The generation of immune cell populations from induced pluripotent stem cells (iPSCs) is a valuable model to study mechanisms that control hematopoietic development; it also is a promising approach to develop immunotherapeutic strategies to treat various diseases, including hereditary, oncological and infectious ones. To date, it has been demonstrated that iPSCs can differentiate into different immune cells, including macrophages, neutrophils, natural killer cells and T cells. However, the protocols suggested so far are experimental, and they require optimization, standardization and scaling. Solution to these tasks requires methods allowing to predict the efficacy of ongoing differentiation at early differentiation stages. Here, we evaluated whether iPSC hematopoietic/myeloid differentiation can be monitored by means of flow cytometry. Human iPSCs were differentiated into hematopoietic/myeloid cells using two protocols previously suggested for the generation of macrophages from iPSCs. The protocols differed by methods used to induce early and late stages of cell differentiation. At early differentiation stages, the protocols differed by approaches used to induce the generation of mesoderm and hemogenic endothelium, i.e., 2D differentiation in the presence of exogenous factors known to promote mesoderm and hemogenic endothelium development (“factor-dependent” protocol) or 3D differentiation in the absence of exogenous cytokines and growth factors (“spontaneous” protocol). At late differentiation stages, the protocols differed by factors added to the cultures to promote hematopoietic/myeloid specification (i.e., SCF, FGF2, IL-6, IL-3 and M-CSF or only IL-3 and M-CSF). At different stages of differentiation, the expressions of antigens known to be expressed by mesoderm, hemogenic endothelium and hematopoietic cells (i.e., CD309, CD34, CD31, CD43 and CD45) were evaluated. At early differentiation stages, the main phenotypic changes observed in cell cultures were an upregulation of the expression of CD309 (a receptor for vascular endothelial growth factor), the appearance of the expression of sialophorin CD43 and the expression of CD34 antigen. In cells cultured in 2D factor-dependent conditions, these changes appeared earlier and were more pronounced as compared with cells cultured in 3D “spontaneous” conditions. The results suggest that CD309 and/or CD43 are valuable markers for an early prediction of the effectiveness of iPSC differentiation into hematopoietic/ myeloid progeny.

During infection with cytomegalovirus (HCMV), the content of so-called adaptive NK cells with the CD57⁺NKG2C⁺ phenotype increases in the peripheral blood, capable of exhibiting specialized functional activity aimed at controlling the infection upon repeated encounter with the antigen. In addition, adaptive NK cells are characterized by antitumor cytotoxic effects and long lifespan. In this regard, HCMV-specific adaptive NK cells are of interest as a therapeutic agent. The specificity of adaptive NK cells to HCMV is determined primarily by the recognition of viral peptides presented by the non-classical class I histocompatibility molecule HLA-E, by means of the activating receptor NKG2C. However, being highly differentiated, adaptive CD57⁺NKG2C⁺ cells tend to proliferate less well in response to soluble stimuli compared to less differentiated NK cells, making their accumulation in vitro difficult. In addition to the activating receptor NKG2C, adaptive NK cells express receptors of the KIR family, but mostly do not express the inhibitory receptor NKG2A, which is also capable of recognizing the HLA-E molecule presenting the HCMV peptide. Despite the fact that, in general, in CD57⁺NKG2C⁺NK cells from HCMV-seropositive donors, NKG2A expression is greatly reduced, in a number of individuals a significant proportion of NKG2A-positive cells was observed in this fraction. Using the example of an individual with a high proportion of NKG2A⁺ in the population of CD57⁺NKG2C⁺NK cells and a high titer of antibodies to HCMV, we showed that when stimulated with IL-2 in combination with K562-mbIl21 feeder cells, NK cells of the CD57⁺NKG2C⁺NKG2A⁺ subpopulation exhibit increased proliferative activity in comparison with CD57⁺NKG2C⁺NKG2A^-, and also have a higher level of expression of the adapter molecule FcεRIγ, taking part in signal transduction of activating receptors NKp30, NKp46 and CD16. Thus, NKG2A-positive CD57⁺NKG2C⁺ cells may be potential precursors of adaptive NK cells and mediate their accumulation during HCMV infection. The data obtained in this work allows us to deepen knowledge in the field of differentiation of HCMV-specific NK cells, as well as expand the range of approaches to the accumulation of highly cytotoxic adaptive-like NK cell effectors in vitro.

Currently, gouty arthritis is considered as a polygenic multifactorial autoinflammatory disease caused by activation of the NOD (nucleotide-binding domain) -like protein receptor 3 inflammasome NLRP3. The two cytokines IL-1β and IL-18 are considered important biomarkers of NLRP3 inflammasome activation. However, usually the concentration of IL-1β in donor sera is extremely low and found to be at the limit of detection level (1-3 pg/ml), while the concentration of circulating cytokine IL-18 in the sera of individual donors varies greatly. This results in difficulty using these biomarkers in the diagnosis of autoinflammatory diseases. We hypothesized that the patient’s blood cells which were sensitized in vivo to the presence of specific factors characteristic of autoinflammatory diseases, in particular, gouty arthritis, would produce increased amounts of the inflammasome-regulated cytokines compared to blood cells obtained from healthy donors. A comparison of the IL-18 cytokine in healthy donors and patients with gouty arthritis was carried out using 2 methods: a) by traditional analysis of the level of serum circulated IL-18 and b) by using a cell-based Hemoassay in vitro developed at the research institute “Biotech” SamGMU. The comparative analysis demonstrated the advantages of using an in vitro cell-based Hemoassay to assess the IL-18 cytokine status of patients. Serum IL-18 values varied widely and showed no significant difference between donors and patients with gouty arthritis. Using the developed cell-based Hemoassay in vitro, significant quantitative differences in the production of the inflammatory cytokine IL-18 produced by blood cells of potentially healthy donors and patients with gouty arthritis were detected. Blood cells of individual patients, sensitized in vivo with specific factors characteristic of gouty arthritis, produce increased concentrations of IL-18 in the cell growth medium in vitro compared to cells from healthy donors. Thus, the in vitro cell-based Hemoassay can be used for a more accurate assessment of the cytokine status of patients.

Currently, a large amount of data has demonstrated that many types of tumor cells, in contrast to their non-transformed forms, are characterized by the translocation of intracellular heat shock proteins 70 kDa (HSP70) to the surface of the plasma membrane. This made it possible to classify HSP70 exposed on the cell surface as a tumor-associated antigen and was the basis for searching the opportunities for the practical use of this phenomenon in clinical oncology. A significant argument in favor of the prospects of such studies was the discovered ability of HSP70, present on the surface of target cells, to enhance the cytotoxic activity of NK cells. In this regard, the work of many research groups is devoted to developing approaches to increasing the level of membrane-associated HSP70 in tumor tissues. At the same time, given the presence of such proteins on the surface of many types of tumor cells, the use of monoclonal antibodies that interact with HSP70 molecules for antitumor therapy can be considered as one of the promising approaches. It is well known that antibody preparations that selectively interact with cancer cells can be used for targeted antitumor immunotherapy. Previously, we obtained a panel of six B cell hybridomas producing monoclonal antibodies to the inducible and constitutive forms of human HSP70, directed to various epitopes of this molecule. It is significant that three varieties of hybridomas produced antibodies with specificity for binding sites on the C-terminal domain of the HSP70 molecule, and the second trio of monoclonal antibodies were specific to epitopes on the N-terminal domain of HSP70, while almost all known commercial antibodies interact only with C-terminal fragments of HSP70. In this work, we conducted a comparative study of the binding of the obtained antibodies to these proteins localized in different types of cells, both in the intracellular space and on the cell surface.

The results obtained allow us to consider the identified varieties of monoclonal antibodies that most effectively recognize surface HSP70 as a promising basis for the creation of new drugs for antitumor immunotherapy.

