Vol 23, No 2 (2020)

CD4⁺T lymphocyte pool is heterogeneous in nature consisting of distinct subsets. The functional activity of each subset is highly influenced by how its energy metabolism is controlled. In naïve, central memory, effector memory, and TEMRA CD4⁺T cells received from healthy volunteers aged 29-42 years we evaluated the level of the major cell energy metabolism regulator: transcriptional coactivator PGC-1α. PGC-1α was shown to be expressed in all CD4⁺T cells. Its level in central memory, effector memory, and TEMRA cells was higher than that in naïve cells for both conventional and regulatory CD4⁺T lymphocytes. Moreover, its level was found to be higher in conventional when compared with regulatory CD4⁺T cells. Our findings suggest that regulatory and conventional CD4⁺T cells rely on PGC-1α to a different extent. Apparently, PGC-1α is an important but not the only factor influencing the functional state of mitochondria in regulatory CD4⁺T lymphocytes.

Thermal skin burns were experimentally modeled in laboratory animals revealed immunotropic effects of new gel metabiotic consisting of complex biological active substances produced by Bacillus subtilis B-3679 strain as part of three gel transcutaneous bases: tizol, eftiderm and organosilicon glycerohydrogel on cellular and humoral immunity. The aim of the study was to evaluate immunotropic activity of a new gel metabiotic on cellular and humoral immunity in experimentally modeled thermal skin burns in 60 white laboratory Wistar rats, weighed 180-220 g, for metabiotic consisting of a complex biologically active substances produced by Bacillus subtilis B-3679 strain containng the three gel transcutaneous bases: tizol, ephtiderm and organosilicon glycerohydrogel (KTGG). Registered, standardized antimicrobial and wound-healing ointment levosin was used as a control. The experimental data obtained confirm the effectiveness of using the drugs studied for treatment of first and second degree thermal burns revealed by faster decreased area of affected area compared to experimental animals treated with levosin. While evaluating therapeutic effectiveness of experimental samples of metabiotic-based gel preparations, their influence on various parameters of cellular and humoral immunity in modeled thermal skin burn was examined. We assessed phagocytic activity of blood neutrophils and peritoneal macrophages, quantified T and B lymphocytes, as well as antibody-forming cells and immunoglobulins of various classes. It was found that in experimental animals reproducing models of traumatic skin injuries, there is an increase in the level of all the studied parameters of humoral immunity. Concentration of immunoglobulin M, circulating immune complexes and immunoglobulin E, as well as various cytokines, increased larger. The level of α-interferon and the receptor antagonist interleukin-1 was elevated. Thus, the study of the humoral status of experimental animals that received test metabiotic-based gel preparations in modeled thermal skin burns, allows to conclude that they significantly influenced various arms both cellular and humoral immunity. More marked effects were observed for metabiotic-based drugs containing tizol and KTGG. In general, based on the experimental data we can conclude that the relatively high therapeutic effectiveness of examined drugs in the treatment of thermal skin burn wounds in experimental animals, use of which provided earlier removal of necrotic masses, accelerated regeneration processes, and prevented development of secondary wound infection in burns.

