Tumor Necrosis Factor (TNF) was discovered almost 50 years ago as “serum factor” detected in mice following infections or administration of bacterial lipopolysaccharide (LPS), with a remarkable anti-tumor effect. Molecular cloning showed that this activity is mediated by a small protein (17 kDa), which belongs to a wide plethora of cytokines. Due to the particular organization of the TNF gene coding sequence, all cells producing soluble TNF also carry a membrane-bound cytokine on their surface. The physiological effects of TNF are mediated by signaling through two types of highly specific receptors. Despite established protective and homeostatic functions of TNF, when overproduced systemically or locally, it can trigger pathologies ranging from septic shock to autoimmune diseases. Therefore, in clinical immunotherapy there were not the TNF agonists, which were expected to induce anti-tumor effects, but rather the antagonistic blockers, that proved effective in a wide range of autoimmune diseases with an inflammatory component. Our studies in mice based on the technologies of reverse genetics and experimental disease models, revealed a paradoxical feature of TNF: some cellular sources of this cytokine (such as myeloid cells) promoted diseases, while other cell types (such as T lymphocytes) produced a protective form of the same cytokine. There are several possible mechanistic explanations for this phenomenon. On the one hand, the "pathogenic" cytokine is produced in a soluble form and can exert systemic effects via broadly expressed TNFR1. On the other hand, protective functions are mediated by the membrane-bound TNF via TNFR2. Systemic anti-cytokine therapy is known to be accompanied by undesirable side effects, which can hypothetically be explained by the neutralization of these protective functions. Thus, we developed prototypes of TNF blockers which limit the bioavailability of this cytokine only from its main “pathogenic” source – myeloid cells. This type of inhibitors, called MYSTI, represent bispecific mini-antibodies binding both TNF and a surface marker on myeloid cells and lacking the Fc domain. MYSTI retain newly synthesized TNF on the surface of the producing cell and then internalize it. This novel type of immunotherapy drug has already shown efficacy in a number of experimental disease models.

NK cells are innate lymphocytes that are able to eliminate altered cells, which makes them promising for the immunotherapy of viral diseases and tumors. The NK cell population is characterized by high phenotypic and functional diversity. In particular, in the pool of highly differentiated NK cells in the presence of cytomegalovirus (HCMV), a population of adaptive cells can be formed, characterized by a high lifespan and high cytotoxicity. However, in order to carry out a cytotoxic reaction, a NK cell must undergo a licensing process, during which it acquires the expression of NKG2A and KIRs. Currently, there are many effective methods of NK cell accumulation for subsequent use in therapy, one of them is the stimulation with IL-2 and K562-mbIL21 feeder cells. Highly differentiated adaptive-like NK cells are able to expand in respond to such stimulation. However, the phenotype of actively expanding NK cells dynamically changes. Loss of inhibitory KIR expression during intense proliferation of NK cells may adversely affect their cytotoxic potential. This work shows that highly differentiated CD56^dimNKG2C⁺ NK cells from HCMV-seropositive individuals have a high proportion of KIR2DL2/3⁺ cells. This may indicate a high stability of KIR receptor expression in this population. We have shown that CD56^dimNKG2C⁺ clonal cultures obtained by stimulation with IL-2 and K562- mbIL21 are characterized by high stability of KIR2DL2/3 expression compared to NKG2C-negative and less differentiated CD56^brightNKG2C⁺. Also, in heterogeneous cultures of adaptive NK cells precursors CD57^- CD56^dimNKG2C⁺, a higher expression level of KIR2DL2/3 was observed in comparison with NKG2C-negative cultures of CD57^-CD56^dimNKG2C^-. Thus, the accumulation of NK cells upon stimulation with IL-2 and K562- mbIL2 feeder cells can lead to loss of expression of KIR receptors and a decrease in their functional activity. However, cultures of highly differentiated NK cells of HCMV-seropositive individuals CD56^dimNKG2C⁺, as well as cultures of precursors of adaptive NK cells CD57^-CD56^dimNKG2C⁺, are characterized by a greater stability of KIR2DL2/3 expression. As a result, stimulation with IL-2 and K562-mbIL21 feeder cells can be used to accumulate adaptive-like cells and their progenitors with stable inhibitory KIR expression and high cytotoxic potential.

Heat shock proteins 70 kDa (HSP70) protect intracellular proteins from the damaging effects of stress factors of various natures. Moreover, HSP70 play an important role in the vital activity of cells under normal physiological conditions, performing chaperone functions. These functions are realized in the intracellular space; however, in some cases, these proteins are also found on the cell surface and in the extracellular environment. The causes and mechanisms of HSP70 translocation to the cell surface and secretion into the extracellular space have not yet been well understood, but such an unusual localization of HSP70 activates the immune system. The surface HSP70 and their extracellular pool stimulate the cytotoxic activity of NK cells. However, direct experimental evidence for the internalization of HSP70 molecules by NK cells has not yet been demonstrated. This paper presents the results of the interaction of the extracellular HSP70 pool with NK cells from the peripheral blood. The results demonstrated the confirmation of the internalization of exogenous HSP70 molecules by NK cells. To this end, fluorescently labeled recombinant stress-inducible human HSP70 were obtained. The electrophoretic data indicated the absence of protein degradation during the labeling process, the purity and stability of the modified protein. To assess the interaction of HSP70 with NK cells, the fluorescently labeled HSP70 was added to an in vitro culture of NK cells isolated by magnetic separation from the peripheral blood mononuclear fraction and analyzed by confocal microscopy. This analysis indicated that living NK cells internalize extracellular HSP70 with localization both in lysosomes and in phagosomes. Our experiments illustrated for the first time the process of penetration of the extracellular form of HSP70 into these cells. The results suggest that the activation of NK cells under the action of exogenous HSP70 could be associated with the internalization of these protein molecules.

Macrophages are involved in the regulation of fibrogenesis and turnover of the extracellular matrix. One way to perform this function is through the production of profibrotic and fibrolytic factors including fibronectin, laminin, collagen, and extracellular matrix proteases. The production of most of them has been well studied in experimental models; however, much remains unclear regarding human macrophages. Therefore, the aim of this study was to study the content of extracellular matrix proteases (MMP-2 and MMP-9, cathepsin L), their inhibitors (TIMP-1), and collagen (type I) in supernatants of differently activated human macrophages. We compared macrophages differentiated by M-CSF or GM-CSF and further polarized in M1 with lipopolysaccharide, in M2a with IL-4, and in M2c with dexamethasone. Macrophages was obtained from peripheral blood monocytes. The content of MMPs, TIMP, cathepsin, and collagen was determined using appropriate ELISA kits. The results obtained demonstrate that differentiation factors are more important for the production of the above factors compared to polarizing stimuli (lipopolysaccharide, IL-4, dexamethasone). Moreover, macrophages differentiated by M-CSF showed predominantly antifibrotic activity because of pronounced MMPs production, while GM-CSF-induced cultures, on the contrary, were characterized by profibrotic properties due to the high level of TIMP-1 and type I collagen. M1, M2a, and M2c, induced by M-CSF, differed only in MMP-2 production, and M2a produced this metalloproteinase more than other subtypes. In the case of GM-CSF-differentiated cells, a higher level of production of TIMP-1 and, to a lesser extent, type I collagen was characteristic of M1, whereas M2c have minimal concentration of them among GM-CSF-induced macrophage subtypes. Concerning the level of cathepsin L production was relatively constant and did not depend on the generation conditions (differentiation and polarizing signals). Thus, the data obtained help to identify macrophage subtypes with anti- or profibrotic potential and may be useful for the development of cell therapy for diseases associated with fibrogenesis dysregulation.

TNFa is a pro-inflammatory cytokine that is signaled through type 1 (TNFR1) and type 2 (TNFR2) receptors. TNFR1 normally mediates apoptosis, cell survival, and cytokine secretion, while TNFR2 selectively mediates cell survival and cytokine secretion. But in some cases, when receptors are activated, the functional response of cells changes to the opposite. Activation of signaling pathways has its own triggers, which differ in the interaction between different forms of cytokine and different forms of receptor complexes, as well as changes in the ratio of different types of receptors. The study of the mechanisms of regulation in the ligand-receptor system is a priority task for many studies. This work shows the dose-dependent effect of TNFa on the expression of cytokine receptors and changes in the functional response of tumor cell lines of various origins. For this, a comparative assessment of the expression and co-expression of receptors, cell cycle phases and apoptosis of cell lines without stimulation and stimulated with TNFa at concentrations of 5 and 50 ng/mL was carried out. It was found that the K562 cell line was characterized by more pronounced changes in receptor co-expression, which were observed at a TNFa concentration of 50 ng/mL compared to both the control group and the 5 ng/mL group. The decrease in the relative content of cells expressing only TNFR1 was combined with a decrease in the percentage of cells in apoptosis, which confirms the literature data on the role of this receptor in the development of apoptosis. At the same time, no changes in expression density were observed for this cell line. For the ZR75-1 cell line, the largest number of effects was also found for a TNFa concentration of 50 ng/mL. An increase in the relative content of cells expressing only TNFR2 was combined with an increase in apoptosis; however, the expression density of this type of receptor was low, which could affect the switching of signaling pathways towards proapoptotic ones. Thus, our study allowed us to reveal the features of changes in the expression and co-expression of TNFa receptors characteristic of cell lines of various origins, as well as changes in the functional response of cells in response to stimulation with different doses of cytokine. All this allows us to expand our understanding of the regulatory mechanisms in the cytokine-receptor system.

The hormone melatonin is involved in regulation of functioning of almost all organs and systems of the organism. In the immune system, T lymphocytes are an important target of melatonin: they express specific melatonin receptors with different affinities – membrane MT1 and MT2 and nuclear RORa, as well as intracellular molecules that nonspecifically bind melatonin at high concentrations. Moreover, many in vitro studies reveal their own production of melatonin by T lymphocytes in response to polyclonal activation and its involvement as autocrine or paracrine factor in the induction of IL-2 and IL-2 receptor (IL-2R) synthesis by T cells, with melatonin receptors involvement in implementation of these effects. Since IL-2/IL-2R-dependent signal is a key event in T lymphocytes proliferative response induction, intrinsic melatonin seems to be directly involved at least in the clonal expansion of these cells. In this work, we investigated the contribution of T cells’ melatonin to regulation of the next stage of T lymphocyte activation, namely, the differentiation of T helper populations Th17 and Treg. It was shown that blockade of both membrane and nuclear melatonin receptors did not cause statistically significant changes in Th17 differentiation, although the trend was fixed for a decrease. Simultaneously, CD4⁺FoxP3⁺Т cells level decreased under the nonselective blockade of membrane hormone receptors, and Treg-associated cytokine TGF-b concentration in activated cultures supernatants decreased both in case of MT1/MT2 nonselective blockade and MT2 selective blockade. The data indicate that melatonin produced by T lymphocytes in culture can contribute to the control of naive CD4⁺T cell differentiation into Treg in vitro, and the hormone effects are mediated by membrane melatonin receptors. The presence of a large number targets with different affinities for melatonin in T lymphocytes determines the key role of the hormone concentration in its effects on these cells. And when interpreting data on melatonin-dependent regulation of Treg, it is important to take into account the hormone’s own production by lymphocytes, since T cells’ melatonin can mask the exogenous hormone effects or interfere with its action due to competitive binding to hormone receptors.