In multiple myeloma (MM), the content of T lymphocytes expressing “checkpoint” molecules PD-1, TIM-3, LAG-3, etc. is increased. Regulatory T cells (Treg) can suppress antitumor immune response and play a sufficient role in MM pathogenesis. Like effector T lymphocytes, some Tregs express checkpoint receptors PD-1, TIM-3, etc., however, the biological meaning of such expression, as well as the consequences of blockade of these receptors, are not clear. The significance of type I regulatory T cells (Tr1), which produce the immunosuppressive cytokine interleukin-10, in MM also remains unexplored. The purpose of this work was to study the content of PD-1- and TIM-3-expressing Tregs and Tr1 in patients with MM. The study included 36 patients with MM and 24 matched healthy donors. The content of CD4⁺CD25^hiCD127-FoxP3⁺Tregs and IL-10-producing CD4⁺IL-10⁺Tr1 populations expressing PD-1 and TIM-3 was assessed in peripheral blood (PB) and bone marrow (BM) by flow cytometry. The relative content of circulating CD4⁺CD25^hiCD127-FoxP3⁺Tregs and IL-10-producing CD4⁺IL-10⁺Tr1 was significantly higher in MM patients compared to healthy donors. A higher relative content of IL-10-producing T lymphocytes was noted compared to Treg. The relative content of Tregs and Tr1 in BM samples did not differ significantly from PB values. The proportion of Tregs expressing PD-1 and TIM-3 in patients with MM did not differ significantly from the values in healthy donors. The content of PD-1- and TIM-3-positive CD4⁺IL-10⁺T cells was significantly higher in PB samples from MM patients compared to donors.

IL-10-producing CD4⁺T cells constitute a significant proportion of T lymphocytes in the PB and BM of patients with MM and may play an important role in the pathogenesis of MM. Their content exceeds that of CD4⁺CD25^hiCD127-FoxP3⁺Treg. A relatively small number of Tregs express the checkpoint receptors PD-1 and TIM-3, no different from donors. The proportion of PD-1-/TIM-3-positive cells is ~20% of CD4⁺IL-10⁺T cells and significantly exceeds the values of healthy individuals.

Chronic inflammation caused by overexpression of IL-6 underlies a number of pathological conditions Mouse models of systemic chronic inflammation with overexpression of human IL-6 (hIL-6) are in demand not only in the context of studying the molecular mechanisms of inflammation, but also in assessing the effectiveness of clinically approved or newly developed IL-6 inhibitors. One experimental approach in addressing such models in mice relies on the induction of systemic acute inflammation in response to lipopolysaccharide (LPS) administration. This work describes mice with tamoxifen-dependent overexpression of human IL-6 in CX3CR1⁺ myeloid cells in the context of systemic inflammation induced by LPS administration. Our study demonstrates that the highest expression of the transgene carrying IL6 was observed in the heart, while high production of this cytokine was detected in the blood serum. In response to LPS administration, the production of hIL-6 in the blood increased in transgenic mice, while the production of mIL-6 also increased and was comparable to that in wild-type mice. The consequences of high systemic production of hIL-6, which in our model originates from CX3CR1⁺ tissue-resident macrophages, were noticeable even in the organs in which these cells are not present. Thus, significant amounts of hIL-6 were detected in tissue lysates of the lungs of transgenic mice after LPS administration. Evaluation of the expression of genes encoding cytokines and markers of tissue remodeling upon injury using quantitative real-time PCR showed significant changes in their expression in the context of LPS-induced systemic inflammation. Thus, this work demonstrates the feasibility of using a mouse model with tamoxifen-dependent transgene activation in CX3CR1⁺ tissue-resident macrophages to study the effects of systemic overexpression of IL-6 and pharmacological blockade of this cytokine with clinically approved or newly developed inhibitors in the context of experimentally induced diseases.

The ability of the CD8⁺HLA-DR⁺ regulatory T lymphocytes population to regulate the immune response was first described several years ago. It is known that the suppressive effects of these cells depend on intercellular interactions and are mediated by the expression of checkpoint inhibitor molecules such as CTLA-4, TIM-3, PD-1 and LAG-3. The CD8⁺HLA-DR⁺ regulatory T cells also share some properties with conventional CD4⁺ regulatory T lymphocytes. Nevertheless, the characteristics and function of this subpopulation remain poorly understood. Furthermore, studying the properties of CD8⁺HLA-DR⁺ regulatory T cells becomes relevant in the light of general age-associated changes in the human immune system and the increased sensitivity of CD8⁺T lymphocytes to these changes. Therefore, the aim of this study was to investigate the age dynamics and search for transcriptional signatures of the CD8⁺HLA-DR⁺ regulatory T cells. For this purpose, flow cytometric analysis of peripheral blood mononuclear cells from 18 donors aged 21 to 85 years was performed. Bioinformatic analysis of single-cell RNA sequencing data was carried out to search for signatures. It was found that CD8⁺HLA-DR⁺ regulatory T cells accumulate with age. The transcriptional signatures of this population consist of genes involved in antigen presentation and cytotoxicity, along with a decrease in the expression of genes encoding proteins of activating protein 1 complex. These data suggest mechanisms of suppressor function of CD8⁺HLA-DR⁺ regulatory T lymphocytes associated with the ability of these cells to present antigens and perform cytotoxic activity against effector T lymphocytes. The accumulation of the studied cells may imply a potential influence of CD8⁺HLA-DR⁺ regulatory T lymphocytes on the efficiency of adaptive immune response in the aging. Further studies of this population may provide insights into its role in age-related changes in the immune system and develop strategies to improve the immune response in the elderly.

The subpopulations of dendritic cells and their functional potential determined by endogenous interferon status in tumor microenvironment is an important stage in tumor-specific T-lymphocytes effector reactions development. In this regard, understanding the patterns of sinonasal neoplasms immune cells changes is important in the context of the search for biomarkers of the tumor process which may open up new opportunities for targeted therapy. This study is the first to provide a comparative description of dendritic cells subsets and the type I and type II interferon production in patients with malignant and benign sinonasal tumors.

Tumor tissue from 30 patients with neoplasms – main group (15 patients with malignant tumors and 15 patients with inverted papilloma) and biopsy material of the mucous membrane from 15 patients with polypous rhinosinusitis (comparison group) were used. Tumor-infiltrating immune cells were isolated from tissue using automated and enzymatic cell dissociation. The surface and intracellular cells phenotype was assessed using monoclonal antibodies and flow cytometry. The production of α- and γ-interferons was determined by enzyme-linked immunosorbent assay. Statistical data analysis was proceeded in Statistica 10.0.

An increase of tumor-infiltrating myeloid dendritic cells number was found in patients with malignant tumors as compared to control group what correlated with the stage of the pathological process (R = -0.67; p = 0.01). There was an increase in both myeloid and plasmacytoid dendritic cells in the neoplasm tissue in patients with inverted papilloma relative to the comparison group. The microenvironment of malignant growth was characterized by an increase in α- and γ-interferons production in combination with a pronounced decrease in IFNγ-producing T cells percent while a decrease in both intracellular and extracellular production of γ-interferons was detected in tumor tissue of patients with inverted papilloma as compared with control group.

In patients with sinonasal tumors changes in dendritic cells subpopulations as well as a decrease in the reserve capacity of γ-interferon synthesis were revealed what may characterize the involvement of these mechanisms in the formation of the antitumor immune response failure and requires further research to establish their pathogenetic role.

The number of patients with autoimmune thyroid diseases (Graves’disease, Hashimoto’sthyroiditis) is increasing globally. The most important part in the diagnosis of Graves’ disease (GD) is the detection of autoantibodies to the thyrotropin receptor (TSHR) in Graves’ patients’ sera. For the differential diagnosis of antibodies to thyroid antigens, it is promising to use tests based on monoclonal antibodies to TSHR, which can be obtained not only as a result of immunization with native or recombinant TSHR protein, but also through DNA immunization with genetically engineered constructs containing fragments of the TSHR gene. Based on mRNA we isolated from the thyroid tissue in GD, a number of fragments of the thyrotropin receptor gene were cloned, suitable for DNA immunization of animals. The purpose of this work is to evaluate the immunogenic properties of one of the constructed vectors, pVAX1-TSHR (1160), in a mouse model. The successful inclusion of the extracellular domain gene fragment of the human TSHR (1160), which was transfected into CHO cells as a part of the pVAX1 vector was confirmed by immunoblotting and ELISA. The immune response formed to the injection of the pVAX1 vector into BALB/c mice, containing a fragment of the human TSHR gene, was detected in different versions of ELISA. Immunization of animals with the DNA vector pVAX1-TSHR according to an experimentally selected scheme was effective for the formation of mouse splenocytes, secreting antibodies to TSHR, which were used for successful hybridization. This was confirmed by the results of determining antibody production to TSHR in murine blood sera. The level of antibody production remained high (titer more than 1:10.000) at the 8th week of the experiment. As a result of selection of individual clones according to the criteria of proliferative activity and stability of antibody production, the most stable cultures secreting mAbs against TSHR were selected.

