This review presents current data on the origin of monocytes/macrophages, the conditions necessary for the differentiation of monocytes into M1 or M2 macrophages. Three subpopulations of peripheral blood monocytes are described: (I) classical – the main subpopulation (85-90%), effectively carrying out phagocytosis; (II) intermediate monocytes (5-10%) – participate in antigen processing and presentation, in angiogenesis, vascular endothelium restoration; (III) non-classical monocytes (10%) - "patrol" vascular network, remove cellular debris, participate in tissue remodeling. The review provides detailed characteristics for each subclass of macrophages: pro-inflammatory (M1) and anti-inflammatory (M2), which play different roles in the initiation and resolution of inflammation; their phenotype, the spectrum of secreted cytokines, the expression of transcription factors, and the functions performed are described. For the M2 population, the features of the subpopulation are described in detail: M2a, M2b, M2c, M2d. The review presents methods and approaches to obtaining polarized macrophages in vitro from both peripheral blood monocytes and cells of transplanted cultures based on signals received by macrophages in vivo; the phenotype, cytokine production and functional properties of artificially polarized macrophages depending on the conditions of their production are given. The review examines in detail the features of contact and distant interaction of macrophages of various subclasses with microenvironment cells on the example of natural killer cells and trophoblast cells, provides information on changes in the phenotype, transcriptional and secretory profile of interacting cells. The mechanisms of trophoblast control of macrophage differentiation into a unique M2 population of decidual macrophages controlling both the development and functioning of the trophoblast and its apoptosis are described. The review examines in detail the currently known variants of the interaction of macrophage subpopulations with natural killers. The influence of Mf on NK cells manifests itself in a change in the expression of transcription factors by the latter, which determine not only their differentiation, but also their functional activity. Macrophages are considered as cells that actively influence the functional state and differentiation of natural killers. The review examines the mechanisms of the relationship of all three types of cells: macrophages, trophoblast and natural killers in the area of uteroplacental contact. The study of the interactions of these cells will shed light not only on the features of intercellular relationships in the area of uteroplacental contact, but also on the relationship of tumor cells with NK cells and macrophages.

In addition to the subsets of T lymphocytes and innate lymphocytes (innate lymphoid cells), the well-known players in adaptive immunity, there is an intermediate group of lymphocytes (innate-like cells) that already possess the T cell receptor, but with a restricted repertoire. This group includes γδT cells, subsets of type I and II NKT cells carrying both T cell receptor and NK-cell receptors, and mucosal-associated invariant T (MAIT) cells. The development of innate-like cells occurs in the thymus, but their positive and negative selection takes place without the participation of thymic epithelial cells. A distinctive feature is that innate-like cells acquire an effector phenotype already in the thymus, and therefore do not require complex activation reactions during antigen recognition. Upon exit from the thymus, noncanonical T cells express chemokine receptors, allowing them to migrate into barrier tissues at an early age. A characteristic feature of the T cell receptor innate-like cells is the recognition of non-peptide antigens presented in non-polymorphic histocompatibility molecules (MHC-Ib). This type of molecule includes the CD1 a/b/c/d/e molecule and the MR1 molecule. These molecules present lipid, glycolipid antigens and metabolites of B vitamins, synthesized by various representatives of the microbiota. The presence of functionally different subpopulations of innate-like cells with an activated phenotype allows them to quickly respond to the antigen by producing cytokines typical of Th1, Th2, Th17. They also exhibit cytotoxic and immunoregulatory activity. These cells are actively involved in regulation of barrier tissue homeostasis and interaction with microbiota. They synthesize growth factors for epithelial cells, fibroblasts, and vascular endothelium, which are required for regeneration of damaged tissues. They also participate in anti-infectious defense, directing the development of the immune response. Moreover, they have been found to be involved in many autoimmune diseases. The special functions of innate-like cells make them a promising target for therapeutic interventions. It has been shown that antibiotics, salicylates and some other well-known drugs exert certain effects on the innate-like cells. Different dietary options also affect the activity of these cells.

