The present review article summarizes the latest world scientific data on the role of receptors for immune mediators in regulating biological effects on the cells. For the main classes of immune regulators (interleukins, interferons, growth factors and tumor necrosis factors), the variants are presented for participation of receptors as components of cytokine/cell interaction, as proven by in vitro and in vivo studies. Ability of the receptors expression to modify characteristics and type of these interactions is shown. The data on participation of receptors for regulatory molecules in development of immune-mediated diseases of various genesis have been analyzed. It was demonstrated that the changes in the receptor expression are of great importance when evaluating functional response of the cells to the mediators and in development of pathological conditions. Current studies confirmed the data suggesting effects of receptor density upon the processes of proliferation and apoptosis, as well as metabolic processes that trigger development of autoimmune, oncological and dystrophic diseases. For all the considered classes of regulatory molecules, the change in the density of receptor expression is one of the key aspects in regulating functional activity of the cells. Thus, studying expression levels of receptors on the cell membrane is important in understanding pathogenesis, whereas changing expression level may be considered as a therapeutic target in the treatment of various diseases.

Antiviral research has focused mainly on viral targets. However, cellular targets involved in the viral life cycle and antiviral response are becoming more attractive for research, providing a variety of opportunities for antiviral therapy. Toll-like receptors (TLR) play an important role in activation of both innate and adaptive immune systems, including a response to respiratory viral infections. In this review we shall discuss TLRs as potential targets for development of novel antiviral drugs including the mechanisms for induction the antiviral response by means of type I interferon production, as well as viral evasion strategies. In addition, we describe several new molecules that have been applied as TLR agonists or antagonists. The safety issues are also discussed.

According to modern concept of the etiopathogenesis of essential hypertension, immune cells play an important role in its development. Mediators produced by immunocompetent cells participate in the initiation and maintenance of chronic systemic inflammation and promote the development of vascular remodeling which is an important part of the pathogenesis of the disease and target organ damage. The immune mechanisms underlying blood pressure elevation include the activation of innate and adaptive immune cells. Endothelial damage triggers an inflammatory cascade, causing migration of the immune cells to the inflammatory site, mediated by chemokines and adhesion molecules. Macrophage infiltration of perivascular tissue contributes to impaired vasodilation and damage to target organs due to the production of active forms of oxygen. Angiotensin II also causes T cell infiltration of perivascular adipose tissue and adventitia and an increased production of tumor necrosis factor alpha and interferon gamma. In addition, T lymphocytes express the mineralocorticoid receptor involved in the development of systemic hypertension. An important role in the progression of hypertension belongs to interleukin-17, which is involved in blood pressure elevation and vascular remodeling. The review also contains data on the effect of gut microbiota on the regulation of blood pressure and the development of hypertension.

We investigated direct effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the surface properties and cytokine-producing activity of human monocytes/macrophages (Mc/Mphs). The CD14⁺ cells were isolated from peripheral blood of healthy donors by positive magnetic separation. The isolated Mc/Mphs were cultured with lipopolysaccharide (LPS, 1 μg/ml) or without LPS for 24 hours. Membrane expression of CD14, CD16, CD119, CD124, and CD197 molecules was assessed by flow cytometry. The contents of tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), IL-6 and IL-10 in culture supernatants were determined by the enzyme immunoassay technique. It was found that GM-CSF at a concentration range of 0.01-10 ng/ml did significantly reduce the number of cells expressing CD197 (CC receptor of chemokine 7), without significantly affecting the percentage of CD14⁺ (coreceptor of LPS), CD16⁺ (low-affinity Fc receptor), CD119⁺ (IFNγ receptor) and CD124⁺ (IL-4 receptor) cells. At the same time, GM-CSF reduced the contents of CD197⁺ macrophages, as well as CD14⁺, CD16⁺, and CD119⁺ cells among the activated cell population, without significantly altering the number of CD124⁺ cells. It was also shown that GM-CSF (10 ng/ml), was able to enhance production of TNFα and IL-6, but not IL-1β and IL-10 by activated Mc/Mphs. The results obtained indicate the ability of GM-CSF to exert both anti-inflammatory and pro-inflammatory effects upon macrophage cell populations. In general, such effects could contribute to the development of adaptive immunogenesis in peripheral tissues.

