secreted into the supernatant of HEK293 cells co-transfected with expression vectors carrying the genes of IgG heavy and light chains.

Conclusion. The method of sequencing Ig genes from single circulating plasmablasts represents a promising approach for the creation of human monoclonal antibodies. This method, in combination with preliminary stimulation of B lymphocytes in vitro, sorting of antigen-specific cells, and the use of immunodeficient mice, opens up new possibilities for the development of targeted drugs. These approaches allow us to significantly accelerate the process of developing new therapeutic antibodies, which is especially important in the fight against emerging infectious diseases.

Introduction. Adoptive cell therapy is aimed at introducing immune effectors with activated natural or artificially created antitumor activity to suppress tumor growth. The main directions for adoptive cell transfer are the use of genetically modified T cells (TCR and CAR T cells). Studying the metabolism of immune cells in in vitro tumor growth models allows us to determine the safety and effectiveness of T cells in the tumor microenvironment.

Aim of the study – to analysis of metabolic activity of different types of genetically engineered T cells in co-culture with tumor cells in the form of 3D spheroids.

Material and methods. Genetically modified T cells were obtained from peripheral blood cells of healthy donors after transduction with gamma-retroviral vectors encoding receptors for recognition of tumor antigens. The intensity of glycolysis and oxidative phosphorylation processes in NY-ESO-1 TCR-T, MAGE-A4 TCR-like CAR-T and GD2-specific CAR-T cells after culturing with 3D spheroids was assessed using the Seahorse XFp extracellular flux analyzer. Metabolic activity of transduced cells during co-cultivation with 3D spheroids of the SK-Mel-37 tumor line was assessed as mitochondrial potential and production of reactive oxygen species using fluorescence microscopy using the IN Cell Analyzer 6000 with a cell culture life support module. The formation of the 3D spheroid structure of the SK-Mel-37 tumor line was assessed using fluorescence microscopy.

Results. Evaluation of glycolysis and oxidative phosphorylation parameters showed that the basal level of OCR in NY-ESO-1 TCR and GD2-CAR T cells upon contact with tumors increases several times, while MAGE-A4 TCR-like CAR T cells showed weak metabolic activity upon contact with tumor spheroids compared to the control group. During daily intravital observation of the joint culture of NY-ESO-1 TCR-T, MAGE-A4 TCR-like CAR-T and GD2-specific CAR-T cells with spheroids, it was shown that in the presence of active oxygen species, they exhibit sufficiently high mitochondrial activity and are capable of suppressing tumor cells.

Conclusion. The resulting MAGE-A4 TCR-like CAR, GD2-specific CAR-T, and NY-ESO-1-TCR-T cells, when co-cultured with SK-Mel-37 melanoma tumor line spheroids, predominantly utilize oxidative phosphorylation and, in doing so, disrupt the 3D structures of tumor cells.

Introduction. Neutrophil extracellular traps (NETs) play a crucial role in the immune response by contributing to pathogen neutralization. However, excessive NETs formation can lead to the development of inflammatory and autoimmune diseases. Interleukin-37 (IL-37) is an anti-inflammatory cytokine capable of inhibiting inflammatory responses, including those mediated by neutrophils. However, data on the effect of IL-37 on NETosis are contradictory.

The aim of this study was to investigate the effect of IL-37 on NETosis in human neutrophils in vitro.

Material and methods. Neutrophils isolated from the blood of healthy donors were stimulated with phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Commercial IL-37 was added to the culture medium at various concentrations (0.5; 2.5; 10 and 100 ng/ml), and its effect on NETosis was assessed. NETs visualization was performed using fluorescent staining and the Celena X system.

Results. In neutrophils activated with PMA but not treated with IL-37, the percentage of NETosis was 92 %. Pre-incubation of neutrophils with IL-37 did not reduce the proportion of cell forming NETs compared to the cells stimulated with PMA alone.

Conclusion. Thus, the obtained results do not support the hypothesis that IL-37 can suppress NETosis by the direct affect on neutrophils.

Introduction. Desquamation of airway epithelium in patients with bronchial asthma is one of the structural signs of epithelial dysfunction due to the trigger action of damaging environmental factors and induced by the proinflammatory influence of cytokines.