Proinflammatory extracellular and intracellular DAMPs are the dominant etiological factors of sterile inflammation in immuno-inflammatory rheumatic diseases. They are generated by systemic progressive disorganization of loose fibrous unformed connective tissue, programmed cell death and cell necrosis. Sterile inflammation is a multi-stage process which is induced by a sequence of reactions mediated by leukocytes and resident cells of the macrophage-monocyte series, aimed at cleansing the focus of inflammation from cellular and tissue detritus, followed by restoration of homeostasis of damaged tissue. An important role in this process belongs to the transendothelial migration of leukocytes to the focus of sterile inflammation and formation of cellular inflammatory infiltrate. The key feature of these events is the reactivity of PRR receptors followed by a cascade of PRR-DAMPs interactions with subsequent launch of molecular and cellular processes causing the local and/or systemic manifestations of sterile inflammation. Activation of innate immunity is the result of PRR-DAMPs interactions which launches the molecular and cellular reactions. Hence, it is possible to attribute the immunoinflammatory rheumatic diseases to the category of systemic sterile autoinflammatory processes. Generalization of the pathophysiological effects of pro-inflammatory DAMPs and, accordingly, the systemic and multi-organ nature of tissue and internal organ damage in immunoinflammatory rheumatic diseases is due to the wide occurrence of receptors for “danger signals”. The most important place in the development of DAMP-mediated sterile inflammation is occupied by the phenomenon of cross-presentation and autophagy. The cross-presentation causes exposition of extracellular DAMPs from internalized proteins with MHC class I molecules to autoreactive CD8⁺ cytotoxic T lymphocytes. Autophagy provides processsing of intracellular peptide DAMPs, their loading onto MHC class II molecules with subsequent induction of adaptive immune response in CD4⁺T cell populations. The innate lymphoid cells (ILC) make an important contribution to these processes. The model of functional coupling and complementarity between ILCs and Th-CD4⁺T cells has expanded our understanding of immune regulation by extending the activity of innate and adaptive immunity to the level of maintaining tissue homeostasis, morphogenesis, repair, regeneration and inflammation. Progression of systemic sterile inflammation may be a result of PRR-DAMP interactions of tissue ILCs followed by switching of ILC/Th-CD4⁺T cell partners. The data presented in this review define the promising molecular and cellular targets aiming for regulation and/or inhibition of sterile inflammation in immunoinflammatory rheumatic diseases.

Arginine metabolism plays an important role in regulating the functions of immune cells in mammals. Pathogenic microbes use the mechanism of arginine depletion to suppress the immune response during infection. Arginine deiminase is a microbial arginine-hydrolyzing enzyme important for survival at low pH in the focus of infection, or in phagolysosomes, as well as under low-glucose conditions. The effect of bacterial arginine deiminase on the functions of adaptive immune cells remains poorly understood. The aim of our study was to evaluate the effect of streptococcal arginine deiminase on the proliferation and autophagy of CD4⁺ and CD8⁺ human peripheral blood T lymphocytes.

The enzyme effects were studied with supernates of ultrasonic lysates from parental Streptococcus pyogenes M49-16, and its isogenic mutant with inactivated arcA gene (Streptococcus pyogenes M49-16delarcA). The study was performed with blood samples of healthy donors. The fraction of mononuclear leukocytes was isolated by centrifugation in a Ficoll density gradient. To evaluate proliferation levels, a method based on the staining of intracellular proteins with vital fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) was used. The level of autophagy was studied using the fluorescent Lysotracker Green DND-26 dye. To analyze the proliferation and autophagy of T helper cells (CD3⁺CD4⁺) and cytotoxic T lymphocytes (CD3⁺CD4^-), the obtained cell suspensions were stained with antibodies against CD4, CD45RA, and CD3. The proportion of necrotic cells was determined by staining with a fluorescent DNA-binding DAPI dye. The normality of the distribution was assessed by Shapiro–Wilk test. The data were analyzed using Kruskal–Wallis criterion, followed by Mann–Whitney criterion for pairwise comparisons and expressed as median and interquartile ranges (Q_0.25-Q_0.75).

When comparing the effects of supernatants from the parental and mutant streptococcal strains, which differed in expression of arginine deiminase gene, we have shown that the bacterial enzyme had no effect on the functions of inactive lymphocytes. However, streptococcal arginine deiminase completely suppressed proliferation of CD4⁺ and CD8⁺T lymphocytes stimulated with anti-CD2/CD3/CD28 antibodies. These effects were accompanied by a decrease in the autophagy levels. At the same time, arginine deiminase did not exert cytotoxic effects upon lymphocytes. L-arginine applied at the doses exceeding physiological levels caused restoration of the cellular functions. There were no differences between the studied parameters of CD4⁺ and CD8⁺T lymphocyte subsets.

The obtained data show that the antiproliferative effect of arginine demimnase may be associated with ability of the enzyme to inhibit autophagy and confirm an opportunity of the bacterial enzyme to suppress host adaptive immune responses.