Regulation of angiogenesis in the utero-placental bed determines adequate trophoblast invasion, placenta formation and development, as well as successful course of pregnancy. Natural killer (NK) cells, macrophages and trophoblast have the most significant effect on angiogenesis. To date, the functions of cells participating in placenta formation have been described in detail, both individually (in vitrо) and in tissues (in situ). However, no models have yet been created that reflect the interactions of NK cells, trophoblast and endothelium during angiogenesis. It remains unclear, how each cell population contributes to placental angiogenesis regulation, and to the cross-regulation of participating cell functions. Therefore, the aim of this research was to study contact and distant effects of NK cells upon formation of tube-like structures through co-culture of endothelial and trophoblast cells influenced by various cytokines (bFGF, VEGF, PlGF, TGF-β, IL-8, IFNγ and IL-1β). Introduction of NK cells to the co-culture of endothelial and trophoblast cells under conditions of both contact and distance-dependent culturing did not change the length of tube-like structures formed by endothelial cells. During contact-dependent culturing of NK cells with co-culture of endothelial and trophoblast cells in presence of IL-1β, the length of tubule-like structures remained unchanged, compared with the length of tube-like structures formed under the same culturing conditions, but without the cytokine added. During distant culturing of NK cells with co-culture of endothelial and trophoblast cells in the presence of IL-1β, the length of tube-like structures increased as compared with those formed under the same culturing conditions but without the cytokine. During contact-dependent (but not distant) culturing of NK cells with the co-culture of endothelial and trophoblast cells in the presence of VEGF, the length of tube-like structures was greater than those formed under the same culturing conditions but without the cytokine. When used in a three-component cell system, the pro-inflammatory cytokine IFNγhad no effect upon angiogenesis. During distant (but not contact-dependent) culturing of NK cells with co-culture of endothelial and trophoblast cells in the presence of TGF-β, the length of tube-like structures was less than the length of tube-like structures formed under the same culturing conditions but without the cytokine. Under conditions of distant culturing, TGF-βtriggered a signal in NK cells that inhibited angiogenesis. Decreased length of tube-like structures under conditions of a three-component cell co-culture in the presence of the following pro-angiogenic factors was revealed: IL-8, PlGF (during contact-dependent culturing only) and bFGF (during both contact-dependent and distant culturing). Thus, the effects of cytokines upon angiogenesis in a three-component co-culture (NK cells, trophoblast and endothelium) differed from those revealed previously in single-component (endothelium only) and two-component (co-culture of endothelium and trophoblast) cell models. The results of these experiments indicated that regulation of placental cell interactions involved both cellular contacts and effects produced by cytokines.

The aim of our study was to perform an association analysis between MMP2, MMP3, MMP9, VEGF gene polymorphisms and development of non-proliferative diabetic retinopathy (DR) in the type 2 diabetic patients (DM).

201 DM patients: 90 cases of DR and 111 subjects without DR features were included into the study. Polymorphic variants of MMP2 (rs2438650), MMP3 (rs3025058), MMP9 (rs3918242), and VEGF (rs699947 and rs3025039) genes were assayed. The genetic typing was carried out by restriction fragment length polymorphism and TaqMan methods.

The analysis of complex genotypes at the five polymorphic positions has revealed some significant findings in positive and negatively incorporated complexes. Increased frequencies of MMP2-1306 CC genotype in the group of patients with “early” development of complication, and more frequent combination of high-level HbA1c with MMP2-1306CC and MMP9-1562CT genotypes were shown in DR patients. Computerassisted modelling with visual reconstruction of network interactions between the genotypes involved into the destruction events and angiogenesis, as well as altered HbA1с levels (an integral parameter of glycemia), has revealed some differences in structural and functional organization of gene-gene and gene- protein interactions between the groups of patients with DR versus those without this disorder. Сonclusion. A design of interactome biological networks based on transcription regulation and metabolic pathways, as well as their topological analysis allows to build and study interactions of genes and proteins, with reference to pathogenetic studies of DM2 complications aiming for development of approaches to personalized prevention and therapy in future times.

Plasma cells are often considered to be among important criteria in histological diagnosis of chronic endometritis (CE). Low plasma cell numbers in endometrial stroma, uneven distribution and similarity to the cells of cytogenic endometrial stroma represent difficulties with their identification by routine histological methods of study, and their absence prevents distinct diagnosis of CE. The paper presents data on comparative ROC-analysis of two immunohistochemical parameters of chronic endometritis: CD20⁺B lymphocytes and CD138⁺ plasma cells (PC). The study group included 937 women with CE, who were examined for infertility and failed IVF attempts. The control group consisted of 103 women without signs of CE who were studied for male infertility factor.

