Vol 25, No 3 (2022)

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.

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.

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 (Q_0.25-Q_0.75) – 12.4 (10-16.1). The numbers of CD4⁺ cells, regulatory T lymphocytes (Treg – CD4⁺CD25^highCD127^low), activated T helpers (Tact – CD4⁺CD25⁺CD127^high), 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.

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.

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.

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.

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.

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.