Vol 25, No 3 (2022)

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Full Issue


Effect of graphene oxide nanoparticles on viability of BAP3 hybridoma cells

Lazarev S.S., Bochkova M.S., Timganova V.P., Rayev M.B.


Graphene oxide (GO) is a promising material, which is likely to find applications in the fields of medicine and biotechnology. However, the current knowledge of its influence on human organism is limited. Even less information is available on the effects of GO on the cell lines widely used in biotechnology. The aim of this work is to describe the interaction between GO nanoparticles and BAP3 hybridoma cells which produce anti-human-PSG1 IgG, in vitro. We studied the effect of GO nanoparticles on cell viability and the intensity of internalization (adhesion) of nanoparticles by the cells. We used GO nanoparticles of different size, with surface being functionalized by linear or branched PEG (GO-PEG). The PEG coating level was 20% (by mass). The following nanoparticle concentrations were used: 5 μg/mL and 25 μg/mL. The BAP3 cells were cultured in a 48-well cell culture plates in serum-free DCCM-1 media in the presence of GO nanoparticles. The cells were cultured for 24 hours at 37 °С and 5% СО2. Cell viability was assessed by a flow cytometer utilizing Zombie Aqua (ZA) staining. Internalization (adhesion) of nanoparticles was monitored using a flow cytometer by GO fluorescense in the samples (λex = 488 nm). Moreover, interactions between hybridoma cells and GO nanoparticles were visualized by EVOS M5000 visualization system, which included an inverted fluorescent microscope.

We demonstrated that GO nanoparticles possess a cytotoxic effect when applied at high concentration (25 μg/mL). The highest cytotoxic effect is caused by GO nanoparticles coated with linear PEG. The degree of nanoparticle internalization (adhesion) was shown to be significantly lower when the particles were present at lower (5 μg/mL) concentration. Internalization (adhesion) of nanoparticles of smaller size was more abundant. Furthermore, these nanoparticles were shown to have a stronger cytotoxic effect compared to larger particles. In general, cytotoxicity of GO nanoparticles decreases with increasing size, which is especially evident if the fact that the mean effective diameter of the nanoparticles coated with branched PEG is considered larger than their linear PEG-coated counterparts. The data obtained allow us to draw a correlation between the cytotoxic effect of GO nanoparticles and the level of their internalization (adhesion) by the cells. In general, this work concerns some novel aspects of interaction between GO nanoparticles and hybridoma cells.

Russian Journal of Immunology. 2022;25(3):245-250
pages 245-250 views

Factors of inflammatory, adhesiveness and thrombosis in acute lower limb ischemia and dexamethasone therapy

Magamedov I.D., Pivovarova L.P., Nokhrin S.P., Soroka V.V., Ariskina O.B., Osipova I.V., Radjabov I.M., Fomin K.N., Potskhor-ogly S.L., Kolichenko L.V., Markelova E.V., Goncharova O.V.


Lymphocyte-to-platelet adhesion during hypoxia, tissue damage, activation of inflammation and coagulation is associated with expression of ICAM-1 membrane molecules by blood and tissue cells. At the same time, the platelet adhesion receptors determine their adherence to endothelium and recruited lymphocytes. Moreover, the role of platelets in pathogenesis of ischemic cardiovascular diseases comprises their ability to modulate both hemostasis and inflammatory reactions, which are accompanied by secretion of inflammatory mediators and some factors that promote recruitment of leukocytes to tissue damage sites. Creatine kinase activity is a sensitive marker of tissue damage and tissue ischemia. The purpose of the present study was to assess the effect of anti-inflammatory therapy with dexamethasone upon the intensity of inflammation and adhesive properties of lymphocytes, number of platelets in peripheral blood of the patients with acute lower limb ischemia (ALLI), as well as to evaluate the effectiveness of treatment.

To study the effect of anti-inflammatory therapy, a group of 32 patients treated with dexamethasone was selected; the control group was represented by 71 patients with basic therapy, the comparison group consisted of 15 volunteers. After revascularization, all patients received antiplatelet and anticoagulant therapy. Dexamethasone infusions were carried out as a course of 4 to 6 days after reconstructive surgery. In all patients, the content of C-reactive protein in blood, the activity of creatine kinase, the content of platelets and, especially, of enlarged platelets were determined. The numbers of lymphocytes expressing ICAM-1 (CD54+) adhesion molecules were counted using immunocytochemical technique. The studies were performed before surgery and 1, 3, 5, 7, 10 days after surgery.

