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Medical Immunology (Russia)

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Vol 25, No 2 (2023)
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REVIEWS

233-252 1136
Abstract

Previously, autophagy was termed as a mechanism used by the cells with a lack of essential nutrients supporting homeostasis. Over the decade of studies, autophagy proved to be a more complex, ambiguous mechanism. Its activation depends on the nature of stimulus, type of immune cells and the final result. Both canonical and non-canonical autophagy, being similar in molecular events, but showing their own distinctive features, are key processes in protecting the body from penetration of intracellular pathogens, maintaining the required level of nutrients in the cell, and removing damaged organelles and cells. Canonical autophagy probably evolved as a homeostatic response to cellular stress and nutritional deficiencies, whereas non-canonical autophagy emerged as a response to suppression of inflammation. Non-canonical autophagy, hereinafter referred to as LC3-associated phagocytosis (LAP), combines the molecular mechanism of phagocytosis with an autophagy mechanism characterized by ingestion of exogenous pathogens, formation of phagosomes (laposomes) and enhanced fusion with lysosomes, followed by degradation of their contents.

Significant differences were found between the processes of LAP- and canonical autophagy, which are similar in its mechanism of action. The presence of PI3K complexes in both processes, utilization and intracellular degradation of the “cargo” which is not required for the cells and organism proceeding in the lysosomes, and involvement of almost the same proteins provide similarity of their mechanisms. However, there are differences in the initiation of the processes, e.g., different types of PI3K complexes (in autophagy, PI3K III class 1 and 2 types; in LAP PI3K III, class 3 type), usage of reactive oxygen species in LAP, different types of regulatory proteins involved (ULK1, FIP200, ATG13 , Ambra1, WIPI2, ATG14 in autophagy; and Rubicon and NOX2 in LC3-associated phagocytosis), different number of layers in the membrane structure in which lysis occurs (double-membrane autophagolysosome and single-layer membrane in laposomes) clearly depict the variety of canonical and non-canonical autophagy. The two pathways are directed for different types of biological objects, i.e., intracellular pathogens, dysfunctional proteins and organelles in autophagy, and extracellular pathogens, apoptotic bodies, bacteria, utilized in LAP, thus making these mechanisms completely different in their significance.

Collectively, the new data indicate that autophagy performed via both canonical and non-canonical pathways, has evolved into a host defense mechanism capable of resisting immunological and pathogenic stress and mediating immunological tolerance to both intra- and extracellular threats. The present review discusses fundamental molecular differences between these mechanisms, as well as their role in immunity, based on the latest literature data.

253-270 609
Abstract

Pregnancy is an immunological paradox, since a fetus carrying paternal antigens is a semiallogeneic transplant that should be rejected by the mother’s body. However, the fetus is completely protected from immune attack, thus suggesting some complex mechanisms of feto-maternal interaction. Hormonal, autocrine and paracrine immune signals and neuronal pathways play an important role in the development and maintenance of pregnancy. Pregnancy is considered a dynamic and actively modulated immunological process at each stage of pregnancy, including embryo implantation, placentation, fetal development, and delivery, being represented by a unique immune status. Studying the mechanisms of maintenance of pregnancy is vital to address the problems of miscarriage of unknown etiology. Successful pregnancy is closely related to the ability of the maternal immune system to properly adapt for each distinct stage of gestation. This review considers the main cell populations, such as regulatory subtypes of T and B cells, T helper cells, decidual natural killers, myeloid suppressors, erythroid nucleated cells which provide feto-maternal tolerance via various intercellular and humoral mechanisms. Maternal immune cells in the placenta do not attack fetal cells (trophoblasts) due to the tolerogenic microenvironment created by regulatory T cells and other immune cells. During pregnancy, each subpopulation of T helper cells plays a key role in promotion of fetal development through the production of angiogenic factors, providing immune surveillance and suppressing aberrant effector cell responses against a semi-allogeneic fetus. Accumulation of myeloid suppressor cells is especially relevant, when the immune tolerance is required for survival. Decidual NK cells closely interact with trophoblast cells and secrete cytokines that promote growth, mediate differentiation, trophoblast invasion, and remodeling of the spiral arteries. The favorable tolerogenic state in utero predisposes the newborn to severe infections, especially those caused by intracellular pathogens. Hence, the fetal tolerance may differ from other types of tolerance due to the presence of various immunosuppressive cells, such as erythroid suppressor cells in newborns. In the course of pregnancy, the properties of these cells change dynamically in order to meet the demands that arise during pregnancy in a timely manner. Understanding the immunological changes induced by pregnancy may not only reveal new therapeutic strategies to improve pregnancy outcomes, but also highlight new aspects of how the immune tolerance works being applicable in other physiological and pathological contexts.