Galectin-9 is a b-galactoside binding lectin with expressed immunoregulatory activity. During pregnancy galectin-9 is produced by trophoblast cells and regulates the function of natural killer (NK) cells at the maternal-fetal interface via binding to Tim-3 (T-cell Ig and mucin domain-containing protein 3) molecules. Natural killer (NK) lymphocytes belong to the innate lymphoid cells, which have a cytotoxic effect on target cells and are capable of producing a large number of regulatory factors (cytokines, chemokines). Decidual NK have a tolerant phenotype and play a leading role in the regulation of invasive trophoblast growth and provide peripheral immune tolerance in the area of uteroplacental contact. Peripheral NK cells express Tim-3 molecules. Galectin-9 concentration is increased in peripheral blood during physiologic pregnancy. At pregnancy phenotype and functions of peripheral NK cells are changed to maintain the maternal–fetal immune tolerance. Peripheral NK cells migrate to the maternal-fetal interface and are transformed into a decidual NK-like phenotype cells. Galectin-9 concentration is decreased in women with a  complicated  pregnancy and miscarriage. However the galectin-9 effects on different NK cell subpopulations of peripheral blood are not investigated. Therefore, we studied the galectin-9 influence on phenotype transformation and Tim-3 expression of NK cells isolated from peripheral blood of healthy non-pregnant fertile women. CD56⁺NK cells were obtained by immunomagnetic separation and cultivated in vitro during 72 hours with cytokines (IL-2 and IL-15). Galectin-9 (5 ng/mL) and anti-Tim-3 (10 mg) antibodies were added to the NK cultures. Galectin-9 concentration is corresponded to its level during first trimester of physiologic pregnancy. The number of regulatory NK (CD16^-CD56^bright), cytotoxic NK (CD16⁺CD56^dim/-) cells and Tim-3 expression on different NK subpopulations were assessed by flow cytometry. It was found that Tim-3 was expressed on all subpopulations of peripheral blood NK cells (CD16^-CD56^brightNK, CD16⁺CD56^dimNK, CD16⁺CD56^-NK). Incubation with galectin-9 increased the expression of Tim-3 on regulatory CD16^-CD56^brightNK cells and did not change on cytotoxic CD16⁺CD56^dim/-NK cells. Galectin-9 reduced the percentage of cytotoxic CD16⁺CD56^dimNK in culture, but did not influence the number of regulatory CD16^-CD56^bright NK and cytotoxic CD16⁺CD56^-NK cells. Thus, galectin-9 regulates Tim-3 molecule expression and NK cell subpopulation distributions in vitro culture.

Itaconate is an immunoregulatory metabolite produced by myeloid cells and plays a key role in the regulation of the immune response. Itaconate, on the one hand, is able to suppress the activity of succinate dehydrogenase (SDH), thereby making a significant contribution to the metabolic reprogramming of the cell. On the other hand, itaconate can regulate the activity of a number of transcription factors and transcription regulators, thereby affecting gene expression. In most experimental studies, itaconate has been characterized predominantly as an anti-inflammatory agent. In particular, itaconate produced by activated macrophages inhibits the production of cytokines TNF, IL-1b, IL-6, IL-10. However, some evidence suggests a pro- inflammatory role for itaconate in a number of mouse disease models. Thus, the deletion of the Acod1 gene responsible for the production of itaconate leads to the suppression of the production of TNF and IL-6 in the mouse polymicrobial sepsis model, which means that in the context of inflammation in vivo, itaconate can act as an inducer of pro-inflammatory cytokines. The mechanism of itaconate regulation of cytokine production in systemic inflammation remains unexplored. In this work, we have shown that injection of itaconate and its derivative dimethyl itaconate into mice, followed by induction of inflammation by bacterial lipopolysaccharide (LPS), leads to changes in the content of cytokines in the blood. Interestingly, the systemic production of IL-6 and IL-10 in response to itaconate is increased, contrary to the results previously obtained in cell cultures. At the same time, IFNg production, on the contrary, is suppressed. Apparently, itaconate regulates the production of cytokines in vivo by suppressing the activity of SDH. Injection of the SDH inhibitor, dimethylmalonate, followed by induction of inflammation in mice, results in similar changes in blood cytokines observed in response to itaconate: increased production of IL-6, IL-10 and suppression of IFNg production. On the contrary, the addition of succinate, a SDH substrate, leads to the opposite effect on cytokine production. Thus, it can be assumed that the observed effects of itaconate on cytokine production in the model of LPS-induced inflammation are mediated by its ability to inhibit SDH. These results help to understand the controversial role of itaconate in inflammation and shed light on a previously undescribed relationship between SDH and cytokine production in inflammation in vivo.

MICA and MICB are non-classical MHC molecules that indicate cellular stress. They act as ligands for NKG2D receptors found on NK cells, thereby triggering a cytotoxic response against damaged, infected, or transformed cells. The production of soluble forms of MICA/MICB occurs via the cleavage of their extracellular domains (ECDs). The expression of MICA/MICB molecules in tumor sections or the levels of their soluble forms in blood have potential as diagnostic tools for cancer. They can predict important clinical outcomes for cancer patients, such as overall and recurrence-free survival. However, their extensive molecular polymorphism complicates the development of monoclonal antibodies (mAbs) for diagnostic use. Therefore, the diagnostic value of mAb-based assays may vary depending on the frequencies of allelic variants in local human populations. We examined the ECD amino acid sequences of more than 280 MICA and 50 MICB allelic variants. Additionally, we identified 172 and 58 single nucleotide polymorphisms (SNPs) located in the coding regions of the respective genes and resulting in amino acid replacements. The most frequent amino acid replacements (> 10%) in the ECD occur at 11 and 4 sites of MICA and MICB, respectively. We found that the frequencies of SNPs in the identified hot spots strongly correlate with each other in different human populations, despite the diversity of allelic variant frequencies. The functional role of only one site is known. The replacement of valine with methionine at position 152 enhances the affinity of MICA to NKG2D receptor. As the hot spots are dispersed throughout the entire ECD sequences, they may play a role other than modulating affinity with the NKG2D receptor interaction. We recommend that Ag sets used to validate anti-MICA/MICB mAbs meet two criteria. First, they should include both MICA and MICB alleles, as these genes have highly similar sequences. Second, the alleles should cover the variability observed in the identified hot spots.

Innate immune cells (monocytes/macrophages, NK) can also develop immune memory, which means that these cells are trained after their first encounter with pathogens so that they exhibit a nonspecific immunological response to the same or another pathogen. Bacilli Calmette–Gu rin (BCG) induces nonspecific innate memory (trained immunity) in innate immune cells. We examined nonspecific innate memory in macrophages of BALB/c mice in response to mycobacteria with or without the RD1 region in the genome. Mice were immunized with BCG vaccine, and peritoneal macrophages were isolated on day 7, and then stimulated with bacterial lipopolysaccharide, CFP-10, or ESAT-6. In addition, mice were immunized with Mycobacterium tuberculosis uro-BCG vaccine (RD1^-) and Mycobacterium tuberculosis strain H37Rv (RD1⁺) subcutaneously or intravenously; peritoneal macrophages were isolated and stimulated with lipopolysaccharide on day 4. Alveolar macrophages were obtained from lung explants of mice infected with Mycobacterium tuberculosis strain H37Rv mice, were expanded to confluence 70-80% and further stimulated with lipopolysaccharide. Lactate, cytokines, and glucose levels were examined in conditioned macrophage medium. Peritoneal macrophages from mice primed with BCG vaccine were shown to increase IL-1b, TNFa, and lactate production in response to CFP-6 and ESAT-10 (p < 0.05). Of note is the fact that lipopolysaccharide also increased production of IL-1b, TNFa, and also increased glucose uptake by peritoneal macrophages primed with BCG vaccine (p < 0.05). Peritoneal macrophages primed with Uro-BCG were shown to increase spontaneous production of IL-1b and decrease spontaneous production of TNFa (p < 0.05). When macrophages were primed by subcutaneous or intravenous administration of Mycobacterium tuberculosis strain H37Rv differentially affected cytokine production, by decreasing IL-1b production and increasing TNFa and IL-10, was observed. In response to lipopolysaccharide, peritoneal macrophages increased IL-1b, TNFa, IL-10 production and glucose consumption (p < 0.05). The mode of priming of macrophages with Mycobacterium tuberculosis strain H37Rv also led to multidirectional levels of cytokine production. Alveolar macrophages were shown to retain trained immunity, as they produced elevated levels of IL-1b, TNFa, and IL-10 (p < 0.05). Thus, mouse macrophages formed a trained immunity phenotype in response to different types of mycobacteria, which persists for a long time after primary contact with the pathogen, particularly in alveolar macrophages.

In recent years, research has revealed a wide variety of erythroid cell functions, including modulation of innate and adaptive immune responses. Anemic or hypoxic stress stimulates a physiological response in the form of stress erythropoiesis, aimed at increasing oxygen delivery to tissues. Stress erythropoiesis activates progenitor cells and uses mechanisms that differ from stationary bone marrow erythropoiesis. To consider the role of erythroid cells in the regulation of hematopoiesis, hematopoiesis-activating states were modeled: chemically induced hemolytic anemia, acute blood loss, hypoxia. A series of experiments was carried out on first-generation hybrid mice CBA C57Bl6. Isolation of erythroid cells was performed using magnetic separation for the CD71 marker. The stages of differentiation of erythroid cells were determined by the combination of expression of TER-119 and CD71 markers and direct light scattering parameters in the population of both CD45-positive and CD45-negative spleen cells. To study the immunoregulatory activity of erythroid cells, we investigated the mediated cytotoxicity of splenocytes against tumor cells of the mouse melanoma B78 line after cultivation with conditioned spleen media after various hematopoiesis-stimulating effects. With various hemopoiesis-stimulating effects, the quantitative and qualitative composition of the spleen cells is reorganized depending on the compensatory mechanism for restoring homeostasis. An analysis of the cellular composition of the spleen showed that under hematopoiesis-stimulating effects, a redistribution of populations with the CD45 marker occurs: during hypoxia, the number of CD45-negative cells sharply decreases and the number of CD45-positive cells increases. The population of basophilic erythroblasts is the least susceptible to quantitative changes under all hematopoiesis-stimulating effects. During hypoxia, the most noticeable change in the cellular composition of the spleen is observed due to the increased accumulation of CD45-positive erythroid cells in the spleen. Mediators of erythroid cells of the spleen of mice after hypoxia do not lead to an increase in the cytotoxic proapoptotic effect of splenocytes on tumor cells, in contrast to the erythroid cells of the normal spleen, spleen with anemia and blood loss. Thus, it is tissue hypoxia that is the process that not only stimulates erythropoiesis, but also leads to the maximum change in the suppressive properties of surrounding cells. We assume that the implementation of compensatory mechanisms under the studied hematopoiesis-stimulating effects is aimed at activating the mechanisms of innate immunity and local immunosuppression to prevent local inflammation, accumulate nutrients, and attract cellular elements to the focus of hematopoiesis to restore homeostatic functions.

Pregnancy is a phenomenon of natural semi-allogeneic transplantation, since the fetus is half alien due to the expression of paternal antigens. It was found that the hypothalamic hormone kisspeptin during pregnancy is produced by the syncytiotrophoblast of the placenta and participates in the formation of a new specific hormonal background. Several forms of the hormone circulate in the blood of pregnant women: kisspeptin-10, kisspeptin-14 and kisspeptin-54 (according to the number of amino acid residues in the hormone molecule), but the main active form is kisspeptin-54. The main mechanism for the formation of immune tolerance during pregnancy is the induction of the expression of the enzyme indolamine-2,3- dioxygenase (IDO) by antigen-presenting cells of peripheral blood, resulting in the catalysis of tryptophan (Trp) to kynurenins (KYN) blocking the activation and causing apoptosis of cytotoxic CD8⁺T lymphocytes in the zone of contact of maternal immune cells with placental-fetal complex antigens. In addition, during pregnancy, an important role is assigned to the process of apoptosis, since activated cells can be potentially dangerous for the developing fetus. Immunocompetent blood cells express a specific membrane receptor of kisspeptin (KISS-1R). Since kisspeptin-54 enters the systemic circulation only during pregnancy, the hormone has an effect on immune cells only during this period.