Single reports were published concerning a minor subpopulation of NK cells with weak coexpression of the B cell antigen CD19 in the patients’ blood and bone marrow. The frequency and relative number of CD56⁺CD19^+dim cells is virtually not assessed, and there is no data on their phenotypic characteristics, as well as the connection of this subpopulation with any disease state. The purpose of the present study was to assess the frequency, relative quantity and phenotypic characteristics of CD56⁺CD19^+dim lymphocytes in blood of patients referred for assessment of the lymphocyte subpopulation profile. Peripheral blood of immunocompromised individuals was taken, and subpopulation composition of lymphocytes was determined using eight-color flow cytometry (markers: CD3, CD4, CD8, CD19, CD25, CD45, CD56, HLA-DR). To estimate incidence of the CD56⁺CD19^+dim subpopulation, we have carried out a retrospective analysis of LMD files on 1210 studies for 935 patients (average age, 39.8±14.7 years old) including 84 children under 18 years old. The study was performed repeatedly for some patients. Phenotyping of CD56⁺CD19^+dim cells was performed using a panel of antibodies to B cell, T/NK cell antigens. The occurrence of blood samples containing CD56⁺CD19^+dim was 1.2%, with a relative content of 2.1±1.9% among total lymphocyte population (0.8±0.6% of leukocytes). Long-term persistence of the subpopulation was noted in the patients throughout the entire observation period. The comparison of specific marker expression by NK CD56⁺CD19^+dim, and CD56⁺CD19^- cells revealed high expression of CD2, CD57, reduced expression density of CD7, CD16, CD38. The phenotype of the studied NK cell subpopulation was as follows: CD56^+dimCD19^+dimCD2^+brightCD7^+dimCD11c⁺CD16^+dimCD38^+dimCD45RA⁺CD57⁺CD94^+dimNKG2D⁺CD3^-CD4^-CD5^-CD20^-CD21^-CD25^-CD45R0^-CD62L^-CD79b^-CD117^-, with variable expression of CD8 and HLA-DR. The phenotype is consistent with activated terminally differentiated adaptive NK associated with cytomegalovirus infection. The individuals with CD56⁺CD19^+dim had a history of CMV-infection and reactivation of chronic EBV-infection at the time of the study. A probable cause of CD19 coexpression may be trogocytosis of B cell membrane fragments by natural killer cells during active EBV-infection. CD56⁺CD19^+dim lymphocytes can reach noticeable values thus altering the results of studies performed by flow cytometry. The errors are most likely to occur upon assessing the minimal residual disease levels in acute B cell leukemias. The minor CD56⁺CD19^+dimNK subpopulation may be detected in routine immunological analysis. Its functional features and association with certain disorders require further studies.

Atopic asthma is a chronic disease characterized by airway obstruction, bronchial hyperresponsiveness, and inflammation. Patients show increased activation of immune cells in the airways, especially T-lymphocytes, leading to chronic inflammation. The lymphocytes of asthma patients are known to have an impairment of the type 1 and 2 programmed cell death, i.e., apoptosis and autophagy, thus contributing to prolongation and intensification of inflammatory process. As compared to apoptosis, autophagy may also contribute to cell survival under stress conditions. Its disruption and hyperactivation leads to exacerbation of allergic responses. Glucocorticoids are the main drugs for the treatment of atopic bronchial asthma by activating the glucocorticoid receptor, thus triggering anti-inflammatory response and apoptosis of the cells. However, some patients exhibit resistance to therapy due to various factors, including single nucleotide polymorphisms of NR3C1 glucocorticoid receptor gene. The highest association between asthma severity and resistance to therapy was found for the GG variant of the NR3C1 Bcl1 polymorphism. Common molecular pathways for glucocorticoid receptor activation and programmed cell death and mediating molecules suggest a significant role for the polymorphic receptor variant in cell death. The aim of our study was to evaluate the effect of a single nucleotide polymorphism (G allele, i.e., Bcl1 polymorphism of NR3C1 gene) of glucocorticoid receptor on expression levels of genes that regulate apoptosis (BCL2, CASP3) and autophagy (BECN1, LC3) in lymphocytes of patients with moderate and severe atopic bronchial asthma. The study was performed with peripheral blood samples of 24 patients aged 20 to 45 years with an established diagnosis of moderate to severe atopic bronchial asthma. Using PCR technique with restriction fragment length polymorphism (RFLP) assay, the patients were distributed according to the genotypes of the BclI polymorphism of the NR3C1 gene: 12 patients with CC genotype, 8 persons with GC genotype, and 4 cases with GG genotype. The lymphocytes were isolated in Ficoll density gradient and cultivated with dexamethasone under the conditions of nutrient depletion. The level of gene expression was determined by real-time PCR. When studying associations between various genotypes of Bcl1 polymorphism and expression of cell death marker genes, the anti-apoptotic reactions were detected in lymphocytes of patients with GG polymorphism under the influence of dexamethasone thus being a potential mechanism for development of resistance to glucocorticosteroid therapy in asthma. Impaired activation of BECN1 gene expression in patients with the GG genotype may suggest deregulation of the autophagy in this group of patients, as a mode of programmed cell death. Moreover, in patients with GC genotype during long-term cultivation, exposure to dexamethasone increases the expression of the LC3 gene, indicating a more pronounced activation of autophagy. Hence, this work demonstrates differences in response of lymphocytes to synthetic glucocorticoid therapy, and probable effect of G allele (Bcl1 polymorphism) on dysregulation of programmed cell death under the influence of dexamethasone.