271-286 858
Abstract

According to the common concept of immune editing, the interaction of malignant tumor cells and immune system is a complex multifactorial process, which may result in both antitumor effector activity and development of suppressor mechanisms that promote tumor growth. Accumulation of scientific knowledge in the field of studying the antitumor immune response and tolerance has led to emergence of many research and therapeutic approaches that use different components of the immune system to combat neoplastic processes. Along with currently available approaches, there are strategies that use the potential of antigen-specific T lymphocytes, the main effectors of adaptive immunity, in order to fight malignant neoplasms which appeared more than a century ago and have built the scientific basis of cancer immunotherapy. One line of evidence of the significant antitumor potential of T cells in immunotherapeutic schemes for the cancer treatment was presented by successful therapy of hemato-oncological diseases, achieved at the end of the past decade. At the same time, however, the therapy of solid malignant neoplasms still faces significant difficulties that limit the efficiency of treatment. In this regard, the main objective of the review is to accumulate up-to-date information on the successes and limitations of T cell immunotherapy in the patients with solid tumors. To date, the phenotype and functionality of T cells is being investigated and modulated both towards enhancing antitumor cytotoxicity, increasing viability and proliferative activity of T cells, and in overcoming the immunosuppressive effect of the tumor and its tolerogenic microenvironment upon T cells, as well as ensuring targeted migration of the effector T cells to the malignant tissues. This review discusses immunotherapeutic approaches exploiting the potential of effector T lymphocytes, e.g., current clinical trials or applied therapeutic regimens for the treatment of solid malignant neoplasms. Antigen-independent approaches aimed at nonspecific enhancement of the T cell responses, i.e., therapy with recombinant cytokines and inhibition of immune checkpoint molecules. Antigendependent, or antigen-specific approaches such as adoptive T cell therapy with endogenous T lymphocytes are also discussed as well as trials on T cells with modified antigen-recognition receptor (CAR-Tcells, TCR-Tcells), like as usage of bispecific antibodies as T cell engagers. The review describes the benefits and disadvantages of these approaches in monotherapy, as well as current results and prospects for their mutual combinations.

ORIGINAL ARTICLES

287-300 453
Abstract

Insulin-synthesizing cells (ISCs) of pancreatic gland are localized both in its islets, and in exocrine portion, as single cells or cellular agglomerates. ISCs differ in their morphological and functional characteristics, depending on characteristics of the microenvironment. Resident macrophages are also involved into formation of their microenvironment. Our purpose was to assess the effect of functional macrophages upon the insulinsynthesizing system (pancreatic islets, cell agglomerates, and separately lying insulin-synthesizing cells) under normal conditions and in alloxan diabetes.

Alloxan diabetes was induced in mature male Wistar rats by intraperitoneal injection of alloxan (30 mg/100 g). Functional activity of macrophages was modeled with anti-inflammatory drug aminophthalhydrazide (AMP). Contents of insulin, glucose, and glycosylated hemoglobin were measured in blood of experimental animals. The levels of IL-1α, TNFα and IFNγ were determined in pancreatic homogenate. The number of macrophages was counted in histological preparations from the insular and exocrine parts of the organ, as well as the number of pancreatic islets, agglomerates, and single ISCs. The amounts of proliferating cells (insulin+Ki-67+), apoptotic forms (TUNEL+insulin+), and insulin content of ISCs at different sites (according to their fluorescence intensity) were determined. All pancreatic islets were divided into 3 types, according to intensity of insulin fluorescence, i.e., islets with high, median and low levels of fluorescence.

In healthy rats, immunomodulation reduced total level of IL-1α in pancreatic parenchyma, without changing the overall parameters of carbohydrate metabolism. In the exocrine part of pancreas, the content of single ISCs in ductal epithelium was increased. Likewise, proliferation of the ISC agglomerates became higher. The intensity of β-cell apoptosis increased in pancreatic islets. The proportion of islets with high-level insulin fluorescence was decreased, along with lower density of macrophages and proliferation rates of β-cells, and higher apoptosis rates, than in intact animals. We have also revealed there an increased ratio of cells with average insulin levels. In the islets with low insulin content, immunomodulation did not cause morphological changes. Administration of AMP in alloxan diabetes contributes to a significantly decreased concentration of IFNγ in pancreatic tissues, stabilizes IL-1α content, along with reduced apoptosis of ISCs and macrophage infiltration in all parts of the gland. In the ductal epithelium, a large number of single ISCs with high synthetic activity was observed, with retained number of agglomerates and their increased cellularity. The number of dividing β-cells is increased in pancreatic islets.

Modulation of the functional activity of pancreatic macrophages under physiological conditions provides a multidirectional effect on the insulin-synthesizing cells, depending on their localization. In exocrine part of the organ, where M2 macrophages are located, we have observed activated differentiation and proliferation of ISC precursors. Meanwhile, in the islets where M1 macrophages are present, apoptosis of β-cells was enhanced. In alloxan diabetes, immunomodulation was associated with reduced destruction of insulinocytes, along with high intensity of their proliferation. Heterogenous response of ISCs to the changes in the microenvironment depends on their synthetic activity. In healthy rats, the islets with high level of insulin fluorescence, the level of apoptosis is increased, and β-cell proliferation is reduced, while the morphological and functional characteristics of islets with low-level insulin fluorescence did not change. In alloxan diabetes, apoptosis prevailed in islets with high fluorescence values, whereas β-cell proliferation predominated in the islets with low insulin contents.

