Vol 23, No 2 (2020)

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Full Issue


PGC-1α in human CD4+T cell subsets

Vlasova V.V., Shmagel N.G.


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.

Russian Journal of Immunology. 2020;23(2):115-118
pages 115-118 views

Endomorphin-1 affecting innate immune cells in vitro

Gein S.V., Kadochnikova Y.A.


Opioid peptides belong to one of most studied groups of regulatory peptides due to exerting extremely wide range of biological activities and play an important role in regulating homeostasis. The endogenous opioid system is involved in the functioning of diverse organs and systems, including the immune system. Currently, an immunomodulatory effect of the three major families of opioid peptides such as endorphins, enkephalins, dinorphins has been thoroughly described. Much less data are available about the endomorphin-related immunomodulatory effects. The aim of this work was to assess effects of endomorphin-1 on phagocytosis, production of reactive oxygen species and IL-1β by various subsets of peripheral blood innate immune cells in vitro.
The leukocytes of obtained from peripheral venous blood of healthy volunteer donors aged 22 to 40 years were used in the study. Endomorphin-1 was used at concentrations of 10-6, 10-8, 10-10, 10-12 М. Blood leukocyte fraction was isolated from heparinized venous blood settled for 2 hours in a thermostat at 37 °С. The neutrophil fraction was isolated by centrifuging in of Ficoll-Urographin (ρ = 1.077) density gradient placing the upper layer of blood plasma with leukocytes. Assessment of leukocyte oxygen-dependent microbicidal activity was carried out by using the reaction of luminol-dependent chemiluminescence (LZHL) by using opsonized zymosan at concentration of 150 μg/ml; 10-5 M luminol was used to probe reaction magnitude. The separation of the mononuclear cell fraction was carried out by centrifuging in of Ficoll-Urographin (ρ = 1.077) density gradient. The monocyte fraction was isolated mechanically. To assess IL-1β production, mononuclear cells and monocytes were cultured for 24 hours followed by measuring its level with enzyme-linked immunosorbent assay. To evaluate monocytes and neutrophil phagocyte activity, FITC-labeled St.aureus uptake method was used. Statistical processing was performed with one-way ANOVA test and paired LSD criterion for post-hoc comparison, with significance set at p < 0.05.
It was found that endomorphin-1 reduced leukocyte spontaneous, but not induced production of reactive oxygen species. In the neutrophil fraction, endomorphin-1 did not affect spontaneous oxygen dependent microbicidity and reduced intensity of the respiratory burst in stimulated neutrophils. In addition, it increased the percentage of monocyte phagocytosis and enhanced spontaneous IL-1β production by mononuclear cells. Thus, endomorphin-1 exhibited multidirectional effects on various parameters of innate immunity. Being typical to other groups of regulatory peptides, modality of endomorphin-1 related effects depended on cell fraction and presence of a stimulating cues.

Russian Journal of Immunology. 2020;23(2):119-124
pages 119-124 views

Pharmacological assessment of immunotropic activity of new gel metabiotic on cellular and humoral immunity in experimentally modeled thermal skin burns

Zabokritskiy N.А.


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.

Russian Journal of Immunology. 2020;23(2):125-132
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Role of WNT signaling morphogenic proteins (sclerostin and β-catenin) in adipogenesis

Kulakova A.S., Snimshchikova I.A., Plotnikova M.O.


Adipogenesis relies on complex and multi-faceted mechanism, as it is influenced by multiple cues, including the components from the WNT signaling pathway. The search for possible markers of developing metabolic diseases associated with obesity accounted for an interest to study the morphogenic proteins sclerostin and β-catenin. The aim of the study was to evaluate activity of the WNT signaling pathway in obese patients by measuring level of serum sclerostin and β-catenin proteins. Materials and Methods. There were enrolled 32 patients with metabolic syndrome featured with progressive forms of obesity (class I-III) lacking diabetes mellitus. Concentration of serum sclerostin and β-catenin was measured by using enzyme-linked immunosorbent assay. Data were presented as absolute and relative (%) number of patients; arithmetic mean; medians, 1 and 3 quartiles – Ме (Q0.25-Q0.75). In obese patients, serum sclerostin level (260 (230-308.75) pg/ml) was increased by 13.5% compared with healthy individuals (225 (220-230) pg/ml, (p < 0.001)); concentration of serum sclerostin tended to increase depending on obesity class, most in parallel with decreased β-catenin level, being in agreement with previous studies that might be considered as a prognostic criterion for assessing course of pathological process in obesity.