The issues of recurrencies in chronic rhinosinusitis with nasal polyps (CRSwNP) still remain unresolved. Therefore, detection patients with uncontrolled clinical course of CRSwNP is required.

The aim of the present study was to assess cytokine profile in nasal polyps as well as clinical characteristics of patients with CRSwNP at varying levels of therapeutic control.

The study included 99 patients with chronic rhinosinusitis. The contents of interferon IFNγ, interleukin IL- 1β, IL-4, IL-5, IL-13, tumor necrosis factor TNFα, transforming growth factors TGF-β1, TGF-β2, TGF-β3 were measured in polyp tissue samples by means of multiplex analysis. The patients were treated according to a stepwise СRSwNP therapeutic algorithm [9]. Following observation for 5 years, all patients were divided into groups, as based on medical control degree. Group 1 included patients with mild CRSwNP, who mainly received stage I and II treatment for the entire observation period. Group 2 was presented by moderate- severity CRSwNP, with stage II or III therapy according to the referred algorithm. Group 3 included the patients with severe CRSwNP who received one or more stage IV courses. The patients underwent repeated SNOT-22 questionnaire, endoscopic examination, clinical assessment. In case of bronchial asthma (BA), ACQ-7 was repeated. Initial cytokine profile of nasal polyps was analyzed by the mentioned clinical groups.

Results:

1. After 5 years of observations in group 1 (mild CRSwNP), we found a minimal decrease in quality of life (SNOT-22), severity of nasal congestion, or smell impairment. Bronchial asthma duration in these patients was significantly lower compared to group 3, the patients had better asthma control level. Cytokine profile of nasal polyps was characterized by the highest IL-4 concentration, average values IL-1β, TNFα, IFNγ and minimal TGF-β1 values.
2. In group 2 (moderate CRSwNP), we noted more pronouced impairment of smell, nasal congestion and quality of life. Bronchial asthma was less controlled than in group 1. The maximal concentrations of IFNγ, IL-1β, TNFα, IL-5, TGF-β1, TGF-β2 were registered.
3. In group 3 with poor CRSwNP control, the highest SNOT-22 scores, severity of difficulty in nasal breathing, impaired sense of smell were revealed. Duration of bronchial asthma was longer, with lowest levels of medical control. In nasal polyps, minimal levels of IFNγ, IL-1β, TNFα, IL-4, IL-5, TGF-β2, TGF-β3 were noted.
4. Treatment of patients depending on the clinical phenotypes of CRSwNP in the presence/absence of allergic rhinitis or bronchial asthma may improve control and reduce incidence of relapses in CRSwNP.

Pathophysiology of severe COVID-19 is characterized by changes in the number, phenotype, and function of neutrophil granulocytes (NG). Among the effector antiviral mechanisms of NG, the neutrophil extracellular traps (NETs) are among the most important features. However, their excessive formation exacerbates inflammation in acute respiratory distress syndrome and contributes to microvascular thrombosis. Their detection and counting may be important in severity grading of COVID-19, for determining correlations with clinical outcome, assessing the risk of developing post-COVID syndrome, and, possibly, for monitoring future targeted therapy. Purpose of our study was to develop a new diagnostic integrative criterion to assess the severity of COVID-19 and the risk of complications in the post-COVID period, including post-COVID signs in peripheral blood. Peripheral blood (PB) samples were studied from 31 patients with acute COVID-19 of moderate (n = 15) and severe degrees (n = 16). Moreover, we observed 52 patients discharged from the hospital after severe COVID-19, with diagnosed post-COVID syndrome (PCS) over the period of 30 to 60 days, and 100 healthy volunteers. The parameters of routine blood counts (MicroCC-20Plus) were evaluated, the leukocyte formula was calculated in PC smears, taking into account the number of formed NETs, and NGs entering pathological apoptosis. Based on the obtained results, an integral diagnostic criterion was calculated using the formula:

$$ IDK = \frac{\%\ unchanged\ NG}{\%NET + \%NG\ in\ apoptosis} $$

A 8.5-fold decrease in IDK index (p < 0.05) was shown in the cases of moderate-severity course of the disease, and a 30-fold drop was seen in severe cases (p < 0.05) compared with appropriate values in the group of healthy individuals. It was also found that, in 88.5% of patients with PCS after the SARS-CoV-2 infection, no morphologically altered NG were detectable in PB samples. At the same time, in 11.5% of patients with PCS, we found NETs and cells with pathological apoptosis, whereas IDC of NG-PCS was 8 times less than in the comparison group, and did not differ from the parameters of patients with moderate COVID-19 (p > 0.05) thus requiring further dispensary observation of such patients. The data obtained in this study indicate that the developed integrative diagnostic criterion allows us to assess both the severity of COVID-19 over acute period, and the risk of post-COVID syndrome. It should be emphasized that the characteristic changes in NG detected in COVID-19 may be readily identified in PB and consistently monitored by the proposed integral diagnostic criterion. A significant decrease in IDC indicates the persisting hyper-activation of NG and a need for targeted immunotherapy aimed at modulating the NG dysfunction.

