Vol 28, No 1 (2025)

Type 2 diabetes (T2D) is a chronic metabolic disease, being one of the leading causes of death and disability worldwide. Immune system disorders play an important role in the T2D pathogenesis, and immune system is exposed to increased stress resulting both of changes in immunometabolic regulation and disruption of tissue homeostasis caused by oxidative stress and chronic inflammation. Opportunities for adaptation of immune organs in T2D are of interest, especially, potential ectopic expression of insulin. Under hyperglycemic conditions, insulin-containing cells were detected in various organs, including the spleen. The aim of the present work was to estimate the number of insulin-positive cells (IPCs) in the spleen of rats with experimental T2D. The disorder was induced in mature male Wistar rats by intraperitoneal administration of 110 mg/kg nicotinamide followed by 65 mg/kg streptozotocin. 30 days later, blood samples and spleens were obtained. The level of glycemia, insulin contents, activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in blood were assessed as well as markers of oxidative stress in the spleen. Biochemical studies revealed an increase in ALT activity in blood, along with increase in malondialdehyde (MDA) levels and decreased levels of reduced glutathione (GSH) in the spleen tissue of rats with experimental T2D. Meanwhile the activity of superoxide dismutase (SOD) and catalase in the spleen did not change. An increase in MDA level associated with unchanged activity of antioxidant enzymes and decreased GSH content suggests the development of oxidative stress in the tissue. Microscopy of spleen tissues was examined by immunohistochemistry using antibodies to proinsulin and insulin, showing an increased number of insulin-positive cells (IPCs) in the spleen tissue of rats with experimental T2D compared with diabetes-free animals. Optical density of the IPC cytoplasm was found to be increased in diabetic rats, thus suggesting increased functional activity of IPCs. Increase of numbers and functional activities of IPCs in the spleen in experimental model of T2D may indicate an adaptive role of extra-pancreatic insulin expression under the conditions of oxidative stress and cytolysis, including those in immunopoietic organs.

The purpose of the present work was to study effects of dehydroepiandrosterone (DHEA) on the oxygen-dependent microbicidal activity of blood leukocytes. The studies were performed on male white nonlinear rats aged 3-6 months (young rats) and adult animals over 1.5 years old (old rats) using an experimental model of zymosan peritonitis. DHEA was administered subcutaneously to old animals on the daily basis (a total of 4 injections of 2 mg/kg body weight, the last injection 24 hours before the end of the experiment). Rats of similar age served as controls and received DHEA solvent (officinal olive oil) according to the same time regimen. 12 hours before the end of the experiment, the animals were administered a sterile suspension of zymosan A intraperitoneally at a dose of 50 mg/kg. The oxygen-dependent microbicidal activity of blood leukocytes was assessed by luminol-dependent chemiluminescence using opsonized and non-opsonized zymosan at concentrations of 15 μg/mL, 150 μg/mL and 1500 μg/mL.

In old male rats, compared to young ones, the chemiluminescence indexes showed a statistically significant increase when blood leukocytes were stimulated with both opsonized and non-opsonized zymosan. Administration of DHEA to old male rats leads to a pronounced decrease in chemiluminescence in samples at all concentrations of opsonized zymosan. When leukocytes of old animals are stimulated with non-opsonized zymosan in vitro, a statistically significant increase in the production of reactive oxygen species was observed, being more pronounced at lower concentrations of inducing agent (15 μg/mL and 150 μg/mL). Meanwhile, this effect was canceled at high concentrations of zymosan. The level of spontaneous chemiluminescence, reflecting in vivo cell activation, was higher in old rats treated with DHEA compared to young animals. The direction of changes in the production of reactive oxygen species in samples with low concentrations of the in vitro activation was similar to the effects of DHEA in old rats tested for spontaneous chemiluminescence. In general, our studies confirm the pronounced immunomodulatory activity of DHEA. In general, we have found that aging of male rats leads to pronounced pro-inflammatory activation of reactive oxygen species produced by blood leukocytes at the peak of inflammation induced by zymosan injections. Administration of DHEA to old male rats significantly reduced the production of reactive oxygen species by blood leukocytes, when the cells were in vitro activated by opsonized zymosan, but stimulates production of oxygen radicals in the samples without zymosan, as well as upon exposition to non-opsonized zymosan at concentrations of 15 μg/mL and 150 μg/ mL.