Russian Journal of Immunology. 2020;23(2):133-138
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Immunoregulatory effects of flavonoid-containing medicinal herbs in human peripheral blood mononuclear cells

Mikhailova I.V., Perunova N.B., Ivanova E.V., Chaynikova I.N., Filippova Y.V., Kuzmicheva N.A.


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.

Russian Journal of Immunology. 2020;23(2):139-144
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Immunophenotypical aspects of lung and spleen macrophages derived animals with the model of alloxan diabetes (type I) and their correction by sodium aminodigydrophtalazindione in vitro

Pozdina V.A., Danilova I.G., Abidov M.T.


Macrophages are found in all tissues and organs and display functional plasticity, which is necessary to maintain homeostasis, tissue regeneration and immunity. The macrophage phenotype is determined by microenvironment signals. Macrophages are traditionally classified into subsets- such as classically (M1) or alternatively (M2) activated macrophages. In the pathogenesis of diabetes mellitus (T1DM), M1 macrophages contribute to damage to the islets of Langerhans, loss of β-cells, causing autophagy, which can result in development of persistent infection increasing the risk of death from influenza or pneumonia in patients with type 1 diabetes. Therefore, it seems Important to study functional response of resident macrophages in organs and tissues not targeted in development of diabetes mellitus, as well as in response to ADPH stimulation that showed modulatory effect on immunocompetent cells.
In this study morphological and functional characteristics of macrophage cell cultures obtained from different sites in intact animal (IA) and modeled type 1 diabetes mellitus (DM1) were investigated. For this, we examined macrophage cell cultures isolated from rat liver and peritoneal cavity to be stimulated in vitro for 24 and 72 hours with a sodium aminodigydrophtalazindione. Cells, nucleus, cytoplasm area were measured and nuclear cytoplasmic ratio (NCR) were calculated. The phenotype was determined by surface expression of CD163 (M2-macrophages) and CD80 (M1-macrophages) receptors. Macrophage cytokine activity was determined by measuring IL-1α, IL-10 и TNFα level. ADPH effects on animal macrophages with DM1 after 24 h of exposure also led to a changedmorphometric parameters (decreased size of the nucleus and cells of the spleen macrophages, increased size of the nucleus of the alveolar macrophages, increased NCR in spleen macrophages) and production activity of the cells (increased levels of IL-1α and TNF α in almost all cell populations). After 72 h of cultivation, the levels of IL-1α and TNFα decreased in alveolar macrophages, splenic macrophages, whereas TNFα level was decreased, but IL-1α asmount was increased. The expression of surface cell markers for M1 and M2 phenotypes was also affected by ADPH so that CD163 expression was increased in stimulated alveolar macrophages isolated from animals with type 1 diabetes.

Russian Journal of Immunology. 2020;23(2):145-152
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Evaluation of triterpenoid miliacin-related anti-mutagenic effect in mice exposed to ionising radiation

Sarycheva Y.A., Tokareva A.A., Shtil A.A., Kolyvanova M.A., Morozov V.N., Panfilova T.V., Frolov B.A.


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.

Russian Journal of Immunology. 2020;23(2):153-156
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Generation of human myeloid suppressor cells in the in vitro experimental model

Timganova V.P., Bochkova M.S., Uzhviyuk S.V., Shardina K.Y., Zamorina S.A., Rayev M.B.


Myeloid suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that generally differentiate into macrophages, granulocytes, and dendritic cells. However, in pathology, these cells acquire a suppressor phenotype, blocking immune response. In particular, MDSC levels increase in many pathological conditions, including inflammation, sepsis, traumatic shock, autoimmune diseases, cancer, and pregnancy. Over the past 12 years, an interest in examining this cell population has been steadily increased [PUBMED: 2008 (65 articles); 2020 (> 650 entries)] that will expand our understanding of immune system functioning. In humans, MDSCs are characterized by HLA-DR-CD33+CD11b+ phenotype, in turn being subdivided into CD15+ or CD66+ granulocytic (G-MDSC), CD14+ monocytic (M-MDSC), and early (e-MDSC) MDSC bearing HLA-DR-CD11b+CD33+CD14-CD66b- phenotype. This work was aimed to develop an adequate experimental model allowing to evaluate cytokine-driven differentiation of human MDSCs from peripheral blood mononuclear cells in long-term in vitro culture system. For this, peripheral blood mononuclear cells were isolated from healthy donors induced to express MDSC phenotype with GM-CSF and IL-6 (40 or 20 ng/ml) cultured for 7, 14, 21 days. In several experiments, LPS (100 ng/ml) was added to the cultured cells 24 hours before immunophenotyping. The percentage of living Zombie Aquanegative cells in cultures (gated on cells according to FSC/SSC) ranged from 90.5-93.9%. No significant differences were observed between cultured cells. In our experimental conditions, the mean percentage of total MDSC subpopulation reached 2-2.3% of total living cells, exceeding that one by 9-10-fold found in freshly isolated mononuclear cells from healthy subjects. Based on the results of our experimental study, we found that induction of e-MDSC derived from human peripheral blood mononuclear cells requires two weeks of co-culture with 40 ng/ml IL-6 and 40 ng/ml GM-CSF. “Mature” MDSCs (M-MDSC + G-MDSC) yield was peaked in the following conditions: co-culture for 3 weeks with 20 ng/ml IL-6 and 20 ng/ml GM-CSF added with 100 ng/ml LPS 24 hours before completing protocol. Overall, further examining factors modulating MDSC differentiation will reveal conditions necessary for generating this suppressor cell subset potentially used in clinical practice.