During exacerbation of the limb ischemia and damage to endothelium, the accumulation of cytolysis products was noted. Expression of adhesion molecules was increased both on endotheliocytes and on inflammation effector cells, i.e., leukocytes and platelets. The adhesion molecules transmit the activating signal inside the cell, thus promoting adhesion of leukocytes and platelets to endothelium, lymphocytic-platelet adhesion, formation of parietal thrombi, and possible occlusion of damaged vessels. Increased expression of adhesion molecules is associated with activation of metabolism, inflammation, coagulation and oxidative stress. It may stimulate all hematopoietic lineages, including platelets. The involvement level of cellular reactions in pathogenesis of the disease affects effectiveness and duration of treatment, risk of recurrent thrombosis and lethal outcome. Anti-inflammatory therapy with dexamethasone contributed to earlier remission, it was associated with lower frequency of infectious and thrombotic complications, decreased mortality, and reduced duration of treatment.

Inflammation, adhesiveness of effector cells and thrombosis are important factors in pathogenesis of acute lower limb ischemia. Additional anti-inflammatory therapy with dexamethasone contributes to earlier remission, decreased proportion of infectious and thrombotic complications, lower frequency of deaths, and reduced duration of treatment.

Russian Journal of Immunology. 2022;25(3):251-258
pages 251-258 views

Comparative effect of ozone exposure and 5-aminosalicylic acid in oxazolon-induced colitis upon the indices of innate immunity

Osikov M.V., Kaigorodtseva N.V., Boyko M.S., Fedosov A.A.


Multiple effects of ozone are a prerequisite for its use in the complex therapy of inflammatory bowel diseases. Our aim was to analyze the effects of ozone and 5-aminosalicylic acid (5-ASA) in oxazolone-induced colitis (OIC) upon the innate immunity functions.

OIC was modeled in 72 male Wistar rats weighing 240±20 g by a two-stage application of oxazolone solution. Ozone at a dose of 0.05 mg/kg, as a part of ozone-oxygen mixture, was injected intraperitoneally every 24 hours. The laboratory indices were studied on the days 2, 4, 6. In blood samples, the number of leukocytes, differential leukocyte counts, absorptive capacity of blood neutrophils and their oxygen-dependent metabolism were studied. In homogenate of the colon mucosa, IL-17 concentration was measured.

In OIC, on days 2, 4, and 6, the number of blood leukocytes was increased, with a predominance of lymphocytes, monocytes, neutrophils and their absorption capacity; the concentration of IL-17 increased in the areas of colonic damage. On days 2 and 4, the activity and intensity of the spontaneous NBT test is increased like as activity and intensity of induced NBT-test on days 2 and 6. Administration of ozone reduced the content of blood leukocytes, lymphocytes, neutrophils on days 2 and 6, like as their absorption capacity on days 2 and 4, along with a decrease in NCT-reducing ability on days 6, and decreased IL-17 concentration in the area of colonic damage on days 4 and 6. The effects of ozone administration in OIC, if compared with 5-ASA, are less pronounced on days 2 and 4, with respect to decreased number of monocytes, neutrophils in blood and their absorption capacity on days 2, like as IL-17 concentration in the area of colonic damage on days 4 and 6.

Russian Journal of Immunology. 2022;25(3):259-266
pages 259-266 views

Evaluation of specific T cell immune response to SARS-CoV-2 in COVID-19 infection and following Gam-COVID-Vac vaccine prophylaxis

Plekhova N.G., Sitdikova T.S., Dubiy A.A., Mikhailov A.O., Prosekovа E.V.


An aberrant immune response during SARS-CoV-2 infection has been shown to determine the clinical features, disease severity, and progression of COVID-19 infection. This work aimed for comprehensive assessment of the immune response by comparative evaluation of diagnostic significance of the antibodies to RBD domain of the SARS-CoV-2 spike protein, as well as detection of effector CD4+ and CD8+T cells specific to SARS-CoV-2 antigens. The study was performed in unvaccinated persons, healthy individuals vaccinated with Gam-COVID-Vac, and in the patients who have had COVID-19 infection. We have found that IgG antibodies to the RBD domain of the SARS-CoV-2 spike protein are detectable at a frequency of 73% to 92% of cases in vaccinated persons and COVID-19 reconvalescents. The numbers of effector CD4+ and CD8+T lymphocytes responding to stimulation with SARS-CoV-2 antigens by producing the IFNã cytokine varied depending on the introduced antigen and tended to be higher in vaccinated individuals. In non-vaccinated healthy persons who contacted with COVID-19 patients, T cell response to the SARS-CoV-2 nucleoproteins was revealed. For adequate assessment of antiviral and post-vaccination immune response to COVID-19, it would be necessary to study not only humoral immune response by the presence of antibodies, but also functionally active specific T lymphocytes directed for SARS-CoV-2 protein antigens.