301-308 377
Abstract

Injectable allogeneic decellularized biomaterials are being developed both as scaffolds for delivery of cellular products and as independent pharmacological agents that affect the cascade of tissue reactions during the period of post-ischemic myocardial remodeling. Biomaterial degradation products can affect cellular processes and modulate cytokine effects, thus determining the healing strategy of damaged tissue. In this work, the influence of biomaterial on the expression of key fibrogenic factors by the cells of tissue bed was demonstrated, and the degree of damage to the myocardium during its ischemic damage was experimentally determined. The aim of our study was to determine the area of myocardial scar degeneration and detection of key fibrogenic factors (bFGF-1, TGFb1, MMP-9), as well as TIMP-2 (MMP-9 antagonist) at the acute and subacute stages of myocardial infarction after implantation of allogeneic powder-like biomaterial in an experimental model.

In the course of experiments, the left ventricular coronary artery was ligated in male Wistar rats (experimental group). All animals were divided into 3 groups: experimental group I (n = 50), experimental group II (n = 50), and controls (n = 50). In experimental group I, the artery ligation was simultaneously accompanied by intramyocardial administration of powder-like biomaterial suspension (2 mg). In experimental group II, the allogeneic powder-like biomaterial was administered 5 days after coronary occlusion, and only physiological saline was administered in the control group. The animals were withdrawn from experiment on days +3, +7, +14, +30, and +45. Standard histological assessment (hematoxylin and eosin staining, according to Mallory) and immunohistochemical examination (MMP-9, TGFb1, bFGF-1, TIMP-2) were made, and statistical evaluation was performed. The cells with positive staining were counted, and the scar area index was calculated.

We have found that administration of dispersed allogeneic biomaterial was followed by a five-fold decrease in the degree of scar degeneration in both experimental groups at the acute and subacute stages of ischemic myocardial damage as compared to the control group. A significantly decreased expression of fibrogenic factors (MMP-9, TGFb1, bFGF-1) by the local cells was found, along with increased activity of metalloproteinase inhibitor (TIMP-2) in connective tissue cells.

Decellularized allogeneic powder-like biomaterial serves as a fibrosis inhibitor and promotes cardioprotection during myocardial remodeling at the initial stages after ischemic injury.

309-318 398
Abstract

Currently, no drugs have been identified that could slow progression of chronic obstructive pulmonary disease (COPD), or have a significant impact on patient mortality. Therefore, research continues aimed at studying the mechanisms of COPD development and searching for drugs that affect its molecular pathogenesis. The aim of our work was to determine the ability of azithromycin combined with corticosteroids to affect the migration of peripheral blood NK cells from the COPD patients. In the present study, we have measured expression of chemokine receptors CCR5, CCR6, CCR7, CXCR3, CXCR4, CXCR6 on the surface of peripheral blood NK cells (CD3- CD56+) by means of flow cytometry in 54 smoking patients with COPD, 21 healthy smokers, and 20 healthy non-smokers. Moreover, the effect of azithromycin (10 µg/mL) and budesonide (10 nM) on the migration of NK cells from COPD patients (n = 8) towards CCL5 (10 nM) and CXCL10 (10 nM) was determined. We found that the percentage of NK cells expressing CXCR3 and CCR5 chemokine receptors was increased in smoking patients with COPD compared with healthy smokers and healthy non-smokers. However, the proportion of these NK cell subsets did not differ between healthy smokers and healthy non-smokers. There were no significant differences in the percentage of NK cells expressing CXCR4, CXCR6, CCR6, CCR7 chemokine receptors between the three groups of subjects. Addition of budesonide to the cell suspensions decreased the migration of blood NK cells towards CCL5 and CXCL10. Azithromycin was also shown to suppress the migration of blood NK cells towards these chemokines. The combination of azithromycin and budesonide was more potent at inhibiting NK cell chemotaxis towards CCL5 and CXCL10 than any of these drugs added alone. Our results demonstrate a change in the chemokine receptor profile of NK cells in COPD patients and indicate the advantages of the combined use of corticosteroids and azithromycin for COPD treatment.

319-330 488
Abstract

Autistic spectrum disorders (ASD) affect about one in every 59 children. It is noteworthy that patients with ASD are more likely to have other comorbidities than the general population. Undoubtedly, they may aggravate clinical course of the underlying disease or affect the diagnostics. The aim of this work was to identify clinical and immunological phenotypes of the ASD clinical course. Patients and methods. The study included children classified in 2 groups: pediatric patients with ASD (n = 100), and clinically healthy children (n = 30). Based on the presence of comorbidities, the children were divided into 3 types of clinical patterns: convulsive, infectious, dermato-respiratory and gastrointestinal phenotypes. Cytokine concentrations in blood serum were determined by ELISA using Bender Medsystems (Austria) for IL-17А and Vector-Best (Russia) for IL-4, IL-6, IL-10, IFNγ. The concentration of spIgG to 111 nutritional antigens (IgG) was determined by a modified ELISA method using the Immunohealth™ technique. Assessment of cognitive and psychophysiological indices in children was carried out using the ATEC questionnaire. As a result of the study, clinical and immunological phenotypes were identified among the ASD patients, being associated with certain types of food tolerance, cytokine profile, clinical severity of psycho-physiological disorders and concomitant comorbid diseases. In all four phenotypes, were have revealed an increased synthesis of specific antibodies associated with humoral immunity for the studied food antigens, increased concentration of total spIgG to food antigens, concentration of spIgG to legumes and casein, and C-reactive protein levels.