In cases of respiratory viral infection, along with innate immunity mechanisms, the adaptive immune system plays a crucial role in the body’s defence. The efficiency of its cellular component is crucial for pathogen elimination. T cell response is detected in almost all cases of COVID-19, being among the key factors of the virus control and resistance to infection, including re-infection. So far, however, many aspects of cellular immune response to SARS-CoV-2 over one year or later after infection remain unclear. The aim of this study was to investigate the dynamics of laboratory parameters of post-infection cellular immunity to SARS-CoV-2 within 16 months from the symptoms’ onset.

Fifteen healthy volunteers and 87 COVID-19 patients were included into the study. The patients were divided into 3 groups depending on the time elapsed from the onset of the first symptoms to the time when blood samples were collected (from 14 to 500 days). For all samples, the number of S- and N-specific T lymphocytes and the cytokines secreting profiles were determined. Also, the Phenograph automatic clustering algorithm was used to discern different functional groups of the cells.

Approximately 1 in 5 × 10³ peripheral blood mononuclear cells was specific for SARS-CoV-2 S-protein, and 1 in 10⁴ was specific for N-protein. Since the first weeks of infection, the number of specific CD8⁺ cells was significantly higher in COVID-19 patients, as compared with the group of healthy volunteers. As the postinfection period increased, the number of virus-specific CD4⁺ and CD8⁺ cells gradually decreased, but remained significantly higher than in control group. Among CD4⁺ cell population, the proportion of IFNγ^-IL- 2^-TNFα⁺ cells decreased and the ratio of IFNγ⁺IL-2^-TNFα^- cells increases. During first weeks of the disease, CD8⁺ lymphocytes are represented predominantly by IFNγ⁺IL-2^-TNFα^- cells and IFNγ^-IL-2^-TNFα⁺ cells by the end of the observation period. The clustering results showed that, in the early post-infection period, virusspecific T lymphocytes were mostly presented by populations of IFNγ- and TNFα-producing CD4⁺ effector memory cells. Meanwhile, in later time period, the most common populations were TNFα-producing CD8⁺ TEMRA and IFNγ-producing CD8⁺ central memory T lymphocytes.

T cell adaptive immunity plays an important role in the control and elimination of viral infections. In this study, we demonstrated that robust cellular immunity against SARS-CoV-2 is present in the vast majority of patients from the first weeks up to 16 months after the onset of the first symptoms of COVID-19. The immune memory to SARS-CoV-2 is provided by production of central and effector memory T cells, and the data on their time dynamics during the study period allow us to hope for a longer duration of cellular immune memory to SARS-CoV-2.

The purpose of this study was to evaluate efficiency of a competitive enzyme immunoassay which specifically detects antibodies that recognize the receptor-binding domain at the S1 subunit of SARS-CoV-2 coronavirus spike protein and block the formation of initiator infection complex between RBD and angiotensinconverting enzyme 2 (pseudo-neutralizing test, PNT) being applied at the stage of preclinical studies of anti- SARS-CoV-2 vaccine. We studied 37 animal blood sera (8 cows, 10 dogs) as well as 19 male and female transgenic mice of the B6.Cg-Tg(K18-ACE2)2Prlmn/HEMI line hemizygous for Tg(K18-ACE2)2Prlmn (Jackson Immunoresearch, West Grove, PA, USA)) immunized with candidate COVID-19 vaccine preparations containing SARS-CoV-2 Spike protein. In this study, 3 techniques were used for detection of antibodies to SARS-CoV-2 virus, as follows: 1) a pseudo-neutralizing test (PNT) to detect antibodies that block interaction between RBD and ACE-2; 2) neutralization test (RN) to detect virus-neutralizing antibodies, and 3) enzyme-linked immunosorbent assay to detect class G antibodies to RBD SARS-CoV-2. The results were expressed, respectively, as the suppression quotients (SC), titers of virus-neutralizing antibodies (VNA), and the positivity index (IP). The data obtained show a pronounced, statistically significant correlation between the results obtained by immunoassay methods with VNA titers determined in the studied animals by the virological neutralization test. E.g., the Spearman correlation quotients for VNA and SC titers, were, respectively, 0.9151; 0.8085, and 0.9207 for dogs, transgenic mice and cows. The Spearman quotient for VNA and PI titers was 0.8854 and 0.8955 for dogs and transgenic mice. Thus, in order to evaluate immunogenicity of vaccine preparations in our study, both methods are adequate and safe analogues to RN-ELISA for determination of IgG to RBD and PNT aoming for detection of antibodies blocking the formation of RBD/ACE-2 complex. However, the advantage of PNT is its versatility, eliminating the need to use different conjugates to detect antibodies in blood sera of different animal species. The data obtained for samples of three animal species (transgenic mice, dogs and cows) well agree with similar data obtained by us and other researchers for human blood sera, thus demonstrating high correlation between the results of PNT-like competitive tests to determine antibodies that block the formation of the RBD/ACE-2 complex, with VNA results in virologic neurtralization test (RN). Therefore, the proposed PNT technique may be used in preclinical and clinical trials of candidate vaccines and drugs.