Russian Journal of Immunology. 2022;25(3):267-274
pages 267-274 views

Association of gene polymorphism and cytokine content in the blood serum in children with allergic bronchial asthma

Prosekova E.V., Dolgopolov M.S., Turyanskaya A.I., Sabynych V.A.


The study of genes controlling cytokine activities of is among important tasks when assessing predisposition and revealing pathogenetic links of initiation and course of clinical disorders. Aberrant production of cytokines and dysregulation of immune response may be considered genetic predictors associated with differentiation and functioning of T helpers, being of decisive importance in pathogenesis of pediatric allergic bronchial asthma. Our objective was to evaluation of associations between polymorphic genotypes and serum levels of cytokines of various T helper profiles in the children with allergic bronchial asthma.

We have observed 175 children aged 3 to 11 years. Of them, we have examined 75 patients diagnosed with allergic bronchial asthma (ABA) as well as 100 healthy children matched for age and gender. All children underwent general clinical and allergological examination. The contents of cytokines attributed to Th1, Th2 and Th17 profiles were determined in blood serum by means of ELISA technique. DNA samples isolated from peripheral venous blood were used for molecular genetic analysis. Using allele-specific PCR technique, the following mutation points were investigated: IFNγ (T-874 A), IL-4 (C-589 T), IL-6 (C-174 G), IL-17A (G- 197 A), TNFα (G-308 A). The analysis of distribution and occurrence of the cytokine gene polymorphisms was carried out, and the odds ratio of the disease risk were calculated. Statistical data processing was carried out using the program “Statistica 10” by methods of descriptive, parametric and non-parametric statistics, comparison of unrelated groups was performed by qualitative characteristics of Hardy–Weinberg equilibrium, and with Chi-square test (χ 2).

These studies have revealed differences in patterns and occurrence of polymorphic genotypes associated with aberrant production of cytokines typical for various Th profiles among the children with allergic bronchial asthma. A comparative analysis of the mutant allele frequencies and cytokine genotypes of various Th profiles, along with determination of the cytokine contents in blood serum of children with allergic bronchial asthma revealed a predominance of homozygous IFNγ 874A, IL-4 589T, IL-6 174G, IL-17A 197A, and TNFα 308A genotypes. Studies of gene polymorphisms, features of production and content of the cytokines specific for T helpers 1, T helpers 2, T helpers 17 profiles in bronchial asthma in the children revealed differences in distribution and occurrence of mutant alleles associated with aberrant cytokine production, variable risk of developing allergic pathology and development of the distinct disease phenotype.

Russian Journal of Immunology. 2022;25(3):275-282
pages 275-282 views

Expression of CD39 and CD73 ectonucleotidases in CD4+ lymphocyte populations in healthy children

Radygina T.V., Kuptsova D.G., Petrichuk S.V., Semikina E.L., Fisenko A.P.


Purinergic system plays an important role in functional regulation of immune system. Extracellular ATP belongs to non-infectious danger signals (DAMP), has a pro-inflammatory effect and modulates the immune response. The end product of ATP breakdown, adenosine, plays an important role in limiting the inflammatory response. The activity of ectonucleotidase CD39 and CD73 supports the balance of ATP and adenosine at the site of inflammation. CD39 and CD73 expression is characterized by high variability. From the literature data, appropriate studies were carried out either in transgenic mice, or with adult healthy donors. The number of cells expressing CD39 and CD73 in T lymphocyte populations has not been evaluated in healthy children. Hence, our aim was to study the features of CD39 and CD73 expression in various subpopulations of CD4+ lymphocytes in apparently healthy children of different ages.

We examined 45 healthy children aged 3.7 to 17.5 years (Me (Q0.25-Q0.75) – 12.4 (10-16.1). The numbers of CD4+ cells, regulatory T lymphocytes (Treg – CD4+CD25highCD127low), activated T helpers (Tact – CD4+CD25+CD127high), and Th17 lymphocytes (CD4+CD161+CD3+) expressing CD39 and CD73 were evaluated by the flow cytometry.

The number of cells expressing CD39 and CD73 depends on specific cell subpopulation. The highest content of CD39+ cells was observed in Tregs, and maximal amounts of CD73+ cells were found among Tact subset. In the Th17 lymphocyte subpopulation, there was no significant difference between the number of cells expressing CD39 and CD73. E have also shown an increase in the relative number of Th17 cells expressing CD73, along with age-dependent decrease in the relative number of Tact cells expressing CD39. An age-dependent decrease in absolute values with age was revealed for Treg, CD39+Treg, CD73+Treg, CD39+Th17, thus being consistent with age-related decrease in absolute numbers of lymphocytes and CD4+ cells.