The aim of this work was to evaluate the effect of kisspeptin-54 in concentrations comparable to its level

during physiological pregnancy on IDO activity and apoptosis of peripheral blood lymphocytes.

Peripheral blood mononuclear cells (PBMC) obtained from 10 healthy non-pregnant women of reproductive age (from 23 to 32 years) were used as the object of the study. Lymphocyte apoptosis was assessed in PBMC suspension by staining with annexin-V and propidium iodide. The determination of the number of cells in the early and late stages of apoptosis was carried out in the isolated gate of lymphocytes. IDO activity in PBMC was determined spectrophotometrically by changes in the concentration of KYN, the first stable metabolite of the Trp decay pathway.

It was found that kisspeptin-54 at a concentration of 4.6 pM corresponding to the second trimester of pregnancy significantly enhances the activity of IDO, increases the number of cells in the early and late stages of apoptosis. Thus, kisspeptin-54 is an important mechanism for controlling these processes during pregnancy, aimed at protecting the semi-allogeneic fetus from adverse immune reactions of the mother and the favorable development of pregnancy.

During pregnancy, the maternal immune system must maintain tolerance to paternal antigens, at the same time being able to eliminate pathogens, which is achieved by the weakening of adoptive immunity and the activation of innate immunity, in particular, monocytes. However, the question about the functional phenotype of monocytes, having not only pro-inflammatory, but also anti-inflammatory activity, remains open. In the given work, we have investigated the expression of M2-associated suppressive markers Arg1 and MerTK in monocyte subpopulations during uncomplicated pregnancy. Fifty-three pregnant women with uncomplicated gestation were recruited, including 14 pregnant in the 1^st trimester, 20 – in the 2^nd and 19 – in the third pregnancy trimester. The comparison group consisted of 15 fertile unpregnant women without aggravated somatic anamnesis, with a history of at least one childbirth. The findings showed that in the unpregnant group circulating Mo express Arg1 and MerTK, and the most relative number of Arg1⁺ and MerTK⁺ cells is concentrated in intermediate and nonclassic monocytes. During pregnancy the expression of researched molecules in monocytes reliably increases. An increase in MerTK expression is manifested by a simultaneous increase in the number of MerTK⁺ cells and the mean fluorescence intensity of this marker; it is observed in the 1^st and 2^nd trimesters and registered in all three monocyte subpopulations. At the same time, an increase in Arg1 expression is manifested either by an enhancement of Arg1⁺ cells, or an increase in receptor density; it is registered throughout pregnancy, including the 3^rd trimester, and is maximally expressed in classic monocytes. There is a direct correlation between the number of Arg1⁺ and MerTK⁺ cells in intermediate Mo, which increases with the progression of pregnancy, and in the 3rd trimester is also detected in classical and non-classical Mo. In general, the revealed increase in the expression of Arg1 and MerTK by monocytes indicates an increase in the anti-inflammatory potential of monocytes during pregnancy, and the involvement of monocytes in the regulation of the inflammatory process at the system level. Moreover, the features of Arg1 and MerTK expression in various monocyte subpopulations during pregnancy suggest that monocytes expressing Arg1 and MerTK can mediate different mechanisms of immune adaptation during pregnancy.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population that primarily suppress T lymphocytes in healthy pregnancies and pathologies. MDSCs are one of the key regulators of immune responses. Finding ways to control them is important for the treatment of cancer, autoimmune diseases, miscarriage, and post-transplant complications. The mechanisms of immune suppression by MDSC are: expression of CD73, ADAM17, PD -L1, production of Arg 1, iNOS, IDO, IL -10 and TGF-b1.

Pregnancy-specific b1-glycoprotein (PSG) has modulatory effects on dendritic cells and macrophages that mediate the shift of T cell phenotypes toward Th2 and Treg. We have previously shown that native PSG suppresses Th17 differentiation and cytokine production, stimulates the production of IDO by monocytes and the differentiation of Tregs.

Considering the immunomodulatory properties of PSG and the key role of MDSCs in pathologies, the aim of our work was to investigate the effect of native and recombinant PSG on the differentiation of MDSCs in vitro.

MDSCs were differentiated from CD11b⁺ peripheral blood cells. Cells were cultured for 7 days and received stepwise GM-CSF, IL-1b, and LPS. Native (n) (1; 10 and 100 mg/mL) and recombinant (r) (1 and 10 mg/mL) PSG were introduced into the cultures three days before the end of incubation. Flow cytometry was used to determine the percentage of MDSC among the cells in culture and the percentage of M-, PMN-, and e-MDSC among the total number of MDSCs.

It was found that rPSG (1 mg/mL) increased the percentage of MDSCs in culture. Both nPSG (1 and 10 mg/mL) and rPSG (10 mg/mL) increased the proportion of M-MDSC, whereas rPSG (10 mg/mL) decreased the number of PMN-MDSC.

Thus, the cytokine background in CD11b⁺ cell cultures favored the differentiation of predominantly M-MDSC, similar to the tumor microenvironment, whereas native and recombinant PSG enhanced this effect. Thus, nPSG and rPSG are able to modulate the differentiation of MDSCs by increasing their number, mainly due to the monocytic subpopulation. This fact opens perspectives for new research on targeted manipulation of MDSCs.

Recently, there has been a steady increase in depressive disorders, which occupy an important place in the structure of the causes of disability. In the pathogenesis of depression, an important role is played by neuroinflammation, which is associated with impaired adult neurogenesis. Notably, neuroinflammation is partially reversible, and the leading role in the initiation and regulation of neuroregeneration is given to macrophages. Opposite states of macrophage activation are classically activated M1 and alternatively activated M2 macrophages, characterized, respectively, by pro- and anti-inflammatory activity. A balance shift towards M2 macrophages has been considered as a new therapeutic strategy of psycho-neurological disorders. One of the inducers of the M2 phenotype is the efferocytosis. We have previously developed an original protocol for the generation of human macrophages under conditions of deficiency of growth / serum factors, in which M2 phenotype is formed through efferocytosis. Macrophages (M2(LS), LS – Low Serum) obtained according to this protocol express M2-associated markers, and are characterized by high production of growth and pro- angiogenic factors (IGF-1, VEGF, BDNF, EGF, FGF-basic, etc.), which can suppress inflammation and stimulate neuroregeneration / neuroplasticity. In the model of stress-induced depression, the antidepressant effect of soluble factors of M2(LS) macrophages was shown, accompanied by a decrease in the level of pro- inflammatory cytokines in certain brain structures. However, the effect of M2(LS) factors on neurogenesis remained unexplored. In the present work, which is a continuation of the aforementioned study, we analyzed the effect of intranasal administration of M2(LS) soluble factors on neuronal density in different brain areas – the frontal cortex and hippocampus – of depression-like mice. The results obtained showed that neuronal density in the frontal cortex, CA1 and CA3 zones of the hippocampus, was significantly higher in mice with intranasal administration of M2(LS) conditioned medium than in depression-like mice, and reached the level of neuronal density in intact animals. These results may indicate the neuroregenerative activity of M2(LS) macrophages in the model of stress-induced depression, which is mediated through soluble factors and manifests itself in an increase in the density of neurons in the brain.

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases leading to dementia. There is no effective treatments for this disease so far, as well as a consensus concerning the mechanisms of its pathogenesis initiation. Obtaining data on them in vivo is possible only by modeling neurodegeneration in laboratory animals. Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative diseases leading to dementia. There is no effective treatments for this disease so far, as well as a consensus concerning the mechanisms of its pathogenesis initiation. Obtaining data on them in vivo is possible only by modeling neurodegeneration in laboratory animals. Among the various theories of the initiation of neurodegeneration, the impact of microglia is vigorously studied recently, as well as inflammaging, which is a term for chronic age-related low-grade systemic inflammation. It manifests in the increasing number of senescent cells with senescence-associated secretory phenotype (SASP). Eventually, it leads to manifestation and progression of age-related diseases, such as AD. The aim of the study was to evaluate age-related changes in microglia, pro- and anti-inflammatory cytokines expression levels in the brain, as well as ones of microglial activation, and also subpopulations of lymphocytes in peripheral blood. We used male Wistar rats of two age groups, which were composed of old (age 24 months) and adult (age 3 months) rodents, without any additional exposure. In the hippocampus, morphological changes in microglia were assessed on preparations stained with antibodies to Iba1. In the prefrontal cortex, RT-qPCR was used to study the level of expression of pro-inflammatory IL-6 and TNFa, anti-inflammatory IL-10 and TGF-b cytokines, as well as microglial activation markers iNOS and MMP-9. In the peripheral blood, the relative numbers of the main subpopulations of lymphocytes and monocyte were measured by flow cytometry. It was shown that, compared with adult rats, old animals are characterized by significant changes in the morphology of microglia, an increase in the level of expression of pro-inflammatory and a decrease in anti-inflammatory cytokines, and an increase in microglia activation markers. With aging, a decrease in the percentage of monocytes and B cells in peripheral blood was observed. These data indicate the development of inflammaging, which displays itself in microglia activation, a shift in the balance of cytokine production towards pro-inflammatory ones, and, as a result, activation of the migration of monocytes and B lymphocytes from the blood into tissues. Thus, it is justified to study the role of inflammation in the development of AD in old animals whose physiological state corresponds to that in humans. Further research in this area will expand the understanding of the mechanisms of initiation and progression of neurodegeneration, which is necessary for the development of novel and effective therapeutic approaches to the treatment of AD.

Depression is one of the leading global health problems worldwide. A significant increase in prevalence among the working-age population, as well as high comorbidity, partial or complete drug resistance in a third of patients determines the need to develop new approaches to the treatment of depression. Violation of mutual regulation of the main homeostatic systems plays an important role in the pathogenesis of depression. Psycho- and immunopathology are closely interrelated: pathological changes in the functioning of both systems occur simultaneously and are interdependent. This determines the prospects for the treatment of depression based on immunological approaches. Caffeine, a drug known for its psychoneuromodulatory properties, is an adenosine receptor antagonist with a pronounced dose-dependent effect. Adenosine receptors are expressed by both CNS cells and cells of the immune system, which determines its immunomodulatory properties. The similarity of both phenotypes and functions of the cellular elements of the immune and nervous systems, as well as the unidirectional effect of most psychoactive drugs on the central nervous system and the immune system, determines the interest in studying the immunomodulatory properties of caffeine for a targeted effect on the functional activity of immune cells, with a view to their subsequent use as model objects for the normalization of neuroimmune regulatory connections disturbed in a depressive state. Previously, we first demonstrated the possibility of editing depression-like behavior by immune cells precultivated with caffeine and showed the central mechanisms of this effect aimed at stimulating neuroplasticity processes and reducing neuroinflammation. The aim of this study was to evaluate the functional phenotype of immune cells in depressive-like animals after in vitro treatment of cells with caffeine, as well as the effects of transplantation of caffeine-precultured immune cells on the parameters of the functional activity of the immune system of syngeneic depressive-like recipients. As a result of the study, it was shown that low concentrations of caffeine increase the spontaneous and mitogen-induced proliferative activity of splenocytes of depression-like male mice (CBA x C57BL/6)F1 in vitro; this changes the spontaneous and mitogen-stimulated production of cytokines TNFa IL-1b, IFNg, IL-2, and IL-10 by these cells. After intravenous administration of the precultured with caffeine depression-like donor’s splenocytes to syngeneic depression-like recipients, stimulation of the humoral immune response was observed in the latter, assessed by an increase in both the relative and absolute number of antibody-forming spleen cells. Stimulation of spontaneous proliferative activity of lymphocytes in splenocyte culture was also registered. The data obtained indicate a positive effect of caffeine in vitro on the immune cell’s functional activity, as well as a positive immunomodulatory effect of the immune cells precultured with caffeine in a depression-like state in vivo.