Graphene oxide offers properties that make it a promising material for numerous biomedical applications, including various cancer treatment methods. Nanomaterials may overcome certain limitations of conventional chemotherapy. In cancer research, cell lines like Jurkat (T cell acute lymphoblastic leukemia cells) serve as models for investigating tumor treatment strategies. Previous studies have explored the effect of pegylated graphene oxide nanoparticles on some parameters of Jurkat cells, such as metabolism and apoptosis. This study aims to investigate how pegylated graphene oxide nanoparticles, varying in size, surface functionalization, and concentration, influence the functional activity of Jurkat cells, including cell viability, IL-2 production, and CD69 expression, both spontaneously, and following external activation.

Jurkat cells were cultured with different types of graphene oxide nanoparticles (100-200 nm and 1-5 ìm; functionalized with linear and branched polyethylene glycol (PEG)) at concentrations of 5 ìg/ mL and 25 ìg/ mL for 24 hours. For assessment of cell parameters under stimulation with different types of particles, phytohemagglutinin (PHA, 50 ìg/mL) was used. Subsequently, the cells were stained with Zombie Aqua and CD69-APC antibody, followed by flow cytometry analysis (CytoFlex S) to determine the percentage of viable cells and CD69-expressing cells. The presence of IL-2 in cell culture supernatants was quantified using ELISA tests.

It was observed that nanoparticles at low concentrations did not induce cytotoxic effects; cell viability improved after PHA stimulation. Small particles (100-200 nm) coated with linear PEG induced IL-2 production and CD69 expression. However, 1-5 ìm graphene oxide modified with branched PEG at a concentration of 25 ìg/mL led to a decrease in CD69 expression following PHA-stimulation.

It was shown for the first time that pegylated graphene oxide nanoparticles affect the functional activity of Jurkat cells. The influence of particles is dependent on the size, concentration, surface functionalization of graphene oxide, and activation by PHA.

Endothelial colony-forming cells (ECFCs) represent a rare population of circulating endothelial progenitor cells (EPCs), which is of interest for tissue engineering, cell therapy, and studying diseorders associated with endothelial dysfunction. Advances in this field imply isolation and pure culturing of endothelial colony-forming cells, which may be obtained from the mononuclear fraction of human peripheral blood. Flow cytometry may provide rather accurate phenotyping and checking purity of the cell cultures. The aim of this study was to obtain a population of colony-forming endothelial cells evaluated by flow cytometry in order to confirm the phenotype and assess the purity of the cell populations. Heparinized donor blood from peripheral vein was used to obtain ECFCs. The mononuclear cell fraction was isolated on a Histopaque 1077 density gradient. The obtained cells were cultured in complete nutrient medium EGM-2 MV with 5% FBS. On the 11^th, 15^th and 18^th days of culture, the cells were detached with trypsin. Some cell aliquots were taken for flow cytometry, and the remaining cells were transferred to a plate for further cultivation until 70% confluency was reached. The phenotype of colonies obtained was evaluated by flow cytometry. Sample preparation of whole peripheral blood, mononuclear fraction and cell culture was performed using two panels of fluorochrome-labeled monoclonal antibodies. Colony growth was visually evaluated by phase-contrast microscopy. Statistical processing of the obtained results was performed using the applied program package “Statistica 6.0”. Up to 9 days, the cell colonies were not detectable visually. During further cultivation, the proliferating colonies of the “cobblestone” type were formed. Two populations were detected by flow cytometry: CD45⁺ and CD45^-. In the course of our study, it was revealed that, with increasing cultivation terms, a decrease of CD45⁺ population was observed, along with progressive increase of CD45^- cells, which may be associated with the gradual displacement of hematopoiesis cells by endothelial colony-forming cells. As a result of our study, the detected CD45⁺ population had a phenotype of hematopoietic lineage cells, whereas the CD45^- population exhibited endothelial phenotype: CD31⁺CD309⁺vWF⁺CD146⁺. By 18 days, the purity of culture reached 97.6%.

