Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. represent ESKAPE group, which is characterized by the greatest resistance to antibiotics. Due to the wide spread of pathogens and their danger to the health care system, the search for new ways to treat bacterial infections remains relevant.

Antibiotics were first obtained in the early twentieth century, but their widespread use began during the Second World War. To date, the range of antibacterial drugs is wide, but despite this, the problem of bacterial resistance to them is acute.

An urgent task of modern science is considered to be the search for overcoming the resistance of bacteria to antibacterial drugs. Since the search for new classes of substances is a long and expensive process, combined drug regimens are used, methods of delivering antibiotics to the source of infection in the body are modified, the structure of the active substance molecules is changed, and adjuvants are used.

NK cells are traditionally considered as part of antitumor or antiviral immunity. However, due to the appearance of data indicating the presence of antibacterial proteins in them and the ability to exhibit cytotoxicity against cells infected with intracellular prokaryotic organisms, today they can be considered as a component of antibacterial immunity.

NK-92 cells reproduce the characteristics of NK cells and have similar properties. In addition, the possibility of their use as a component of antitumor therapy is being actively studied, and clinical trials are being conducted at different stages. In combination with the antibacterial properties of NK cells and the facts described above, it becomes possible to use NK-92 cells as an adjuvant in the antimicrobial therapy of infections caused by antibiotic-resistant bacteria.

The review presents data on the possibility of using the NK-92 cell line and the microvesicles produced by them to combat antibiotic-resistant bacteria of ESKAPE group. Currently, there is not enough research in this area, but data on NK cells presented in the review allow us to propose a cell line reproducing their characteristics and the MV produced by them as a promising adjuvant of antibacterial therapy.

Angiogenesis refers to the formation of new blood vessels from existing ones and is considered a crucial stage in vascular development. Two distinct types of angiogenesis have been identified: branching and non-branching angiogenesis. This process is involved in both normal physiological and pathological conditions, making it an area of significant interest for biomedical research. The development of angiogenesis involves a series of six stages, each controlled by various microenvironmental factors, such as cytokines and growth factors. These factors can either stimulate (pro-angiogenic) or inhibit (anti-angiogenic) the angiogenesis process. Additionally, ligand-receptor interactions between cells in the vicinity of the site of vessel formation play a role in regulating this intricate process. To date, the precise molecular mechanisms underlying the effects of cytokines, growth factors, and intercellular interactions in angiogenesis have not been fully elucidated. However, studies have demonstrated that signals received by endothelial cells during angiogenesis trigger a series of reactions within these cells, leading to alterations in gene expression and affecting the cellular phenotype and function. These changes influence the nature of vascular formation. The purpose of this review is to summarize current understandings of angiogenesis and its associated molecular mechanisms. The review presents data on the characteristics of endothelial cell populations in growing vessels, the stages of angiogenesis, and the factors that control vascular formation. It highlights the latest findings on signaling pathways such as PI3K/Akt, MARK/ERK, mTOR, RhoA, Ras, Notch, Smad2/3, Smad1/5/8, STАT3, STАT5, NF-κB, and molecules induced in endothelial cells during their interactions with cytokines and growth factors, as well as activation of certain endothelial surface receptors (VEGFR1, VEGFR2, VEGFR3, Tie1, Tie2, FGFR, PDGFR-α, PDGFR-β, TNFR1, TNFR2, VE-cadherin, and TßRI and TßRII. Special attention is given to the interaction between TGF-β signaling and other pathways during angiogenesis. Special attention is given to the description of the interaction between TGFβ signaling and other signaling pathways in angiogenesis. The review also provides current data on inhibitors of these signaling pathways, which can be used in the study of angiogenesis and, if necessary, in its correction during therapy. The review summarizes information on 29 such inhibitors at various stages of development – from in vitro research to clinical applications.