The sensitivity of CD20⁺B cell assays for the diagnosis of CE was 98% (Se = 0.98), thus being 1.44 times higher compared to the sensitivity of CD138⁺PC, that was, respectively, 68% (Se = 0.68). The specificity of both indicators for the diagnosis of the absence of CE was high, i.e., 98% (Sp = 0.98), and 99% (Sp = 0.99), respectively. The threshold quantitative values for the two endometrial parameters (cutoff point) for the CE diagnosis were also determined when using CD138⁺PC-1 cell/5mm² and CD20⁺B lymphocytes-5cell/5mm². Analysis of linear regression showed a highly significant (p = 0.00053) relationship of CD138⁺ PC from CD20⁺B lymphocytes: With increased numbers of CD20⁺B lymphocytes, there is a trend for higher CD138⁺PC numbers which confirms their direct pathogenetic relationship. The topographic and quantitative features of CD20 B lymphocytes in the endometrium determined during immunohistochemical examination allow us to recommend this approach as a highly sensitive and significant early diagnostic indicator of chronic endometritis.

Purulent inflammatory diseases of various types and etiology comprise major causes of death among the HIV-infected individuals. The purpose of this work was to determine a variety of communityacquired pathogens causing pneumonia, their antibiotic resistance profiles, and dependence on the CD4 lymphocyte levels, as well as identification of methicillin-resistant Staphylococcus aureus species and their molecular genetic characteristics in HIV-infected patients from the Krasnoyarsk City. Over the period of 2012 to 2016, we have examined 152 HIV-infected patients at the Clinical Pulmonology Department with a verified diagnosis of community-acquired pneumonia. Sputum specimens, bronchoalveolar lavage, pleural fluid, washings, pleural pus, as well as nasal and pharyngeal smears were studied for microflora, and blood tests for sterility were performed in these patients, by means of bacteriological techniques. Antibiotic sensitivity was determined by the disc diffusion method; drug sensitivity of staphylococci was performed by screening, PCR technique, serial dilution in semi-solid medium, according to the CLSI and EUCAST recommendations. PCR, M-PCR, and gene sequencing were applied for genotyping and determination of their genetic features. The results were processed with WHONET digital program (WHO). The significance level was p < 0.05. During the entire study period, the yeast-like fungi of Candida genus (30.4 and 35.6%) were consistently isolated from HIV-infected patients. These microorganisms were isolated in a pure cultures at etiologically significant amounts from one-third of the HIV-infected cohort. At the same time, they formed active associations, mostly with Enterobacteriaceae family members. At the same time, Candida fungi were most frequently detected in the lower respiratory tract of those HIV-infected persons who showed severe immunodeficiency (CD4 cell levels < 200 cells/µl). We have also isolated non-fermenting Gram-negative bacteria (12.9%), staphylococci (8.9%), and Enterobacteriaceae (4.4%). The microorganisms were characterized by polyresistance to antimicrobial agents. The MRSA clone circulating in the HIV-infected cohort was characterized as ST239/spa3(t037) /agr1/SCCmecIII.1.1.2 (IIIA)/coaIV/tst⁺ with high virulence and multiresistance levels. Hence, we have found a number of poly-resistant microorganisms playing a role for development of community-acquired pneumonia in HIV-infected patients, i.e., Candida spp, Gram-negative microorganisms, MRSA, often presenting a component of microbial associations. Candida fungi were detected most often in the HIV-infected individuals with severe immunodeficiency, at the CD4 level of < 200 cells/µl. High detection frequency of such microflora requires some modifications of antimicrobial therapy in HIV-infected subjects affected by the community-acquired pneumonia.

Sarcoidosis is a disorder of unknown etiology characterized by development of necrosis-free epithelioid cell granulomas in various tissues. There are two main phenotypes of pulmonary sarcoidosis (PS): Lofgren’s syndrome (LS) is an acute form with favorable outcome, while non-Lofgren’s syndrome (nLS) is a chronic type of disease that can lead to pulmonary fibrosis in 20% of cases.

Our study was aimed at investigating changes in the main cell-surface differentiation antigens on peripheral blood regulatory T cells (Tregs) from the patients with first diagnosed PS without treatment (LS, n = 11) and nLS (n = 46) compared to healthy volunteers (HC, n = 26).