The obtained data concerning specific pattern of ectonucleotidases expression in functionally different populations of CD4+ lymphocytes should be taken into account when studying children with immune-mediated diseases from different age groups.

Russian Journal of Immunology. 2022;25(3):283-290
pages 283-290 views

Relationships between the indices of innate immunity in sepsis depend on clinical outcomes

Savochkina A.Y., Poltorak A.E., Nikushkina K.V., Zotova M.A.


At the present time, sepsis is primarily considered a disorder of the immune system caused by dominant infection, manifesting with individual response reaction of the host organism. Hence, sepsis should not be classified as a distinct pro- or anti-inflammatory syndrome, but rather as a variable continuum of overlapping immune mechanisms. The role of innate immunity in sepsis is probably the leading one, since it exerts an early and nonspecific response to any foreign agent. The nature of appropriate relationships may determine current state of the immune system. To perform a deeper study of the anti-infectious protective mechanisms, we have evaluated the relationships between the immunological parameters of peripheral blood in patients with sepsis, depending on the outcome of the disease. Total number of leukocytes, and neutrophils was determined in whole peripheral blood of the patients with sepsis, along with calculation of leukocyte counts and numbers of neutrophil extracellular traps. Serum levels of procalcitonin, peptidyl-arginine deiminase 4, and cytokines were studied by ELISA assay. To study the relationship between the indices of innate immunity, we used correlation analysis, which was carried out by the Spearman rank criterion. When conducting the statistical analysis, strong correlations between the immunological parameters of peripheral blood in patients with sepsis were not revealed, regardless of the outcome of the disease. In cases of favorable outcome, 10 relationships were identified, with an unfavorable outcome, 7 relationships were registered. All the relationships were of medium strength. Regardless of clinical outcome of sepsis, the significant relationships were established between the number of leukocytes and the level of neutrophil extracellular traps; positive relationships were also found between pro-inflammatory cytokines IL-1 – IL-6, TNF – IL-18. However, these relationships were significant in cases with favorable outcome, while becoming weaker and losing their significance for the group with lethal outcomes.

Among the revealed relationships, the most interesting finding was an association between favorable outcome of sepsis with IL-10 contents, which is an anti-inflammatory cytokine coordinating the innate immune response. In lethal outcomes, such relationship with IL-10 was not revealed. IL-10, a cytokine with anti-inflammatory properties may limit the immune response to pathogens and, thus, potentially prevent damage to the host. Therefore, the relationship between IL-10 and other immunological parameters of peripheral blood in sepsis may affect the outcome of this condition.

Russian Journal of Immunology. 2022;25(3):291-294
pages 291-294 views

Cytokine system in the patients with type 2 diabetes mellitus non-infected with SARS-CoV-2

Savchuk K.S., Simbirtsev A.S.


Diabetes mellitus (DM) is a common chronic non-communicable disease, being the most significant comorbidity in SARS-CoV-2 viral infection. The proportion of DM patients among those with COVID-19 is up to 25.0% in the Russian Federation. In presence of DM, clinical course of COVID-19 is characterized by greater severity and persistence of pulmonary damage, an increased need for immunosuppressive, glucocorticoid and combined antiviral therapy in COVID-19 patients, and with prolonged rehabilitation period. Pathogenetic effects of DM on severe course of the SARS-CoV-2 viral infection are being actively studied. The following factors were considered, e.g., negative impact of hyperglycemia on the course of infection; direct cytotoxic and indirect damage to pancreatic β-cells with further activation of pro-inflammatory mechanisms; cumulation and progression of generalized inflammation common to DM and COVID-19 including impaired production of cytokines; influence of SARS-CoV-2 virus on the renin-angiotensin-aldosterone system causing inhibition of insulin secretion and increased insulin resistance. Chronic inflammation and impaired immune response may be among the main mechanisms of association between type 2 DM (T2DM) and COVID-19. It is important to identify systemic inflammatory disorders in patients with type 2 diabetes, which may be associated with greater disease severity, being of negative prognostic value. The aim of the present work was to investigate concentrations of some serum cytokines in the patients with type 2 diabetes not infected with SARS-CoV-2.

The study included 20 patients with type 2 diabetes; the control group consisted of 11 clinically healthy volunteers. The serum concentration of 13 cytokines was assessed by multiplex analysis on a MAGPIX-100 immunoanalyzer using a Merck multiplex analysis kit (Germany), in accordance with the manufacturer’s instructions.