Platelets are circulating anucleated structures derived from megakaryocytes. Intercellular adhesion molecules, Toll-like receptors, chemokine and cytokine receptors are represented on their surface. Platelets contain biologically active molecules, including chemokines, cytokines, and growth factors. Due to its cytological features (membrane system and secretory granules), platelets are capable of fast activating and interacting with other cells. Platelets are involved in hemostasis, immune reactions, and angiogenesis.  Platelets can be activated by components of subendothelium, complement system proteins, products of secretion of other platelets. Activation process is mediated through calcium. When activated, platelets change their morphology, release secretory granules and produce microvesicles – a relatively new target of biological research.  The aim of this review is a comparative description of platelets and their microvesicles. Platelet-derived microvesicles perform platelets functions and communicate with other cells, including endothelial cells. Microvesicles is a promising object of research, and the possibility of their use as a diagnostic and therapeutic agent is being actively studied. Majority of circulating microvesicles has platelet origin. Platelet-derived microvesicles contain cytokines and other proteins, lipids and nucleic acids (DNA, mRNA, microRNA). Microvesicles has  the surface markers of the parent cells; phosphatidylserine is represented on their membrane, which additionally participates in clotting due to accumulation of coagulation factors. Under the influence of microenvironment signals, the composition, phenotype of platelet microvesicles, as well as their functional abilities towards the endothelium may vary depending on the stimulus. The effect they have on angiogenesis and regeneration has not been sufficiently elucidated, experimental data demonstrate controversial effects. In pathologies accompanied by endothelial dysfunction an increase in the level of platelet microvesicles is observed, which indicates their possible participation in the pathogenesis. The effect of platelets and their MV on the endothelium, including activation of various signaling pathways in the endothelium, remains the subject of further research.

Abstract

The primary function of γδT cells is to regulate the responses of innate and adaptive immune systems. These cells also play a role in antibacterial, antiviral and antitumor immune responses, regulate inflammation, maintain homeostasis in barrier tissues, control cell interactions in the uteroplacental interface, monitor pregnancy progression, contribute to the pathogenesis of autoimmune disorders, participate in wound repair, and maintain epithelial integrity. In recent years, a large amount of data on the diversity of subpopulations of γδT cells and the role of these subpopulations in physiological and pathological processes was presented, sometimes opposing, or even antagonistic. Therefore, the purpose of this review was to systematize data on the biology of γδT cells, including their origin, phenotype, functions and methods of application in the clinic. The review presents modern conceptions regarding the origin of γδT cells, the stages of their intrathymic differentiation, and the possibilities of extra-chemical transdifferentiation of some subpopulations into others. The review presents a modern classification of human γδT cell subpopulations based on the expression of γ- and δ-chains of the T cell receptor, the phenotype and describes the properties of the most common populations of Vδ1, Vδ2, Vδ3 T cells. The classification of human γδT cells based on their cytokine production and expression of intracellular messengers is given, the properties and functions of the most studied subpopulations are described in detail: γδT1, γδT17, γδNKT, γδTreg, γδTAPC, γδTfh. The review pays special attention to the phenotype of various populations, their secretion of cytokines, and provides data on the expression of surface receptors of human γδT cells and their functions. In particular, the structural features and ligands of the γδT cell receptor, as well as the receptors controlling their activity (LIRB1/ILT2, KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DL2, KLRD1, NKG2A, NKG2C, NKG2D, NKG2F, NKp30, NKp44, NKp46, KLRC3, DNAM1, KLRG1/MAFA, FcyRIII, BTLA, PD1, TIGIT, VISTA, LAG3, TIM3, CTLA-4, 2B4, NK1 (NK28), KLRB1, TLR1, TLR2, TLR3, TLR5, TLR6, TLR7, TLR8), cytotoxicity against target cells, chemokine CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, cytokine and adhesion receptors. The review provides information on the participation of subpopulations of human γδT cells in various physiological and pathological processes, and their role in the supervision of tumor growth. Based on the described data, information about possible prospects for the use of γδT cells in the treatment of certain diseases is also provided.

Abstract

The aim of the work: to summarize the existing results in antitumor RNAi therapy, as well as to highlight the role of siRNA as a remodulator of the immune response in oncological diseases.

Materials and methods: the literature review includes an analysis of scientific papers from the PubMed, Embase, eLIBRARY, CyberLeninka and Web of Science, CNKI and MEDLINE databases

Results: The importance of cancer problem is due to the immune tolerance of tumors, their drug resistance, and a number of limitations in the use of traditional treatment methods. RNA interference (RNAi)-based approaches, which can be implemented using small interfering RNA (siRNA) molecules, can offer promising therapeutic tactics that will combine a immunomodulatory effect and targeted suppression of gene expression important for tumor growth. Inhibition of tumor regulatory pathways will disrupt tumor proliferation and metastasis, while mobilizing antitumor immunity through stimulation of Toll-like receptors, maturation of dendritic cells, and infiltration of cytotoxic T lymphocytes into the tumor. A study of this approach on an in vivo model in laboratory conditions led to a decrease in the volume of melanoma, breast tumors and hepatocellular carcinoma up to 5 times, suppression of metastasis and an increase in overall survival.