Polypous rhinosinusitis is a chronic inflammatory disease of the nasal mucosa and paranasal sinuses. Recombinant interferon has antiproliferative activity and corrects the deficiency of endogenous regulatory molecules, which allows us to consider this class of immunotropic drugs as a promising component of conservative immunotherapy for polyposis rhinosinusitis. Purpose of the study: To select the optimal composition of the experimental composition of interferons and evaluate their pharmacokinetic parameters in laboratory animals.

For laboratory evaluation of the effectiveness of the auxiliary components of the proposed composition in creating a local effect, Wistar rats in the amount of 30 animals were selected, to which the proposed form of the drug (IFN1) was administered once intranasally. The control group included 30 animals that were alternately administered intranasally with a similar dose of IFNα2b and IFNγ dissolved in water for injection (IFN2). Quantitative determination of interferon concentration in blood samples was carried out using the enzyme immunoassay method. To assess the dependence of changes in concentrations in the blood of animals on the time elapsed after their administration, standard pharmacokinetic models were used. Next, the integral from the initial moment of time to infinity was determined, which corresponded to the area under the pharmacokinetic curve and made it possible to calculate a number of pharmacokinetic characteristics.

The following indicators were obtained: 1) Area under the pharmacokinetic curve (AUCt) ng/ml/min – IFN1 = 683.0; IFN2 = 1707.7. 2) Absorption constant (Kp) – IFN1 = 0.13096; IFN2 = 0.03836. 3) Suction constant (Kel) – IFN1 = 0.00177; IFN2 = 0.00317. 4) Clearance (Cl) ml/min – IFN1 = 129.35; IFN2 = 51.73.

Based on the differences in the values of pharmacokinetic parameters (AUCt) for the IFN1 and IFN2 preparations, it can be concluded that the use of a composition based on chitosan, succinic acid and DMSO leads to a pronounced retention of interferon in the nasal mucosa, which provides a pronounced local therapeutic effect and allows for fewer systemic adverse reactions. This composition is suitable for further clinical studies of the effectiveness of immunotherapy for polyposis rhinosinusitis.

Today, the proportion of elderly and senile people is steadily growing throughout the world. The most important factors in the first line of immune defense of the mucous membranes of the upper respiratory tract are β-defensins, which are a group of secretory proteins with antimicrobial activity. The aim of this study was to study the expression of genes for antimicrobial peptides β-defensins and the composition of the microbiome of the mucous membranes of the upper respiratory tract in elderly people and long-livers with various aging phenotypes.

The main study group included 67 centenarians and 49 elderly people, who were further divided into two subgroups depending on the course of aging (pathological and successful aging). Nucleic acids were isolated from nasopharyngeal scrapings and the expression levels of the DEFB1 and DEFB4 genes were determined using real-time polymerase chain reaction. The composition of the microbiota in nasopharyngeal swabs was determined by MALDI-TOF mass spectrometry.

In analyzing the expression of the DEFB1 gene in elderly people and centenarians with successful and pathological aging phenotypes, no difference was revealed between the groups. Expression of the DEFB4 gene was increased in centenarians with pathological aging compared to centenarians with successful aging and in the elderly group. Excessive production of antimicrobial peptides is dual in nature; on the one hand, they provide the first line of defense against microorganisms, and on the other, they are cytotoxic to their own cells. An increase in the expression of the DEFB4 gene during aging may be due to an increase in the number of pathogen-associated molecular patterns, which can be one’s own microbiota and/or components of microbial metabolism. Analysis of the microbiota composition showed an increase in biodiversity in individuals with a successful aging phenotype compared to a pathological phenotype. Particular attention is paid to Staphylococcus spp., the species composition of which depends on the aging phenotype. In the pathological aging group, the frequency of St. aureus colonization is significantly higher than in the successful aging group.

Thus, overexpression of the DEFB4 gene and changes in the composition of the microbiota of the mucous membranes of the upper respiratory tract may be one of the mechanisms explaining the increased susceptibility to infections in various aging phenotypes.

The key problem of introducing nanoparticles into clinical practice for diagnostic and therapeutic purposes is their safety. Connective tissue, an important component of which are mast cells, reacts actively in response to foreign particles. The reaction of mast cells can be an indicator of the biocompatibility of foreign particles. The study was conducted on male Wistar rats. Iron-carbon nanoparticles in the FeC modification stabilized in an aqueous medium using an auxiliary substance were used. Tissue examination (liver, lungs, heart, thymus, kidneys) was performed 1, 7 and 30 days after injection. After the injection of nanoparticles, the largest quantity of them is found in the liver and lungs, a smaller quantity is found in the heart, kidneys and thymus. The liver and lungs are the main organs of excretion of nanoparticles due to phagocytes. The accumulation of nanoparticles in the liver leads to the development of destructive processes and to the activation of compensatory and adaptive mechanisms in the form of cellular and intracellular regeneration of hepatocytes. In organs with a low content of nanoparticles, structural changes are poorly expressed. The mast cells of the studied organs react differently to the introduction of nanoparticles. The first to react are the mast cells of the liver by reducing the quantity and increasing degranulation. The population of lung mast cells reacts unidirectionally by sharply activating degranulation without changing the amount. An increase in the secretory activity of mast cells in the lungs and liver indicates the participation of mast cells in the regulation of particle elimination through intercellular signaling pathways of interaction with the system of phagocytic mononuclear cells. Heart mast cells are involved in maintaining the inflammatory process in the early period of the experiment, contribute to the return of myocardial parameters to homeostatic norm in the late period of the experiment. Mast cells can be considered as indicators of biocompatibility nanoparticles. The absence of an inflammatory process and the preservation of the structural and functional characteristics of the tissues where nanoparticles accumulate, as well as the reaction of mast cells in them, indicate the relative safety of the particles under study.

Changes of the immune state in patients with fibroadenoma (FA) or other benign lesions of the breast, as well as the involvement of immune checkpoints in pathogenesis of these lesions, remain underexplored. The aim of this study was to compare the expression level of the key immune checkpoint markers CD279/PD-1, CD274/PD-L1, CD366/TIM3, and CD223/LAG3 in total circulating lymphocytes, T cell population as well as in its CD4 and CD8 subsets in peripheral blood from women with breast FA and healthy controls. Blood samples were taken from 12 women diagnosed with FA of the breast (aged 23-54 years, FA group) and 15 healthy women (aged 22-52 years, control group). Sample uptake was performed immediately before surgery, and samples were further analyzed by multicolor flow cytometry using monoclonal antibodies CD3-VioBlue, CD4/CD8-FITC, PD1-PE, PD-L1-PerCP-Cy.5.5, and TIM3/LAG3-APC. Each sample was incubated with 4 antibody combinations: CD3/CD4/PD1/PD-L1/TIM3, CD3/CD4/PD1/PD-L1/LAG3, CD3/CD8/PD1/PD-L1/TIM3, and CD3/CD8/PD1/PD-L1/LAG3. First, mono-expression of each of the 4 immune checkpoint markers was evaluated in the lymphocyte gate from both investigated groups. In FA samples, a significant increase in PD-L1 expression (assessed as percent of positive cells and fluorescence intensity change) was observed. Regarding expression of immune checkpoints in CD3⁺ T cells, along with significantly increased %PD-L1⁺, elevated numbers of PD1⁺T cells were detected. As for the differences in immune checkpoint expression changes between CD4⁺ and CD8⁺ T cell subsets, FA patient group demonstrated a more prominent increase in the amount of CD8⁺PD1⁺T cells relative to CD4 subset. The profiles of PD-L1 changes in CD4 and CD8 subpopulations were comparable showing, in both cases, a significant increase in FA sample group. We also analyzed changes in co-expression of any two immune checkpoint markers in CD4⁺ and CD8⁺ T cell subsets. The most noticeable was as increase in the prevalence of PD1⁺PD-L1⁺ phenotype in both T cell subpopulations from FA patients compared to the controls. With respect to co-expression of 3 checkpoint markers in CD4⁺ and CD8⁺ T cells, a significant increase in the percentage of PD1⁺PD-L1⁺TIM3⁺ cells among CD4 T helpers was found in FA. Thus, specific changes of T cell phenotype related to (co-)expression of immune checkpoint regulators may occur in systemic circulation of women with breast FA.