Increased serum concentrations in T2DM patients were found, as compared with the control group for some key pro-inflammatory cytokines: CX3CL1, TNFα, IFNγ, IL-8, IL-17A, MIP-1α, and MIP-1β. We have also revealed a decrease in serum concentrations of IL-4. Serum immunoregulatory cytokines in the T2DM were found to be changed in different directions: a decrease in IL-5, along with increase of IL-12p70 and IL- 17, whereas the serum contents of IL-2, IL-13 did not change.

A comprehensive analysis of serum cytokine concentrations may increase clinical significance of assessing serum cytokine concentrations as prognostic and diagnostic markers, as well as therapeutic targets in type 2 DM, like as in SARS-CoV-2 infection.

Russian Journal of Immunology. 2022;25(3):295-298
pages 295-298 views

Mast cells as biomarkers of inflamm-ageing

Sadek A., Khramtsova Y.S., Yushkov B.G.


Most mechanisms of ageing are believed to be more or less associated with inflammation. With age, a unique form of chronic inflammation develops which is termed as inflamm-ageing. The mechanisms of this process are still not fully clear due to the lack of reliable assessment criteria. Immune system is among those involved in accelerating age-related changes in the body. It also directly participates in the process of inflammation. In its pathogenesis, the reaction of mast cells may be of great importance. The role of mast cells in tissue remodeling deserves special attention, since the latter event is among the main features associated with ageing. Hence, the “inflamm-ageing” is considered a sufficient indicator of ageing, and the mast cells could provide biomarkers of this process. In order to test the proposed hypothesis, the present study was conducted to determine age-related morpho-functional changes in mast cell populations in various organs in rats. Some morpho-functional parameters of mast cells (number, synthetic and functional activity, degree of maturation) in different animal organs were evaluated in male Wistar rats of different ages (4 months and 2 years). We have found the age-dependent changes upon examination of thymus, adrenal glands, and skin, i.e., a decrease in the number of mast cells and their synthetic capacity, along with significantly increased functional activity. In the stomach, small and large intestines, at the constant number of mast cells, we revealed a decrease in their synthetic ability, and increased functional activity. These changes were accompanied by enlargement of blood vessels in the studied organs. Liver is the only organ which did not exhibit any changes in mast cell populations with age. The detected changes in mast cell populations may play an important role in formation of “inflamm-ageing” events, which accompany the ageing processes, because these cells are an integral component of inflammatory response. The progression of “inflamm-ageing” leads to accumulation of cytokines and pro-inflammatory mediators in tissues, which, in turn, activate the mast cells. At the same time, increased degranulation of mastocytes may promote the process of “inflamm-ageing”. The oberved mutual influence of mast cells and “inflamm-ageing” makes it possible to consider mastocytes as potential candidates for searching the biomarkers in “inflamm-ageing”.

Russian Journal of Immunology. 2022;25(3):299-304
pages 299-304 views

Limited mutagenesis of myelokaryocytes following acute external irradiation as a protective mechanism of miliacin in radiation-induced immunosuppression

Sarycheva Y.A., Tokareva A.A., Shechtman A.G., Panfilova T.V., Pimenova Y.S., Mitrofanov R.A., Frolov B.A.


Antimutagenic effect of the plant triterpenoid miliacin was studied, in order to characterize its protective properties in a model of acute irradiation immunosuppression using outbred male mice. Ionizing irradiation at different doses (0.5; 1.0; 2.0; 4.0 Gy) was used for experimental (miliacin-treated), and control animals that received the miliacin solvent. Miliacin was administered three times intraperitoneally at a single dose of 4.0 mg/kg with 24-hour intervals between injections. The last dose was applied 1 day before irradiation. Myelokaryocytes served as test objects, the analysis of which was carried out 24 hours after irradiation. Miliacin had a certain protective effect by limiting the post-radiation myeloablation, reducing the number of aberrant cells and the total number of aberrations. Protective effect of triterpenoids showed inverse relation to the radiation dose, being most pronounced at the dose of 0.5 Gy. Higher values of chromatid aberrations at radiation doses of 1.0 and 2.0 Gy in animals from the experimental group versus control mice, probably, due to anti-apoptotic effect of the triterpenoid, thus ensuring higher survival rates of mutated cells with severe damage to their genome. The protective effect of miliacin at 24 hours after radiation exposure may indicate its effect on the primary radiochemical stage of radiation injury. It is suggested that the mechanism of protective action of triterpenoid is mediated by its previously shown antioxidant activity, due to its ability to stabilize membranes and normalize expression of genes encoding antioxidant protection enzymes. Thus, the antimutagenic activity of miliacin after irradiation is an important characteristic of its immunoprotective effect during the radiation-induced immunosuppression. With respect to its ability to limit the mutagenic effect, miliacin may be classified as a weak radioprotective antimutagen with a protection efficiency of 20-40% at the dose range of 0.5 to 1.0 Gray.