Moreover, in convulsive phenotype (concomitant epilepsy and convulsions), the maximal concentrations of spIgG are shown for Solanaceae products, the concentration of IL-10 is increased, IL-4 amounts are reduced, and the content of serum iron and ferritin is also lowered. In the infectious phenotype (frequently ill children) the spIg’s to grain and fermented products are detected, IL-10 and IFNγ concentrations are increased and IL-4 contents is reduced, along with increased absolute and relative number of lymphocytes and fibrinogen. In the dermato-respiratory phenotype (skin rashes) – to dairy products, the concentrations of IL-4 and IL-17A are increased. In the gastrointestinal phenotype, the highest number of elevated IgG responses to the largest range of food antigens was found in presence of changing cytokine profile , i.e., an increase in IFNγ in IFNγ/IL-4 and IFNγ/IL-10 ratios. Thus, the identified phenotypes of the ASD course are associated with the influence of food antigens and reflect a special variant of the immunological inflammatory pathogenesis, which makes it possible to personalize elimination diets, propose measures for correction and individual prevention, and, probably, to predict clinical course of the disease.

331-338 369
Abstract

Abnormal expression of matrix metalloproteinases (MMP) in watery moisture in patients with glaucoma may affect regulation of intraocular pressure (IOP). MMP activity is regulated by tissue metalloproteinase inhibitors (TIMP). The imbalance between tissue metalloproteinase inhibitors and matrix metalloproteinases may contribute to the development of glaucoma. Genetic factors, including polymorphism of matrix metalloproteinase genes and their inhibitors genes, can regulate the level of their expression, thereby affecting susceptibility to disease. Our aim was to perform comprehensive analysis of the MMP2 (rs243865), MMP3 (rs3025058), MMP9 (rs3918242) polymorphisms, and TIMP1 (rs4898), TIMP2 (rs8179090) tissue inhibitor genes polymorphisms in the patients with stage II (advanced) primary open-angle glaucoma.

99 patients (52 men and 47 women) with a verified diagnosis of stage II primary open-angle glaucoma were examined. The comparison group consisted of 100 age-matched persons (81 women and 19 men) without ophthalmic disorders. The single-nucleotide polymorphisms in promoter regions of MMP2, TIMP1, TIMP2 genes were analyzed by the TaqMan method, the MMP3 and MMP9 genes, by means of restriction fragment length polymorphism technique. Statistical evaluation was carried out using the specialized package of IBM SPSS Statistics 23 programs. The critical level of significance was assumed to be 0.05.

The differences in the distribution of MMP2 rs243865 allelotypes with decreased frequency of TT genotype were found in the patient group and, vice versa, increased heterozygosity rates were revealed among them. In addition, the frequency of TIMP1 rs4898 heterozygous genotype was decreased in this group as compared to control sample. Four MMP/TIMP complex genotypes are positively associated with the development of pathology. Two of them were of bilocus type, i.e., MMP2-1306TC:TIMP2-418GG, and MMP3-11715A6A:TIMP1 372CC whereas two three-locus constellations were revealed, i.e., MMP2-1306TC:MMP9-1562CC:TIMP2- 418GG, and MMP3-11715A6A:MMP9-1562CC:TIMP1 372CC. There are nine MMP/TIMP complexes, the frequency of which in patients with glaucoma was significantly reduced when compared with control group.

Polymorphism of regulatory regions of MMP2, MMP3, MMP9 genes and distinct gene variants of their inhibitors (TIMP1, TIMP2 genes) can be considered potential markers of the POAG development associated with an imbalance of MMP/TIMP activities.