Mast cells (MCs) are a required component of the thymus microenvironment. They affect intercellular interactions and permeability of the hematothymic barrier through cytokine production. There is speculation that the thymus is the site of MCs formation and deposition. MCs are under complex neuro-endocrine control and they can play an important role in the process of acute transformation of the thymus in the formation of a stress reaction, affecting the extrathymic migration of cells. The purpose of this study is to assess the functional involvement of MCs in the process of the thymus acute transformation at various hyper- and hypodynamic effects during the formation of stress response and without it.

The study was conducted on male Wistar rats. The stress factors were physical activity (swimming) of different intensities and immobilization, which represent two opposite states of dynamic stress. MCs were classified on histological preparations; a degranulation coefficient and a mean histochemical coefficient (synthetic activity) were calculated.

In groups with preserved adrenal glands after exposure a significant decrease in the thymus mass coefficient is noted, which indicates a weakening of its functional activity in response to the development of stress. At the same time, MCs of the thymus quickly respond to neuro-endocrine factors under stress. These cells are involved in a general reaction: their activity consists in a synchronous decrease of the synthesis of granules in the cytoplasm and an increased release of active substances accumulated earlier. The mass and structure of the thymus remain unchanged in groups with removed adrenal glands after immobilization. No changes in morphofunctional indicators of mast cells were detected either. Experiments with hypo- and hyperdinamic loading of animals with preserved and removed adrenal glands indicate that the MCs response is largely determined by the hypothalamic-pituitary-adrenal axis of the endocrine system. Removal of the adrenal glands (inability to release glucocorticoids) leads to a lack of functional response from the thymus MCs. The stimulating effect of adrenal glucocorticoids on MCs under stress is carried out in combination with other neuro-endocrine factors (catecholamines, corticotropin-releasing hormone, adrenocorticotropic hormone). When this axis is activated and a full-fledged stress reaction is formed by the body, MCs are actively involved in the process of acute transformation of the thymus through cytokine secretion. These is an important condition for the development of adaptation mechanisms by the immune system.

Endogenous opioid peptides are a large group of physiologically active compounds with a pronounced affinity for opioid-type receptors, capable of showing pronounced analgesic activity, as well as having additional effects on the periphery, due to their wide distribution on the cells of many organs and tissues. Little studied representatives of this group are endomorphins, which due to their structure and properties, are capable of producing a strong antinociceptive effect after central administration, which means that, in the future, they can be considered as potential substitutes for low molecular weight opiates. The aim of this study is to evaluate the effect of endomorphins on the humoral immune response, the production of Th1/Th2/Th17 cytokines and apoptosis of CD4⁺, CD8⁺ lymphocytes in vivo. The splenocytes of Swiss white mice were used as the object of the study. The number of antibody-forming cells in the spleen was assessed using the method of local hemolysis in agarose gel according to Jerne. Quantitative determination of cytokines was carried out by enzyme-linked immunosorbent assay using kits (R&D, USA) according to the method proposed by the manufacturer. Apoptosis was assessed using Annexin V-FITC/7-AAD kit reagents (Beckman Coulter, USA) according to the manufacturer’s instructions by flow cytometry on a CytoFLEX S flow cytometer (Beckman Coulter, USA). In the course of the study, it was found that endomorphins enhance the antibody genesis of the spleen, and the preliminary blockade of opiate receptors with naloxone led to the cancellation of the stimulating effect of peptides. Endomorphins didn’t affect splenocyte production of IL-2, IL-4, and IFNg, however, the introduction of endomorphin-2 naloxone-independent enhanced the induced production of IL-17. Evaluation of the effect of endomorphins on apoptosis of splenocytes in 24-h cultures showed that endomorphin-2 in unstimulated cultures of naloxone-dependently increased the percentage of late apoptosis of CD8⁺ lymphocytes, however, in stimulated cultures, both endomorphins increased the apoptotic activity of CD8⁺ lymphocytes, regardless of the preliminary blockade of opioid receptors. In summary, we can say that in the in vivo system, endomorphins have a wide range of multidirectional immunomodulatory effects, which may be of interest for practical use in the future.

It is known that there are individual differences in resistance to hypoxia, which can determine the predisposition to the development and severity of various diseases, including infectious, inflammatory and tumor. There are no standardized methods for assessing resistance to hypoxia in experimental animals and humans without hypoxic exposure. The search for molecular-biological markers, identifying people with different resistance to oxygen deficiency under normoxic conditions or under moderate hypoxic exposure is undoubtedly efficient. It is possible that the assessment of the basic resistance to hypoxia can help to predict the development and severity of the course of diseases, the mechanisms of which are associated with oxygen deficiency. One of the methods to assess organism resistance to hypoxia without exposure in a decompression chamber or in highland conditions can be modeling hypoxia in vitro. The aim of the study was to characterize the phagocytic activity of peripheral blood monocytes in tolerant and susceptible to hypoxia Wistar rats under normoxic conditions, as well as after hypoxic exposure in vitro and in vivo. The resistance of rats to hypoxia was determined by the gasping time at an altitude of 11.500 m in a decompression chamber. A month after determining the resistance to hypoxia, one group of rats was placed in a decompression chamber at an altitude of 5,000 m for 1 hour to simulate the hypoxic state in vivo. Blood from the tail vein of the other group of rats was placed in 1% oxygen for 1 hour to simulate the hypoxic state in vitro. The phagocytic activity of peripheral blood monocytes was assessed by flow cytometry. It was demonstrated that phagocytic activity of monocytes did not differ in tolerant and susceptible to hypoxia rats under normoxic conditions. The phagocytic activity of monocytes after in vitro and in vivo hypoxic exposure was higher in tolerant to hypoxia animals in comparison to susceptible ones. An increase in the phagocytic activity of monocytes compared to normoxia conditions was observed only in tolerant rats under in vitro conditions of hypoxic exposure. The obtained results indicate that tolerant and susceptible to hypoxia organisms differ in the phagocytic activity of monocytes under conditions of oxygen deficiency, which can determine the course of inflammatory and tumor diseases. The data obtained will be the basis for further experimental investigations organism hypoxia resistance markers.

Recurrent urinary tract infections (UTIs) are associated primarily with the ability of Escherichia coli to form biofilms. The interaction of neutrophils, factors of innate immunity, with microorganisms in biofilms is difficult compared to planktonic forms due to the lack of direct contact, as well as due to the antiphagocytic action of the extracellular matrix of biofilms. The purpose of this study was evaluation of neutrophils phagocytic and oxidative activity during interaction with biofilms of uropathogenic E. coli (UPEC) DL82 and R44. Peripheral blood neutrophils from healthy men were isolated using ficoll-urographin double gradient, incubated for 1 h with bacterial cells from biofilms or their supernatants, then leukocytes functional activity was evaluated. Phagocytic activity of neutrophils was determined by the degree of bioluminescence inhibition of bioluminescent strain E. coli K12 TG1 lux⁺ (pXen) upon their absorption by neutrophils. Production of extracellular reactive oxygen species (ROS) was analyzed by the intensity of luminol-dependent chemiluminescence in spontaneous and stimulated by E. coli K12 variants. Significance of differences was determined using Student’s t-test at p < 0.05. It was found that neutrophils interaction with UPEC biofilm cells or supernatants did not affect the phagocytic activity. E. coli DL82 supernatants reduce neutrophils spontaneous ROS production compared to control and biofilm cells. E. coli R44 supernatants with a low virulence potential did not affect ROS production, while biofilm cells stimulated it. When assessing stimulated ROS production, exposure to R44 strain supernatants did not cause a decrease in neutrophils activation in response to an external stimulus (E. coli K12 cells). Preliminary contact of neutrophils with E. coli R44 bacteria resulted in a high and prolonged level of ROS production compared to the control. Neutrophils interaction with DL82 cells resulted in a higher level of ROS compared to supernatants, however a subsequent rapid depletion of neutrophils oxidative potential was observed. Thus, cells and supernatants of UPEC biofilms can determine the activation of neutrophils.

Rythmometric markers can be identified within a day during the study of biological characteristics in order to be used for differential diagnostics of pathogens of different patients’ physical condition. These principles are based on analysis of clinical isolates С. albicans, С. tropicalis and C. krusei allocated from the vaginal microbiota at Candida dysbiosis condition. Control examples were the master samples from the American Type Culture Collection (ATCC). Detailed research was conducted on physiological characteristics through the formation of biofilms by yeast pathogens. Biological activity of Candida sp. biofilming was observed within 2 days with 4 hours interval in winter. Daily cultures were used for the experiment to correspond to their maximum adhesion to the glass surface. It was important to obtain 6 measurements per day with 3-5 times repetition of experiment conditions during the specified timeline. In order to determine the periodicity of the parameters studied, the data was statistically processed by Student’s t-test, using Mann–Whitney criteria and nonparametric method of least square method.

It was found out that biofilming activity during 24 hours (р < 0.05) of fungi exists and that all species have many principles in common. It was attested that the main rhythmometric parameters of diagnostic significance are the rhythm period and amplitude-phase stability. It was found that the daily dynamics of C. albicans 24433 biofilm formation from American Type Culture Collection was characterized by an ultradian (about 12-hours) contribution of the rhythm in the morning – 4 A.M and in the evening – 4 P.M. Significant circadian (approx. daily) rhythms of adhesion glass surface activity were revealed in C. non-albicans from American Type Culture Collection. The dynamic of biofilm formation isolates of yeast from female reproductive organs with Candida pathology was characterized by reliable ultradian (about 12-hour) harmonics which biological significance defines resistance to external impact and the ability to adaptively respond to periodic stimuli.

To sum up, implementation of the chronobiological approach has opened up new prospects for studying the physiology of Сandida sp., as it enables us to predict the dynamics of microbial states and takes into account the specificity of emergency and long-term adaptation to different environmental factors. The detection of the circadian rhythm of biofilm formation activity of different Candida sp. strains provides the possibility to manage the vitality of the Society of Bacteria and Fungi and predicts its resistance to various antibiotics.

As is known, bacterial polyamines, which include cadaverine and putrescine, are capable of influencing the activity of immunocompetent cells in many ways. In particular, this situation is observed in long-term inflammatory diseases, especially with intensive reproduction of microorganisms capable of producing polyamines. It is of interest to study the production of one of the main anti-inflammatory cytokines, IL-10, under the influence of bacterial polyamines. For research, a population of mononuclear leukocytes was isolated from the peripheral blood of healthy donors by gradient centrifugation. The cell suspension was placed in a round-bottomed plates with preliminarily added polyamines at concentrations of 5, 25, 50, 75, and 100 mmol/L. Wells not containing polyamines were used as a control. After incubation for 72 h at 37 °C and 5% CO₂, the supernatants were harvested and used to determine the concentration of IL-10. We used a kit for determining the concentration of IL-10 using the enzyme immunoassay method (Russia). Statistical analysis was performed using the Statistica 6.0 software package. In the case of a distribution close to normal, Student’s t-test was used; in the rest, the Mann–Whitney test was used to assess the significance of differences. Studies have shown that leukocytes in the presence of concanavalin A produce IL-10 at a concentration of 17.13±6.08 pg/mL. It has been established that under the influence of polyamines of bacterial origin, the production of IL-10 is enhanced only if putrescine and cadaverine are at concentrations of 50 mmol/L and higher. At low concentrations of polyamines, no significant increase in IL-10 production was detected. Since IL-10 is an anti- inflammatory cytokine, for which the analgesic effect is also known, it should be expected that with an increase in its concentration in the focus of invasion of opportunistic bacteria, the inflammatory process will develop latently, when the symptoms are mild. In general, it can be expected that polyamine-producing bacteria will contribute to the maintenance of few symptomatic inflammation.