Conclusions: Cancer - most significant problems of modern medicine, characterized by high mortality, mechanisms of immune tolerance and frequent emergence of resistance to existing therapeutic approaches. Despite the progress in modern oncoimmunology, the use of checkpoint inhibitors and targeted antitumor agents, the effectiveness of current approaches islimited by the immunosuppressive environment of the tumor, heterogeneity of malignant cells, side effects and toxicity of the drugs themselves. RNAi is a promising approach that can simultaneously solve several key problems of oncoimmunology, such as suppression of the expression of critical oncogenes; blocking tumor signaling pathways; as well as activation of innate immunity. The dual effect of RNAi, which consists of a direct effect on tumor cells and immune modulation of the tumor environment, makes RNAi an excellent tool for overcoming tumor immunotolerance and providing a direct cytotoxic antitumor effect.

Abstract

A global study shows that valvular heart disease still occupies one of the leading places in the structure of mortality from cardiovascular diseases, being one of the leading causes of heart failure, including among the working population. Xenogeneic tissues are widely used in cardiac surgery, both in biological prosthetic heart valves and in vascular and intracardiac patches. Modern methods of chemical treatment of xenogenic tissue aimed at the eliminating of its immunogenicity do not completely remove xenoantigens from the tissue. It is suggested that residual animals’ carbohydrate antigens are a trigger of immune response to xenotissues. At the same time, the role of the immune response to xenogeneic antigens in the induction of inflammation, valve dysfunction, and calcification are discussed.

The aim of this review was to summarize scientific research data on the immune response to xenogeneic tissue implanted in the heart and to find ways to overcome this immune conflict.Modification of the pericardium of large animals by various methods does not remove carbohydrate epitopes of the extracellular matrix and cell membranes, which are recognized by pre-existing antibodies of class M and G. The highly dynamic functioning of xenogeneic biological prostheses increases their antigenicity by reducing the primary cross-linking of the extracellular matrix and activating the alternative complement pathway with adsorption on xenogeneic tissue complement component iC3b, as an opsonin for micro- and macrophages. Inflammatory endotypes of individuals are determined by genetically determined increased synthesis of certain cytokines. In particular, rheumatic heart disease, as the basis for the formation of pathology of the native mitral heart valve, is characterized by an increase in TNF-a, INF-g and IL-6. All of these cytokines may be targets for biological therapies aimed at limiting the constitutional inflammatory endotype. OMICS technologies applied to various options for biological xenogeneic heart valve prostheses degradation, taking into account their implantation and a wide clinical examination of patients, can help to find new variants of immune-inflammatory endotypes leading to dysfunction of bioprostheses and identify target molecules through which the antixenogeneic immune response can be inhibited.

Abstract

Sepsis is a heterogeneous and life-threatening condition caused by a dysregulated immune response to infection. The most severe form of sepsis is septic shock, characterized by arterial hypotension, impaired tissue perfusion, and hypoxia. Despite new findings in antimicrobial and intensive care therapy, the incidence and mortality rates of sepsis remain high, which underscores the relevance of further investigation of its pathogenesis. In recent years, research has shifted from clinical signs to the analysis of immunological and molecular mechanisms, which has led to the identification of  specific phenotypes and endotypes of the disease. Sepsis phenotypes are based on clinical manifestations and biomarkers, while endotypes are defined by molecular mechanisms, including immune gene expression patterns.

This article reviews key aspects of the innate and adaptive immune responses in sepsis, including the activation of proinflammatory cytokines, the development of coagulopathies, and disruptions of endothelial integrity and microvascular regulation.Moreover, the importance of mechanisms such as hyperinflammation, the simultaneous development of immunosuppression and functional exhaustion of immunocompetent cells is emphasized. Thus, immunological biomarkers are considered as a promising tool for patient‘s stratification, prognosis prediction and personalized therapy. Current immunodiagnostic methods are also considered in this article, including quantitative analysis of cytokine levels and assessment of innate immune dysfunction markers.