Plant-derived medicinal products provide a prominent advantage due to their low toxicity to humans and combined effects of biologically active substances, mainly presented by polysaccharides, flavonoids and terpenoids. One of the mechanisms undedrlying effects from medicinal plants on the immunoregulationrelated events is mediated via controlled production of certain cytokines. Here we examined immunoregulatory activity of water extracts derived from medicinal plant raw materials (LRS) containing polyphenolic compounds – flavonoids (rutin, quercetin, called P-vitamins). The aim of the study was to assess profile and level of cytokines secreted by human peripheral blood mononuclear cedlls exposured to flavonoid-containing LRS water extracts. LRS (1:10) water extracts of the following species were used: black currant leaves (Ribes nigrum L.), field horsetail grass (Equisetum arvense L.), common yarrow grass (Achillea millefolium L.), licorice roots (Glycyrrhiza uralensis Fisch.), sand immortelle flowers (Helichrysum arenarium (L.) Moench), wild strawberry leaves (Fragaria vesca L.), fruit common bird cherry (Padus avium Mill.), tansy flowers (Tanacetum vulgare L.) and oat grass (Avena sativa L.) (all purchased at the pharmacy). Production of pro – (TNFα, IL-8, IL-1β) and anti-inflammatory (IL-10) cytokines was measured by using ELISA kits (“Cytokine”, Russia) in mononuclear cell culture supernatant treated with / without LRS (experiment and control group, respectively). Amount of flavonoids contained in flowers and leaves was quantified aftedr complexation reaction with aluminum chloride on UV-3600 spectrophotometer (Shimadzu, Japan). It was found that LRS water extracts predominantly inhibited production both of pro- (TNFα, IL-8, IL-1β) and anti-inflammatory (IL-10) cytokines so that magnitude of such suppressive effect ranged from 51.5±3.4 to 99.5±4.1% compared to untreated control samples (p < 0.05). Total flavonoid level in the LRS samples diirectly correlated with intensity of related immunoregulatory activity on cytokine secretion particularly TNFα (r = 0.65), IL-8 (r = 0.4), IL-1β (r = 0.48) and IL-10 (r = 0.68). The data of our study allow to conclude that extracts from the examined medicinal plant raw materials can be considered as promising components while developing new drugs with exhibiting immunoregulatory and antiflogogenic effects.

Triterpenoid miliacin-related anti-mutagenic effect aftert exposure to isonizing radiation was studied to assess its protective properties against radiation-induced immunosuppression. (CBAxC57Bl6)F1 mice were subdivided into four groups: 1) intact mice; 2) irradiated mice; 3) miliacin-pretreated irradiated mice; and 4) miliacin-solvent-pretreated irradiated mice. Irradiation of animals was performed on the X-ray device “RUST-M1” (4 Gy; exposure time 288 sec.). Twenty four hours post irradiation myelokaryocytes were isolated for analysis. Chromosome preparations were examined by light microscopy. Injections of miliacin (4 mg/kg daily for 3 consecutive days) attenuated the irradiation-triggered mutagenic effect assessed by counting cells with chromosomal aberrations as well as number of aberrations per 100 metaphase plates. Miliacin exerted a protective effect on radiation-induced chromosomal aberrations, although degree of protection was less pronounced compared to chemically induced mutagenesis. These data indicate about limited potential for miliacin to protect central immune organs such as red bone marrow upon radiation exposure.

Here we present the data on examining protein A-gene-positive and -negative Staphylococcus aureus daily broth culture supernaturants for production of cytokine-like substances to determine non-specific activity by using enzyme-linked immunosorbent assay with specific kits (JSC “Vector-Best”, Novosibirsk; LLC “HEMA” (Moscow) to diagnose infections and hormone levels. We found no non-specific cross-activity in all test ELISA kits assessing various Staphylococcus aureus strain supernatants, producing cytokine-like substances identical to human ones, except for HBsAg kit. It may evidence about high specificity for remaining test kits examined and indirectly confirm an assumption that staphylococci are able to secrete proteins specifically reacting mainly with antibodies against human cytokines, and can cross-react with other antigens, but not with antibodies. Cytokine-like or nonspecific activity does not depend on presence or absence of the protein A in Staphylococcus strains examined by us.

Along with functioning in the nerve system, the endogenous opioid peptides as the ligands of mu-, delta-, and kappa-opioid receptors exert multiple effects on immune cells. Moreover, experimental evidence showed that morphine as an exogenous agonist of mu-opioid receptors affects immune cell viability. Such effects were discovered in experiments with cultured cells and laboratory animals. Hence, we studied effects of endogenous opioid peptides dynorphin A and beta-endorphin on viability of human peripheral blood mononuclear cells in vitro. For this, we used samples of peripheral blood cells collected from the fourteen healthy volunteers, who provided with signed informed consent and might request any information regarding the research. Mononuclear cells were collected from the heparinized blood samples according to standard protocol and cultivated in the humid atmosphere for 72 hours. Two μCi ³H-Methyl-thymidine was added into each test tube at 18 hours before the end of the cultivation period. Scintillation counting was performed by using Guardian liquid scintillation analyzer (Wallac, Finland) expressing the data as count per minute. To assess apoptosis, the cells were cultured for 24 hours in similar conditions except for adding radioactive probe. Next, the cells were stained with Annexin V-FITC/7-AAD kit (Beckman Coulter, USA) according to the manufacturer’s instructions for further recording apoptotic cells in flow cytometers BD FACSCalibur (Becton Dickinson, USA) or CytoFLEX S (Beckman Coulter, USA). The lymphocyte gate set by light scatter parameters was shown in typical Annexin V-FITC vs 7-AAD plot followed by counting Annexin V⁺/7-AADcells. All data were expressed as means ± S.E. Statistical significance was assessed by using Student’s t-test. It was found that physiologic concentrations of mu- and kappa-opioid receptor agonists beta-endorphin and dynorphin A exerted multidirectional effects on proliferation of human peripheral blood lymphocytes. In particular, dynorphin A increased basal proliferation and proliferation in response to suboptimal mitogen stimulation. Moreover, beta-endorphin enhanced effects of mitogen stimulation at suboptimal concentration but profoundly suppressed proliferation in maximally activated cells. The modulating effects of beta-endorphin and dynorphin A on in vitro proliferation are not associated with augmented cell apoptosis.