Russian Journal of Immunology. 2020;23(2):157-162
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ELISA-based comparative analysis of non-specific activity for cytokine-producing protein a gene-positive and negative Staphylococcus aureus strains

Fayzullina A.I., Zurochka A.V.


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.

Russian Journal of Immunology. 2020;23(2):163-168
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Non-specific activity of Staphylococcus aureus strains producing and not producing cytokine-like substances assessed by immuno-enzyme analysis

Fomina L.O., Zurochka V.A., Dobrynina M.A., Gritsenko V.A.


A cytokine profile covering cytokine level in various biological fluids isa of great importance un ummunbe-based diagnostics, examinign parameters and stated bof functional asctivity of immune system organs. While using ELISA asw oner of the most common diagnostics tools, it is worth taking into account origin of biological samples. Daily Staphylococcus aureus broth culture supernaturants, positive and negative for cytokine-like substances, were used to determine non-specific activity by using enzyme immunoassay using kits pruchased from the JSC “Vector-Best” (Novosibirsk) as well as kits of LLC “HEMA” (Moscow) to diagnose some infections and hormone levels. We found that in supernatants from some Staphylococcus aureus strain producing cytokine-like substances identical to human, the absence of non-specific cross-activity assessed by ELISA was documednted exсept for the HBsAg test kit, which may indicate high specificity of the other kits studied and indirectly confirm an assumption that staphylococci secrete proteins specifically reacting mainly with antibodies against human cytokines, but can also cross-react with other antigens, but not antibodies.

Russian Journal of Immunology. 2020;23(2):169-174
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Effects of beta-endorphin and dynorphin A on in vitro apoptosis in human peripheral blood lymphocytes

Yuzhaninova S.V., Gileva S.G.


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.

Russian Journal of Immunology. 2020;23(2):175-180
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Evaluating T and B cell lineage differentiation stage and related prognostic impact in miscarriage

Baturina I.L., Loginova Y.V., Nikushkina K.V., Emelyanov I.V., Zotova M.A., Nikonova T.I.


The WHO (World Health Organization) defines miscarriage as two or more pregnancies documented in woman medical history ended up with spontaneous miscarriage at stage of up to 22 weeks of pregnancy from conception. Habitual miscarriage of pregnancy (HMP) is one of high priority problems for infertile marriages comprising 5-20% among other causes of miscarriage. As many as 80% of all idiopathic HMP cases are associated with immune-related disorders. Currently, many researchers have being paying more attention to examining effects of T and B cells at varying differentiation stage in developing immune response against allogenic graft and formation immunological tolerance. However, it is worth noting that pregnancy-associated human hormone chorionic gonadotropin (HCG) exhibits a marked immunosuppressive effect by regulating regulatory T (Treg) cell functional activity. It has been proven that HCG contributes to increasing Tregs level in periphery and recruiting them to the embryo implantation zone, thus establishing immunological tolerance during pregnancy. However, no data regarding HCG effects on T and B cells at various differentiation stage were obtained during HMP. A correlation analysis between frequency of T and B cells at various differentiation stage and level of serum HCG in women with HMP was performed. It was found that low level HCG was associated with decreased naive T and B cell frequency paralleled with elevated percentage of effector memory CD8+ (Tem) that may serve as a poor prognostic sign especially in early stage of pregnancy.

Russian Journal of Immunology. 2020;23(2):181-186
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Detection of immunoregulatory rheumatoid factor

Beduleva L.М., Sidorov A.Y., Abisheva N.N., Gilmanova L.U., Gorbushina A.N.


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.

Russian Journal of Immunology. 2020;23(2):187-190
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Analyzing IL6 gene polymorphism in patients with various clinical forms of pulmonary tuberculosis

Belyaeva S.V.