Thus, the current understanding of sepsis as an immunologically heterogeneous syndrome enables researchers to expand existing concepts of its pathogenesis. In contrast to the classical concept which is based on a shift from inflammation to immunosuppression, increasing data highlights the simultaneous presence of both processes in the same patient, making it necessary to reconsider existing diagnostic and therapeutic approaches.

Abstract

Recent studies in immunology highlight the critical role of mechanical factors in shaping the immune response. Mechanoimmunology, as an emerging interdisciplinary field, investigates the influence of mechanical stimuli on immune cell behavior, particularly T-lymphocytes. It has been demonstrated that microenvironment stiffness, mechanical interactions with the extracellular matrix, and changes in membrane tension can modulate T-cell activation, migration, proliferation, and effector functions. An optimal mechanical environment enhances T-cell activity, whereas increased stiffness of the microenvironment and alterations in extracellular matrix properties can reduce their functional capacity. Key molecules such as Piezo 1, integrins, and Yes-associated protein serve as central regulators of mechanotransduction in immune cells. The gradual expansion of knowledge regarding their role in the immune response indicates a high degree of interconnected modulation, forming a system that enables a coordinated reaction to mechanical stimuli. Mechanomodulation alters the intracellular environment, acting as a determinant of the metabolic profile of T-cells. Additionally, studies indicate that mechanosensitive signaling pathways may regulate intercellular interactions and adaptive immune responses, offering broad opportunities for modifying immune reactions. Understanding mechanotransduction mechanisms provides new prospects for the development of therapeutic strategies. Mechanical signals can be leveraged to enhance the efficacy of CAR-T cells by optimizing their activation, proliferation, and infiltration into tumor tissue, which is particularly important in treating malignant neoplasms, especially solid tumors, where CAR-T cell therapy faces significant limitations. Mechanoimmunological approaches are also being explored in the context of autoimmune disease treatment. It is hypothesized that mechanosensitive pathways may regulate excessive T-cell activation, preventing autoimmune processes and pathological hyperactivation of the immune system. Moreover, the development of effective methods for preventing graft-versus-host disease and transplant rejection, as well as strategies for treating chronic infections, remains an important goal. The spectrum of potential pharmacological interventions includes the use of activators and inhibitors of Piezo 1, integrins, and Yes-associated protein. Bioengineering approaches are also being actively developed. One promising direction involves the use of nanomotors for ex vivo T-cell activation, which may improve the efficacy of cellular immunotherapy in various diseases. Furthermore, fine-tuning immune responses through mechanical properties could enable precise regulation of immune activity based on the specific characteristics of pathological processes.

Abstract

Study Objective. To review current advances in transcriptome technologies focused on single cell analysis with emphasis on their application in the study of the tumor microenvironment and immune landscape in prostate cancer.

Methods. PubMed, Medline, Web of Science, and Embase scientific databases were analyzed.

Results. Prostate cancer (PCa) is an androgen hormone-dependent malignant neoplasm that affects the male genitourinary system. Evidence shows that in men under 40 years of age, PCa is extremely rare, while the highest number of cases occurs in the 50 to 70 age group. Today, PCa is one of the most common cancers among men and represents one of the leading causes of cancer-related deaths. According to the Global Cancer Observatory (GCO), there were 1,467,854 new cases of cancer worldwide in 2022, resulting in 397,430 deaths associated with the disease. PCa ranks fourth in terms of incidence and second in terms of mortality among all cancers in men. In Russia, PCa ranks second in incidence among all cancers in men, with 52,712 cases registered as of 2022, and fourth in mortality, with 14,635 cases. A thorough understanding of the mechanisms of the pathogenesis and progression of PCa  is key to effective diagnosis and treatment development. This review presents an analysis of transcriptome-based techniques in single cell resolution in the study of cellular heterogeneity in prostate cancer. The methodology of the analysis is also presented in detail, cellular heterogeneity in prostate cancer is characterized, current research in the field of single cell transcriptomics in prostate cancer is described, and promising directions of application of the results in clinical practice are outlined. The results of research in this area have significant potential for use as both prognostic and diagnostic markers of tumor processes. Thus, the work emphasizes the importance of studying cellular heterogeneity to improve the methods of prostate cancer diagnostics and therapy.