These indexes might be used as immunological markers for predicting severity of this disorder. Flow cytometry analysis of peripheral blood cell samples demonstrated that the nLS patients had decreased relative numbers of CD3⁺ cells vs healthy controls, as well as diminished CD3⁺CD4⁺ cells vs HC and LS patients. Furthermore, the relative and absolute Treg numbers were also decreased in nLS group vs HC (2.83% (2.47; 3.36) vs 3.33% (2.79; 3.84), p = 0.021), and 37 (29; 52) cells vs 50 (42; 65), p = 0.004, respectively) per one microliter of peripheral blood. Relative number of CD39-positive Тregs in chronic vs acute sarcoidosis patients was associated with 51.02% (38.20; 61.62) vs 48.64% (41.46; 63.72) that was significantly (p < 0.001 and p = 0.007, respectively) higher than in HC (39.52% (11.55; 46.34). We have found that “naïve” (CD45R0^-CD62L⁺) Тregs did not significantly differ in percentage of CD39- and CD73-positive cells in all the groups tested. Moreover, CD45R0⁺CD62L⁺ Тregs in LS and nLS patients contained significantly more CD39-positive cells (69.66% (61.92; 79.34) and 67.62% (61.92; 79.34), respectively, compared to 47.55% (15.74; 65.32) in HC (p < 0.001 and p = 0.004, respectively). In case of CD45R0 + CD62LTregs able to exit from the circulation and migrate to the site of inflammation, an increased percentage of CD39-positive subset was noted only in patients with chronic sarcoidosis and HC (61.79% (55.12; 73.09) and 57.27% (16.03; 66.98), p = 0.006). Enhanced CD39 expression on Tregs seems to be related to chronic immune response, so that antigen elimination becomes impossible due to Treg overactivation, as shown in patients with sarcoidosis and some other chronic autoimmune and infectious disorders.

The etiology of sarcoidosis is not completely understood. A hypothesis exists about the relationship between sarcoidosis and a complex of pathological autoimmune reactions that occur under the influence of triggering factors. In this study, specific immune complexes in the blood plasma of patients have been determined, which can indirectly reveal the causes of the disease.

The study included 33 patients with lung sarcoidosis (I group), compared to 24 healthy donors who served as a control group (II group). The patients underwent standard examination. Their blood plasma was investigated by the dynamic light scattering method with addition of tuberculosis antigens (ESAT-6/SFP-10) and “lung healthy tissue extract”. Statistical analysis was performed using the Statistica 7.0 program. Test results were considered significant at p < 0.05.

Аccording to the data obtained, addition of ESAT-6/SFP-10 to patient’s blood plasma almost did not lead to the formation of immune complexes in most samples. Meanwhile, development of such complexes after addition of “lung tissue extract” was revealed in all the patients. The immune complexes were not detected in any donor from control group after stimulation with both kinds of antigens (p < 0.01).

The data on distinct formation of immune complexes with the addition of “lung healthy tissue extract” in patients with lung sarcoidosis may be considered an indirect evidence for occurrence of autoimmune reaction under the influence of some pathogenic factors. Absence of de novo immune complex formation after addition of tuberculosis antigens (ESAT-6/SFP-10) makes it unlikely any direct effects of tuberculosis bacteria upon development of sarcoidosis.

Respiratory muscle (RM) strength was studied in 85 men with exacerbations of chronic obstructive pulmonary disease (COPD). The strength indicators of expiratory (MEP) and inspiratory pressure (MIP, SNIP) in oral cavity were registered by means of the MicroRPM device (CareFusion, UK), as well as intranasal pressure levels by SNIP test. The measured MEP, MIP и SNIP values were compared to the proper indices. Serum concentrations of cytokines (IL-4, IL-6, IL-10, IL-17A, IL-21, TNFα, IFNγ and TGF-β) were determined. The results of the study were processed by means of canonical analysis and by clustering methods. Expiratory RM dysfunction was recorded in mild COPD, expiratory-inspiratory RM dysfunction was recorded in moderate COPD and the diaphragm dysfunction was recorded in severe COPD. Three groups of patients with different combinations of RM strength indicators and immune parameters were identified by means of cluster analysis. The cytokine profile in the first cluster was characterized by maximal concentrations of IL-17A, IL-21, TNFα and TGF-β, whereas RM strength indexes showed minimal values. In the second cluster, a decrease of RM strength indicators by 25-40% against control was associated with a sharp rise of IL-6, along with moderate increase of IL-21 and TGF-βconcentrations. In the third cluster, maximal levels of IL-6, IL-10 and IFNγwere registered, along with low levels of IL-17A, IL-21 and TGF-β concentrations, whereas MEP, MIP и SNIP values did not sufficiently differ from their levels in second cluster. The results of canonical and correlation analysis indicated to interconnections between either certain cytokines, or their pool with the RM strength indicators, dyspnea severity and functional state of COPD patients, thus suggesting involvement of cytokine-mediated mechanisms in pathogenesis of the respiratory muscle dysfunction.