Regulatory rheumatoid factor (regRF) represents a separate rheumatoid factor population that might be potentially used for clinical diagnostics, whereas regRF-producing lymphocytes serve as a promising therapeutic target for autoimmune diseases. Hence, it is worth outlining conditions allowing to reliably detect regRF in humans and laboratory animals. The method of agglutination with tanned IgG-loaded red blood cells was used to measure level of regulatory rheumatoid factor, because it allows to identify solely regRF as compared to the latex fixation method detecting both the regulatory rheumatoid factor as well as the rheumatoid factor associated with rheumatic diseases. It was found that fresh or frozen serum should be used to detect regRF in rats. Freshly purified rat blood plasma stabilized with heparin, sodium citrate or EDTA did not allow to detect regRF, potentially being linked to the presence of fibrinogen known to demonstrate IgG binding activity. In addition, heparin itself also complicates regRF detection in rats. In humans, regRF was reliably detected in pre-frozen plasma stabilized with sodium citrate. Finally, false-negative results were obtained in a small number of cases while using blood serum for detecting regRF in humans.

The aim of the study was to assess pathogenetic significance of TP53 gene mutations in adult acute myeloid leukemia (AML) patients. Clinical observation was carried out on 114 AML patients at the Sverdlovsk Regional Clinical Hospital No. 1 (Ekaterinburg), including 56 males and 58 females. The average age of subjects was 53.3±2.8 years.
Morphologically, AML was previously verified in all cases at specialized laboratories by using standard cytological, cytochemical, immunophenotypic, histological and immunohistochemical methods. The study included the following variants of AML: M0 – 5, M1 – 9, M2 – 47, M2baso – 3, M2eo – 2, M3 – 8, M4 – 25, M4eo – 3, M5 – 3, M6 – 4, M7 – 1, acute myelofibrosis – 1, blastic plasmacytoid dendritic cell neoplasm – 2. Samples of peripheral blood and bone marrow aspirates from patients were examined. Exons 4-11 within the TP53 gene were tested for molecular damage by using sequencing method. In addition, 81 samples, including 22 AML with normal and 23 with an unspecified karyotype were examined for gene mutations by using molecular genetic and immunohistochemical methods. cDNA sequencing was carried out on automatic genetic analyzer in forward and reverse sequences. The sequencing results were processed by using the MEGA X software and statistical hypothesis that they may be described by a binomial distribution. The statistical hypothesis was tested by using Fisher’s exact test and χ² test.
According to the results of cytogenetic and PCR studies, a favorable prognosis was determined in 25 cases (21.9%), intermediate – 24 (21.1%) and unfavorable – in 33 (28.9%). No genetic abnormalities could be detected in 32 samples (28.1%) with standard cytogenetics and real-time PCR, and prognosis option for such patients was not specified.
TP53 missense mutations were revealed as C292T, A377G, A659G, C817T transitions (4 cases) and C569G, G733T, G841C transversions (3 cases); synonymous A639G substitutions were also determined (1.8% ) and C891T (0.9%), in codon position 3, providing no pathogenetic significance. In one sample (0.9%), a deletion of thymidine at position 645 of the coding sequence was determined, leading to produced shortened mutant protein. All the above mutations were localized in the region of the DNA-binding domain. Also, in one case (0.9%), a tandem duplication of 19 nucleotides at position 960 of the coding sequence of the NLS domain protein located in acetylation site. Non-synonymous C215G transversion, which is a polymorphic gene variant, was determined in 94 samples (82.5%). Clinically, all TP53-positive AML were characterized by unfavorable prognosis and primary resistance to standard chemotherapy. The average age of such patients was 63.0±5.4 years, with average follow-up reaching up to 3.1±0.9 months.