Discussion. Technologies for studying the transcriptome of single cells provide unique opportunities for in-depth understanding of the molecular and cellular mechanisms underlying the immune response in cancer. The data obtained may become the basis for the development of new directions in fundamental immunology, the development of innovative therapeutic strategies and the introduction of a personalized approach to prostate cancer treatment, which opens prospects for improving the effectiveness of therapy.

AbstractThe article analyzes the effect of viral infections such as SARS-CoV-2 and lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, on the development of osteonecrosis through the pathological lung-joint axis. Viral infection causes damage to lung cells and vascular endothelium, which leads to inflammatory and coagulation disorders, increasing the risk of bone ischemia. LPS, interacting with TLR4 receptors, enhances the inflammatory response and disrupts the blood supply to bones, stimulating resorption and preventing bone formation. In addition, the article highlights the role of lung dysbiosis caused by viral infection, which increases inflammation and increases the permeability of barriers to endotoxins. Information was searched for the keywords "osteonecrosis and lipopolysaccharide", "Covid-19 virus and lipopolysaccharide-binding protein", "Viremia and osteonecrosis", "Lung microflora and LPS" in foreign and domestic scientometric databases such as e-Library and PubMed. The presented data suggest that the combination of imbalance of “lipopolysaccharide-binding systems”, impaired pulmonary barrier, viral aggression and LPS is an important aggravating pro-inflammatory factor. This combination forms a predisposition or full-fledged osteonecrosis, which makes the search for mechanisms of influence on these conditions, both individually and in combination, a promising scientific and clinical direction. This study focuses on the variety of mechanisms through which viral infections and bacterial lipopolysaccharides can affect bone tissue. Research shows that influencing these mechanisms can open up new avenues for the development of therapeutic strategies. The prospects of using targeted therapies to mitigate the negative effects of these interactions are also being considered. These data emphasize the importance of an integrated approach in the study and treatment of osteonecrosis, taking into account both infectious and inflammatory components of the process.

Abstract

Ischemic stroke is one of the most common diseases worldwide, with a high incidence and mortality rate. In the pathological process of ischemia of nervous tissue, neuroinflammation is an important factor that determines the functional prognosis of the outcome of the disease. During the formation of an ischemic focus, microglial cells and astrocytes are activated, which leads to the launch of a cascade of neuroinflammatory reactions that play an important role in the pathophysiology of ischemic stroke. Activated microglial cells and astrocytes are able to form a variety of phenotypes depending on the corresponding parameters of the microenvironment. These phenotypes can have both neurotoxic and neuroprotective effects. On the one hand, when nerve tissue is damaged, glial cells contribute to the removal of cellular debris, maintain ionic homeostasis, regulate the extracellular content of neurotransmitters and ensure the trophism of neurons. On the other hand, microglia and astrocytes can acquire a pro-inflammatory phenotype characterized by the secretion of inflammatory cytokines, which contributes to the progression of neuroinflammation and tissue damage. Thus, astrocytes and microglia undergo both morphological and functional rearrangements, thereby actively participating in neuroinflammation due to the release of pro-inflammatory or anti-inflammatory factors. It is important to note that these rearrangements are associated with metabolic reprogramming, which leads to a change in the activity of metabolic pathways to compensate for the lack of energy and building materials caused by impaired cerebral blood flow. The pro-inflammatory phenotype of microglia is characterized by activation of glycolysis, the pentose phosphate pathway, synthesis of fatty acids and glutamine, whereas the anti-inflammatory phenotype demonstrates increased oxidative phosphorylation and oxidation of fatty acids. Reactive astrocytes are characterized by increased glycolysis, glycogenolysis and reduced glutamate uptake. Recently, there has been increasing evidence that manipulation of glial cell homeostasis can be used to switch from a neurotoxic phenotype to a neuroprotective one. A comprehensive understanding of the basic mechanisms of switching metabolic phenotypes can potentially allow targeted reprogramming of glial cells during the pathological process, which can be used in therapeutic approaches for the treatment of the consequences of ischemic stroke. This review presents current ideas about metabolic reprogramming in astrocytes and microglial cells in the context of pathophysiological processes in cerebral ischemia.