K. pneumoniae is one of the leading microorganisms causing nosocomial infections among premature newborns. The ineffectiveness of immune defense, morphofunctional immaturity, length of hospitalization and invasive procedures create the prerequisites for the implementation of the infectious process in a hospital setting. The question of the reasons for the development of infection, the etiological agent of which colonizes the intestines, remains open. Purpose of the study: to evaluate the expression of CD14⁺CD282⁺, CD14⁺CD284⁺, CD14⁺HLA-DR⁺, and CD14⁺CD11b⁺ receptors on blood monocytes and the level of sIgA in coprofiltrates in premature infants with intestinal colonization by K. pneumonia with different genetic profiles. We examined 11 children with the uge gene (group 1), 20 newborns with the uge + fim genes (group 2), and 12 children with the kfu + uge + fim genes. Microbiological examination of feces included identification and antibiotic sensitivity of microorganisms. Detection of the uge, fim and kfu genes in K. pneumoniae strains was carried out by PCR. The expression level of monocyte activation markers was determined by flow cytometry. Gestational age and anthropometric parameters did not differ between newborns. Children identified with the fim gene in combination with other genes were more often discharged home with K. pneumoniae than with the uge gene. In these children, a decrease in the level of expression of CD14⁺CD282⁺, CD14⁺CD284⁺, CD14⁺CD11b⁺, CD14⁺HLA-DR⁺ receptors at birth and upon reaching postconceptional age, and a low sIgA content in coprofiltrates during 24 days of life were recorded. Thus, a decrease in the expression of CD14⁺CD282⁺, CD14⁺CD284⁺, CD14⁺CD282⁺ and CD14⁺HLA-DR⁺ receptors by blood monocytes and insufficiency of sIgA production in the large intestine determine a long period of colonization of K. pneumoniae strains with the presence of the fim gene in combination with other genes (from 15 to 180 days), as well as the possibility of Klebsiella infection occurring in subsequent periods of the child’s life. Much more often, children with the combination of genes uge + fim and kfu + uge + fim were discharged from the hospital with a diagnosis of anemia; only in these groups of children was the development of bronchopulmonary dysplasia recorded.

The purpose of the study was to compare and analyze the levels of KREC (kappa-deleting recombination excision circle) and TREC (T cell receptor excision circle) levels, which indirectly reflect impaired maturation of T and/or B lymphocytes, in children of the medical and biological risk group and the comparison group (patients considered relatively healthy, relative to the population).

The medical and biological risk groups were:

1) 15 children with operated congenital heart defects with combined thymectomy and 9 without it; the average age is 5 months ±4 months and 7 months ±3 months, respectively; and

2) 27 children with relatively frequent morbidity of the respiratory tract (acute respiratory viral infection more than 8 times a year) aged 1.6±1.4 months, among whom the proportion attending a preschool institution is 20 people (74%).

The comparison groups were:

1) 16 relatively healthy children (health group 1) aged 1.7±1.6 years, among whom the proportion of those attending a preschool institution was 13 people (81%); and

2) 48 apparently healthy newborn children, whose average age was 15±12 days.

Quantitative determination of TREC and KREC was carried out using a multiplex test system developed at the Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences (Novosibirsk). The average concentrations of TREC and KREC were determined in apparently healthy newborns. Children with a high incidence of infectious respiratory morbidity (more than 8 cases of acute respiratory viral infection per year) had a significantly higher risk of developing disorders of both T cell and B cell immunity, compared with the healthy population. Children who underwent total thymectomy during surgical treatment of congenital heart defects had a higher risk of developing immunodeficiency conditions, affecting predominantly T cell immunity, in comparison with the group of children operated on with preservation of the thymus gland.

We present a unique clinical case of an atypical hemolytic-uremic syndrome in a child. The mutation in exon 6 of the CD46 gene (chr1:207940532G>C) leads to a homozygous or hemizygous missense substitution. An 8-year-old girl was urgently hospitalized with symptoms of hemorrhagic syndrome and acute kidney injury. The child from the second pregnancy with an aggravated obstetric anamnesis, the first operative labor at the 38th week, who has an aggravated genealogical anamnesis. Initially, the disease developed in the guise of gastroenterological pathology: the girl had dyspeptic disorders, most likely associated with pathology of carbohydrate metabolism, also frequent infectious diseases and a lag in physical development. From the age of 4 years, the girl suffered from persistent hypoalbuminemia, hypoproteinemia and hypogammaglobulinemia, requiring replacement therapy with intravenous immunoglobulins. She was repeatedly examined in gastroenterological departments in Yekaterinburg and Moscow in 2021-2023, but no data concerning protein-losing enteropathies or primary immunodeficiency was obtained. Also, the whole-exome sequencing with result validation hasn’t confirmed an inherited etiology of the presumed congenital immune error. However, a CD46 gene mutation, associated with the development of hemolytic-uremic syndrome in some publications, was identified in the study. The signs of thrombotic microangiopathy were preceded by fever of unspecified etiology: microangiopathic hemolysis, thrombocytopenia, hyperazotemia, C3 consumption, proteinuria, and macrohematuria; hypercholesterolemia, protein metabolism disorder, increased transaminases, ferritin were also noted. According to the ultrasound of the kidneys there were diffuse changes in the parenchyma, decreased velocity indices of blood flow in both renal arteries at all levels. Differential diagnosis was carried out with thrombotic thrombocytopenic purpura, autoimmune hemolytic anemia, antinuclear form of atypical hemolytic-uremic syndrome, viral and bacterial infections, systemic pathology, antiphospholipid syndrome, and hemoblastosis. The girl didn’t need a renal replacement therapy. When complement-blocking therapy with eculizumab was initiated on vital indications, the signs of the disease were gradually eliminated. The prolonged absence of nephropathy in the child may indicate the diversity of the CD46 gene’s functions and different phenotypic manifestation of its mutations. Analysis of literature sources and detection of CD46 “environment” genes (CYBA, LYST, ARPCIB) by full-exome sequencing suggest ambiguity and polymorphism of phenotypic manifestations of complement-mediated mutation.

Atopic dermatitis (AD) is a genetically determined chronic inflammatory skin disease characterized by itching, relapsing course, age-related features of the lesion morphology and localization. The etiological factors contributing to the development of AD are: allergic reactions, inflammatory reactions in the skin, leading to a barrier disfunction, the affect and interaction of genetic and environmental factors. Epigenetic mechanisms also play a key role in immune regulation. The aim of this work is to study the genome-wide methylation profile in the skin of children with AD in the acute stage, as well as to determine changes in the expression level of immune response genes associated with common signaling pathways during therapy. Whole-genome sequencing was conducted to examine methylation in the skin samples from AD patients and controls. The stages of bioinformatics data processing were carried out. Changes in the expression levels of the TLR2, TLR9, IL4, IL13, CAMP, and DEFB1 genes before and after treatment were carried out using RT-PCR-RT on samples of mononuclear blood cells. When comparing samples from patients with AD and healthy children, changes were shown in the methylation of 2364 regions of genomic DNA with their corresponding specific genes. Among the identified genes, those related to processes associated with the pathogenesis of AD were selected. At the next stage, changes in the expression of the described immunity factors (TLR2, TLR9, IL-4, IL-13, DEFB1, CAMP) were assessed over time according to the type of treatment. For this purpose, patients were divided into 2 groups depending on the prescription of systemic therapy. Three months after treatment, the level of expression for all studied immune factors decreased. However, a significant decrease in expression for almost all parameters (except for the DEFB1 gene) was recorded in the group in which systemic therapy (dupilumab) was used in addition to topical treatment. At the same time, the SCORAD index after treatment improved by an average of 63% and amounted to 17±6.0 points. Thus, systemic therapy aimed at suppressing signaling of IL-4 and IL-13 cytokines indirectly leads to normalization of the expression levels of innate immune factors. Studying the regulation of molecular genetic mechanisms of immunity involved in the processes of inflammation in AD helps to clarify the immunopathogenesis of this disease, determine diagnostic markers and targets for drug therapy.