A decrease of nonspecific body resistance, an imbalance of local and systemic immunity and a free-radical oxidation abnormality substantially contribute to the pathogenesis of community-acquired pneumonia (CAP).

Purpose: To study the efficiency of including immunomodulators into the comprehensive treatment of nonsevere community-acquired pneumonia and assess the long-term effects of the treatment conducted.

Patients (n = 55) with non-severe CAP (41 (31-48) years old, with CRB-65 score of 0.15 (0-1)) are included in the study. Group 1 (control) received only standard CAP therapy; the other two groups received immunomodulators concurrently with the standard therapy: bacterial lysate (BL) for group 2 and azoximer bromide (AzB) for group 3. TNFα and IL-6 concentration was determined on the day of visit, on day 13 and day 60 of follow-up. During 2 years, the incidence of low respiratory tract infections (LRTI) was studied in the same patients with CAP in past (n = 55). All patients (n = 55) had clinical signs of non-severe community-acquired pneumonia. The overall duration of all symptoms was lower in immunomodulators groups as compared to the control group: 12 (11-13) days in BL group (p < 0.001) and 12 (11-12) days in AzB group (p < 0.001) with no statistically significant difference between intervention groups (p = 0.36). During treatment, TNFα and IL-6 concentration decreased on day 13 and day 60 in all patients; in patients who received immunomodulators, TNFα and IL-6 were reliably lower as compared to the control. Changes of TNFα and IL-6 concentration in the groups on day 60 of the study as compared to the baseline showed a decrease in BL group by 85 (-89 – -82) % and 86 (-90 – -85) % (p < 0.001; p = 0.001 and control); in AzB group by 82 (-86 – -80) % and 86 (-88 – -84) % (p = 0.002; p = 0.007 and control). Intensity of IL-6 concentration decrease on day 60 in BL and AzB groups did not differ (p = 0.72). Gender- and age-adjusted odds ratio for the development of low respiratory tract diseases (during 2 years after CAP) in AzB group was 0.15 (0.02-0.93) (p = 0.04) suggesting its protective effect. Inclusion of immunomodulators in basic treatment of non-severe community-acquired pneumonia reduces duration of symptoms and is associated with improvement of the proinflammatory cytokine profile. In 2 years of follow-up, long-term effects of the immunomodulatory therapy showed statistically significant lower incidence of low respiratory tract infections in AzB group only.