Abstract. Pregnancy represents the state with particularly activated constituents of hemostasis and immune systems. Hyperactivation of platelets and monocytes may be a causative factor for pregnancy complications including preeclampsia. The pathogenetic role of platelet-monocyte complexes (PMC), recognized as diagnostic marker and therapeutic target, is poorly investigated. The aim of the study was to determine quantitative changes in the peripheral blood PMC level and antigenic phenotype in preeclampsia, and to evaluate effects of platelets on the expression of monocyte surface marker proteins in normal and pathological pregnancy. The tested groups included third trimester pregnant women diagnosed with severe preeclampsia (35-41 weeks of gestation) and women with uncomplicated (physiological) pregnancies (33-41 weeks of gestation). All participants were between the age of 24 and 42 years. PMC levels and CD62P, CD11b, CD86, CD162, HLA-DR, TREM-1 expressed by PMC and free circulating cells were determined by flow cytometry in the peripheral blood total monocytes and monocyte subpopulations.It was found that PMC level increased (29.2% of total monocyte population) when compared to uncomplicated pregnancy (17.5%), and this augmentation was ensured by two PMC-forming monocyte subpopulations: classical and intermediate. Moreover, expression levels of platelet and monocyte activation markers CD62P, CD162, HLA-DR, CD86, TREM-1, CD11b were significantly higher in preeclampsia. The fractions of classical, intermediate and non-classical monocytes differently contributed to preeclampsia-associated changes in the expression levels of monocyte activation markers. Comparison of PMC and free circulating monocytes demonstrated that observed changes in the surface antigenic phenotype of monocytes within PMC were ensured by platelets and other factors. In preeclampsia, platelet-induced augmentation of monocyte inflammatory and adhesive capacities displayed itself in the increased TREM-1 and CD11b expression. In contrast, increased levels of HLA-DR and CD86 in monocytes were not induced by the interaction with platelets. The results of the study suggest that preeclampsia is accompanied by increased peripheral blood PMC levels and activation of monocytes within PMC, demonstrate immunomodulatory effect of platelets, and provide a rationale for the evaluation of expression patterns of PMC surface antigenic markers with diagnostic and therapeutic purposes.

Recurrent pregnancy loss is a significant clinical problem that affects 1-5% of the population, and in more than half of cases the cause of premature pregnancy loss remains unknown. One of the possible reasons is an imbalance in the maternal hemostatic system, leading to thrombosis of the uteroplacental vessels, decreased placental perfusion and hypoxia. Changes in the morphofunctional features of monocytes and the aggregates formed by them and activated platelets can be factors causing various complications of pregnancy, in particular, miscarriage. However, the role of platelet-monocyte complexes (PMC), which are of interest as a diagnostic marker and as a therapeutic target, is virtually unknown in the pathogenesis of recurrent pregnancy loss. The purpose of the study was to determine quantitative changes in the content and phenotypic characteristics of the peripheral blood PMC in patients with recurrent miscarriage, and to assess the effect of platelets on the expression of monocyte surface marker proteins during the physiological and pathological pregnancy. The study groups consisted of women aged 24-42 years diagnosed with recurrent miscarriage, having a current pregnancy of 6-12 weeks, and women with an uncomplicated (physiological) pregnancy of 7-12 weeks. In the total population and subpopulations of peripheral blood monocytes, the PMC content and expression of platelet and monocyte surface antigens CD62P, CD11b, CD86, CD162, HLA-DR, TREM-1 were determined using cytoflorimetric analysis. It was found that in recurrent pregnancy loss, the level of PMC was increased (26.5%) compared to uncomplicated pregnancy (15.3%), and all three subpopulations of monocytes (classical, intermediate and non-classical) contributed to the increase. At the same time, a decrease in HLA-DR expression and increase in CD11b expression was observed in the total PMC, while the expression of CD62P, CD162, CD86 and TREM-1 did not change significantly. Monocyte subpopulations differently contributed to the changes in the expression of activation markers associated with recurrent miscarriage, and the changes seen in subpopulations were not always evident in the toatl monocyte population. A comparison of PMC and free monocytes showed that changes in the surface phenotype of monocytes aggregated with platelets, were caused by both the influence of platelets and other factors. In cases of recurrent miscarriage, a platelet-induced increase in the adhesive properties of monocytes was observed, which was manifested in an increase in CD11b expression. In contrast, the decrease in the level of HLA-DR expression in monocytes was not associated with their interaction with platelets. The results obtained suggest that recurrent miscarriage is accompanied by an increase in the content of peripheral blood PMC and changes in the antigenic phenotype of platelet-associated and free monocytes, demonstrate the immunomodulatory effect of platelets, and also provide justification for the importance of determining the expression patterns of surface antigenic markers of PMC for diagnostic and therapeutic purposes.