Aim – to determine the relationship between lung function, conjugated bronchial inflammatory response and IL-4, IL-17A and IFN-γ content in blood in AD patients with cold airway hyperresponsiveness (CAH) and in combination with increased bronchial response to hypoosmolar stimulus.

Material and methods. 66 asthma patients were examined, categorized into 3 groups on the basis of airway responsiveness (drop in forced expiratory volume in 1 sec – DFEV₁) during bronchoprovocation tests with isocapnic hyperventilation with cold air (IHCA) and ultrasonic inhalation of distilled water aerosol (IDW): group 1 – with verified combined bronchial hyperresponsiveness to cold and hypoosmotic stimuli; group 2 – with hyperresponsiveness only to cold stimulus; group 3 – with absence of hyperresponsiveness to both stimuli. Lung function, airway responsiveness were evaluated by spirometry and bodyplethysmography. The concentration of IL-4, IL-17A and IFN-γ in peripheral blood serum was determined by multiplex analysis. Cytological examination of induced sputum was performed.

Results. In patients of groups 1 and 2 lower values of bronchial patency indices, higher content of desquamated bronchial epithelium and neutrophils in sputum, IL-4, IFN-γ and IL-17A levels in blood were registered. The amount of desquamated epithelium in sputum correlated with the degree of bronchoconstriction during provocative tests (with ∆FEV_{1 IHCA} r = -0.57; p = 0.004 and ∆FEV_1IDW r = -0.61; p = 0.027, and bronchial patency indices (with Raw ex r = 0.67; p = 0.025 and FEV₁ r = = -0.68; p = 0.002). IL-17A content in blood correlated with ∆FEV_1IHCA (Rs = -0.32; p = 0.048), RV/TLC (Rs = -0.66; p = 0.0015), IL-4 content in serum (Rs = -0.66; p = 0.0015).

Conclusion. Asthma patients with isolated CAH and combined airway reaction to cold and hypoosmolar stimuli have increased content of desquamated epithelial cells and neutrophil count in sputum, which is associated with increased concentration of IL-4, IFN-γ and IL-17A and with worsening of airway patency.

Introduction. Convalescent plasma (ССР) of recovered patients has been widely used for the treatment of patients with COVID-19. Its clinical effectiveness is associated with the presence of virus-neutralizing antibodies and immunomodulatory properties determined by many proteins, including cytokines.

The aim of the study was to evaluate the significance of the level and ratio of cytokines’ levels of ССР for change in clinical and laboratory parameters during its transfusion to patients with COVID-19 with different disease outcomes.

Material and methods. The study included the results of a survey of 111 patients with COVID-19 who received treatment using immune anti-COVID-19 plasma (CCP) produced by the Republican Scientific and Practical Center for Transfusiology and Medical Biotechnologies in 2020–2022, Minsk, Belarus. Cytokines in ССР samples were determined by enzyme immunoassay. Laboratory parameters of peripheral blood were measured 2–3 days before and 5–7 days after CCP transfusion and included leukocytes, lymphocytes, neutrophils, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), D-dimer and others. To analyze the data obtained, the programs STATISTICA 10.0, MedCalc 22.009, and the software package R 4.3.1 were used.

Results. After the use of CCP in the group of patients who subsequently deceased, lymphopenia and neutrophilia with consistently high values of ESR, CRP and D-dimer have developed, unlike the group of patients who subsequently recovered, they developed lymphocytosis and decreased levels of ESR and CRP. Higher levels of IL-6 and INF-γ, ratio of IL-6/IL-10, IL-6/IP-10, INF-γ/IL-10, INF-γ/IP-10, INF-γ/MCR-1 levels were observed in the CCP transfused to patients of the deceased group as compared to those in the CCP transfused to patients of the recovered group. The level of blood oxygen saturation had a negative correlation with the level of IFN-γ in CCP transfused to patients of the deceased group (r = -0.5519; n = 14; p = 0.041), but not of patients of the recovered group. The levels of IL-6 and INF-γ, ratio of IL-6/IL-10, IL-6/IP-10, INF-γ/MSR-1 levels in ССР had high sensitivity and specificity in assessing the prognosis of the outcome of the disease based on ROC analysis of the data obtained. Direct correlation was found between the level of IL-6 in the CCP and an increase in the level of CRP in the blood of recipients, as well as the level of INF-γ in CCP versus an increase in the content of leukocytes and neutrophils in the peripheral blood of recipients with an unfavorable outcome of the disease. An inverse correlation was established between an increase the level of IL-10, IP-10, MCP-1, the ratio of cytokines INF-γ/IL-10 and INF-γ/MCP-1 levels in ССР and a decrease the level of ESR, D-dimer, leukocytes and neutrophils, lymphocytes, the level of ESR in the peripheral blood correspondingly of the recipients with a favorable outcome of the disease.