In recent decades, the frequency and prevalence of allergic diseases among children have increased dramatically. The aim of the current study was to investigate features of hapten specific sensitization and immune status in different student age groups during high-intensity educational process. A number of parameters related to specific sensitization to metals and organic compounds were studied: 67 first grade children and 35 6-7 grade children attending school with a high-intensity educational process simultaneously chronically exposed to exogenous chemical haptens (the observation group No. 1 and observation group No. 2, respectively); in 20 primary school students and 27 6-7 grade students educated in the absence of excessive exposure to negative factors (the comparison group No. 1 and comparison group No. 2, respectively). Measurement of serum IgE immunoglobulins specific to manganese, IgE specific to nickel, formaldehyde as well as IgG specific to benzene, lead, phenol was carried out by using enzymeallergosorbent test; level IL-4 (Th2) was assessed by enzyme-linked immunosorbent assay; level of CD19⁺ receptor expression on lymphocytes was estimated by flow cytometry. A simple linear regression analysis was used to analyze the data. To test null hypotheses about equality of mean values between two independent groups with a normal distribution, a two-sample Student’s t-test was used. It was found out that the degree of specific sensitization changed with age in students chronically exposed to exogenous haptens. The level of specific sensitization to organic compounds increases markedly in high school students vs. to primary school children (IgG level to phenol elevated by 1.5 times) paralleled with profoundly increased production of Th2 cytokines (IL-4 – by 1.8-fold) as well as activated humoral immunity (percentage of CD19⁺ lymphocytes increased by 1.3-fold). Probable causative link was found between amount of serum exogenous phenol hapten and increased concentration of phenol-specific IgG (F = 140.81; R² = 0.53; p < 0.001). Hapten-specific sensitization in school students increases progressively with age or duration of hapten exposure.

Radiation-induced changes in the immune system develop quite early after the onset of radiation exposure and persist over a long time after it's removal. In case of chronic radiation exposure at dose rate lower than 0.1 Gy/year, the threshold of annual dose to suppress red bone marrow hemato- and immunopoiesis reaches 0.3-0.5 Gy. It was shown that adaptation mechanisms are triggered under the chronic impact of ionizing radiation in the hematopoietic system. In our study we quantitatively and qualitatively analyzed relationships between individual arms of the immune system which is important for understanding features of homeostasis and the adaptation capacity of immune system in chronically irradiated subjects at later time points. The main group included 376 persons exposed to chronic irradiation due to 1949-1960 industrial pollution with radioactive waste residing in Techa River basin. Average radiation dose for the red bone marrow in this group was 1.08±0.04 (0.08-4.46) Gy. The comparison group included 162 unexposed persons. The mean age of people in the main and comparison group was 70.3±0.3 (58-88) and 69.3±0.5 (58-90) years, respectively. The Kendall correlation analysis identified 82 statistically significant correlations (correlation coefficient higher than 0.3, p < 0.05) between individual immune parameters versus 65 similar correlations found in the comparison group. The majority of identified correlation links in both groups ranged from 0.3 to 0.5 (main group – 57 correlations, comparison group – 41 correlations). There were found 16 and 14 correlations in the main and comparison group, respectively, with a coefficient ranged from 0.5 to 0.7. The correlation coefficient value higher than 0.7 was noted for 9 correlations in the main group and for 10 – in the comparison group. The χ-square analysis revealed no significant differences between total number of correlations and number of correlations of varying strength both in the main and comparison groups. The obtained data are consistent with previous studies and confirm that delayed changes in the immune system of subjects exposed to chronic low-rate irradiation were mild and evidenced about developed feedforward and feedback compensatory mechanisms.