Human cytomegalovirus (hCMV) is a prevalent virus that affects a large proportion of the population worldwide. Natural Killer (NK) cells are essential immune cells that play a crucial role in controlling hCMV infection. Despite the wide spread of hCMV infection, there is still not enough data related to the association between innate and adaptive immunity. This study investigated the coordination between some of the NK cell markers expression and humoral immune response during hCMV infection. Thirty-three samples obtained from different healthy donors were investigated. The anti-hCMV IgG antibody titer was measured in serum samples, and expression of NKG2C, HLA-DR, CD57, KIR2DL2/DL3, and KIR2DL1 were analyzed in CD56⁺CD3^- cells in PBMC samples by flow cytometry. To evaluate the dependence of proportions of different NK cell subsets on IgG titers, cluster analysis was first performed on all the obtained data, resulting in the identification of four main clusters. The identified clusters demonstrated a dependence on the levels of hCMV antibodies, according to which clusters corresponding to seronegative and low-positive were grouped. The results confirmed that hCMV infection leads to an expansion of NK cell populations expressing the NKG2C marker, which correlates with higher levels of IgG response to hCMV. Besides, we identified increased HLA-DR⁺ and decreased of KIR2DL1⁺ NK cells proportions in the middle anti-CMV-IgG level group compared to samples obtained from seronegative and low-positive donors. Moreover, the statistically significant negative correlation was found between KIR2DL1⁺NK cell percentage and anti-CMV IgG antibody titer, while the positive correlation between HLA-DR⁺NK cell proportion and the IgG level was noticed only without the cluster corresponded to high level of anti-hCMV IgG. In this cohort, we did not find any association between KIR2DL3 and CD57 expression in NK cells and levels of IgG response to hCMV. This may indicate that different subsets of NK cells may have distinct roles in regulating humoral immunity to hCMV. Overall, the results of the study provide valuable insights into the coordination of NK cell marker expression and IgG response in hCMV infection.

Currently, there is an active search for exogenous stimulators of repair and regeneration processes. In the recent decades, some data on the immunotropic activity of bifidobacteria have been accumulated. The key role in healing of wound defects belongs to fibroblasts due to the secretion of the extracellular matrix components, metabolites, signal factors for the surrounding cells, and tissue metabolism regulation. The paper presents the results of the study of the effect of Bifidobacterium bifidum supernatant (10 ml/mL) on the morphological and functional properties of human fibroblasts in real time during the in vitro experiment. In our work, we used the reference strain B. bifidum 791 (All-Russian Collection of Industrial Microorganisms of the State Research Institute for Genetics and Selection of Industrial Microorganisms “Genetika”, Deposit No. AS-1247) used in the production of the probiotic product “Bifidumbacterin” (ZAO “Ecopolis”, the city of Kovrov), and adult human fibroblasts (cell line LECH-4 (81)) (laboratory of cell cultures ENIIVI, the city of Yekaterinburg). Structural and functional studies were conducted on co-culture days 1, 3, 7, 14, 21, and 28. The products of B. bifidum secondary metabolism have a stressful effect on the morphological and functional state of fibroblasts on the first day. The processes of proliferation are stimulated in the culture in the experiment (2.67±0.24) compared with the control group (0.75±0.15) (p < 0.01) without blocking apoptosis in the cell. This leads to the increase in the production of extracellular matrix proteins, both collagen (pg/mL) (400±19 against 110±25 in the control group), and elastin (ng/mL) 395±30 and 125±29). Co-culture of fibroblasts within 24 hours in the experimental sample leads to a massive “release” of the CD44 receptor (p < 0.05), compared to the control group which is confirmed by phenotypic changes (r = 0.66). The decrease of CD105⁺, CD44⁺ receptors (p < 0.05), compared with the control group and the increase of CD29⁺ expression (p < 0.05) is observed on days 1 and 3. Activated fibroblasts have an altered secretory phenotype that produces cytokines of various types such as TGF-b (r = 0.78), IL-6 (r = 0.57), IL-1b (r = 0.75), IL-8 (r = 0.63). The maximum adaptation of cells in the experimental system is registered on the 7th day, which correlates with morphometric (r = 0.59) and cytometric (r = 0.71) studies. The received data contribute to understanding of the mechanisms of the immunoregulatory influence of normal biota (in the bifidobacteria model) on the repair and regeneration processes.

The gut is inhabited by a trillion bacteria that produce up to 60% of the host’s metabolites. The gut microbiome plays an important role in regulating host immune function. A lot of research concerned the effect of probiotic on the pathologies associated not only with dysbiosis and metabolic disorders, but there is breakthrough in the treatment of inflammation, oncology and neurodegenerative disorders. Animals with mutation of the genes leading to pathology used to assay probiotic effect. To understand direct action of probiotics, cells derived from control mice or cell culture of tumor genesis in vitro studies are used. However, there is little research of the probiotic effect on cells derived from mice with pathology. In this study, we assessed the phenotypes of dendric cells derived from Muc2^-/- mice with chronic inflammation and assessed the effect of L. johnsonii on the dendric cells. It is known that the key features of IBD models are thinning of mucin layer and changes in the intestinal microbiome. We compared the efficiency of maturation and activation of dendric cells derived from the bone marrow of Muc2^-/- mice and dendric cells obtained from healthy C57BL/6 mice free from specific species pathogens. We evaluated the expression of co-stimulatory molecules, the proliferative index, and the ability to trigger the T regulatory response of dendric cells, which were stimulated with the probiotic L. johnsonii. Markers of dendritic and T cells were assessed by flow cytometry using antibodies to extra- and intracellular proteins. The proliferative activity of splenocytes was assessed using the WST test. It was shown that dendric cells derived from the Muc2^-/- had an immature phenotype. Dendric cells of Muc2^-/- mice could not effectively stimulate the proliferation of allogeneic and syngeneic T cells. L. johnsonii was able not only to stimulate the maturation of dendric cells derived from Muc2^-/- mice, but also to increase the expression of FoxP3 on CD25⁺ T cell that were co-cultured with DCs. Thus, we believe that this probiotic bacterium can reduce signs of inflammation and reduce pathological processes in animals of an experimental model of IBD in vivo.

Interleukin-6 (IL-6) is a broad-spectrum cytokine involved in the immune, nervous, and endocrine regulation of many biological processes. IL-6 performs both homeostatic and pathogenic functions. It is one of the key factors in the cytokine storm in COVID-19, and it also controls the production of acute phase proteins during inflammation. IL-6 is involved in the maintenance of intestinal homeostasis and is required for both the induction of inflammation and the repair of the injured intestinal tissue. In turn, the commensal microbiota, represented by eukaryotes, prokaryotes, and viruses, is one of the key factors modulating the immune response in the gut. The predominance of certain groups of commensal microorganisms is associated with the development of intestinal inflammation, while probiotics and antibiotics are successfully used to control inflammatory bowel disease. IL-6 is also necessary to maintain the barrier function of the intestine by modulating the proliferation of intestinal cells, which is necessary for their timely renewal both in homeostasis and inflammation. It has been established that the genetic inactivation of IL6 contributes to the development of intestinal inflammation, while the involvement of IL-6 in the control of the gut microbiota composition remains unclear. To investigate this issue, we analyzed stool samples from wild-type naive mice and mice deficient in IL6 (IL-6 KO) generated on the C57Bl/6 genetic background. It has been determined that IL-6 KO shows significant changes in some taxonomic groups of commensals, which may explain the sensitivity of IL-6 KO to the development of colitis. Interestingly, the relative contents of Firmicutes and Clostridiales are significantly reduced, whereas Bacteroides are increased in IL-6 KO as compared with wild-type mice. Our data on the reduction of Firmicutes, Lactobacillaceae, and other large taxa in IL-6 deficient mice suggest that the microbiota composition of IL-6 KO mice is somewhat similar to that of mice with chronic intestinal inflammation. Our study serves as a perspective for further research on the contribution of IL-6-mediated changes in the microbiota composition to the maintenance of intestinal homeostasis and the development of chronic gut inflammation.

This brief report is devoted to the experimental study of the immunotropic activity of a new compound, a metabiotic, based on metabolites (biologically active substances, BAS) produced by the saprophytic and safe standardized strain of Bacillus subtilis B-9909. The aim of the study was to experimentally evaluate the immunotropic effect of metabolites produced by probiotic microorganisms of the genus Bacillus of the culture of probiotic microorganisms of the Bacillus subtilis B-9909 on laboratory animals when modeling their toxic lesion. Metabolites were isolated from the culture fluid of the bacterial culture Bacillus subtilis, strain RNCIM (Russian National Collection of Industrial Microorganisms) B-9909, during its deep cultivation in a medium consisting of hydrochloric acid hydrolysate of soy flour or pancreatic hydrolysate of casein. The study of humoral status indicators in experimental groups of animals in assessing the therapeutic efficacy of an experimental metabiotic sample, in relation to the group receiving the ursosan comparison drug, was carried out by determining such quantitative serum parameters as the titers of immunoglobulins M, G, A, E, the IFNa titer and the concentration of circulating immune complexes. Liver damage was studied by modeling acute toxic hepatitis in white rats. Thus, the studies conducted to study the humoral status of laboratory animals receiving metabolites produced by probiotic microorganisms of the genus Bacillus of the culture of probiotic microorganisms of the Bacillus subtilis B-9909 on laboratory animals when modeling toxic damage in them, give grounds to conclude that the test sample of BAS has a significant immunomodulatory effect, in comparison with ursosan. This circumstance allows us to recommend this compound as promising for the creation of a new hepatoprotector drug candidate with an immunotropic effect.

Membrane molecules PD-L1 and PD-L2, ligands of T lymphocytes PD1 receptor, perform immunoregulatory functions. Their binding to the receptor leads to inhibition of proliferation, reduction of cytokine production, cytotoxic response, and apoptosis of T lymphocytes. The cells of many tumors, regardless of their histogenesis, express PD-L1 molecules, thus limiting the development of an anti-tumor immune response. Glioblastomas are highly malignant recurrent tumors of the central nervous system. The main sources of glioblastoma recurrence are resistant tumor cells initially present in gliomas with heterogeneous cellular composition, as well as resistant cells that are formed during therapy. Increasing the dose of cytostatic drugs or radiation during relapse therapy is not effective in glioblastomas. It has been shown for a number of tumors, including ovarian cancer and non-small cell lung cancer, that drugs preventing PD-L1/PD1 interaction are effective in the treatment of neoplasms resistant to chemo- and radiotherapy. Immunotherapy using drugs that inhibit the binding of PD-L molecules to their receptor is considered as a way to overcome the resistance of glioblastomas to therapy. The aim of this work was to assess the level of PD-L1 and PD-L2 gene expression in resistant glioblastoma cells lines A172, R1, T2 and T98G, which resumed proliferation after exposure to the maximum for each line, sublethal doses of cytostatic drugs (fotemustine and temozolomide), fractionated or single gamma irradiation. A172 line belongs to glioblastomas that are highly sensitive to these influences, T98G is a highly resistant cell line, while R1 and T2 lines occupy an intermediate position. In intact glioblastoma A172, R1, and T2 cells the level of PD-L1 and PD-L2 gene expression was equally high, while in T98G cells it was significantly lower. Exposure of A172 and R1 glioblastoma lines to cytostatic drugs or irradiation did not significantly change the level of PD-L1 and PD-L2 genes expression typical for intact cells. In T2 glioblastoma cells, and especially in T98G cells, a significant increase in expression of these genes was found, most pronounced for PD-L2 gene. This increase in expression may indicate an enhanced malignancy of resistant T2 and T98G cells. High expression of the genes responsible for the production of PD-L1 and PD-L2, which limit the cytotoxic response against tumor cells, is a prerequisite for the use of drugs targeted against PD-L1 and PD-L2 for the elimination of resistant cells in glioblastoma.