Conclusion. The correlative link of the level of cytokines IL-6, INF-γ, MCP-1, IP-10, cytokine levels ratio with their participation in the plasma of the immune anti-COVID-19 and changes in the content of leukocytes, neutrophils, ESR, CRP, D-dimer in the peripheral blood of patients with COVID-19 recipients of CCP was established. The nature of such link was differed for groups of recovered or subsequently deceased CCP recipients. The data obtained indicate for participation of the above mentioned cytokines in the immunomodulatory effect of CCP.

Introduction. Chronic rhinosinusitis (CRS) is one of the most common pathological processes in the structure of ENT pathology. The chronic course of this disease is manifested by persistent inflammation of the mucous membrane of the nasal cavity and paranasal sinuses, often due to the presence of several etiopathogenetic factors, which significantly complicates the choice of treatment tactics. In recent years, more and more data have been accumulated on the role of neutrophils and, in particular, such an immune response mechanism as the release of neutrophil extracellular traps (NETs) in the development of diseases of the upper and lower respiratory tract.

Aim – to study inflammatory changes associated with NETs and the processes of NETosis in the mucous membrane of the paranasal sinuses in patients with CRS without polyps.

Material and methods. The study included 60 patients diagnosed with CRS and 15 patients of comparison group without rhinosinusitis who were recommended for surgical treatment because of another ENT pathology. All participants of the study were examined for the mucous membrane of the paranasal sinuses using immunohistochemical analysis.

Results. The presented work describes the results of immunohistochemical analysis of distribution of NETs and intact neutrophils and in the mucous membrane of the nasal cavity and paranasal sinuses in patients with CRS without polyps and healthy donors. The study revealed accumulation of NETotic and intact neutrophils in the histological material of the inflamed mucous membrane of the paranasal sinuses of patients with CRS without polyps and their absence in healthy donors (p < 0.001).

Conclusion. The data obtained in the presented study may contribute to the expansion and reorientation of existing therapy of CRS without polyps in order to suppress activated neutrophils, in particular, NETs.

Introduction. For a long time, the causes of early recurrent pregnancy loss (RPL) were associated with the characteristics of the parents’ histocompatibility according to the HLA system, and the studies were aimed at analyzing the allelic characteristics of the HLA genes. At present, there is an opinion that it is insufficient to limit the studies of the immunological causes of RPL only to the HLA system.

Aim – to estimate the possible influence of the genetics of killer cell immunoglobulin-like receptors (KIR) of NK cells and HLA class I molecules (HLA‑I), which are their ligands (KIR‑L), on the outcomes of pregnancy that occurred during treatment of RPL using immunocytotherapy (ICT).

Material and methods. The study included 67 patients diagnosed with RPL. The patients were divided into two groups by simple randomization: a group of patients (n = 39) whose standard treatment was supplemented with ICT, and a group of patients (n = 28) who did not undergo ICT. ICT was performed twice outside pregnancy and twice during pregnancy. Maternal peripheral blood, newborn cord blood, and abortive chorionic tissue were used for typing of KIR genes and HLA‑I genes. Typing was performed using high-throughput NGS sequencing.

Results. No differences in the frequencies of KIR genotypes and KIR‑L representation were found in mothers with different pregnancy outcomes when treated RPL without and with ICT. However, after both treatment methods, miscarriages differed from pregnancies that resulted in the birth of a child by the absence of KIR‑L with the A3 epitope in all fetuses. In addition, when treated with ICT, fetuses in miscarriages were more often carriers of KIR‑L with the Bw4‑80I epitope on the HLA‑B molecule.

Conclusion. The absence of the HLA-A3 epitope in the fetus, as well as the presence of the HLA-Bw4-80I epitope, can be considered a prerequisite for RPL and the low effectiveness of its treatment.

Introduction. Despite numerous studies aimed at identifying proteolytic fragments of proteins as potential biomarkers for cancer, it has not yet been possible to develop a panel of proteolysis products with sufficient sensitivity and specificity to improve cancer diagnostics.