Despite a significant amount of works specifying immune mechanisms of bronchial asthma (BA), different phenotypes observed in this pathology need to be studied. The aim of present study was to analyze functional activity of Th1, Th2 и Th17 lymphocytes, and to determine features of inflammation in controlled and partly controlled asthma.

We examined eighty-four BA patients that were divided into 2 groups, depending on the control of symptoms and the clinical course of BA. Group I included 45 patients with controlled BA, whereas group II included 39 patients with partially controlled asthma. The subsets of Th1, Th2, and Th17 lymphocytes were assessed by serum cytokine levels (TNFα, IFNγ, IL-2, IL-4, IL-6, IL-10, IL-17A) using flow cytometry technique.

The results of this study were as follows: we have shown a combined T-helper (Th) immune response in asthma patients, with its origin depending on the degree of the disease control. Th2 (62%), Th1/Th2 (20%) and Th1 (18%) types of immune response have been detected in the patients with controlled BA. Th2/Th17 (49%), Th1/Th17 (13%) and Th17 (37%) types of immune response have been identified in the patients with partially controlled BA. It has been shown, that Th1 immune response in patients with controlled asthma is induced by intracellular infection. The formation of the Th1/Th2 phenotype is associated with a site of chronic bacterial infection revealed, and with persistence of viral infection in the body. This phenotype can be used as an indicator of asthma worsening. Further studies in the role of prevalent immune response type in the development of partially controlled BA have shown that activation of Th17 lymphocytes is associated with prolonged course of the disease. Irrespectively of initial phenotype, the development of Th17-dependent immune response seems to result from a durable systemic persistent inflammation.

The views on the key role of Th1/Th2 balance in the development of asthma are accomplished by evidence of Th17 lymphocyte involvement into the process, and Th1/Th17, Th2/Th17 phenotypes seem to be the polar features of the disease. Estimation of intensity and phenotype of inflammation in BA will permit a more objective evaluation of the therapy applied, and to choose further management strategies.

Bronchial asthma is a chronic inflammatory disease of the respiratory tract. T-lymphocytes play a key role in pathogenesis of this allergic disease. The reduction in number of naïve T cells and the accumulation of memory T cells in bronchial asthma are accompanied by dysregulation of T lymphocyte function. In present study, we have investigated the contents of different T lymphocyte subpopulations in peripheral blood as well as in resting and PHA-stimulated cultures, along with their proliferative capacity in patients with bronchial asthma and healthy donors. The study included 10 patients with bronchial asthma (age 45.4±11.8 years). One-half of patients was in remission state, the others having been at the stage of clinical exacerbation. The group of donors was formed by healthy individuals matched by gender and age to the patients. Based on expression of cell surface markers CD45R0, CD62L and CD197 (CCR7), the CD4⁺ and CD8⁺T lymphocytes were divided into central (Tcm) and effector memory cells (Tem), naïve T lymphocytes (Tnaïve) and terminally differentiated effector cells (Temra) using flow cytometry technique. The proliferative activity of Tcm, Tem and Tnaïve was evaluated in response to PHA as a functional marker of T cells. We have found that the percentage of peripheral CD4⁺TemCD62L⁺ and CD8⁺TemCD62L⁺ cells in the patients with asthma exacerbation was significantly reduced, if compared to the donors. Following PHA stimulation, these differences in T cell subsets between the groups of patients and donors were not detectable. We performed a correlation analysis between the memory T cell contents and age of the subjects studied. It was shown that the relative amounts of CD4⁺ and CD8⁺ memory cells increased with age in asthmatics, but not in healthy donors. Analysis of mitogen-induced proliferation showed that Tcm and Tnaïve cells proliferated more actively than other subpopulations in both groups. Meanwhile, the proliferative activity of CD4⁺T lymphocytes and subsets of CD8⁺Tcm, CD4⁺Tcm and CD4⁺Tem62L was higher in the group of asthma patients in remission state than in the patients with exacerbating disease, and healthy donors. The revealed increase in the relative number of memory T cells with age suggests that these cells participate in development of bronchial asthma. Proliferative response of the studied subpopulations, which was comparable to the donor values, suggests a functional maintenance of memory T cells and naïve T lymphocytes in bronchial asthma. The increased proliferation of some T-cell subpopulations in asthmatics in remission suggests an activated state of memory T cells. The observed decrease in the number of CD4⁺TemCD62L⁺ and CD8⁺TemCD62L⁺ in patients with asthma exacerbation may be, by our opinion, associated with an active inflammatory process in the airways.