In recent decades, there has been an increase in the incidence of autoimmune diseases (AID) among adults and children. The immunopathogenesis of AID is based on an imbalance between autoaggressive and regulatory cells (Tregs), which is regulated by metabolic signaling pathways and the cytokine microenvironment. Understanding the mechanisms of immunometabolism opens up new possibilities for the treatment of patients with AID. The aim was to evaluate the activity of lymphocyte dehydrogenases associated with OXPHOS and glycolysis, depending on the level of proinflammatory and anti-inflammatory cytokines in children with AID.

324 children with AID were examined: 80 – Crohn’s disease (CD), 53 – ulcerative colitis (UC), 89 – psoriasis (PS), 66 – multiple sclerosis (MS), 36 – autoimmune hepatitis (AIH). Activity of mitochondrial dehydrogenases (succinate dehydrogenase (SDH) and glycerol-3-phosphate dehydrogenase (GFDH)) were evaluated by immunocytochemical method using flow cytometry. The level of cytokines (CС) in blood sera was determined by multiplex analysis.

In each studied group of children, CС with the highest values in exacerbation and remission of the disease were identified. The maximum values of CС were in patients with exacerbation: CD, UC, PS, MS – IL-23; AIH – IL-27. Evaluation of cytokine complexes associated with cells showed significant differences between patients in exacerbation/remission: CD, UC and PS – M1(IL-1+IL-6+TNFα), cTh1(IFNγ+IL-12p70+TNFβ+IL-2), cTh2 (IL-4+IL-5+IL-10+IL-13+IL-17E/IL-25+IL-33), cTh17 (IL-1β+IL-6+IL-17A+IL-17F+IL-21+IL-22+IL-23); MS – M1, cTh1, cTh2; AIH – cTh2. SDH activity in AID remission differed between pathologies in CD4+ cells, Th17 and Tregs. In exacerbation of AID, there were differences in Tregs between patients with UC and PS. The highest GPDH activity in exacerbation was observed in CD4+ lymphocytes, Th17 and Tregs in CD. The ratio of SDH/GPDH in T-lymphocytes in children with CD in exacerbation and remission was lowest and significantly lower than in UC, PS, MS, AIH and apparently healthy children. In the group of children with a low SDH/GPDH ratio, the levels of CCL20/MIP3α, IFNγ, IL-12p70, IL-13, IL-17A, IL-1β, and TNFα were significantly increased. Conclusions. Informative cytokine complexes were identified in children with AID. The relationship between the metabolic activity of lymphocytes and the level of circulating cytokines is shown.

Schizophrenia is a mental illness of complex etiology. Recently, there has been increased interest in the role of the immune system in the pathophysiology of mental disorders. The concept of neuroinflammation in neurodevelopmental disorders is gaining widespread interest, including the role of Toll-like receptors.

Schizophrenia is associated with an increase in the concentration of cfDNA in human blood, and the composition of cfDNA fragments changes significantly compared to cellular DNA: GC-rich fragments of the ribosomal repeat accumulate and base oxidation occurs. Similar changes, but less pronounced, also occur for cfDNA from healthy donors.

To confirm the hypothesis about the possible participation of cfDNA in the inflammation induction, we studied the effect of cfDNA samples on cultured mononuclear cells.

Unlike cellular DNA, cfDNA(SZ) and cfDNA(K) stimulate transcription of the TLR9 gene in mononuclear cells. After 1 hour the amount of TLR9 RNA increases by 2.9 and 3.3 times compared to the control. After 24 hours, the TLR9 RNA level decreases slightly, but is still 2-3 times higher than the control level. After 1 hour, TLR9 protein increases by 1.5 and 1.7 times, respectively, and further increased after 24h of culture.

An increase TLR9 protein expression correlates with an increase of the transcription factor NF-kB in lymphocytes and is accompanied by an increase in proinflammatory cytokine IL8 RNA, the transcription of IL8 is controlled by the NF-kB factor.

Thus, cfDNA(SZ) and cfDNA(K) stimulate the TLR9-NF-kB-proinflammatory cytokine signaling pathway in lymphocytes. The effect of cfDNA also depends on the concentration of these fragments in the extracellular environment. Since the concentrations of cfDNA in the blood of patients with schizophrenia are significantly increased compared to healthy donors, we should expect a much higher level of activation of the TLR9-NF-kB signaling pathway in the body cells of sick people.

Samples of cfDNA from patients with schizophrenia have a pronounced biological effect on cells of the immune system, stimulating the synthesis of pro-inflammatory cytokines by activating the TLR9-NF-kB-proinflammatory cytokines signaling pathway. High levels of cfDNA in blood plasma may be one of the reasons for the induction and maintenance of low-level inflammation in schizophrenia.

Atherosclerosis is a chronic disease in which lipids, cells and various proteins accumulate in the walls of the arteries, forming atherosclerotic plaques. The growth of plaques leads narrowing of the lumen of the blood vessels. Atherosclerosis is accompanied by local inflammation, while the number of hematogenous macrophages are derived from monocytes increases in the vascular wall. The reasons why the inflammatory reaction cannot be completed and becomes chronic are not clear. To resolve inflammation and protect tissues from high concentrations of cytokines that can cause apoptosis, there is a mechanism of immune tolerance of innate immunity. Lipopolysaccharide (LPS) tolerance of monocyte-macrophages is a phenomenon in which cells reduce their sensitivity to repeated exposure to LPS. This condition is characterized by a decrease in the ability of macrophages to produce proinflammatory cytokines and promotes resolution of inflammation. We hypothesized that in atherosclerosis, tolerance violations in monocyte-macrophages are possible. The study included patients who were admitted to the department of cardiac surgery, Moscow Regional Research and Clinical Institute (MONIKI). Patients were divided into patients with coronary atherosclerosis (CAD) with detected stenosis in 2 or more arteries and healthy controls without stenosis in the arteries according to the results of coronary angiography. In the present study, we examined the ability of macrophages from 13 patients with CAD and 11 patients without CAD to develop tolerance to LPS. To do this, we isolated CD14⁺ monocytes from the blood by positive selection using immunomagnetic separation and subjected them to two sequential LPS stimulations, immediately after cell isolation and after 6 days of culture. The secretion of cytokines TNFα, IL-1b, IL-6, IL-10, IL-8, and CCL2 was measured in cell culture supernatants using by ELISA. Our results showed impaired macrophage tolerance for CCL2 secretion and improved tolerance for IL-8 secretion in macrophages from patients with CAD compared with patients without. Since IL-8 and CCL2 are chemoattractants for other immune cells, it can be assumed that the observed impairment of macrophage tolerance to LPS in atherosclerosis increases the infiltration of other monocytes into the inflammatory site, contributing to the chronicity of inflammation.

Type 2 diabetes mellitus is characterized by a mild inflammatory reaction in the pancreas, which affects the structure and function of the pancreatic islets: the number of β-cells decreases and the number of α-cells increases. The work examined the features of β-cell differentiation in the development of experimental type 2 diabetes mellitus and while reducing the inflammatory process. Biochemical, histological methods, enzyme-linked immunosorbent assay, immunohistochemical methods were used using primary antibodies to insulin, glucagon, proliferation marker Ki-67 and secondary antibodies labeled with fluorescent dyes. Streptozotocin and nicotinamide were used to model type 2 diabetes mellitus, and the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione was used to reduce the inflammatory response. Previous studies have shown that it changes the macrophage phenotype from proinflammatory M1 to anti-inflammatory M2. In type 2 diabetes mellitus, against the background of a decrease in the number of macrophages with the CD163 marker and the concentration of the cytokine TGF-β1, which have an anti-inflammatory effect, in the pancreatic islets, a decrease in the number of β-cells and their functional activity was observed, while the content of α-cells synthesizing glucagon increased. After administration of the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione, the opposite picture was observed in the pancreatic islets: against the background of an increase in the number of CD163⁺ macrophages and the content of TGF-β1, the number of β cells increased and the number of α cells decreased-cells. The increase in the number of insulin-synthesizing cells was not accompanied by their mitotic activity. It is likely that a decrease in the number of CD163⁺ macrophages and the level of the antiinflammatory cytokine TGF-β1 in the islets are factors contributing to changes in the cell microenvironment and, as a consequence, the differentiation of β-cells into α-cells. On the contrary, an increase in the number of CD163⁺ macrophages and TGF-β1 against the background of administration of the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione presumably promotes reverse differentiation of α-cells into β-cells and restoration of insulin synthesis pancreas. Targeted effects on the microenvironment of cells in the pancreatic islet in type 2 diabetes mellitus may be a new approach to treating the disease.