339-348 597
Abstract

Helicobacter pylori (H. pylori) increases the risk of diseases associated with mucous membrane inflammation of gastrointestinal tract, in particular, gastritis, stomach ulcers, and duodenal ulcers. It may also induce a chronic immune response, causing damage to the mucous membrane and development of these diseases. In addition, the role of H. pylori in the initiation of a wide range of autoimmune diseases is discussed. The aim of this study was to assess the level of autoantibodies – markers of various autoimmune diseases in the blood of H. pylori-infected patients with chronic gastritis. We used samples of whole peripheral blood from 267 primary patients with chronic gastritis in the acute stage. The presence of H. pylori in gastric juice from patients was determined using real-time PCR. The level of autoantibodies to double-stranded and single-stranded DNA, autoantibodies to thyroglobulin, thyroid peroxidase, concentration of rheumatoid factor, IgG autoantibodies to the cyclic citrullinated peptide, IgM and IgG autoantibodies to beta(2)-glycoprotein were determined by the enzyme immunoassay. The average level of rheumatoid factor in blood serum was similar for H. pylori-infected and non-infected patients, and did not exceed the normal values. The level of antibodies to cyclic citrullinated peptide, one of the sensitive markers of rheumatoid arthritis, was increased in all patients, being, however, significantly lower in H. pylori-infected patients compared with non-infected persons. Autoantibodies to thyroglobulin, thyroid peroxidase are considered classic markers of autoimmune diseases of the thyroid gland. In blood of H. pylori-infected patients we have found an increased concentration of autoantibodies to thyroglobulin and thyroid peroxidase in comparison with non-infected ones, but the average level of these antibodies did not exceed the normal range. Any differences in the levels of systemic lupus erythematosus serological markers, i.e., autoantibodies to double-stranded and single-stranded DNA, were found between H. pylori-infected and non-infected patients. The levels of thrombosis risk marker in patients with systemic lupus erythematosus (IgG and IgM autoantibodies to beta(2)-glycoprotein) were also within the normal ranges. However, in H. pylori-infected patients, it even turned out to be statistically significantly lower than in non-infected ones. Thus, no data have been obtained on increased levels of the tested markers of autoimmune pathology in blood of H. pylori-infected patients with chronic gastritis at the acute stage. However, this does not allow us to make an unambiguous conclusion that the influence of H. pylori does not affect the development of immunological changes associated with autoimmune diseases.

349-356 297
Abstract

Varicocele is a vascular disease characterized by abnormal tortuosity and dilation of the veins in pampiniform plexus that drains the testis. Due to difficult outflow of blood via the altered veins, the intratesticular blood flow becomes impaired, leading to pathological changes in the testicular tissue caused by hyperthermia, ischemia, hypoxia and development of inflammatory reaction. Seminal plasma contains numerous proteins, molecules, a wide range of cytokines, chemokines, growth factors. Their properties and levels largely determine the stages of post-testicular maturation of spermatozoa. At the same time, cytokines are an integral part of the inflammatory effect and are synthesized by various immunocompetent cells present in the male reproductive tract. Increased cytokine levels in ejaculate may act as a marker of local inflammatory process, being a significant factor of male infertility. The purpose of our study was to assess cytokine profile of ejaculate in adolescents with varicocele.

The level of cytokines IL-1β, IL-6, IL-8, TNFα, IL-4, IL-10, VEGF was determined in ejaculate of adolescents aged 17 years. The main group consisted of 100 adolescents with II-III degree varicocele; the comparison group included 30 adolescents without varicocele.

Adolescents with varicocele have statistically higher levels of all studied pro-inflammatory cytokines, except of IL-8 and IL-10, which may suggest presence of a local inflammatory process. We did not find significant differences in the levels of cytokines between groups with grade II and III varicocele. However, the levels of pro-inflammatory IL-1β, IL-6 cytokines in ejaculate proved to be increased in the patients with grade III varicocele. When comparing the results between both groups, depending on the period after varicocelectomy and in the comparison group, statistically higher levels of IL-1β, IL-6, TNFα were detected in patients with more recent surgical correction and IL-10 levels in both subgroups with varicocele. This finding, given the progressive course of this disease, may be considered an unfavorable factor, since the pro-inflammatory status of testicular tissue returned, at later terms after surgery.

In adolescentswith varicocele, increased levels of pro-inflammatory cytokineswere revealed in the ejaculate. There were no statistically significant differences in the level of cytokines in ejaculate of the patients with II and III degrees of varicocele. At the longer postsurgical period, an increased level of pro-inflammatory cytokines was revealed in the ejaculate samples.

357-366 767
Abstract

Metastasis is the leading cause of death in patients with breast cancer (BC). It is known that the lesion of regional lymph nodes by tumor cells is more common in tumors with higher proliferative activity. Moreover, there is literature evidence on effects of cytokines and proteins upon the migration potential of the tumor. The aim of our work was to study the correlation between the concentrations of cytokines, proteins, and expression of Ki-67 proliferation marker in breast cancer with histology of non-specific invasive carcinoma.

On the basis of pathological findings, 16 patients had metastases in regional lymph nodes (group I), and 18 patients had no detectable metastases (group II). Solid-phase enzyme immunoassay was used to determine concentrations of 14 cytokines in the supernatants of immunocompetent blood cells, i.e., IL-2, IL-6, IL-8, IL-10, IL-17, IL-18, IL-1β, IL-1ra, TNFα, IFNγ, G-CSF, GM-CSF, VEGF and MCP-1, and concentrations of 6 proteins were determined in blood serum: estrogen and progesterone receptors, cadherin-E (CDH1), plasminogen activator type 1 (PAI-1), mucin 1 (MUC1), heat shock protein 90αA1 (HSP90αA1). Immunohistochemical study of Ki-67 expression was performed in paraffin sections of tumors using monoclonal antibodies.

The study showed that Ki-67 expression in tumor tissues and blood concentrations of IL-6, IL-8, IL-1β and TNFα were higher in group I patients. On the contrary, blood concentrations of CDH1 and PAI-1 were higher in group II patients. It was found that Ki-67 showed both inverse correlations with CDH1 and PAI1, and direct correlations with IL-8 and TNFα. CDH1 had a direct correlation with PAI1, and inverse correlations with IL-6, IL-1β and TNFα. The studied cytokines showed direct correlations with each other. The analysis of ROC curves showed good quality and optimal values of the cut-off points for Ki-67 expression, cytokine and protein concentrations, thus allowing best prediction for detectable lymphatic metastasis.