Diagnosis of specific T cell immunity to the antigenic determinants of SARS-CoV-2 in patients seems to be an increasingly important task due to accumulation of data about the role of T cell immune response in course of coronavirus infection and clearance of SARS-CoV-2 in case of secondary infection. Previously, we designed the recombinant CorD_PS antigen for evaluation of T cell antiviral immunity, containing conservative and immunogenic sequences of structural proteins of the SARS-CoV-2 coronavirus. E. coli CorD_PS producing strain with stable expression of the recombinant CorD_PS antigen was obtained. Aim of the present work is to design a laboratory technology for the production of recombinant antigen CorD_PS, to control the quality of the obtained chimeric protein and to study its immunological activity. Development of the cultivation conditions for E. coli CorD_PS cells was carried out in conical flasks in LB-M_Km medium at 37 °C, then scaled in a fermenter with a volume of 30 liters. Expression was induced by the addition of IPTG. Expression was controlled in culture lysates in 12% SDS-PAGE. The resulting biomass was lysed using an ultrasonic disintegrator with followed by centrifugation. The compositions of lysing and solubilizing buffers were selected, as well as conditions for refolding of the recombinant protein. Cation exchange (SP-sepharose), hydrophobic (Butylsepharose) and exclusive (Sephacryl S-200 HR) chromatography were used sequentially to purify the dissolved protein. Protein impurities in the preparation were determined by reverse-phase HPLC and electrophoresis in 12% SDS-PAGE, residual lipopolysaccharides were determined using a gel-thrombin variant of the LAL test, residual proteins of the producer strain were determined by enzyme immunoassay, residual DNA of the producer strain was determined by binding with PicoGreen dye. Specificity was controlled by indirect enzyme immunoassay. The evaluation of cytokine production by CD4⁺T lymphocytes in response to their stimulation by recombinant antigen ex vivo was performed on a flow cytofluorimeter. The biomass yield during cultivation of E. coli CorD_PS in a 30L fermenter was up to 20 g/L for 4 hours of 0.1 mM IPTG induction. Sequential washing of inclusion bodies from bacterial cellular components and their subsequent solubilization in a buffer containing 8 M urea allowed to obtain a solution of denatured antigen with a concentration of 10 mg/mL. The efficiency of refolding by dilution was 75%. After three stages of chromatographic purification, protein samples with a concentration of 1.2-1.4 mg/mL, HPLC purity of 98.43%, corresponding to key quality parameters according to the OFS.1.7.1.0007.15, were obtained. The recombinant antigen showed specific binding to a sample of the First WHO International Standard and sample Company Reference Standard No. 3 of anti- SARS-CoV-2 human immunoglobulins in a specified concentration range. CD4⁺T lymphocytes effectively responded to recombinant antigen treatment by increasing IFNγ production. The optimal concentration of recombinant coronavirus antigen was 5 μg/mL. The developed technological process makes it possible to obtain 5-7 grams of coronavirus recombinant CorD_PS antigen in one cultivation cycle in 30 liters of fermentation medium with key quality parameters according to the OFS.1.7.1.0007.15. As a result of specific immunological activity studies of the recombinant coronavirus antigen CorD_PS, the concept of its possible use as a diagnostic tool for determining the formation of a T cell immune response was confirmed.

Hypersensitivity pneumonitis (HP) is a complex interstitial pulmonary syndrome. This clinical entity is characterized by sensitization to a specific antigen. Early detection of this antigen is associated with an increased likelihood of a favorable outcome. Increased mortality in hypersensitivity pneumonitis is associated with the development of lung fibrosis. At the same time, clinical interventions do not significantly improve the prognosis of the disease due to a lack of understanding the mechanisms underlying the development of this type of fibrosis. Using reliable biomarkers that objectively reflect biological processes in lung fibrosis may improve clinical decisionmaking. Various biomarkers are now beginning to play a critical role in diagnosing and treating a variety of human diseases. Unfortunately, hypersensitivity pneumonitis is an exception to this general trend. There is still a great deal of research to be done in this area in the search for diagnostic biomarkers. The aim of this study was to identify biomarkers of lung fibrosis development in patients with hypersensitivity pneumonitis. We used mature serum microRNAs, which may regulate inflammation and fibrosis, as such diagnostic markers. Patients with a diagnosis of hypersensitivity pneumonitis (with and without lung fibrosis) as well as healthy individuals without chronic diseases (control group) were included into the study. Clinical and laboratory parameters were assessed in all patients. The miScript miRNA PCR Array Kit (QIAGEN) was used for gene expression profiling of mature serum miRNAs. The data obtained were verified using real-time PCR. Our research has identified a number of mature microRNAs that are likely to be involved in lung fibrosis and inflammation (miR-22, miR-150 and miR-106b). Following an extended study, including monitoring of disease progression over time, the applied diagnostic kit may be used in clinical practice to determine disease activity and development of fibrosis formation in lung tissue in patients with different variants of hypersensitivity pneumonitis.