Cystic fibrosis (CF) is one of the most common autosomal-recessive inherited diseases. The primary genetic defect in CF is aligned CFTR gene mutation which encodes a membrane protein functioning as cAMP-depended chloride channel. Classic phenotypical manifestations of CF include chronic obstructive pulmonary disease with bronchiectasis, persisting infection (St. aureus, Ps. aeruginosa, B. cepacia) and aberrant inflammatory response, as well as exocrine pancreatic insufficiency with malabsorption, hypotrophy and growth retardation. CFTR deficiency is also accompanied by β-cell pancreatic dysfunction, causing glucose metabolism disturbances and CF-related diabetes. The aim of the study was the comparison of inflammatory markers dynamics in patients with normal and disturbed glucose metabolism during pulmonary exacerbation treatment. The study included 10 patients with impaired glucose tolerance (Group 1) and 24 patients with normal carbohydrate metabolism (Group 2). Patients of the two groups did not significantly differ in demographic characteristics, pulmonary function test and body mass index parameters, as well as in the number of F508del mutation carriers and in the number of those who were infected with Ps. aeruginosa and B. cepacia complex. Blood sampling was performed twice: before and after a routine course of antibiotic therapy. Plasma levels of biomarkers including the antibodies to single- and double-stranded DNA (ss-DNA-IgG, ds-DNA-IgG, respectively), the hormones (dehydroepiandrosterone (DHEA) and DHEA sulfate), C-reactive protein (CRP), Mn-dependent superoxide dismutase (Mn-SOD), and the cytokines (tumor necrosis factor-α (TNFα), interferon-γ (IFNγ), IFNα, tissue growth factor-β1 (TGF-β1), interleukin-4 (IL-4), IL-6, IL-10, IL-17A) were assessed using commercial immunoassay kits. Our study shows that antibiotic treatment did not have a sufficient influence on levels of inflammatory markers in patients with disturbances of glucose metabolism while patients with normal glucose tolerance demonstrated a significant reduction in inflammatory marker values after the therapy. The data may suggest both impaired effectivity of antibiotic treatment and aberrant inflammatory response in patients with glucose intolerance.

It is relevant to study of angiogenesis mediators and the mobilization of early endothelial progenitor cells (EPС) from bone marrow into the blood in patients with coronary heart disease (CHD), suffering and not suffering from ischemic cardiomyopathy (ICMP).

СHD patients: 30 people with ICMP and 22 people without ICMP, 15 healthy donors. The content of early EPС (VEGFR2⁺CD34⁺CD14⁺) was determined in the blood and bone marrow by flow cytofluorometry, the concentration of MSP-1, SDF-1, VEGF-A – by multiplex analysis, of HIF-1α – by ELISA.

Тhe content of SDF-1 and HIF-1α in peripheral blood in patients with CHD without cardiomyopathy was higher than in healthy individuals (respectively 60.00 (50.00-80.00) pg/mL and 6.00 (5.00-6.20) ng/mL versus 30.00 (5.00-45.00) pg/mL, p = 0.049 and 4.60 (3.28-5.11) ng/mL, p = 0.049), with ICMP corresponded to the norm. Тhe concentration of SDF-1 in the bone marrow was higher, and the level of HIF-1α was less than their content in the bloodstream, regardless of the presence of ICMP (respectively 130.0 (90.0-170.0) pg/mL, p = 0.005 and 0.97 (0.80-1.11) ng/mL, p < 0.001). The level of MCP-1 in the blood varied within the normal range in patients with CHD of both study groups (190.0 (168.0-215.0) pg/mL), and in the bone marrow was higher only in patients with ICMP (406.5 (265.0-583.0) pg/mL, p = 0.028). Regardless of ICMP presence, the content of VEGF-A in the blood of patients with CHD corresponded to the norm (3.80 (1.00-6.50) pg/mL) and in myeloid tissue. The number of EPC was increased in the blood of patients with CHD without cardiomyopathy (0.70 (0.46-1.23) and 0.19 (0.13-0.32) %, p < 0.001) and corresponded to their number in the bone marrow. And in patients with ICMP, normal values of the indicator were recorded in the blood with the accumulation of EPC in myeloid tissue (0.57 (0.45-0.98) %, p = 0.019).

The development of ICMP is associated with the accumulation of early EPC in myeloid tissue due to their increased retention by an excess of MCP-1 in the bone marrow with weak involvement in the bloodstream due to the lack of a surplus of SDF-1 and HIF-1a in the blood.

The aim of the research was to study the population composition of the splenic lymphoid cells, to assess the functional activity of lymphocytes as well as the state of the gastrointestinal tract microbiota in experimental modeling of metabolic syndrome (MS).

The studies were conducted using two experimental models of MS and hyperlipidemia (HL), based on prolonged drinking of animals with 20% aqueous fructose solution with added cholesterol and intraperitoneal administration of Poloxamer 407 to mice, respectively.

The results of the experiments indicate a change in the population composition of splenocytes (decrease in CD4⁺ and CD8⁺T cells, activation of CD4⁺CD25⁺FoxP3⁺Thed cells), accompanied by a decrease in T cell activity and increased proliferation of B lymphocytes, impaired production of IL-15 and IL-22, as well as lipid and carbohydrate metabolism (adiponectin, leptin, insulin), which serves as a prerequisite for the development of chronic inflammation, which is a pathogenetic sign of MS.

We found changes in the intestinal microbiota of mice characteristic of the manifestation of metabolic dysbiosis – an increase in the representation of Firmicutes bacteria (staphylococci, streptococci, enterococci) in the biomaterial, changes in the content of facultative (E. coli) and transient (Enterobacter) microflora.

In order to develop a new kind of medicine for therapy and prevention of HL and MS, we used a combination of sodium polyprenyl phosphate (PP) and beta-sitosterol (BSS), polyisoprenoid derivatives of plant origin.

More pronounced changes were found in the splenocyte population composition and activation parameters of Treg cells in HL modeling compared with the MS model. The introduction of PP and BSS has an immunocorrective effect during treatment.

The therapeutic effect of this drug, as well as the prevention of the MS symptoms, is accompanied by normalization of the microbiota state.

The data obtained indicate the prospects of using PP and BSS for the prevention and treatment of HL and MS in order to influence the leading links in the pathogenesis of metabolic disease.

Scientific and technological progress contributes to the discovery and production of innovative materials. The emergence of graphene is a clear example of this. Graphene is considered a promising material for use in nanobiomedicine and nanobiotechnology. It is therefore important to understand how it affects human immune cells. In a study, the effects of 5 and 25 μg/mL graphene oxide nanoparticles with lateral sizes of 100-200 nm and 1-5 μm, modified with linear and branched polyethylene glycol, on human neutrophils were investigated. The formation of reactive oxygen species was evaluated with a lucigenin as a chemiluminescence activator.Inaddition, we investigated theeffect of a 60-minute incubation of neutrophils with pegylated graphene oxide nanoparticles on the viability of these cells by staining with trypan blue and a 30-minute incubation on the uptake of fluorescein isocyanate-labelled E. coli. The percentage of neutrophils which engulfed E. coli and the uptake index were determined. Samples without added nanoparticles served as controls.

A decrease in lucigenin-enhanced chemiluminescence of neutrophils was observed under the influence of two types of graphene oxide nanoparticles: 1-5 μm in size coated with linear polyethylene glycol, and 100-200 nm in size coated with branched polyethylene glycol, at a concentration of 25 μg/mL in the zymosan-stimulated version of the assay. No dependence of the effect on the particle size and the type of polyethylene glycol was observed. The indicators for spontaneous chemiluminescence of neutrophils did not change with the addition of PEGylated graphene oxide nanoparticles.