On the basis of these results, a quotient was proposed, which represents a ratio of CDH1 contents to the sum of IL-1β and TNFα concentrations in blood samples, which can help identification of the patients with breast cancer at risk for lymphatic metastasis.

367-376 474
Abstract

Vaccination is the only guarantee for elimination of measles infection. Healthcare workers have a 13- to 19-fold higher risk for contracting measles than the general population. The number of individuals in the population who did not respond to vaccination is up to 10%, and their accumulation may lead to an outbreak of the infection. The aim of our research was to find potential predictors of arising post-vaccination measles antibodies in the panel of biochemical and immunological serum markers in healthcare workers. The group of healthcare workers (n = 76) aged from 19 to 51 years, with proven absence of pre-existing anti-measles antibodies were twice vaccinated 3 months apart with live measles culture vaccine (SPA “Microgen”, Russia). Measles-specific IgG, total IgG, IgM, IgA, IFNγ, IL-6, CRP, total protein, ALT, AST, total bilirubin, urea, creatinine, protein fractions were determined before vaccination, 1 month after vaccination, 1 month following revaccination, 1 year after revaccination. ROC analysis was used to gain access to the diagnostic performance of quantitative variables in predicting a categorical outcome. Development of a predictive probability model for the binary outcome was carried out using logistic regression. IFNγ, total IgG, IgM, total bilirubin, ALT activity at various post-immunization stages may be considered potential laboratory predictors of measles vaccination failures in healthcare workers. Meanwhile, the contents of pre-vaccination IFNγ, and IgG to measles virus after first vaccination proved to be most informative indexes, which formed the basis for the development of regression models predicting the risk of both primary and secondary vaccination failures. These models allowed to develop algorithm for predicting failures of the measles vaccination in healthcare workers that can be used for detection of persons at risk for non-forming specific humoral immunity. This algorithm is primarily focused on search for the persons who have not responded to measles vaccination, including subjects with probable immunodeficiency conditions. We do not exclude that, on the basis of revealed predictors following measles vaccination, it would be possible to build prognostic models of vaccination efficiency for other vaccinemanaged infections.

377-386 443
Abstract

Studies of real opportunities for physical skills of athletes sufficiently depend on their adaptive potential for increasing physical loads. Extreme physical and psychoemotional loads may lead to overwork and decreased physical ability in professional sportsmen. These adaptation processes are regulated by the main biochemical systems of the body. A special role belongs to the factors of humoral immunity, i.e., natural antibodies, which are a component of innate immunity. They circulate in blood of healthy persons in absence of obvious antigenic stimulation. Analytical techniques for measuring the level of natural antibodies that reflect the state of the system of endogenous bioregulators involved into the molecular mechanisms of adaptation process have been developed. An important role among them is played by the regulators of the opioid system β-endorphin and orphanin. The biochemical and immunological parameters were determined in 10 athletes active in figure skating (Master of Sports), whose average age was 16±0.4 years, and sport experience of 9±1 years. The duration of the study was divided into 5 stages and was 62 days. During the dynamic observations in the course of intensive training, no clear shifts in biochemical parameters were revealed towards adaptation stress and delayed recovery. The level of natural antibodies to orphanin and beta-endorphin was measured in the athletes blood serum by ELISA techique. It is found that each athlete is characterized by individual immune profile. At the initial stage of the examination, the level of antibodies to beta-endorphin was within normal ranges, except for its decrease in one athlete. The level of antibodies to orphanin in majority of cases was higher than normal, probably, due to inhibitory control of the pain signal. Further study in time dynamics revealed that the immunological parameters, natural antibodies to opioid peptides, change in accordance with the state of adaptation resources in the athletes. These indexes reflect psycho-emotional potential and pain tolerance threshold for athletes from the start of training and throughout the entire period. Therefore, from a prognostic point of view, it is important to monitor the content of natural antibodies to beta-endorphin and orphanin in athletes in the course of training. Such individual monitoring of the athlete’s immunological indices allows us to select a more effective, personal training program.