Throughout the world, tuberculosis still remains one of the most important public health problems. According to numerous publications, TB infectrion is considered as a genetically determined disease, and host genetical polymorphism underlies a mechanism resulting in disease progression from primary infection to clinical manifestations. Currently, an association between TB infection and IL6 gene polymorphism at position -174 responsible for low cytokine production has been extensively investigated. Previously, we evaluated the association between IL6 gene alleles and genotypes and predisposition/resistance to tuberculosis in Russian descendants residing in the Chelyabinsk Region. Here, we conducted a comparative analysis for prevalence of IL6 gene alleles and genotypes in patients differed in lung damage degree such as infiltrative, focal, and fibrous-cavernous forms of pulmonary tuberculosis by isolating whole blood DNA samples and subsequent genotyping of IL6 gene polymorphisms with polymerase chain reaction and agarose gel electrophoresis. We found that patients with a focal TB form were characterized by prevalence of IL6(-174)*C/C genotype associated with low cytokine production, whereas patients with severe fibrous-cavernous carried IL6(-174)*G/C genotype accounting for modedrated IL-6 production.

Russian Journal of Immunology. 2020;23(2):191-194
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Pathogenetic value of TP53 point mutations in adult acute myeloid leukemia patients

Vinogradov A.V., Rezaykin A.V., Litvinova D.V., Loboda A.N., Sazonov S.V., Sergeev A.G.


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.

Russian Journal of Immunology. 2020;23(2):195-202
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Experience of applying flow cytometry to assess immunologic reactivity in subjects with increased blood vanadium concentration

Dianova D.G., Dolgikh O.V., Alekseev V.B.


Environmental pollution with external vanadium results in its accumulation in human biological media and alters ability of human body to properly react to negative impact after exposure to haptens. Aim of the work was to assess immunologic reactivity in subjects bearing increased blood vanadium (hapten) level by using flow cytometry. There were examined adults bearing various blood vanadium concedntration. The test group was comprised by adult subjects (n = 50) having blood vanadium concentration exceeding that one for the upper limit of the reference level and mean reference group (n = 42) corresponding to reference range by 3.7- (р < 0.001) and 4.3-fold, respectrively, It was found that subjects with excessively vanadium-contaminated serum were featured with stronger lymphocytes activation, increased count of effector helper T cells, lowered count of regulatory lymphocytes and decreased humoral immune arm, and suppressed p53-dependent control of cellular cycle. Thus, flow cytometry allows to assessing immunologic reactivity in subjects with varying level of blood exogenous chemicals as well as determining body adaptation potential to ewffects exerted by environmental chemical cues.

Russian Journal of Immunology. 2020;23(2):203-208
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Features of hapten specific sensitization and immune status in different student age groups

Dolgikh O.V., Dianova D.G.


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.

Russian Journal of Immunology. 2020;23(2):209-106
pages 209-106 views

Sensitization to inhaled and food allergens in children

Emelina Y.N.


A retrospective analysis of the annual allergen sensitization detected during skin testing in children aged 3-17 years was performed. Significant positive data were more frequently (p < 0.01) detected after testing for indoor (70.2%), food (72.7%) and pollen allergens (67.9%) compared to epidermal allergens (58.6%). Sensitization to house dust (43.6%) and library dust (25.7%), guinea pig hair (22.6%), cat hair (21.8%), horse dandruff (19.6%), sheep wool (16.8%), as well as pollen of trees (82.4%), meadow grasses (64.0%) and weeds (64.1%) was most common in group of inhaled allergens.
Skin testing in the group of food allergens revealed that children were more sensitized to chicken eggs, fish, milk, chicken and wheat. Among food cereals, peak percentage of positive reactions was observed for wheat (9.9%) and barley groats (9.1%) compared to oatmeal (4.4%), rye flour (5.0%) and buckwheat (5.6%).
While assessing the data in all allergen groups it was found that low positive reaction with 2-3 mm blister in diameter was most common. The frequency of detected positive data for all allergen groups varied markedly on different calendar months and also depended on allergen preparation batch. Intensity of reactions did not significantly differ in children with allergic diseases vs. those at risk of developing allergopathology.

Russian Journal of Immunology. 2020;23(2):217-224
pages 217-224 views

Delayed correlated parameters of adaptive and innate immunity in chronically irradiated subjects

Kodintseva Е.А., Akleyev А.А.


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.

Russian Journal of Immunology. 2020;23(2):225-230
pages 225-230 views


Козлов Владимир Александрович (к 80-летию со дня рождения)


Russian Journal of Immunology. 2020;23(2):231-232
pages 231-232 views

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