In response to damaging factors, a cascade of cytokines is released, triggering the processes of inflammation and fibrogenesis. The purpose of our study was to evaluate the levels of cytokines (IL-6, IL-10, IL-18, TNFα) in blood plasma under conditions of tobacco intoxication and with intramuscular administration of aminophthalhydrazide. The experiment was performed with 40 outbred white male rats. Intact group (n = 10) were in a priming chamber in absence of tobacco smoke. Experimental groups (1, 2, and 3) – n = 30, 10 animals in each group. The first and second groups of animals were exposed to tobacco smoke for one and two months. The third experimental group underwent intramuscular injections with sodium aminodihydrophthalazindione for 3, 7, 14 days. The content of IL-6, IL-10, IL-18, TNFα in blood plasma was determined by enzyme immunoassay technique. In 1st group, the TNFα level in blood plasma increased to 452.14±176.18 pg/mL (in intact animals, 15.23±3.13 pg/mL); in the 2nd group, this index decreased to 9.8; in group 3, these values showed a sharp increase (to 167.44±5.93 pg/mL). The level of IL-6 showed maximal values after 4 months of tobacco intoxication (group 3). The following IL-10 changes were observed: in the 1st group, its amounts increased by 1.2-fold; in the 2nd group a sharp decrease in the indindex was noted (13.7 times); in the 3rd group a decreased content of the cytokine was found (28.8 times) as compared with intact animals. After administration of aminodihydrophthalazinedione for 3 and 7 days, the content of TNFα showed sharp increas (14.8 times and 7.6 times, respectively); after 14 days of treatment, the level of TNFα decreased by 1.3 times compared to intact animals. We noted fluctuations in the levels of pro-inflammatory cytokines (IL-6 and IL-18). The level of IL- 10 in blood plasma had a statistically significant increase (5.3-fold at 3 days after drug administration); 8.2-fold 7 days after administration of the drug, and 23-fold after 14 days of treatment, as compared with the 3rd group. The plasma concentrations of TNFα, IL-6, IL-10, IL-18 showed sufficient changes under the conditions of tobacco intoxication. The immune response is characterized by increased levels of pro-inflammatory cytokines in blood plasma. Upon corrective therapy with sodium aminodihydrophthalazindione, an increase in the antiinflammatory IL-10 cytokine occurs, along with systemic decrease in the TNFα, IL-6, and IL-18 contents.

Allogeneic hematopoietic cell transplantation is the standard of care for patients with highrisk acute leukemia. Cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cells for patients who need transplantation but do not have a related donor. The purpose of our study was to evaluate the characteristics of leukocyte pool (cellular composition, immunophenotype) of umbilical cord blood (UCB) cell concentrates, which were harvested for public long-term cryostorage for the needs of transplantology. A study of 1096 samples of umbilical cord blood of full-term newborns before and after processing [isolation of hematopoietic stem cells (HSC) concentrate] was carried out. The number of cells in the pooled concentrate of HSC at a volume of 25 mL, after long-term cryostorage was as follows: leukocytes, 17.41±0.36 × 10⁸; HSCs with CD34⁺ immunophenotype, 5.25±3.6 × 10⁶; natural killer cells (CD3-CD16⁺CD56⁺), 1.65⁺0.7 × 10⁸. Analysis of the NKT cell population revealed 6.22±2.1% of cells with CD3⁺CD16/CD56⁺ immunophenotype. The contents of CD3⁺CD56⁺ cells was 2.69±2.4%, the relative amount of CD3⁺CD16⁺ was 2.53±1.2%. In the pooled NKT cell preparations, 5.15±2.1% of CD56⁺CD16 cells were detected, and two minor subpopulations were also identified, which differ in CD56 and CD16 antigen expression: the level of CD56_-CD16⁺ was 2.91±1.2%, and the ratio of CD56⁺CD16⁺ cells was 0.69±0.3%. The obtained data on relative amounts and characteristics of cellular immunophenotype as well as the heterogeneity of the NK and NKT cell population in the HSC UCB concentrate, subjected to long-term cryostorage must be taken into account when selecting the HSC UCB concentrates for the purpose of transplantation in oncohematological diseases.

Thymectomy in a number of clinical situations is an unavoidable step of cardiac surgical treatment of congenital heart defects, but the issue of its effects on development of immunity in early ontogenesis remains open. There is still debates among scientists about the risk of immune deficiency states in children who underwent thymectomy at an early age. One may see completely different opinions, both in domestic and foreign publications. A tool for resolving this issue may be provided by morpho-functional studies of the removed thymic tissue by means of modern scanning electron microscopy techniques. The objective of our study was to assess the possibilities of EMbedding and backscattered scanning electron microscopy for morphological and functional evaluation of the thymus, which was forcedly removed in the infant patients with congenital heart defects over the first weeks of life. The thymus of a newborn infant (postnatal day 27) with congenital heart disease: ventricular septal defect was examined using EMbedding and backscattered scanning electron microscopy imaging after embedding in epoxy resin. The mass of thymus gland was 15.7 g, the dimensions of thymus were: transverse, 3.4 cm; longitudinal, 4.1 cm; thickness, 1.7 cm; volume, 12.4 cm3. The study showed ability of EMbedding and backscattered scanning electron microscopy in morphological and functional assessment of thymus gland considered the central organ of the immune system. Step-bystep visualization from low to high magnifications, from tissues to cells and intracellular structures, as well as layer-by-layer examination of thymic cortex, medulla, interlobular septa and vessels, allows you to effectively assess the functionality of the thymus. This research method is sufficient for scientific research of the forcedly removed thymus since it enables us to visualize its microanatomy, allowing cell phenotyping at different layers of the thymus, studying intercellular interactions of thymocytes with reticulo-epithelial cells, subtle features of Hassall’s bodies and, finally, the process of T lymphocytes’ release from thymus gland.