By the beginning of May 2023, the epidemic situation regarding COVID-19 was assessed by WHO as favorable, thus enabling to cancel the international emergency state and declare the end of the pandemic on May 5, 2023. Currently, COVID-19 is becoming a seasonal infection. COVID-19 is often accompanied by imbalanced immune response including decreased number of white blood cells and functional changes of immune cells, e.g., affecting activity of neutrophils, including their phagocytic abilities. About 48% of patients who suffered from COVID-19 developed a post-COVID syndrome which is referred to a number of persistent immune disorders which persist for more than 6-12 months after acute infection. Post-COVID-19 disorders of the cellular component of the immune system can manifest with decreased levels of various lymphocyte subpopulations, e.g., NK cells, T cytotoxic lymphocytes, reduced expression of pan-leukocyte CD46 marker on T lymphocytes, as well as changed number and functionality of neutrophils involved in the antiviral immune response. The neutrophils, as a key population of immune system, play an important role in the response to the infection. So far, however, the mechanisms responsible for their functional changes in the context of the post-COVID syndrome remain poorly understood. The results of the study showed that, in patients with post-COVID syndrome, the neutrophil-lymphocyte ratio (NLR) remains within normal range in patients with lung damage of less than 50%. In patients with lung damage of more than 50%, there is a trend for increase of this index, which may suggest a need for in-depth study of immunity in these groups of patients. Statistically significant differences in the innate immunity indexes were detected with respect to phagocytic and bactericidal activity of neutrophils in patients over post-COVID period who showed different extent of lung damage during the acute phase of coronavirus infection.

The long-term consequences of COVID-19 still remain among priority issues. Special attention should be paid to the state of health of newborns whose mothers suffered a coronavirus infection during pregnancy. The aim of our study was to evaluate clinical and immunological parameters in full-term newborns from mothers who underwent COVID-19 during pregnancy.

We have performed a prospective cohort comparative study. A group of full-term newborns from mothers who had mild or moderate COVID-19 at different stages of pregnancy (n = 134) was compared with a group of newborns from mothers who were not exposed to SARS-CoV-2 infection during their pregnancy (n = 84). Individual health indices and immunological characteristics were also evaluated at the age of 3 months of infants’ life. For statistical analysis, the Microsoft Excel computer software package (2010), SPSS Statistics version 22.0 (IBM Microsoft, USA) was used. Statistical hypotheses about the absence of intergroup differences in quantitative characteristics were tested using the Mann–Whitney U criterion, the Student’s criterion, and binary characteristics were assessed with Chi-square test (÷²). Qualitative data are presented as an absolute value (n) and percentages. The results were considered reliable at a significance level of p < 0.05.

It was found that, in the neonatal period, there were no statistically significant differences in gender characteristics, anthropometric characteristics and Apgar scores between the groups. There were no differences in the incidence rate in the neonatal period. At the age of 3 months of life, there was a higher incidence of somatic and neurological morbidity in children whose mothers had suffered a new coronavirus infection during pregnancy (OR = 3.78 (1.5; 9.51); p < 0.001). It was found that the levels of hematocrit (p = 0.001), lymphocytes (p < 0.001), platelets (p < 0.001) were statistically significantly lower in newborns in group I. When analyzing the levels of umbilical cord blood cytokines in the two groups, it was found that the levels of TNF, IL-6, VEGF-A, IL-10, IL-1á, IL-1â, IL-8 (p ≤ 0.001) were higher in group I compared with group II. The levels of cytokines in their mothers showed a positive correlation with appropriate indexes in umbilical cord blood. Strong positive associations were found in the mother-child pairs on the 3^rd trimester of pregnancy, in terms of TNFá, IL-1â, moderate positive correlations were revealed for IL-8, IL-10. Hence, despite the absence of clinical manifestations of infectious process and other disorders of early neonatal adaptation, the laboratory changes in general blood counts and active expression of proinflammatory cytokines in newborns whose mothers suffered COVID-19 during pregnancy may suggest the presence of a fetal systemic inflammatory response. An increased morbidity at the age of 3 months may be the result of low-level inflammation.

From December 2019 to March 2020, the human population faced a new coronavirus pandemic, COVID-19, caused by SARS-CoV-2, which led to global morbidity and high mortality worldwide. Typical features of coronavirus infection tended to resemble a seasonal infection over time. Despite this fact, it has been proven that, after acute disease, numerous disorders are detected in the patients, manifesting both with general health disturbances and as complications of pre-existing chronic diseases, including allergic syndrome. On the basis of these findings, the study of post-COVID syndrome became an important aspect of the ongoing COVID-19 research. It has been shown over recent decades that the number of people suffering from various types of allergic reactions is growing every year. Therefore, the studies in allergic pathology in the structure of post-COVID syndrome are among the most pressing issues. In addition to studying exacerbations of allergic reactions, a thorough analysis has shown that adverse reactions caused by coronavirus infection are viewed from new aspects, being characterized by functional disorders of acquired and innate immune response, causing increasingly severe consequences for the immune system and the whole body. Currently, the features of humoral immune response in post-COVID syndrome have not been sufficiently studied. In this respect, we have evaluated the features of humoral immunity in patients with post-COVID syndrome, with clinically confirmed allergic anamnesis. The study revealed changes in humoral immune system, indicating that the most significant changes are observed in post-COVID patients with a burdened history of allergies, thus requiring a more detailed study of immune status in order to develop an individualized approach to immunocorrection of these disorders in this group of patients.