Russian Journal of Immunology. 2022;25(3):305-312
pages 305-312 views

Assessment of lymphocyte populations and their subsets in the patients with acute coronary syndrome

Safronova E.A., Ryabova L.V.


We examined 23 patients aged 40 to 65 years (mean age 54.52±6.72) with a diagnosis of acute coronary syndrome (ACS) at admission, who underwent emergency or delayed coronary stenting a day later. All patients had arterial hypertension as a concomitant disease. Upon additional examination, blood troponin levels were determined, ECG was performed in the time dynamics. Acute myocardial infarction with ST elevation was diagnosed in 7 patients, infarction without ST elevation, in 6 patients, the unstable angina rest, in the rest of this group (Grace risk from 75 to 150 points, on average, 107.7±27.16 points). To assess the immune status, especially, lymphocyte populations and subsets we used standardized techniques, i.e., flow cytometric assays with Navios cytofluorimeter (Beckman Coulter, USA). The following subpopulations were determined: CD45+ (panleukocyte marker for gating lymphocytes), CD45+, CD3+ (T cells), CD45+, CD3+, CD4+ (helper inducers), CD45+, CD3+, CD8+ (cytotoxic T cells), CD45+, CD3+CD16+, CD56+ (TNK cells) CD45+, CD3-, CD16+, CD56+ (natural killer cells), CD45+, CD3-, CD19+CD5+ (B cells), CD45+, CD3+, CD4+, CD25+ (activated helpers, early activation phase), CD45+, CD3+, HLA-DR (activated T lymphocytes – late activation phase). The data obtained indicate that the relative indices of T helper subpopulations, T cells at early and late activation step, and B lymphocytes were increased in the patients with acute coronary syndrome, compared with control group. At the same time, there is a trend for increasing absolute values of these indexes. The subpopulation of TNK lymphocytes proved to be significantly increased both in relative and absolute values, whereas percentages of CD45+CD3+CD19- (p < 0.01) and T cytotoxic lymphocytes (p < 0.001) were decreased and showed the same trend in absolute terms. The ratio of CD4/CD8 lymphocytes was almost doubled (p < 0.001), due to increased content of T-helpers and decrease in cytotoxic T lymphocytes. In clinical blood analyses of ACS patients a tendency for leukocytosis was shown, (10.15±5.22), with a shift to the band forms.

Russian Journal of Immunology. 2022;25(3):313-320
pages 313-320 views

Immune response against DNA- and mRNA vaccines encoding artificial influenza virus immunogens

Starostina E.V., Sharabrin S.V., Rudometov A.P., Litvinova V.R., Borgoyakova M.B., Bazhan S.I., Ilyichev A.A., Karpenko L.I.


Constant antigenic drift of circulating influenza viruses leads to inefficiency of seasonal influenza vaccines, thus requiring annual re-design of these vaccines. Therefore, the development of a universal influenza vaccine is of particular relevance. A promising line of research in this area is to design the immunogens consisting of conserved protein fragments from different influenza viral strains. The aim of this work was to assess immunogenicity of DNA vaccines and mRNA vaccines encoding artificial antigens consisting of conserved hemagglutinin stem fragments and conserved M2 protein. We have obtained DNA vaccine constructs encoding artificial immunogens AgH1, AgH3, and AgM2, which contained conserved fragments of the hemagglutinin stalk from the two subtypes of influenza A – H1N1 and H3N2, and conserved M2 protein. These DNA vaccines were used as templates for the synthesis of mRNA vaccines. To assess immunogenicity of the obtained constructs, BALB/c mice were immunized with DNA and mRNA vaccines by i/m administration. Assessment of the humoral immune response was carried out by ELISA, using influenza viruses A/Aichi/2/68(H3N2), A/California/07/2009 as antigens and the ULTRIX vaccine containing purified antigens of H1N1 and H3N2 influenza viruses. T cell immune response was assessed using two methods: intracellular cytokine staining (ICS) and ELISpot. ICS was performed to determine CD8+ and CD4+T-lymphocytes producing IFNγ. ELISpot was carried out using the mouse IFNγ ELISpot kit (BD). A peptide mixture which included composition of the target antigens, was used for cell stimulation. The results showed that the designed DNA vaccine constructs induce virus-specific humoral and cellular responses in immunized BALB/c mice. Intramuscular administration of the naked mRNA vaccine constructs induced a weak humoral immune response, thus suggesting a need for further work to improve the delivery approaches.