One of the modern methods of treating patients with primary and recurrent brain tumors is radiosurgical irradiation using Gamma Knife, which allows therapeutic doses to be delivered to tumors not exceeding 2.5 cm in diameter in 1–2 sessions. Tumor cells on the periphery of this tissue volume that receive lower radiation doses can resume proliferation and serve as a source of recurrence. The increase of radiation dose may cause necroses formation and a worsening prognosis. The properties of glioblastoma cells that survive and resume proliferation long after stereotactic irradiation are still poorly known. The aim of the work was to evaluate the expression of IL-6 and IL-8 by glioblastoma A172, R1, T2, and T98G cell lines that resumed proliferation after sublethal Gamma Knife irradiation. Cells were irradiated once at doses ranging from 6 to 16 Gy, and then cultured for 40 days. Cell number was counted weekly; lethal and sublethal irradiation doses for each glioblastoma cell line were determined. In cultures descendant from proliferation of single most resistant cells, the level of IL-6 and IL-8 secretion after 96 hours cultivation (ng/1000 cells) was determined by ELISA. The cells of all four glioblastoma lines secreted IL-6 and IL-8 into culture medium. The highest production of cytokines, never before demonstrated for glioblastomas, was discovered in R1 cells. Glioblastoma T2 also had high interleukin production levels. In contrast to these lines, glioblastoma A172 (highly sensitive to the action of cytostatic drugs and radiation) secreted IL-6 at 30 times lower level than R1 cells. Glioblastoma T98G (highly resistant to the action of cytostatic drugs and radiation) also exhibited low interleukins production level. R1, T2, and T98G glioblastoma cells that resumed proliferation after irradiation had increased secretion of IL-6 and, to a lesser extent, IL-8. The dependence of cytokine production increase on irradiation dose for these cells was not linear. In contrast, A172 cells reduced IL-6 and IL-8 secretion under irradiation. The multidirectional changes in IL-6 and IL-8 production by cells of different glioblastoma lines were long-term and persisted for more than a month. The presented results cast doubt on the possibility to use IL-6 and IL-8 production by glioblastoma cells as potential biomarkers for early diagnosis, therapy monitoring as well as prognostic markers of the disease course.

To date, much remains unclear about the pathogenesis of asthma, one of the most common chronic and highly heterogenic diseases of the respiratory system. The lack of specific and highly effective therapy in case of certain asthma subtypes requires the search for new approaches to treatment. One possible approach would be to influence the metabolism and immune functions of myeloid cells. This approach finds its application in the treatment of cancer and other diseases in the pathogenesis of which macrophages play an important role. It was shown that the pathogenesis of allergic asthma in response to one of the most common allergens, house dust mite, is due to a metabolic TNF-mediated reprogramming of alveolar macrophages. This suggests that influencing the process of TNF production or metabolic adaptations with specific blockers may also lead to a reduction in the symptoms of the course of the disease as a whole. In this work, we experimentally tested whether the previously obtained phenotype that occurs in macrophages in response to HDM cultured in DMEM is preserved if cells are cultured under more physiologically relevant conditions: in a medium closely related in composition to blood plasma. We also analyzed open databases of alveolar macrophages sequencing obtained from patients with asthma or from the lungs of mice in an HDM-induced asthma model in order to correlate specific immunometabolic changes. It was found that macrophages cultured under conditions close to physiological, simultaneously increase the rates of respiration and glycolysis, and also produce TNF in response to HDM. The observed phenotype is consistent with transcriptomic analyzes performed on human and mouse samples, which revealed an increase in the expression of genes related to glycolysis, oxidative phosphorylation, and the TNF signaling pathway. Thus, the data confirm the relevance of the phenotype obtained in vitro to the changes occurring in the in vivo system. However, functional verification at the level of metabolites, proteins and changes in metabolic activity is also required. In addition, it remains to be established how the blocking of individual metabolic pathways affects the features of the functional macrophage phenotype that occurs in response to HDM, and whether this effect can alleviate asthma symptoms.

Daily inhaled antigens induce cellular immune response in the airways. In case of allergens, allergic airway inflammation is usually represented by eosinophils, however, neutrophil infiltration is also observed during severe asthma. Animal models contribute to investigation of the mechanisms that involve the switching to eosinophil- or neutrophil-mediated inflammation. Data about the spatial location of eosinophils and neutrophils in the airways are necessary for both the understanding of allergic airway inflammation mechanisms and the drag potential estimation, however, not completely investigated. In the present study, we characterized the model of Aspergillus fumigatus extract-induced allergic airway inflammation that allowed investigating the early stage of inflammation development. The model adequacy was confirmed according to the blood and bronchoalveolar lavage eosinophilia. Using immunohistochemical staining of conducting airway as a whole-mount and confocal laser scanning microscopy, we estimated neutrophil and eosinophil spatial location: in the luminal side of the epithelium, in the airway wall or in the submucosal compartment close to the smooth muscle layer. An allergic airway response activation was detected upon significant elevation of blood eosinophil percentage compared to intact mice. Simultaneously, the number of eosinophils in the bronchoalveolar lavage was also significantly increased compared to the intact mice. At this time point, eosinophils predominated both in bronchoalveolar lavages and in conducting airway mucosa compared to neutrophils. Spatial location of conducting airway mucosal cell analysis demonstrated that eosinophils mostly located in the submucosal compartment, in a lesser extent in the airway wall, and a few eosinophils were detected in the luminal side of the epithelium. Neutrophils mainly infiltrated the luminal side of the epithelium, and a few neutrophils were detected in the submucosal compartment, while no neutrophils were detected in the airway wall. The data suggests that in response to the further allergen challenge, evidently eosinophils but not neutrophils will migrate through the airway wall to the airway lumen. Thus, eosinophils can be expected to damage airway epithelium in allergic airway inflammation development. Simultaneously, neutrophils located in close proximity to the smooth muscle layer together with eosinophils can contribute to bronchoconstriction induction.

Neurodegenerative ophthalmopathology is one of the main causes of irreversible blindness and disability in the world. In the pathogenesis of diseases of this group, more and more attention has recently been paid to the role of local inflammation caused by the activation of innate immunity and the mechanisms of its genetic regulation. In recent years, works have appeared in the field of experimental ophthalmology that have demonstrated the possibility of NLRP1, NLRP3 inflammasome complexes assembling when exposed to hyperglycemia, oxygen deprivation of retinal cells, as well as modeling compressive stress similar to that in glaucoma [15]. However, the mechanism of inflammasome involvement in the development of neurodegenerative eye diseases remains unclear. The aim of the study was to investigate the local expression of genes encoding proteins of the NLRP3 inflammasome complex (NLRP3, CASP-1) in an experimental model of retinal degeneration in rabbits. The studies were performed on samples of tissue complex (TC) of the retina/retinal pigment epithelium (RPE) (retina/RPE TC), isolated from the eyes of 14 New Zealand albino rabbits, in which degenerative retinal lesion was modeled by a single subretinal injection of 0.01 mL of 0.9% sodium chloride solution, and 7 healthy rabbits without eye damage. The formation of retinal degeneration was judged on the basis of changes in morphofunctional parameters obtained during specialized ophthalmological research methods (optical coherence tomography, fundus autofluorescence, electroretinography) at follow-up periods of 1, 3 and 6 months. The level of expression of NLRP3 and CASP-1 genes in the retina/RPE TC was evaluated by reverse transcription polymerase chain reaction (RT-PCR). According to the results of the study, a statistically significant increase in NLRP3 gene expression (p < 0.001) was noted in the retina/RPE TC of experimental animals, which may indicate the involvement of NLRP-3 inflammasome components in the development of neurodegenerative retinal lesions. At the same time, the expression of the gene encoding CASP-1 was detected only in the retina/RPE TC of experimental eyes and is probably due to local inflammatory mechanisms in the retinal tissue.

The high level of NLRP3, CASP-1 mRNA, detected in all retina/RPE TC samples of experimental eyes at late stages of the experiment (3 and 6 months), allows us to assume the formation of mechanisms (for example, activated glial phenotype) that support inflammation in retinal tissue. This should be taken into account in actively developing transplantation methods for the treatment of retinal degeneration.

The hypothesis that atherosclerotic processes are mostly caused by immune (autoimmune) mechanisms has recently been gaining attraction. At the same time, the autoimmune hypothesis of atherogenesis has not become generally accepted and requires additional evidence. Previously, we were able to induce changes in the aortic wall similar to those observed in the early stages of human atherosclerosis, and also to produce visceral obesity in normocholesterolaemic Wistar rats by a single immunization with human native high- or low-density lipoproteins. We also found that the immune response to native human HDL causes atherosclerosis-like lesions in the rabbit aorta, such as adipocyte and chondrocyte metaplasia, proteoglycan deposits, and leukocyte infiltration. Atherosclerosis-like lesions developed in the aorta of hnHDL-immunized rabbits against a background of normal blood LDL-cholesterol level. Thus, an immune response against HDL or LDL may be an independent cause of atherogenesis. The aim of this study was to test whether immunization with human HDL apoproteins (apoA1 and apoE proteins) would induce atherosclerosis-like lesions in the aorta of normocholesterolemic Wistar rats. HDL apoproteins were isolated from human or rat plasma. Wistar rats (n = 5) aged 2 months were used for immunization with human HDL apoproteins. HDL apoproteins were administered as a single intradermal injection of 100 mg per rat in incomplete Freund’s adjuvant. Control rats were injected subcutaneously with incomplete Freund’s adjuvant (n = 5). Rats were dissected 25 weeks after immunization. Rat aorta sections were stained with hematoxylin and eosin for light microscopy. T lymphocytes infiltration was determined by immunohistochemical staining with FITC-labeled antibodies specific to rat CD3. CD3⁺T lymphocytes were detected using an Olympus BX53 fluorescent microscope. The level of antibodies to human and rat HDL apoproteins was determined by indirect enzyme-linked immunosorbent assay. Immunization with HDL apoproteins induced a T cell mediated immune response without production of autoantibodies to HDL apoproteins. The aortic intima and adventitia were infiltrated with T lymphocytes in rats immunized with HDL apoproteins. Pronounced T lymphocytic infiltration was found in all layers of the vein wall in rats immunized with human HDL apoproteins. Thus, immunization with HDL apoproteins causes T cell mediated inflammation of the aorta and venulitis.