Chronic exposure to multimodal stress, e.g., allostatic overload leads to dysadaptive response of various organs and triggers pathophysiological mechanisms that contribute to development of post-stress disorders in veterans of modern military conflicts. Brain is a key stress response organ, and sustained matching of blood flow to metabolic demands of the brain is ensured by signaling mechanisms of neuroimmunovascular interaction. The purpose of the study was to assess the contents of humoral neuroimmunovascular factors, and the state of microcirculation at the ocular bulbar conjunctiva in post-traumatic stress disorder (PTSD) in veterans of modern military conflicts. Materials and methods: 35 veterans of a special military operation on the territory of Ukraine with a documented diagnosis of PTSD (ICD-10: F43.1; ICD-11: 6B40) aged from 25 to 60 years particioated in the study. Biomicroscopy of the ocular bulbar conjunction was performed using a slit lamp SLR 100 (K. Zeiss, Germany). The levels of following vasoactive factors were determined in blood by ELISA method: vascular endothelial growth factor VEGF (eBioscience test system, BenderMedSystems, Austria); endothelin-1 (BioMedica, Austria). The concentration of nitrites was determined by Gries reaction using the technique of N. Emchenko. The blood cytokine profile was studied with a Luminex Magpix 100 immunoanalyzer (USA), using Bio-Plex multiplex test system (MERZ, Germany), in order to determine IL-6, TNFá. The data were evaluated with SPSS program. Results and discussion: a study of blood microcirculation at the bulbar conjunctiva of veterans with PTSD revealed the predominance of spastic-atonic changes, mainly due to the capillary-venular component, emergence of arterial-venous anastomoses, aneurysms contributing to increase of hypoxic-ischemic changes in brain tissue, dystrophic changes, reduction of cerebral blood flow, being a consequence of vascular remodeling and neovascularization. There is an imbalance of vasoactive molecules in blood, with predominance of vasoconstriction effects associated with increased levels of endothelin-1, a vascular endothelial growth factor along with decreased concentrations of nitrite ions. We have shown increased systemic circulation of pro-inflammatory cytokines IL-6 and TNFá which may promote neuroinflammatory and neurotoxic processes in brain tissue. Conclusion. Development of neurocognitive post-stress disorders occurs in presence of accumulating “allostatic cargo”, being accompanied by maladaptive changes in the microcirculation of brain tissue along with disturbances in angioarchitecture, imbalance of vasoactive molecules, and increased contents of pro-inflammatory cytokines.

Maintenance of intestinal homeostasis suggests dynamic interaction between the host immune system and local microbiota. Impaired immune response, genetic predisposition and changes in microbiota composition lead to chronic gut inflammation, which is the basis for the development of immune pathology accompanying inflammatory bowel disease (IBD). Resident enteric viruses also have immunomodulatory effects in IBD. Toll-like receptors recognizing PAMPs penetrating intestinal barrier are an important component of the inflammatory process in ulcerative colitis (UC). Viral double-stranded RNA is recognized by endosomal TLR3. Our goal was to identify the association between alleles, genotypes of the TLR3 gene and its haplotypes formed with TLR2 genes, TLR1, TLR6, and UC in Russian population of the Chelyabinsk Region. The study groups included 96 patients with UC and 86 healthy individuals. DNA was isolated from whole blood using a column method, polymorphic gene regions were amplified using allele-specific PCR and RFLP, amplification products were detected by gel electrophoresis in a 3% agarose with UV-visualization. The SNP 1234C>T (Leu412Phe) of the TLR3 gene were typed. Linkage disequilibrium parameters were evaluated for the mentioned TLR3 SNP and other SNPs, i.e., 1805T>G (Ser602Ile) in TLR1 gene, 2258G>A (Arg753Gln) in TLR2 gene, 745C>T (Ser249Pro) in TLR6 gene. The frequency analysis of alleles and genotypes of TLR3 SNP 1234C>T showed a statistically significant increase of the mutant T allele frequency (p = 0.019; OR = 1.72; 95% CI: 1.09-2.71), and higher frequency of homozygous TT genotype among the patients with UC (p = 0.011; OR = 4.72; 95% CI: 1.31-17.05). By assessing the parameters of linkage disequilibrium, two haplotypes were discovered that may be predisposition factors for UC, i.e., haplotype 1234*T ~ 2258*A, with linkage of mutant alleles of SNPs 1234C>T TLR3 and 2258G>A TLR2 (p = 0.006; OR = 12.42; 95% CI: 1.61-95.97), as well as haplotype 1234*T ~ 1805*T, with linkage of the mutant allele of SNP 1234C>T TLR3 to wild allele of SNP 1805T>G TLR1 (p = 0.009; OR = 2.94; 95% CI: 1.35-6.42).