Russian Journal of Immunology. 2022;25(3):321-326
pages 321-326 views

Comparative assessment of genetic polymorphism of Toll-like 2 and 6 receptors predisposing for non-specific ulcerative colitis and irritable bowel syndrome in Russians from Chelyabinsk Region

Stashkevich D.S., Belyaeva S.V., Evdokimov A.V.


Ulcerative colitis and irritable bowel syndrome are multifactorial disorders with genetic predisposition. Recent studies suggest that the mucosal immune activation, increased intestinal permeability, and altered host-microbiota interactions may modulate innate immune response, thus contributing to immunopathogenesis of these diseases. Toll-like receptors (TLR) are considered to play the main role in genetic susceptibility to the conditions. The mechanisms for regulating activity of Toll-like receptors are represented by single-nucleotide gene polymorphisms (SNPs), thus producing allelotypes with different biological effects. Among all known TLRs, TLR2 is the most actively studied. The TLR2 gene is located on the long arm of the chromosome 4 and contains the genetic variant leading to the substitution of arginine for glutamine (Arg753Gln) in TLR2 protein. Meanwhile, the most studied SNP of TLR6 is located at the C745T position causing Pro249Ser amino acid substitution in the protein. The present work aimed for analysis of distribution of alleles, genotypes and haplotype combinations of the TLR2 and TLR6 SNPs, and their associations with predisposal for ulcerative colitis and irritable bowel syndrome in Russians from Chelyabinsk Region. The following methods were used in the study: isolation of DNA samples from whole blood, genotyping of the studied gene polymorphisms using PCR with electrophoretic detection. The frequencies of two-locus haplotypes formed by SNPs TLR2 – TLR6 were calculated with Arlequin ver 3.5 software. Comparison of two populational samples for predisposition to UC and IBS was carried out using standard immunogenetic criteria. Significance of differences was set at p ≤ 0.05. Results: Analysis of the data showed the association of specific alleles and genotypes, but not TLR2 – TLR6 haplotypes, with predisposition to the studied diseases. The Arg753Gln gene polymorphism of TLR2 was shown to be significant for a predisposition to ulcerative colitis, and SNP Pro249Ser TLR6 is associated with susceptibility to irritable bowel syndrome in Russians from the Chelyabinsk Region.

Russian Journal of Immunology. 2022;25(3):327-332
pages 327-332 views

Morphological and functional characteristics of LPS-stimulated microglial cells under the action of orexin A

Synchikova A.P., Korneva E.A.


Interest to the orexin-containing neurons is caused by their recent discovery and perspectives of their usage for treatment of different diseases. The studies in this area were launched recently and are of special interest since the opportunity of modulating functional activity of the brain immune system is of pivotal significance for therapy of various central nervous system (CNS) disorders providing novel ways of search and promising data on therapeutic effects of orexins in inflammatory, autoimmune diseases as well as malignant tumors.

Some data from literature show that orexins may exert therapeutic effects in different disorders caused by altered neuroimmune interactions. Participation of this neuromediator system is shown in pathogenesis of narcolepsia, obesity, multiple sclerosis, Alzheimer disease, intestinal disorders, septic shock and cancer, due to involvement of orexins in functional regulation of various components of immune syste, e.g., microglial cell populations. Despite only scarce data on these effects, some experimental results obtained over last years, add to our understanding of orexin effects upon functional activity of the brain immune system.

A number of previous studies allowed to assess the orexin effects on morpho-functional features of microglial cells activated by lipopolysaccharide (LPS), thus presenting a prospective for development of novel approaches to therapy of infectious, inflammatory, neurodegenerative and autoimmune disorders affecting CNS. In the present study, we aimed for detecting the effects of neuromediator orexin A upon functional traits of of microglial cells activated by LPS (M1 phenotype) as evaluated by changes of their size and length of their processes, as well as density of cell distribution.

We have studied the changes of microglia cell numbers following intraperitoneal LPS injection. It was shown that, the LPS causes higher activation degree of these cells, i.e., the contents of microglial cells becomes increased in somatosensory area of the brain cortex. A series of these studies allowed us to demonstrate that intracerebroventricular injection of orexin A in animals following LPS injection does not cause detectable changes of the processes initiated by LPS. The comparative analysis did not detect any changes in length of microglial processes localized in somatosensory or motor cortical areas, and corpus striatum. Other parameters of the microglial cell activation will be studied in future.