To evaluate the immunity indexes in the children who were born with bronchopulmonary dysplasia (BPD) of varying severity at very early delivery terms (22-27 weeks), dynamic examinations were performed in 35 infants: 17 children were with severe BPD; 18, with mild-severity and moderately severe BPD. The comparison group consisted of seven children born at 22-27 weeks of gestational age without signs of BPD. Relative numbers of lymphocyte subpopulations (CD3⁺, CD4⁺, CD8⁺, CD19⁺, CD16⁺CD56⁺, CD4⁺CD25⁺), and monocytes (CD14⁺CD64⁺, CD14⁺HLA-DR⁺) were determined by flow cytometry. The level of cytokines (IL-6, IL-8, IL-4) was measured by enzyme immunoassay technique. The features of immune status in children with BPD of severe, mild and moderate severity were discerned. It was found that the predictors of severe BPD development in the children born at very early terms, are: increased content of IL-8 at birth and at the age of 1 month, reduced level of expression on monocytes (CD64, HLA-DR) on the 1 month of life, and CD14⁺CD64⁺ cells at 38-40 weeks post conception. The revealed features of immune status in newborns with BPD can be used to assess the effectiveness of the therapy, which requires further research in this direction.

In this work, we used a reference population of newborns and sampled dried blood spots on Guthrie cards of 2,739 individual samples to determine the reference intervals for TRECs and KRECs values, in order to diagnose primary immunodeficiency by means of neonatal screening. The median absolute values for TRECs and KRECs were 195 (CI95%: 185-206) and 185 (CI95%: 176-197) copies per μl, respectively; the normalized value for TRECs was 2780 (CI95%: 2690-2840), and for KRECs, 2790 (CI95%: 2700-2900) copies per 2 × 10⁵ copies of the albumin gene or 10⁵ cells. The reference interval was calculated for 99 and 99.9 percentiles of total TRECs and KRECs individual values. Due to asymmetric distribution of data, the outliers were filtered off, using the Tukey’s criterion applied after logarithmic transformation of the data. When analyzing absolute values for TREC/KREC (per μL of blood), no “drop-down” TRECs values were identified; for KRECs, 18 experimental values were excluded from further analysis (from 9.8 to 13.5). The outlying values were not identified among the normalized values of TRECs/KRECs. The obtained reference values for TRECs and KRECs (at the 0.1 percentile level) were, respectively, 458 and 32 per 10⁵ cells, or 23 and 17 per μl of blood samples from neonates.

Cancer-testis antigens (CTA) can be used as a target for immunotherapy of various malignant tumors, including cervical cancer. However, immunotherapy is often used in combination with anthracycline chemotherapy, in particular, doxorubicin (DXR). Their effects upon expression of CTA genes have not been yet studied. Therefore, we studied the effects of doxorubicin at different concentrations and exposure time upon transcriptional profile of 17 cancer-testicular (CT) genes of HeLa CCL-2 cells. A long-term line of human cervical cancer cells (HeLa CCL-2 line) was used in this work. Culturing of HeLa CCL-2 cells was carried out in sterile culture flasks with adhesive surface and ventilated lids at 5% CO₂, and 95% humidity at 37 °C, in RPMI-1640 medium with 10% fetal bovine serum, supplied with gentamicin (50 μg/ml), and different concentrations of doxorubicin: 0 μg/ml (control), 2 μg/ml, and 4 μg/ml. Expression levels of 16 RT-genes were determined by quantitative RT-PCR using a Bio-Rad CFX96 thermal cycler. Normalization of results was performed against a reference gene, and expression of tested genes in the control samples. We have found that the time of in vitroexposure, and concentration of doxorubicin exert a significant influence upon expression of MAGEA1, MAGEA3, MAGEA4, MAGEB1, MAGEB2, GAGE1, GAGE3, BAGE, CTAG1B, XAGE3, NY-ESO1, PRAME1 and SYCP1 genes, however, without affecting the SSX2, MAGEA2, GAGE4 and MAGEC1 expression, and DXR concentration as a single factor did not affect MAGEB1 and MAGEB2 expression. Time of response to DXR effects enabled us to discern early cancer testicular genes with increased expression (MAGEA1, MAGEA3, MAGEA4, NY-ESO1, SYCP1), reduced expression (GAGE1 and BAGE), and late inducible testicular genes (GAGE3 and XAGE3). These results must be taken into account when carrying out immunotherapy based on the dendritic-cell vaccine technology.