As found in clinical and laboratory studies, platelets not only play a key role in the processes of coagulation and thrombosis, but are also able to actively participate in other pathophysiological processes, including the development of immune reactions. It has been shown that changes in the immune system leading to systemic lupus erythematosus (SLE) are often accompanied by changes in the number of platelets and their activity in the peripheral blood of SLE patients, which correlate with the severity of the clinical manifestations of the disease. Earlier we have studied the standard experimental model of SLE in detail, based on the induction of chronic graft-versus-host disease (cGVHD) in the semi-allogeneic system DBA/2 → (C57Bl/6 x DBA/2)F₁. However, the participation of platelets in this immunopathological process has not been studied. There are no data in the literature on the behavior of platelets in cGVHD or on their relationship with the state of Th1/Th2 balance. It can been expected that the platelet count changes according to the development of cGVHD in the used experimental model by analogy with the development of SLE in humans.

In the experiments, we used female mice of the DBA/2 strain and (C57Bl/6 × DBA/2)F₁ hybrids. Chronic GVHD in a semi-allogeneic system was induced by injecting DBA/2 mouse splenocytes into B6D2F₁ hybrid mice: 60-70 × 10⁶ cells intravenously twice with an interval of 6 days. The studied parameters were evaluated three months after the start of the experiment and the formation of lupus-like glomerulonephritis in animals with Th2-dependent cGVHD variant.

A decrease in the number of erythrocytes and hemoglobin, a decrease in hematocrit and a parallel increase in the number of reticulocytes in the blood of mice with cGVHD are in good agreement with our earlier conclusion that these animals have autoimmune hemolytic anemia. It was found that, platelets increase significantly with the development of cGVHD unlike other blood cells. Secondary thrombocytosis is observed in the case of the Th2-dependent variant of сGVHD in this model of SLE, while in the group with the Th1-dependent variant of сGVHD, the average number of platelets in the blood does not differ from the control group.

Since the jawbone tissue is injured at the stage of metal dental implant introduction, the problem of inflammatory complication development leading to a breakdown in osseointegration remains relevant. Of interest are the immunological mechanisms of inflammatory process development during the emission of metal nanoparticles, as well as the mechanisms of its subsidence after the removal of a metal object. Microscopic and elemental analysis of the bone tissue of the Wistar rat lower jaw after artificial traumatization was carried out. During the experiment, the situation of presence of a metal foreign body in the bone bed was simulated. An insulin needle was injected into the connective tissue of the lower jaw, followed by its removal after seven days. Microscopic analysis of bone tissue was performed using a Tesscan Vega 4 scanning electron microscope. The teeth alveoli cortical layer surface, connection of the jaws with a gap, stratification of the cartilaginous layer were determined at low magnification in direct projection. Using higher magnification in the direct projection there are visible dense-structural crystalline inclusions, foci of necrosis in the area of junctions of the jaws alveolar processes. The elemental composition of bone tissue was obtained by atomic emission spectroscopy by iCAP 6300 Duo. In the test sample, the quantitative ratio of calcium and phosphorus was 1.68, which slightly exceeds the optimal value of 1.67. An upward change in this ratio indicates a decrease in phosphorus level, which can be interpreted as local osteoporosis. In addition, the following elements were found: Bi, Ga, Pb, Ti, Zn in the amount of 0.03-0.06 mass percent. The list of these elements corresponds to the chemical composition of an insulin needle, indicating the penetration of metal particles into bone bed tissues. The emission of nanoparticles and their subsequent association to micro- and submicron sizes, their persistence, as well as biocorrosion in areas of active bone formation can be a trigger for the development of an aseptic inflammatory process. This effect is due to both a direct damaging factor and an indirect effect through specific signal molecules produced in response to tissue damage.

The pathogenesis of inflammatory bowel diseases has not been fully studied, and the therapies used have side effects that limit their use.

The purpose of this study is to conduct a clinical and immunological analysis of the effectiveness of vitamin D₃ in the original rectal suppositories in experimental colitis (EC).

EC was modeled with oxazolone. Original suppositories with vitamin D₃ in group 3 and 5-ASA in group 4 were used per rectum. The clinic was evaluated on the Disease activity index scale. The expression of MPO and TNFa, the content of neutrophils, lymphocytes, eosinophils, histiocytes, plasmocytes, fibroblasts, ulcerative defect, tissue damage index were determined in the focus of colon injury. The study was carried out on days 2, 4 and 6.

With EC, DAI increases for the entire day, MPO and TNFa increase in the lesion, ulcerative defect is

fixed, neutrophil-lymphocytic infiltration increases, and TDI increases. When comparing the morphometric parameters of the alteration zone in EC under the conditions of vitamin D₃ use, in contrast to the use of 5-ASA, a decrease in the number of lymphocytes, an increase in fibroblasts was revealed on day 2, a decrease in the number of plasmocytes and an increase in fibroblasts on day 4, an increase in the number of histiocytes and fibroblasts on day 6. The diameter of the ulcerative defect and the TDI index have no significant differences between the compared groups. When comparing the effectiveness of vitamin D₃, in contrast to the use of 5-ASA, the MPO content is higher on day 6; the TNFa content is higher on day 4.

In EC, the effects of using rectal suppositories with vitamin D₃ on clinical signs, the size of the ulcerative defect, the content of MPO and TNFa in the lesion are comparable to the effects of using rectal suppositories with 50 mg of 5-ASA; more pronounced with respect to the dynamics of the cellular composition of the lesion of the colon.

An important role in restoration of damaged organs and tissues is played by mesenchymal stem cells (MSCs) and microvesicular particles (MV) produced by them. They can be a source of cytokines, anti- apoptotic and growth stimulating factors. In addition, MVs carry out transport of mRNA, miRNA, and signal proteins into damaged tissues. This increases the ability of cells to regenerate and to inhibit apoptosis, promote to angiogenesis and stimulate cell proliferation. The aim of our research was to study the immunoregulatory and pro-regenerative properties of mesenchymal stem cell microvesicles (MSC-MV) in a model of glycerol- induced acute renal failure (ARF) in mice. The experiments were carried out on CBA mice aged 3-4 months. AKI was induced by a single intramuscular injection of 50% glycerol. MSCs were obtained from the bone marrow of healthy animals and cultivated under standard conditions. Microvesicles were obtained by centrifugation at 12000g of MSC supernatant after induction of their apoptosis by culturing under oxygen deprivation conditions and in serum-free medium. MSC-MV was injected intravenously into the retroorbital sinus one day after induction of ARF. The MV dose was calculated as equivalent to (derived from) 1 million MSCs, which was 100 mL per mouse. Animals were taken out of the experiment on days 4 and 11 after MSC-MV injection. Blood plasma was taken to determine the level of creatinine, urine – for albumin analysis, kidneys – for histological examination. It has been shown that MVs induced by MSCs dose-dependently stimulated splenocyte proliferation in both spontaneous and Con-A induced tests. The addition of MV caused a decrease in doxorubicin-induced apoptosis of splenic lymphocytes in mice. Probably, in this case, MV produced by MSCs had an immunostimulatory and antiapoptotic effect. Also, MVs had a positive impact on the restoration of structure and function kidneys in a model of ARF in mice. The use of MSC-MV in treatment of acute renal failure induced by a single injection of 50% glycerol contributed to decrease albumin level urine and restoration of creatinine level in blood serum of animals. Morphological studies have shown decrease in the height cell and collecting duct diameter in the medulla and a decrease in the largest transverse diameter of superficial glomeruli in the renal cortex of sick mice. Thus, the obtained results indicate significant therapeutic and pro-regenerative properties of MSC-MV, which require further study.

The vaccine strain F. tularensis 15 NIIEG induces long-lived cell-mediated immunity but exhibits a certain reactogenicity and genetic instability. Progress in development of a vaccine against tularemia has been limited by a lack of information regarding the mechanisms required to protect against this disease. The BALB/c mouse is the most commonly used animal to study tularemia due to its relatively low cost, well-characterized genetics, available immunological tools and mouse infection with virulent F. tularensis recapitulates human disease.

CD4⁺ and CD8⁺T cells are known to be critical for the formation of protective immunity but the relative roles of memory T cell subpopulations in long lived protection against virulent strains of F. tularensis are not well established. We hypothesized that this immunity depends on central (T_CM) and effector memory (T_EM) T cells and their functional activity. In this study we have dissected the T cell immune response in BALB/c mice 30, 60 and 90 days after subcutaneous vaccination with 15 NIIEG.

Multiparametric flow cytometry were used to characterize in vitro recall responses of splenocytes to F. tularensis antigen. T_EM cells were identified as CD3⁺CD4⁺CD44⁺CD62L^- and CD3⁺CD8⁺CD44⁺CD62L^-, T_CM cells as CD3⁺CD4⁺CD44⁺CD62L⁺ and CD3⁺CD8⁺CD44⁺CD62L⁺, respectively. The functional activity of memory T cells was assessed by the following parameters: the level of expression of the activation marker CD69 and cytokine-producing activity by staining with the intracellular cytokines IFNg and TNFa.

Thus, development of a long-lived vaccine directed against F. tularensis is dependent on identifying not only the correlates of immunity present early after vaccination, but also those that persist in the host after the effector phase has ended. The maintenance of long-term protective immunity initiated by vaccination with F. tularensis strain 15 NIIEG has been shown to require the presence of antigen-specific CD4⁺ and CD8⁺ memory T cells producing IFNg and TNFa and expressing the activation marker CD69. A decrease in count and functional activity of CD8⁺T_CM and CD8⁺T_EM was detected in the long term after vaccination. The detected parameters of functional activity of memory T cells can be used as criteria for evaluation of protective immunity against virulent strains of F. tularensis.

The relevance of the search for new vaccine adjuvants is growing along with the increase in the number of current vaccine preparations, especially those developed on the basis of proteins. Some cytokines are known to exert adjuvant properties. The present work is devoted to the study of adjuvant activity of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) and constructs based on it. Earlier, we developed a technology for isolation and purification of GM-CSF from the E. coli SG20050/p280_2GM producer strain, as well as a technology for conjugating polyglucin:spermidine complexes with rhGM-CSF. Double-stranded RNA was used to obtain molecular constructs on the basis of rhGM-CSF conjugate. To assemble constructs, the ratio of the components was calculated for one dose of the preparation to contain 5-40 mg of rhGM-CSF and 100 mg of double-stranded RNA. The effectiveness of the formation of molecular constructs was evaluated by dsRNA electrophoretic mobility shift in a 1% agarose gel. The effectiveness of the resulting adjuvants was determined in ELISA assays by measuring the titers of specific antibodies in mouse sera against ovalbumin or recombinant receptor-binding domain of the surface S protein of the severe acute respiratory syndrome coronavirus 2 (Delta variant (B.1.617.2). The experiments were carried out in 100 male BALB/c mice weighing 16-18 g. Mice were immunized twice, with a 14-day interval, by intramuscular injection of 200 mL per animal. Recombinant receptor-binding domain of the surface protein of SARS-CoV-2 was administered at a dose of 50 mg/animal, ovalbumin – at two doses – 1 mg or 5 mg/animal. Corresponding antigen was used as a positive control, a saline solution – as a negative control. It was shown that the maximum effect was achieved by immunization with a construct based on double-stranded RNA and rhGM-CSF conjugated to polyglucin-spermidine. The use of a conjugate without double-stranded RNA as an adjuvant also improved humoral response. The use of native rhGM-CSF did not increase the titers of specific antibodies. Thus, it was found that rhGM-CSF being a part of a polysaccharide conjugate or a molecular construct exerted an ability to enhance the humoral immune response to protein antigens.