It is known that plasmin (Pm), the active form of plasminogen (Pg), can cleave immunoglobulins, particularly IgG, resulting in fragments with C-terminal lysine residues. This is of particular significance in the context of the Pg structure, where the heavy chain (Pg-H) contains lysine-binding sites located in the kringle domains.

Previously, we proposed the concept of a diagnostic test for detecting proteolytic fragments of IgG using the heavy chain of Pg. This approach is based on the ability of the lysine-binding sites of Pg to specifically interact with the C-terminal lysine residues of IgG fragments, which are generated as a result of Pm’s proteolytic activity.

Aim of this study – to evaluate the level of IgG degradation products in the serum of gastric cancer patients.

Material and methods. Pg was isolated from pooled human blood plasma by affinity chromatography on Lys-Sepharose 4B in the presence of aprotinin. The heavy chain was obtained by activating Pg with urokinase, followed by reduction of S–S bonds between Pm chains under conditions preventing autolysis. It was then separated from other reaction products by affinity chromatography on Lys-Sepharose 4B in the presence of aprotinin. The purity of preparations was verified by SDS-electrophoresis in 12 % PAGE. IgG degradation product levels were assessed using Pg heavy chain adsorbed on a 96-well plate. Statistical analysis was performed using Statistica 12 (StatSoft Inc., USA). Intergroup differences were analyzed using the Mann–Whitney U test. ROC analysis was conducted to determine the informativeness of IgG fragment levels. Differences were considered statistically significant at p < 0.05.

Results. ROC analysis demonstrated that in groups of patients with gastric cancer, the sensitivity was 68 % and the specificity was 85 %. The level of IgG fragments bound to Pg-H was expressed as the ratio of the sample’s optical density in ELISA to the threshold value (IgG-LysK coefficient). The blood IgG level in gastric cancer patients with positive IgG-LysK coefficients was 22 % higher than in patients with false-negative values. Comparison of prothrombin time revealed a significant decrease in the group with false-negative (IgG-LysK-) coefficients compared to positive (IgG-LysK+) coefficients. The activity of α₂-antiplasmin in the blood plasma of gastric cancer patients with positive IgG-LysK coefficients was 2-fold lower than in patients with false-negative values. No differences in protein C activity were observed between positive and false-negative samples.

Conclusion. The obtained results indicate active proteolysis of IgG in the tumor focus of patients with gastric cancer. Proteolytic fragments of immunoglobulin G enter the general circulation. In patients with gastric cancer at all stages of the disease, the level of these fragments in the bloodstream significantly exceeds that in the comparison group and the group of patients with distant metastases. In the group of patients with distant metastases, there was a noted decrease in the levels of total IgG, an increase in α₂-antiplasmin activity, and a reduction in prothrombin time compared to these indicators in patients without metastases.

Introduction. To develop new conjugate vaccines against Streptococcus pneumoniae, capsular polysaccharides (CPS) free from the impurity of common pneumococcal polysaccharide antigen (CPPA) are required, so studying of structural features of CPS and design of new methods for their isolation and purification seems to be an important problem.

Aim – development of a new method for the isolation and purification of modified S. pneumoniae CPSs with a 2.5-anhydromannose residue at the reducing end (m-CPSs), as well as a comparative analysis of СPS and m-СPS using HPLC and spectral methods, a study of the immunogenicity of m-CPS in an animal model and a comparison of the cross-reactivity of sera to СPS and m-СPS.

Material and methods. S. pneumoniae CPSs were isolated from the supracellular fluid (SC) using ultrafiltration and purified by treatment with nucleases and proteinase K. m-CPSs were obtained as a result of the CPS deamination reaction. The structure of the obtained substances was studied using high performance chromatography (HPLC) and ¹H-nuclear magnetic resonance spectroscopy (NMR). The immunogenicity of the resulting preparations was assessed by the production of specific antibodies of the IgM and IgG classes in mice as a response to immunization with m-CPS.

Results. Immunologically active, purified from OPAP impurities S. pneumoniae m-СPS with an aldehyde group at the reducing end were obtained. This allows to use m-CPS for the synthesis of m-CPS-containing vaccine conjugates.

Conclusion. The developed method for obtaining S. pneumoniae m-CPS purified from OPAP impurities makes it possible to obtain physico-chemically characterized and immunologically active preparations, which can lead to the creation of new pneumococcal vaccines with high immunological characteristics.