Regulatory framework and methodological approaches to evaluation of immunological effects of vaccination against brucellosis are not established, and the degree of immunological post-vaccinal rearrangement is not yet developed. Due to leading role of cellular immunity in formation of immune protection against brucellosis, evaluation the cellular response in response to antigenic stimulation may be considered the most informative and objective approach to analysis of immune changes in the body during vaccination. In order to develop the most diagnostically informative methods for design of antigen-stimulation cell tests in vitro, a careful selection of a stimulating agent (antigen) is required, which should have a sufficient activating potential, thus providing specificity of reaction under in vitro conditions. The aim of the present study is to study the in vitro specific activity of a protein-polysaccharide antigenic complex from the Brucella abortus 19 BA strain (BrAg), and an opportunity of its application in order to assess the formation of post-vaccinal cellular immunity against brucellosis.

The study was performed with white laboratory mice (n = 50) immunized with the Brucella abortus 19 BA strain. The control group (n = 50) consisted of laboratory mice that received a sterile saline solution in a volume of 0.5 ml. Blood samples were taken from immunized and control animals before vaccination, and 7, 14, 21, and 30 days after immunization. By means of flow cytometry, the activation molecules CD25, CD69, MHC II and CD95, expressed on T lymphocytes (CD3⁺CD69⁺, CD3⁺CD25⁺, CD3⁺CD95⁺, CD3⁺MHC⁺) were determined. To observe the development of immunity, the intensity of expression of T lymphocyte activation markers was calculated using the stimulation quotient. BrAg was used for specific in vitro stimulation of T lymphocytes. The liquid brucellosis allergen (brucellin) was used as an antigen for comparison, when studying opportunity of BrAg usage for assessing the postvaccinal immunity development.

The following results were obtained: BrAg has pronounced specific activity, it did not cause non-specific in vitro reactions (activation) of T lymphocytes, thus enabling its application as a test antigen when evaluating development of adaptive vaccine immunity against brucella.

Experimental testing of brucellosis antigen for carrying out the in vitro antigen-stimulated cellular reactions, aiming for evaluation of post-vaccinal immunity development against brucellosis, showed that the usage of BrAg promotes increase in diagnostic sensitivity of cellular reactions under in vitro experimental conditions. The applied experimental antigen is a quite promising tool for development of laboratory algorithms for brucellosis diagnostics, and assessment of actual vaccination efficiency in cohorts previously vaccinated against brucellosis.

Since 1924, BCG vaccine is used to protect children from the most severe forms of tuberculosis. At the same time, the protective effect of BCG in adults is variable. The potential for revaccination with live vaccine is further limited by the rapid spread of HIV infection. The early-secreted Mycobacterium tuberculosis proteins have been used extensively in TB vaccine development, due to their high immunogenicity and have shown protective effect in animal models. The aim of our study was to evaluate the opportunity to increase the anti-TB resistance in experimental animals by re-vaccination with a new subunit vaccine preparation following primary immunization with BCG. To perform such boost vaccination, we used a combination of the Ag85B-TB10.4-FliC chimeric protein, and the plasmid DNA encoding Ag85A antigen. Efficiency of the boost vaccination was evaluated in a model of M. tuberculosis H37Rv aerosol infection of C57BL / 6 laboratory mice, either in the intact animals, or those vaccinated with BCG only, or BCG followed by revaccination with the test vaccine. The data concerning mycobacteria outgrowth from the organs, and life-span of animals after infection were subject to comparative analysis. We have demonstrated that additional boost vaccination with the vaccine under study, as compared with conventional BCG vaccination, leads to further inhibition of mycobacteria dissemination from the site of infection, and significantly prolonged survival of infected animals.