Russian Journal of Immunology. 2022;25(3):333-338
pages 333-338 views

Comparative characteristics of cytokine content in neutrophil supernatants after incubation with daily cultures of S. aureus with or without spA gene

Fomina L.O., Gritsenko V.A., Fayzullina A.I.


The study was aimed for assessment of cytokine profile in the whole blood neutrophil supernatants stimulated by Staphylococcus aureus, with and without the spА gene.

20 clinical isolates of S. aureus were tested in vitro. Ten of them carried spА gene and other 10 were without the spА gene. We selected the strains with very high ability to secrete cytokine-like substances (CLS), both in terms of the level and spectrum of the cytokines. The S. aureus strains were divided into two groups depending on the spА status according to the data from the spА genotyping databank (http://spa.ridom.de/). The neutrophils of donors were isolated in a double gradient of Ficoll-Verografin, according to the standard method, at a concentration of 5 × 106. The bacteria were taken at a ratio of 1:20 to neutrophils (at a concentration of 108 bacteria/mL). The effect of staphylococci on cytokine secretion by neutrophils was determined with a Magpix-100 device (USA), using immunofluorescence multiplex kits from BioRad (USA) for the detection of 17 cytokines (G-CSF, GM-CSF, IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17A, TNFα, MCP-1, MIP-1β) after 1 hour incubation with the neutrophils. Results: It has been shown that staphylococci with a high degree of CLS activity, regardless of presence or absence of the spА gene, affected the ability of neutrophils to secrete cytokines. At the same time, S. aureus carrying the spА gene caused increased secretion of IL-1β, TNFα, IL-6 (pro-inflammatory cytokines), but, at the same time, it reduced secretion of IL-8, MIP-1β (chemokines). S. aureus strains devoid of spА genes have a more diverse spectrum and more pronounced effect on cytokine secretion of proinflammatory cytokines by neutrophils, e.g., IL-10, IL-12p70, IL-17A, IL-1β, IL-2, TNFα and the G-CSF growth factor. Both variants of staphylococci caused reduction of IL-6 secretion.

Russian Journal of Immunology. 2022;25(3):339-344
pages 339-344 views

Effect of mifepristone under acute stress on thymocyte apoptosis in mice

Shilov Y.I., Shilov S.Y., Barkov S.Y., Shilova N.A.


In 1936, the Canadian pathologist Hans Selye, in experiments with adrenalectomy, has shown adrenal cortex hormones to be involved in development of thymic acute involution under stress. By 1970s, the idea of lysis of lymphoid cells by glucocorticoids dominated among researchers as the main mechanism of these changes. Later on, increased migration of T lymphocytes from thymus as well as decreased migration of bone marrow precursors to the thymus were considered. Since 1990s, apoptosis induced by glucocorticoids was also studied in this respect. To elucidate the in vivo contribution of glucocorticoids these events, a convenient tool was developed in experimental endocrinology and pharmacology, i.e., treatment of male rats or mice with antagonist of glucocorticoid and progesterone receptors mifepristone, also known as RU-38486 or RU-486. The purpose of this work is to investigate the effect of mifepristone on thymocyte apoptosis in mice under acute stress. Experimental studies were performed in male white noninbred mice. Mifepristone was injected once subcutaneously 30 min before immobilization at a dose of 50 mg/kg body weight prepared in olive oil solution. Control mice and the animals from comparison group were injected once subcutaneously with an equivalent amount of the drug solvent. A classical model of 24-hour immobilization stress in a plastic restrainer (supine position) was used for experimental simulation of acute stress. Thymocyte apoptosis was assessed by flow laser cytometry with BD PE Annexin V Apoptosis Detection Kit I (BD Pharmingen™) reagent kit adapted for Guava EasyCyte flow laser cytometer by Millipore Corporation. It was found that acute stress induced by 24-hour immobilization of mice leads to an increase in the total relative number of 7-AAD-positive cells as well as proportion of cells stained with 7-AAD, but not annexin V-PE (nuclear debris, annexin V(-), 7-AAD (+)). Administration of mifepristone alleviated these changes, thus confirming involvement of glucocorticoids in increasing number of necrotic thymocytes. The number of thymocytes in early apoptosis (i.e., annexin V-PE-positive, 7-AAD-negative), as well as total relative number of cells with phosphatidylserine exposed at the surface (annexin V-PE-positive thymocytes) under acute stress and at stress on the background of the introduction of mifepristone did not differ from the control.

Russian Journal of Immunology. 2022;25(3):345-350
pages 345-350 views

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