Introduction. The development and use of approaches for the analysis and isolation of antigen-specific cells is a necessary step towards a deeper understanding of the mechanisms of the immune response, and is also necessary for the development of new therapeutic strategies. At the moment, there are several approaches to identifying and sorting antigen-specific cells, which have their advantages and disadvantages, which allows you to choose the most optimal option for each task. The main principle of all methods is based on the use of a T-cell receptor ligand, which is an MHC/peptide complex.

The aim of the study is to describe various technologies for the direct detection of antigen-specific cells in the practice of developing cell technologies.

Material and methods. Peripheral blood of conditionally healthy donors was used as the material for the studies. To increase the proportion of antigen-specific cells, protocols for stimulation and clonal expansion using dendritic cells developed in the laboratory were used. The following reagents were used to detect antigen-specific cells: tetramers to identify receptors that recognize the NY-ESO-1 epitope; streptamers with the HER2 protein peptide; multimers based on Flex-T monomers for receptors recognizing MAGE-A3 epitopes; dextramers to detect cells with receptors that recognize epitopes of the E6 and E7 proteins of the human papillomavirus. All reagents recognize epitopes of antigens in complex with HLA-A*0201.

Results. The content of antigen-specific T-lymphocytes was assessed using reagents obtained by 4 technologies. Tetrameters with the NY-ESO-1 epitope allowed us to effectively assess the level of expression of the specific receptor after retroviral transduction of mononuclear cells. To assess the biological effects of cytotoxic lymphocytes, we used the streptamer technology with the HER2 protein epitope, which allowed us not only to analyze antigen-specific cells, but also to sort the pure population of HER-2 specific T-lymphocytes, which showed a cytotoxic response against target cells. The Flex-T technology allowed us to obtain multimers conjugated with the desired peptides and confirmed their effectiveness for the analysis and sorting of cytotoxic T-lymphocytes specific to the epitopes of the MAGE-A3 protein. For use in the analysis of the transcriptome of single cells, the technology of Dextramers was used as molecules with epitopes of the E6 and E7 proteins of HPV, which made it possible to assess the clonality of receptors in culture with the induction of clonal expansion.

Conclusion. Multimeric staining is an important and valuable tool both for studying the formation of a cellular immune response and for the development of modern protocols for cellular immunotherapy of oncological, viral and autoimmune diseases, as well as for assessing the effectiveness of the therapy. The existence of a number of technologies allows you to choose the necessary tool for each research task.

Malignant neoplasms are one of the main causes of death worldwide. Despite advances in traditional treatments such as surgery, radiation and chemotherapy, the effectiveness of these approaches is significantly reduced in the presence of metastatic and recurrent tumors. In recent years, immunotherapy, and in particular chimeric antigen receptor (CAR) therapy, has offered a new, promising and personalized approach to cancer treatment. CAR-T therapy is based on the genetic modification of the patient’s T lymphocytes for the expression of CAR directed against specific tumor antigens. Despite the impressive results achieved in the treatment of hematological malignancies using CAR-T therapy, its use for the treatment of solid tumors is associated with a number of difficulties. The key obstacles to the introduction of CAR-T therapy for the treatment of solid tumors are the problems of delivering CAR-T cells into the tumor microenvironment, local immunosuppression, as well as tumor heterogeneity. This review examines strategies aimed at improving the effectiveness and safety of CAR-T therapy in solid tumors.

Cardiovascular diseases remain the leading cause of death in the Russian Federation. The Ministry of Health of the Russian Federation is implementing national and federal projects to achieve the priority task of reducing mortality in stroke and myocardial infarction. Effective and safe treatment is crucial for preserving the life and health of patients. Thrombolytic therapy (TLT) can reduce long-term disability and mortality after a vascular catastrophe. Recombinant proteins are the basis for biotechnological preparations used in TLT. Immunogenicity of medicine with a foreign protein as the active ingredient may lead to a reduced clinical effectiveness and the development of adverse reactions after the first administration. Therefore, the medicinal product’s high immunogenicity presents a significant risk to the health and life of patients and requires a thorough evaluation. This review analyzes the immunogenicity of the most frequently used drugs for TLT in patients with ischemic stroke and myocardial infarction in the Russian Federation.