The use of modern subunit vaccines involves adjuvant introduction into their composition. Currently, the search for new and improvement of existing adjuvant systems is actively underway. Squalene- based adjuvants are well-known and approved in a number of countries for clinical use in influenza vaccines. Our study was devoted to the development of an adjuvant composition on the basis of squalene. The resulting adjuvants were composed in a form of oil emulsion containing a hydrophilic and hydrophobic phase. The stability of the emulsion was achieved by treating it with ultrasound at a frequency of 22 kHz. Particle sizes of the obtained emulsions were examined with the use of an electron microscope. The particle size was calculated to be 50-80 nm for the majority of particles (84%). Adjuvant activity was evaluated in 100 male Balb/C mice, weighing 16-18 g. To assess the humoral immune response, immunization was performed twice, with a 14-day interval, by intramuscular injection of 200 mL per animal. The receptor-binding domain (RBD) of the surface S protein of the severe acute respiratory syndrome coronavirus 2 (Delta variant (B.1.617.2)) or ovalbumin (OVA) from chicken eggs were used as antigens. RBD was administered at a dose of 50 mg/animal; OVA was administered at two doses (1 mg or 5 mg/animal). An antigen with aluminum hydroxide was used as a positive control; a saline solution was used as a negative control. The effectiveness of the obtained adjuvants was determined by measuring the titers of specific antibodies in mouse sera in ELISA assays using the recombinant RBD of SARS-CoV-2 S-protein or ovalbumin from chicken eggs. It was shown that the use of squalene-based adjuvants increased the antigens’ immunogenicity. The average titers of specific antibodies against RBD in the experimental group were 4 times higher than in the group immunized with RBD adjuvanted with aluminum hydroxide. An increase in immunogenicity of the antigen adjuvanted with squalene was also observed in the experimental OVA-group. Thus, it was shown that the developed squalene-based adjuvant compositions could be an alternative to the traditional adjuvants based on aluminum salts.

Modern medicine allows us to study and develop materials and methods of restorative treatment that would be based on the immunological mechanisms of bone repair. One of the promising directions in guided bone regeneration is the use of mesenchymal stem cells. Interest in MSCs is associated with their ability to regulate the inflammatory process, and directly participate in the formation of new bone structures, thereby providing a physiological repair process. The effector impact of MSCs on the inflammatory process due to their ability to form a specific microenvironment. Low expression of MHC-II and CD80/CD86, the production of PGE2 and NO determines their low immunoconflict, and the production of TGF-b1, IDO and IL-10 has an immunomodulating effect. The ability of MSCs to differentiate into an osteogenic phenotype is accompanied with the synthesis of ALP, BSP and, subsequently, Gla-protein and OPN determine the synthesis of the extracellular matrix and its subsequent mineralization. This process is provided by the action of Runx2, which activates the differentiation of MSCs along the osteogenic pathway. These effects of MSCs were taken as the basis for the development of a new method for the treatment of bone atrophy. To accomplish the task set, a model of bone tissue atrophy and a drug containing MSCs was developed, and an experimental study was conducted to evaluate the effectiveness of the developed methodology. As the main criteria, data from clinical and laboratory studies were taken. Visual changes in the studied area were taken into account, compared with a similar area in the developed model of atrophy, the parameters of the complete blood count (CBC) were evaluated. The performed study allows us to determine the developed treatment method as capable of fully recreating the conditions of bone repair processes, taking into account the optimization of the body’s immune reactions and repair processes, without additional external influence, to obtain predictable and controllable results.

Tumors are a leading pathology in the population. Chemotherapy cannot provide adequately and effectively to cure patients. Some medicine, such as cytostatic, are characterized by a wide range of side effects and resistance of solid tumors to chemotherapy by these medicines. In recent research, the mechanisms of action of cytotoxic agents have been described, and the most appropriate causes of resistance have been suggested. Drug delivery system based on Cucurbit[7]uril (CB[7]) was used to minimize side effects and overcome resistance. CB[7] has ability to form host-guest supramolecular complexes with oxaliplatin and carboplatin.

It is important to consider the immune system maintain to a great role, and platinum compounds are able to have an immunomodulatory effect on immunocompetent cells. There is convincing evidence about the cytotoxic response against tumor cells is also associated with immunomodulating properties. A specific immune microenvironment with high frequency of suppressor cells is made by tumors. FoxP3⁺ regulatory T cells are recruited by the tumor, an increased number of these cells and expression levels of CTLA-4 and PD-1 on them contribute to the progression of the tumor process. These markers correlate with recurrence and poor survival of the patients. Therefore, it is necessary that antitumor therapy agents have an effect on a subpopulation of regulatory T cells and their functional activity. This study evaluated the effects of cucurbit[7] uril, platinum compounds, and supramolecular complexes on FoxP3⁺ regulatory T cells and the expression of immune checkpoint molecules.

In this study peripheral blood cells from volunteers (n = 8, average 29.0±2.4) were used. Mononuclear cells obtained in the standard protocol were incubated for 72 h at concentrations of 0.3 and 0.1 mM for carboplatin and oxaliplatin, respectively, as well as complexes and CB[7] in equivalent dosages. Next, the samples were labeled with monoclonal antibodies to determine the phenotype and expression of immune checkpoint molecules by flow cytometry.

We obtained the following results: The CB[7]-carboplatin complex in stimulated and non-stimulated cultures significantly reduced the number of FoxP3⁺ regulatory T cells compared to the control. At the same time, carboplatin and the CB[7]-carboplatin complex reduced the expression of CTLA-4 in an non-stimulated culture compared to CB[7].

Complexes of Cucurbit[7]urils with platinum compounds are a perspective antitumor agent with immunomodulatory properties.

The article presents the results of a study of the effect of anthocyanins on cellular immunity in rats on a model of alimentary obesity. The aim of the study was to study the effect of an anthocyanin- enriched diet on cellular immunity in diet induced obesity in rats. The study was carried out on male Wistar rats with an initial body weight of 108±2 g. The animals were randomized by body weight into 3 groups (8 pcs. in group). For 12 weeks, rats of the 1^st (control) group received a complete modified diet of AIN93M; rats of the 2^nd group consumed a high-calorie choline-deficient diet (HCChDD), the fat content of which was 45%, fructose – 20% of the energy value of the diet; rats of the 3^rd group received HCChDD with the addition of standardized blueberry and blackcurrant extract (30% anthocyanins) at an average daily dose of 11 mg anthocyanins/kg body weight. The expression of differentiation markers of peripheral blood lymphocytes was carried out by flow cytofluorimetry. As a result of the study, it was found that in rats of the 2^nd group with alimentary obesity, the relative content in the peripheral blood of T helpers (CD3⁺CD4⁺) was increased (p < 0.05) (75.75±1.11% versus 70.07±0 49% – group 1, 72.14±0.91% – group 3) and reduced (p < 0.05) content of T cytotoxic lymphocytes (CD3⁺CD8⁺) (22.54±1.14% versus 28.09±0.72% – 1^st group, 26.07±0.87% – 3^rd group). The CD3/CD4 ratio in rats of the 2^nd group exceeded (p < 0.05) this index in rats of the 1^st and 3^rd groups (3.44±0.25 versus 2.47±0.09 – 1^st group, 2.79±0.13 – 3^rd group). Enrichment of the HCChDD with the blueberry and blackcurrant extract led to the normalization of these parameters of cellular immunity. The number of B lymphocytes (CD45R⁺), Т lymphocytes (CD3⁺) and NK cells (CD161⁺) in the rat peripheral blood of all experimental groups had no statistically significant differences. The results of the study of cellular immunity in rats with alimentary obesity indicate the presence of metainflammation. The received data indicate the prospect of using biologically active substances.

Treatment of consequences of traumatic brain injury (TBI) remains one of the current problems of medicine. To increase the effectiveness of treatment of post-traumatic complications, various drugs are recommended, including the peptide with neuromodulatory activity Semax.

The present study aims to determine the presence of neuro- and immunoprotective properties of the synthetic peptide PR5, composed of fragments of proline-rich antimicrobial peptides.

The work was performed on male Wistar rats weighing 300-350 g. The “falling weight” model of mechanical brain injury was used, which mainly causes diffuse brain damage. The synthesized peptide PR5, composed of fragments of known proline-rich peptides of animal neutrophils, and the peptide preparation Semax in the form of a 1% aqueous solution were used. The drugs were administered intranasally 1 hour after TBI, then twice a day for 4 days at a dose of 100 mg/kg body weight. Control animals received physiological saline in the same regimen as the peptide preparations.

TBI led to a significant decrease in body weight, but in rats receiving the peptide preparation Semax, the decrease in body weight was significantly less than in control animals, and the PR5 preparation completely prevented the decrease in body weight after TBI. After TBI, the proliferative activity of lymphocytes was suppressed and the cytotoxicity of NK cells decreased. In animals treated with peptide preparations, there was no significant suppression of NK cell cytotoxicity, and the proliferative activity of lymphocytes was restored to the level of control animals by day 14 after TBI. Both peptide preparations used contributed to higher locomotor activity, and in animals treated with the PR5 peptide, this type of activity reached the parameters of control animals. The reduction in freezing duration in groups treated with peptide preparations indicates the presence of a sedative effect.

The peptide preparation PR5 was active in this series of experiments, showing immunotropic and neuroprotective activity comparable to the Semax preparation. Further studies aimed at confirming the identified types of activity of the peptide preparation PR5 may justify its prospects for clinical use as a new nootropic agent.

The prevalence of thermal trauma, the high risk of infectious and non-infectious short- and long- term complications, and the limited effectiveness of the therapeutic approaches used are prerequisites for the search and pathogenetic justification of new therapies, among which the endogenous homeostasis regulator with pleiotropic properties melatonin attracts attention.

The aim of the work is to investigate the immunological aspects of intraperitoneal use of melatonin (MT) in experimental thermal trauma (TT).

The work was performed on 158 rats of the Wistar line, grade III TT and a relative area of 3.5% were simulated by skin immersion in water at 98-99 °C for 12 s. MT was administered intraperitoneally daily at a dose of 10 mg/kg for 5 days. The quantitative composition of blood cells was evaluated on a hematological analyzer. Plasma concentrations of IL-4, TNFa, IFNg, and CRP were determined on an automatic enzyme immunoassay using rat-specific test systems, and MT by capillary electrophoresis.

With experimental TT, against the background of a progressive increase in the number of leukocytes in the blood from 5 to 20 days due to neutrophils, monocytes, basophils, the number of lymphocytes decreases. With TT, the concentration of CRP increases in serum on days 5 and 10. The content of TNFa, IL-4 increases on days 5, 10 and 20 in the absence of significant changes in the concentration of IFNg. The concentration of serum MT does not change significantly. Intraperitoneal use of MT in TT leads to a partial restoration of the number of lymphocytes in the blood on day 5. Evaluation of the cytokine profile in serum revealed a decrease in the concentration of TNFa on days 10 and 20, no significant changes in the concentration of IL-4 and IFNg were recorded, the concentration of CRP decreased on day 5. The concentration of serum MT increases by 5 days.

With TT on the 5^th, 10^th, 20^th day of the experiment, the number of neutrophils, monocytes, basophils in the blood increases, decreases – lymphocytes, the serum content of CRP, TNFa, IL-4 increases, the content of IFNg and melatonin does not change. Intraperitoneal use of MT in TT partially restores the number of lymphocytes in the blood, the concentration of CRP, TNFa. A decrease in serum concentrations of TNFa and CRP in TT under the conditions of MT use suggests a limitation of the acute phase response as a consequence of the antioxidant, anti-inflammatory effect of MT, which can accelerate healing and reduce the area of the lesion of TT.