Vascular Endothelial Growth Factors (VEGFs) are a group of proteins that involved in the development of various cell types, including endothelial cells, monocytes, macrophages, stem cells, tumor cells, vascular smooth muscle cells, trophoblast cells, and other cells that express VEGF receptors.  Pathological conditions, such as abnormalities in placental development, can be caused by disruptions in the production and signaling of VEGFs. Trophoblast cells play a significant role in placental formation and are essential for angiogenesis due to their secretion and reception of VEGF. However, there is a lack of information in the literature regarding the influence of VEGF signaling in trophoblast cells on their functional characteristics. Maternal immune cells, particularly natural killer (NK) cells, have been shown to affect the activity of trophoblasts during pregnancy. Given the high abundance of NK cells in the decidual tissue, it is important to consider their potential influence on the phenotypic changes in trophoblast cells. In this study, we investigated the expression of MICA, MICB, and CD105 proteins by NK cells and trophoblast cells. MICA and MICB are stress markers that allow us to assess cell viability. CD105 is a receptor that is expressed on the surface of various cell types and plays a role in signal transmission from TGFβ family proteins. In particular, endoglin has been shown to regulate signaling from TGFβ by directing signals through the SMAD2/3 or SMAD1/5/8 pathways. According to the literature, endoglin inhibits signaling involving SMAD3. However, it has not yet been determined whether endoglin plays a similar role in NK cells and trophoblasts. The investigation of changes in endoglin expression is a significant issue, as signals from TGFβ are essential for the differentiation of trophoblast cells. Disruption of TGFβ signaling can lead to pregnancy complications and miscarriage. We have demonstrated that VEGF plays a role in regulating the activity of trophoblasts and NK cells. In particular, treatment with neutralizing monoclonal antibodies to VEGF-A resulted in inhibition of the expression of CD105, a VEGF coreceptor, on trophoblasts and NK cells under co-culture conditions. However, pretreatment of trophoblasts with anti-VEGF antibodies did not alter their resistance to the cytotoxic activity of NK cells. Taken together, these findings suggest that inhibition of VEGF signaling results in significant changes in the reception of TGFβ family proteins by trophoblasts and natural killer cells.

Chronic hepatitis C (CHC) represents a significant public health concern. In the majority of cases, the infection progresses to a chronic form, which is characterised by the development of fibrosis and cirrhosis of the liver. A plethora of cytokines and chemokines are generated as a consequence of inflammatory processes within the liver. These can exert a dual effect, both protective and damaging, particularly in relation to the death of hepatocytes and the progression of liver fibrosis. Furthermore, a number of growth factors have been identified as playing a role in the pathogenesis of CHC. The objective of the study was a comprehensive evaluation of a wide range of cytokines, chemokines and growth factors in the blood plasma of patients with CHC at varying stages of liver fibrosis. The study cohort comprised 63 patients diagnosed with CHC, who were divided into three groups according to the stage of liver fibrosis. The control group comprised healthy individuals (n=32). Concentrations of the following cytokines were determined in plasma: Interleukins and some cytokines (IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IL-17-E/IL-25, IL-17F, IL-18, IL-27, IFNα, IFNγ, TNFα, TNFβ); chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL7/MCP-3, CCL11/Eotaxin, CCL22/MDC, CXCL1/GROα, CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, CX3CL1/Fractalkine) and growth factors (EGF, FGF-2, Flt-3L, G-CSF, M-CSF, PDGF-AA, PDGF-AB/BB, TGFα, VEGF-A) by multiplex analysis based on xMAP technology. Nonparametric statistics methods were used for statistical analysis. As a result of the study, increased concentrations of cytokines IL-12 (p40), IL-15, IL-17E/IL-25, IL-27, IFNγ, TNFα, chemokines CXCL9/MIG and CXCL-10/IP-10 and growth factors FGF-2 and M-CSF were found at all stages of liver fibrosis. Elevated concentrations of cytokines IL-1α, IL-1β, IL-2, IL-6, IL-9, IL-10, IL-17F, IFNα, TNFβ, chemokines CCL2/MCP-1, CCL11/Eotaxin, CCL22/MDC and growth factors G-CSF, TGFα, Flt-3L were found in severe liver fibrosis/cirrhosis. Correlation analysis revealed a relationship of high significance between the severity of liver fibrosis and the content of cytokines IL-6, IFNγ, TNFα, IL-7, chemokines CCL2/MCP-1, CCL11/Eotaxin, CXCL9/MIG, CXCL10/IP-10, CXCL1/GROα, growth factors TGFα, PDGF-AA, PDGF-AB/BB. Thus, a certain profile of cytokines characteristic for CHC was revealed, cytokines, chemokines and growth factors significant for liver fibrosis in CHC were found. 