A thirty-minute incubation of human neutrophils at 37 °C with PEGylated graphene oxide nanoparticles with lateral dimensions of 100-200 nm and 1-5 μm had no effect on the viability of these cells and on the percentage of neutrophils that engulfed E. coli. However, 1-5 μm graphene oxide modified with linear polyethylene glycol at a concentration of 25 μg/mL increased the amount of E. coli engulfed by neutrophils per cell.

Thus, in the absence of cytotoxicity, PEGylated graphene oxide particles have multidirectional immunomodulatory effects on neutrophils. In this case, their concentration is decisive and not the size of the graphene oxide particles and the type of polyethylene glycol.

One of the modern approaches to the treatment of cancer is the creation of targeted delivery systems for anticancer drugs, which allows increasing the concentration of the delivered substance in the right place and preventing its accumulation in healthy organs and tissues. At the same time, one can also expect an increase in the duration and effectiveness of the drugs, as well as a reduction in side effects during therapy. The hyaluronic acid receptor CD44, which, according to the literature, is highly expressed in many types of tumors and regulates metastasis, is a promising target for targeted delivery of anticancer drugs. The purpose of this study was to evaluate the effect of a supramolecular delivery system based on hyaluronic acid with nanosized cavitand cyclodextrin on the antitumor properties of oxaliplatin in vitro. Cell lines 1301, SK-MEL-28 and B16 were used as tumor cells. Cells were cultured in the presence of a delivery system based on hyaluronic acid (HACD), oxaliplatin (OX), and their complex (HACD-OX) at various concentrations in complete culture medium RPMI-1640 containing 0.3% L-glutamine, 4% gentamicin and 10% inactivated FBS serum for 48 hours in a humidified atmosphere of 5% CO₂ at 37°C. The effect of the studied compounds on the viability of cell cultures was assessed using the WST test. It was shown that in the case of the T-cell lymphoma cell line 1301, the HACD delivery system did not affect the ability of OX to reduce the viability of tumor cells of this line; the effect of free oxaliplatin and the complex was comparable. However, in the case of melanoma cells (B16 and SK-MEL-28), the HACD-DOX complex has a more pronounced antitumor effect, causing a statistically significant decrease in the viability of B16 and SK-MEL-28 cells compared to free oxaliplatin.

Dysfunctions of the immune system, in turn, of neutrophil granulocytes (NG), are the cause of the emergence and progression of the focus of infection in bone tissue and bone marrow in acute hematogenous osteomyelitis (AHO). When colonizing bones, S. aureus, osteoblasts, osteocytes and macrophages secrete chemoattractants and cytokines, which initiate the influx of large amounts of NG into the site of infection, which, in turn, secrete cytokines and form an inflammatory microenvironment that promotes the formation of osteoclasts that resorb bone. It is known that NG activated by cytokines undergo functional and phenotypic changes. In this regard, detecting the cytokines that lead to the transformation of the NC phenotype into an antigen-presenting cell (APC) in AHO is of interest. Goal, to determine the levels of neutrophil-associated serum cytokines IL-8, IL-17A, TNFα, IFNγ and their relationship with numerous and phenotypic characteristics of subpopulations CD66b⁺CD16⁺CD33⁺HLA-DR^-, CD66b⁺CD16⁺CD33⁺HLA-DR⁺NG in localized and septicopyemic forms of CSO in children.

Children (N = 28) aged 8-15 years with AHO were studied: group 1 – 20 children with a localized form; group 2 – 8 children with septicopyemic form; and a comparison group of 13 conditionally healthy children. The number of NG subpopulations CD66b⁺CD16⁺CD33⁺HLA-DR⁺, CD66b⁺CD16⁺CD33⁺HLA-DR^-, receptors mean expression intensity (MFI) (FC 500, mAb Beckman Coulter, USA); serum cytokines IL-8, IL-17A, TNFα, IFNγ ELISA (ASCENT, Finland), test systems Cloud-Clone Corp. (USA).

The appearance of two activated subpopulations in the peripheral blood (PB) of children with various forms of AHO severity were found: CD66b⁺CD16⁺CD33⁺HLA-DR⁺ with the APC phenotype, capable of presenting the S. aureus superantigen for T lymphocytes and a subpopulation with high cytotoxic activity CD66b⁺CD16⁺CD33⁺HLA-DR^- while concentrations of serum cytokines IFNγ, IL-17 are high and levels of TNFα are increased. Due to the fact that the NG activation range correlates with the range of inflammatory tissue damage, including bone tissue, determination of the level of IFNγ, IL-17 can be useful for assessing the severity of AHO, and also possibly for monitoring infectious and inflammatory processes occurring in the bone tissue.

Taking into account the leading role of NG in various inflammatory reactions, determining the expression of HLA-DR, in order to identify the manifestation of the APC-NG subpopulation in peripheral blood, may have diagnostic value not only for AHO, but also for other manifestations of infectious and inflammatory diseases.

Psoriatic arthritis (PsA) is a chronic immune-mediated inflammatory joint disease, often associated with psoriasis. Interactions between immune cells, mainly T lymphocytes, and cells of the osteoarticular system are central in the pathogenesis of PsA. Th1, Th17 cytokines associated with the development of PsA contribute to an increase in the production of cytokines, chemokines, matrix metalloproteinases, adhesion molecules by immune cells, chondrocytes, fibroblasts, and induction of osteoclasts. That leads to the destruction of cartilage and bone tissue. Among the cytokines potentially involved in the pathogenesis of PsA, IL-7 is of particular interest. IL-7 is a T cell survival factor. However, it can promote the production of IFNγ and TNFα by T cells. IL-7 may indirectly promote osteoclast maturation and cartilage degradation. The aim was to investigate the effect of IL-7 and blockade of the α-chain of the IL-7 receptor (IL-7R) in vitro on the production of IL-5, IL-13, IL-2, IL-6, IL-9 IL-10, IFNγ, TNFα, IL-17A, IL-17F, IL-4, and IL-22 by T cells in norm and PsA.

The study included 14 patients with PsA in the acute stage of the disease and 8 healthy individuals. To determine cytokines concentration, multiplex analysis was performed using flow cytometry.

It was shown that IL-7 enhances the production of Th1, Th2, Th17, Th22, and Th9 cytokines in both patients with PsA and healthy individuals. The exception was IL-2 for both groups. Under the blockade of IL-7R with monoclonal antibodies, the level of IL-6 increases and production of IFNγ, TNFα, IL-17F, IL-10, IL-5, and IL-9 by donors’ cells and production of IFNγ, TNFα, IL-22, IL-2, IL-4, IL-5, IL-13, and IL-9 by cells from patients with PsA decreases relative to production of cells stimulated with IL-7. In donors, the blockade contributed to a change in the balance of Th1/Th2 cytokines: level of IL-4, IL-13 did not change on the background of a decrease in production of IFNγ, TNFα. In patients with PsA, under blockade of IL-7R the production of IL-10 remained at an increased level and concentration of IFNγ, TNFα and IL-2, which are actively involved in the tissue damage mechanisms, decreased. The obtained data indicate the prospect of using the IL-7R as a target for the treatment of psoriatic diseases.

Animal tumor models are used for preclinical studies of drugs and cancer therapy. The aim of this work was to analyze the growth of murine pancreatic tumor cells Pan02, carrying GFP marker, injected subcutaneously (s. c.), intraperitoneally (i. p.) or orthotopically into the pancreas (ortho) of C57BL/6 mice. Mice were injected with 2 × 10⁵ cells: s. c. in the right flank; i. p. with a syringe into the abdominal cavity, or ortho surgically under the pancreas capsule. The weight of mice was determined in the dynamics of tumor growth, and blood serum was taken to analyze the antibody response to the GFP reference protein. At the 2^nd and 4^th weeks of tumor growth, some mice were slaughtered and the expression of GFP by the tumor cells, as well as the composition of the immune cells in the tumor, were analyzed by flow cytometry and confocal microscopy. It was shown that with the different localization, the pancreatic tumors grew at different rates and lethality. When the tumor was injected i. p., mice lost weight with rapid tumor growth. In the ortho model, the mice increased their weight. Mortality in the s. c. and i. p. groups was comparable. In the s. c. model, the tumor grew slowly to a volume of 200-400 mm³ and stopped growing. There was no mortality in this group during the follow-up period (2 months). The same antibody response to GFP was formed with all injection schemes. The subpopulation composition of immune cells varied greatly in the different models of tumor cell administration. Regardless of the type of immune response, Pan02-GFP cells rapidly suppressed GFP gene expression in vivo. The data obtained showed that murine pancreatic tumor Pan02 is immunogenic and causes the formation of an adaptive immune response. Regardless of the presence or absence of an immune response and elimination of GFP⁺ cells, the tumor continued to grow in the i. p. and ortho models, but not in the s. c. one, and caused the death of mice. When conducting preclinical studies, it is necessary to use several ways of tumor cell injection to obtain a more objective result.