SHORT COMMUNICATIONS

387-394 406
Abstract

Ribonucleic acids (RNA), in particular, double-stranded RNAs, due to their ability to modulate innate immune responses, are of undoubted interest in view of their usage as vaccine adjuvants. However, despite the fact that dsRNA preparations have been known for a long time, the issues of cellular interactions and orientation of immune response upon their exposure have not yet been properly studied. The aim of this work was to evaluate the in vitro response of mouse splenocytes to dsRNA exposure in cell cultures, and after drug administration in vivo. The studies were carried out in female Balb/c mice. Activation status of various splenocyte populations after treatment with yeast dsRNA and reference substance (PolyI:PolyC) was assessed by means of flow cytometry by expression of CD69 and CD86 activation markers on CD19+B lymphocytes and CD11c+ dendritic cells (DC). During in vitro studies, the splenocytes were incubated in DMEM medium containing 10% fetal calf serum for 22 hours following addition of the yeast dsRNA preparations, or PolyI:PolyC (2.5 μg/mL) preparation. Single-stranded high-polymer RNA (hpRNA), which is a component of the substance, was used as an additional control at the dose of 16 μg/mL. Our study has shown that the activating effect of dsRNA and PolyI:PolyC on expression of CD86 and CD69 markers upon the cells of the entire pool of splenocytes, B lymphocytes and DC. Highly polymeric RNA increased the total number of CD86+ cells in the population without changing the expression level of these markers upon B lymphocytes and DCs. When performing the in vivo studies, yeast dsRNA substance was administered intravenously into mice at a dose of 2.5 mg/kg, and hpRNA was used at a dose of 16 mg/kg. The number of CD69+ and CD86+ splenocytes was assessed 4 hours after drug administration. The highest stimulating effect of dsRNA was registered with CD69 expression marker: significantly increased numbers of CD69+ cells were registered for B lymphocytes and the entire cell population. The stimulation of CD86 co-receptor expression on B lymphocytes was less pronounced, but statistically significant. The ability of single-stranded and double-stranded RNAs to cause significant increase in CD86+ cell numbers was demonstrated among dendritic cell population. The results of the study made it possible to evaluate the effect of dsRNA on the immune cell function, with respect of their interaction, maturation, and migration. This approach may be useful for developing optimal strategies for selection and screening of new nucleic acid-based adjuvants.

395-402 399
Abstract

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a myelopoietic growth factor that exerts pleiotropic effect not only on the differentiation of immature progenitor cells into polymorphonuclear neutrophils, monocytes/macrophages and dendritic cells, but also controls the functioning of differentiated cells. GM-CSF is currently being investigated in clinical trials as an immunomodulator and adjuvant. However, a wide range of biological activities and, sometimes, paradoxical effects of this cytokine require more thorough studies of its action, in order to predict its efficacy under different conditions of immunotherapy. In this work, we have studied the effect of recombinant human GM-CSF on metabolic activity of mouse peritoneal exudate cells in primary cell cultures. Metabolic (redox) activity of the cells was assessed by their ability to reduce nitroblue tetrazolium (NBT) in the course of MF- and Fc-dependent phagocytosis triggered by addition of opsonized zymosan, or sheep erythrocytes to the culture medium. We have shown the dose-dependent stimulatory effect of GM-CSF on the oxidative metabolism of phagocytic peritoneal macrophages and neutrophils. Upon culturing the pepton-elicited cells at wide range of GM-CSF concentrations (5 to 40,000 ng/mL) for 2 and 24 hours, a more pronounced effect of the substance was observed for neutrophils. The GM-CSF preparation caused a significant increase (by 13-17%) in the redox activity of neutrophils induced by opsonized zymosan that persisted at a low dose range, and was retained after 24 hours. The stimulatory effect of GM-CSF on macrophages with NBT index increase by 16% was observed in the short-term cultures. In general, the elicited cells of both types showed a more pronounced response to lower concentrations of GM-CSF (5-125 ng/mL), and weaker effect at higher doses of the preparation. A similar dependence was found when studying the resident macrophages. Culturing of resident cells with GM-CSF at the doses of 5,000 to 40,000 ng/mL for 24 hours caused a significantly increased redox activity of the cells induced by zymosan, or sheep erythrocytes (by 33-52%). In both cases, the maximal response was detected at a dose of 5,000 ng/mL and decreased with increasing dose. The stimulatory effect of GM-CSF upon resident macrophages was more pronounced as compared to elicited cells, which was characterized by the prolonged period of cell activation (up to 24 hours of culture). The data obtained are of interest, in view of prospective usage of GM-CSF as a component of immunomodulatory and adjuvant therapy for various infectious diseases.

403-408 296
Abstract

Severe burn injury (BI) is accompanied by disturbed microcirculation, water-electrolyte and acidbase imbalance within 2-3 days after the accident, and the development of toxemia within 4-12 days. The severity of toxemia depends on the area and depth of the lesion, resorption of tissue decay products, and development of a systemic inflammatory response syndrome. In the patients suffering with deep BI sepsis, it develops in 15% of cases. Pathogenesis of critical conditions is related to the functional activity of myeloid cells, including neutrophilic granulocytes (NG). Тhe determination of NG’s dysfunctions in patients with BI is important, both for prediction of septic complications and administration of rational therapy. The aim of our work was to study the functions of neutrophils in patients with severe BI and to determine early predictors of burn-associated sepsis. The study involved 53 patients with severe BI at the mean age of 43 years (32 to 52); the area of damage was 43% (17 to 63) of the body surface, with deep-burn area of 17 (13 to 27) %. The severity of BI was assessed using the Frank index, at the average value of 74 conventional units (62 to 89). Тwo groups of patients were identified: 24 persons without sepsis, and 29 people with sepsis and severe sepsis. The studies were carried out upon admission, on the 1st, 3rd, 5th, 10th , and 20th day of the burn disease. We determined the numbers of NGs expressing CD18+, CD14+, defensin+; serum contents of soluble defensins (sDеf), IL-6, IL-8 levels (ELISA); procalcitonin, as well as luminol-mediated spontaneous аnd induced NG chemiluminescence. Тhe results of this study showed a relationship between the amounts of NGs containing antimicrobial peptides, contents of NGs expressing CD18+ adhesion molecules, activation of oxidative metabolism, IL-6 overproduction, and development of sepsis in patients with burn injury, as well as with severity of burn trauma.