Liver diseases cause about 2 million deaths per year worldwide, with cirrhosis accounting for 2.1% of this number. The cytokine and chemokine balance determines outcomes of immune response. Many cytokines are involved in progression and control of various liver diseases via regulation of cellular activity.

Our aim was to assess the level of circulating cytokines and chemokines depending on the stage of fibrosis in children with chronic liver diseases.

51 children with chronic liver diseases were examined (32 children with autoimmune hepatitis, 12 children with Wilson’s disease, 5 children with Gaucher disease and 2 children with glycogen storage disease). All children were assessed for the stage of liver fibrosis (AF) using METAVIR scale (FibroScan F502). The contents of circulating serum cytokines, chemokines, growth factors and angiogenesis was determined by the MILLIPLEX^® Human Cytokine/Chemokine/Growth Factor Panel A Magnetic Bead Panel, an immunoassay based on Luminex^® technology. Statistical evaluation was carried out using the program “Statistica 10.0”.

The levels of EGF, Fractalkine, IFNá, IL-10 and MIG increased significantly from F0 stage to F4 stage. A significant decrease from stage F0 to stage F4 was revealed for eotaxin, IL-5, IL-8, IL-17A. Some cytokines were characterized by nonlinear dynamics: the concentrations of IL-4 and MDC increased significantly from the F0 stage to the F2-3 stage, and then decreased to the F4 stage; the level of IL-18 showed a significant decrease by stage F2-3 relative to F0, then being significantly increased by stage F4. The level of TNFá was increased at all stages of liver fibrosis and reached its maximum values at stage F2-3 of AF.

Our data confirm the significant role of cytokines and chemokines in the pathogenesis of chronic liver diseases. The identified changes in circulating cytokines in the blood serum in children with CKD, depending on the stage of fibrosis, are characterized by differently directed disturbances thus presuming involvement of both pro- and anti-inflammatory mechanisms in the immunopathological process in the course of liver fibrosis formation. Further research is required in order to study the participation of cytokines and chemokines in formation of liver fibrosis for development of targeted therapy for liver diseases.

The study of biologically active inflammatory mediators is important in the diagnosis of allergic rhinitis. The aim of the present study was to evaluate the levels of secretory immunoglobulin A (sIgA) in nasal secretions in the upper respiratory tract allergies in patients living in various cities of the Russian Federation. Patients and methods. We examined 86 patients aged 28 to 40 years (men-to-women ratio was 1:1 in patients and control group) with a persistent form of allergic rhinitis living in various cities of Russian Federation (Chelyabinsk, Moscow). Group 1 included 36 patients with newly diagnosed allergic rhinitis who did not take any medications (subgroup 1a included 18 persons living in Chelyabinsk; 1b consisted of 18 patients living in Moscow). Group 2 included 30 patients who received long-term intranasal steroid medications (4.9±0.9 months): 2a, patients who lived in Chelyabinsk (n = 15), and 2b lived in Moscow (n = 15). Control group 3 included twenty healthy volunteers aged 28 to 40 years. The disease symptoms were documented in the patients, nasal secretions were collected by endoscopy, and the samples were analyzed by enzyme immunoassay (ELISA). It has been shown that the levels of secretory immunoglobulin A (sIgA) in nasal secretions showed a significant (p < 0.05) unidirectional decrease in adult patients with allergic rhinitis living in various cities of the Russian Federation.