Allergic rhinitis (AR) is an inflammatory disease of the upper respiratory tract (nasal mucosa). AR affects up to 40% of the world’s population; in the Russian Federation, the incidence is 18% to 30%, depending on the region. Despite the fact that AR is not a severe pathology, it causes significant economic burden. Another threat associated with this disease is that in 40% of cases, patients with AR eventually develop a more severe disabling pathology – AD. Widespread prevalence and significant economic disadvantages caused by AR determine the importance of developing new ways of prevention and control of this disease, as the existing methods of therapy are insufficient. However, the search for new ways of therapy is impossible without a detailed investigation of the molecular mechanisms of AR pathogenesis. For a long time it was considered that this allergic inflammation is formed by Th2-dependent mechanism with involvement of Th2-lymphocytes, B-cells and eosinophils and pro-inflammatory cytokines: IL-4, IL-5 and IL-13. However, experimental evidence has now accumulated on the role of epithelial cells of the respiratory tract and the proinflammatory cytokines they secrete (IL-25, IL-33 and TSLP) in the pathogenesis of AR and AD. IL-25 has been shown to induce the production of IL-4, IL-5 and IL-13, directing a Th2-type immune response. At the same time, mice with inactivated IL-25 developed barely any Th2-immune response. Inactivation of IL-33 significantly reduces inflammation (mediated by eosinophils) of the respiratory tract. Mice knockout for the cytokine receptor TSLP did not develop nasal hyperreactivity in response to allergen, but the level of nasal mucosal inflammation remained high. Currently, work is actively progressing on the development of new drugs capable of specifically blocking the activity of the listed cytokines; first of all, drugs based on neutralizing monoclonal antibodies. However, there are other technologies that can be used to regulate the activity of genes, such as the technology based on the RNA interference. It can be used to suppress the expression of any gene with a known nucleotide sequence, including genes encoding pro-inflammatory cytokines.

Considering the above, the aim of this work was to design synthetic miRNA molecules and study their ability to specifically block the expression of genes encoding proinflammatory cytokines IL-25 and TSLP in experiments in vitro.

The T cell immunoglobulin domain and mucin domain-containing molecule-3 (TIM-3), an inhibitory checkpoint receptor, has been identified as a crucial regulator of cellular immune responses. TIM-3 has been discovered as a receptor involved in the negative regulation of T cells. Recent studies have demonstrated that TIM-3 is expressed on innate immune cells, including dendritic cells (DCs), even at a higher level than T cells. In the tumor microenvironment, the majority of DCs have a monocytic origin. Models for studying such DCs in vitro are DC cultures generated from monocytes in the presence of growth factors. The present study aimed to investigate the expression of TIM-3 in IFNα-induced monocyte-derived DCs (IFN-DCs) and the impact of DC activation on TIM-3 expression. DCs were obtained by culturing the adherent fraction of mononuclear cells from healthy donors for 4 days in the presence of GM-CSF and IFNα, followed by LPS addition for 24 hours. Human double-stranded DNA (dsDNA, 5 μg/mL) was added as an activation stimulus to intact IFN-DCs at the stage of maturation, along with LPS. Expression of the membrane TIM-3 molecule was determined by flow cytometry, and the level of expression of TIM-3 mRNA – by real-time RT-PCR with reverse transcription. Intact donor IFN-DCs expressed the membrane TIM-3 molecule at a high level (more than 70% of cells). The addition of LPS as a maturation stimulus almost halved the expression of TIM-3 (p_W < 0.05) without affecting the expression of HAVCR2/TIM-3 mRNA. Exogenous dsDNA (along with LPS) increased the expression of HAVCR2/TIM-3 mRNA by more than three times (p_W = 0.05) with a decrease in the number of TIM-3⁺DCs (p_W = 0.003). Our findings indicate the presence of mechanisms that support expression of this inhibitory checkpoint receptor under conditions of DC activation. Further studies of the regulation of TIM-3 expression by monocyte-derived dendritic cells will expand the understanding of the biological significance of inhibitory receptors on DCs from the point of view of the immune response, as well as, in the future, increase the effectiveness of current approaches in cancer immunotherapy using IFN-DCs and inhibitors of checkpoint molecules.

Monocytes and macrophages play an important role in the development of inflammatory diseases, including sepsis, etc. In addition to their role as “scavengers,” macrophages secrete various substances (mainly cytokines and chemokines), which have a systemic effect and modulate the microenvironment of the inflammatory focus. Disruption of their normal function can cause various immune pathologies and changes in tissue homeostasis. Macrophages are able to transmit signals to other cells in the area of inflammation, including various cytokines and other compounds. Usually, they are secreted directly into the extracellular space. However, the lifetime and range of action of these substances may be limited. An alternative method of intercellular communication is the packaging of substances into extracellular vesicles, which can help in signal propagation. Extracellular vesicles are small particles with a bilayer lipid membrane that transport various biologically active substances. There are different types of extracellular vesicles, each of which can have its own specific effect on other cells. Due to the presence of specific receptors on the surface of the vesicles, they can make selective delivery of biological molecules to certain cells. Vesicles can contain various components, such as nucleic acids, proteins, lipids, and even individual cell organelles. Therefore, it is not surprising that vesicles are able to modulate physiological processes in the body and be involved in the pathogenesis of various diseases. Establishing the mechanisms of vesicle participation in the development of inflammatory diseases, including chronic ones, is extremely important. The aim of the present study was to determine whether extracellular vesicles are capable of modulating the immune response. To do this, we assessed cytokine secretion by macrophages treated with vesicles from LPS-stimulated monocytes and macrophages. It was found that cells that received vesicles from LPS-activated monocytes and macrophages secrete more pro-inflammatory signaling molecules: the cytokine IL-6 and the chemokine IL-8. The results demonstrate that intercellular interaction and transmission of the inflammatory signal of monocytic cells also occurs through extracellular vesicles. In the future, additional research is needed to understand the full picture of what substances in the extracellular vesicles of monocytes and macrophages allow them to have an immunomodulatory effect not only on each other, but also on other types of cells.

The purpose of the study was to evaluate the clinical manifestations of osteoarthritis (OA) in combination with MS (OAMS) and their relationship with concentration of several circulating proinflammatory cytokines, the level of lipids in peripheral blood serum. Forty women patients with knee OA were examined: 19 patients from the experimental group in whom OA was combined with metabolic syndrome (MS), 21 patients with OA without MetS. All patients were elderly and overweight. In the first subgroup of patients, the absolute majority of people were obese, while in the second subgroup, overweight patients predominated. Patients in the experimental subgroup showed a statistically significant increase in waist circumference compared to patients without MS. The duration of OA did not differ in both subgroups. It has been established that the metabolic phenotype of gonarthrosis – OA in combination with metabolic syndrome – differs from patients with OA without MS in greater severity of pain, a decrease in the level of daily activity, an increase in the burden of the disease and other symptoms of OA. These core characteristics are associated with poor quality of life and clinically significant symptoms of depression. The metabolic type of gonarthrosis is characterized by more pronounced laboratory signs of systemic low-grade inflammation in comparison with patients without MS, as evidenced by a threefold increase in CRP content and an increase in the level of IL-6, IL-18 in PC serum. In addition, in patients with the metabolic phenotype of OA, a fivefold increase in the level of a specific humoral immune response to type 2 collagen (Col2Ab) and dyslipidemia – an increase in the content of LDL cholesterol and triglycerides, with a comparable reduced level of HDL cholesterol – were revealed. It is concluded that the phenotype of OA in combination with MS is probably due to the pathogenetic similarity of OA and MS (syntropy), which is based on low-grade inflammation. Studying the pathogenesis of the OAMS phenotype and developing new principles for the treatment of multimorbidity should be based on patient-oriented approaches.