409-414 541
Abstract

The studies were carried out in 24 sexually mature male guinea pigs. They were used as model of immediate-type allergic reaction (active cutaneous anaphylaxis). The animals were divided in 2 groups: control and experimental. The control group was housed in the mountain valley during the study, and the experimental group stayed for 45 days in high mountains (Anzob, 3375 m above sea level). On the day +30, all animals were sensitized with horse serum. On the 12th day of sensitization, blood was taken for analysis, and on the 15th day, an allergic reaction was provoked. We have revealed that, under high-altitude conditions, the severity of reaction was 1.5 times lower than in controls. Moreover, the animals kept in highlands exhibited lower contents of T and B lymphocytes, and IgE antibodies than in the control group. On the contrary, the numbers of phagocytically active neutrophils, as well as total effect of phagocytosis, proved to be higher in this group. This shift may be facilitated by hypoxia, since aerobic processes are known to prevail in the energy metabolism of lymphocytes, and anaerobic processes dominate in neutrophils. The allergic conditions are developed in three stages. Immunological stage is the first and main one, and allergic restructuring of the body immunity largely depends on it. What is the matter of reconstruction under the high-altitude conditions? We suggest, that, along with Haeckel–M ller biogenetic rule, there is also an immunogenetic law: “The activation sequence of events in the body’s defense system is a reproduction of phylogenesis, i.e., switching of subsequent link in defense system follows the evolutionary principles of complication and improvement. Namely, with respect to failing of previous link to completely eliminate the antigen in defense system”. One may conventionally consider that the first protective barrier against antigens is the most evolutionarily ancient structure, i.e., skin and mucous membrane; the second represents factors of nonspecific defense (phagocytosis, lysozyme, interferon, etc.); the third barrier is presented by cellular immunity (T effector cells, etc.), with humoral immunity serving as the fourth barrier. The fifth protective barrier provides evolutionarily late defense, i.e. allergic reaction, which is triggered by Ig E antibodies. On the mentioned basis, one may state that the decreased allergic reaction (the 5th barrier) in the animals from the experimental group was caused by adaptive changes in protective mechanisms of body. One may suggest that, under the high-altitude conditions an increase was observed in functional activity of phagocytes (2nd barrier), along with a decrease in activity of lymphocytes, i.e., cell populations responsible for the evolutionary later protective barriers.

415-422 313
Abstract

In connection with the increasing incidence and prevalence of inflammatory bowel disease (IBD), the search for prognostic markers of the effectiveness of therapy is an urgent problem. An imbalance between Th17 lymphocytes and regulatory T cells (Treg) is a major defect in the immune system leading to IBD. Extracellular ATP produced during tissue damage, rebound pro-inflammatory effects, and activates Th17 cell differentiation. Ectonucleotidase CD39 catalyzes the dephosphorylation of ATP to AMP, followed by conversion to adenosine by CD73. CD39 is expressed in various cell types, including Treg. Aim – evaluate the functional activity of CD39+ in Treg in children with IBD using the luciferin-luciferase method.

68 children with IBD were examined. Of these, 28 children were in remission, 40 were in exacerbation. The number of Tregs (CD4+CD25highCD127low) expressing CD39 was estimated by flow cytometry. The ATP concentration in supernatants and cells was determined using the luciferin-luciferase test. Results are presented as median (Me) and quartiles (Q0.25-Q0.75). The significance of differences between groups was assessed using the nonparametric Mann–Whitney U test.

The relative number of CD39+Treg in patients in remission of IBD was significantly higher than in patients in a state of exacerbation. A decrease in ATP concentration under the influence of CD39+Treg in patients with IBD occurred immediately upon the addition of exogenous ATP. ATP in patients in remission decreased by 44.5% (Me 54.5 (41.5-65.9)), in patients in exacerbation – by 32.5% (Me 67.5 (59.7-71.3)). At the same time, in patients in remission, the decrease in the ATP content after 5 minutes of the reaction was significantly higher than in patients in the state of exacerbation (p = 0.01), after 30 minutes of the reaction, no significant difference was found. It was shown that samples with a smaller number of cells and a lower intensity of CD39 expression in Treg had a higher activity of CD39 ectonucleotidase.

For efficient ATP hydrolysis, in addition to the amount of CD39 in Treg, their functional activity is important. The assessment of the catalytic activity of CD39 in Treg in patients with IBD is most informative in the first minutes after the addition of exogenous ATP. In patients in remission, the catalytic activity of CD39 in Treg was higher than in patients in a state of exacerbation.



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ISSN 1563-0625 (Print)
ISSN 2313-741X (Online)