The aim of present work was to assess the role of immunological disorders, hypoxia and lipoperoxidation in development and progression of erosive/ulcerative lesions of duodenum accompanied by chronic cerebrovascular insufficiency. We have studied a cohort of 125 patients with erosive and ulcerative lesions of duodenum associated with chronic disorders of cerebral circulation, aged from 48 to 74 years old. They underwent outpatient treatment and care at the Clinical Hospital No.5 (Saransk) over 2015-2018. A comparison group consisted of 39 patients with chronically impaired cerebral circulation, who did not show signs of erosive/ulcerative lesions of stomach and duodenum over the study period. Patients received conventional anti-ulcer therapies for 14 days. The cytokine profile, indexes of hypoxia, and serum markers of lipid peroxidation were evaluated. The study has shown an increase in the level of pro-and anti-inflammatory cytokines in cases with erosive/ ulcerative duodenal lesions associated with chronic impairment of cerebral circulation throughout the observation period, thus indicating to evolving imbalance of immunoregulatory system. The development of hypoxic changes in the blood plasma was observed, which was confirmed by increased contents of lactic and pyruvic acids, like as the hypoxia coefficient at all the observation terms. The lipid peroxidation processes were also activated in the course of evolving disorder, as judged by increased contents of diene conjugates, triene conjugates and the malonic dialdehyde levels upon admission, as well as on day 7 and 14 of observation, associated with decrease in the antioxidant potential of blood plasma, estimated by the superoxide dismutase index. A trend for a decrease in the indexes of diene conjugates, triene conjugates, malonic dialdehyde and an increased levels of superoxide dismutase was registered as late as after 30 days of observation. Hypoxia and lipid peroxidation may be viewed as predictors of chronic inflammation. Developing imbalance between pro-inflammatory, anti-inflammatory cytokines, and chronic hypoxia are of significant value for pathogenesis of erosive-ulcerative lesions of duodenum, being associated with chronic impairment of cerebral circulation. Under such conditions, oxidative stress is observed, with a shift of equilibrium towards pro-oxidants, along with exhausted potential of antioxidant defense system. In general, these disturbances determine the pathogenetic mechanisms of damage that occur in erosive and ulcerative lesions of duodenum, being associated with chronic cerebrovascular insufficiency, which have a mutually negative influence in the course of disease progression.

A generally underestimated problem of immunoassays is its susceptibility to various interferences, being the Achilles heel of these assays (interventions in the analysis). The presence of interfering substances in the patient’s specimen can cause erroneous test sample result, which may lead to incorrect diagnosis and catastrophic consequences for the patient. Hence, one should pay particular attention to identifying possible interferences in the test systems used and, when possible, develop and apply methods to overcome them. The issue of avoiding interference is particularly important for immunoassays of biomarkers in human plasma or serum. In order to reduce possible interferences, it would be desirable to have an opportunity of specific and adaptable pretreatment of blood samples for a specifically assayed marker protein, as applied to a specific test system. We assume that such pretreatment may be done by combining the immunosorbents based on the use of special single-domain antibodies (nanobodies).

The nanobodies are recombinant proteins, derivatives of single-domain antigen-recognizing variable fragments of specific antibodies, consisting of a dimer of truncated heavy chains in the complete absence of light chains. Such specific antibodies are detectable in the normal samples taken from members of the Camelidae family (Camelids), and in some species of cartilaginous fishes, along with the common antibodies. The special properties of nanobodies can provide certain advantages in their use, compared with antibodies of traditional structure and their derivatives.

In this paper, we have shown for the first time, that the immunosorbents based on certain combination of single-domain antibodies used as ligands able for specifically binding and removal of specific high-abundance human blood proteins, may be selected for a given marker blood antigen in such a way that they will be an effective tool for blood pretreatment, aiming to reduce possible effects of interference and increased sensitivity in the diagnostic “sandwich” enzyme immunoassay. A new method of plasma pretreatment is demonstrated with human plasma samples (at a dilution of 1:40) of two patients. A previously developed model system for detection of lactoferrin protein was used to analyze plasma samples. It is shown that a significantly increased ratio of total detected signal to the nonspecific background signal could be obtained after drastic reduction of this background by affinity removal of 3 major protein fractions, i.e., fibrinogen, IgG alpha-2-macroglobulin from blood plasma samples, using appropriate immobilized single domain antibodies.