An increased content of platelet-leukocyte aggregates indicates an elevated thrombogenic and inflammatory activity of periphery blood cells. The aim of this study was to investigate the percentage and properties of platelet-monocyte and platelet-lymphocyte aggregates in patients with coronary atherosclerosis. The study included 19 patients with coronary artery disease and coronary atherosclerosis (15 men; 4 women; 59.0 (55.0; 69.0) y.o.). A comparison group consisted of 8 high cardiovascular risk patients without coronary atherosclerosis. The severity of atherosclerosis was assessed by coronary angiography and Gensini Score. Platelet-leukocyte aggregates were analyzed by imaging flow cytometry. We assessed the percentage of platelet-monocyte and platelet-lymphocyte aggregates; the percentage of P-selectin (CD62P)+ aggregates; the number of platelets aggregated with each individual leukocyte (either monocyte or lymphocyte). A significantly lower number of monocytes formed small aggregates, consisting of 1 monocyte and 1 platelet, in patients with coronary atherosclerosis compared to patients without atherosclerosis (Gensini Score>0) (78.8 (68.1; 86.2) versus 84.7 (83.8; 87.1)% (p=0.039)). At the same time, in patients with more sever atherosclerosis (Gensini Score³42.5), the percentage of lymphocyte aggregates with more than 3 platelets tended to increase (0.6 (0.3; 1.6)%) compared to patients with Gensini Score<42.5 (0.1 (0; 0.8)%, p=0.075). The proportion of large platelet-lymphocyte aggregates (with 3 or more platelets) directly correlated with Gensini Score, IL-1b concentration, systemic inflammatory indices, the ratio of triglycerides to glucose and triglycerides to high-density lipoprotein cholesterol (insulin resistance indices), and inversely correlated with high-density lipoprotein cholesterol concentration. The percentage of small aggregates (1 lymphocyte with 1 platelet) inversely correlated with the severity of coronary atherosclerosis, IL-1b concentration, and insulin resistance index. Thus, the distinguishing feature of patients with coronary atherosclerosis appears to be not the increased number of platelet-leukocyte aggregates, but the increased size of heterotypic aggregates. An unfavorable sign is the formation of large aggregates (with 3 or more platelets), which is also associated with the intensity of systemic inflammation and metabolic imbalance.

Recurrent pregnancy loss is a significant clinical problem affecting 1-5% of the population, while in more than half of the cases the cause of premature loss of pregnancy remains unknown. Changes in the morphofunctional properties of monocytes can be factors leading to various pregnancy complications, in particular, to miscarriage. However, the role of monocytes in the pathogenesis of recurrent pregnancy loss has not been sufficiently studied. The aim of the study was to determine the quantitative changes in the content and antigenic phenotype of platelet-free (not bound to platelets) monocytes at the level of the entire population and individual subpopulations of peripheral blood monocytes in recurrent miscarriage compared to uncomplicated pregnancy. The study groups included 6-12-week pregnant women aged 24-42 years diagnosed with recurrent pregnancy loss and women with uncomplicated pregnancy (7-12 weeks). Monocyte content and expression of CD11b, CD86, CD162, HLA-DR, TREM-1 were determined in the total population and subpopulations of peripheral blood monocytes using cytofluorimetric analysis. It was found that the proportion of platelet-free monocytes decreased (74.6%) compared to uncomplicated pregnancy (83.4%), with all three subpopulations of monocytes (classical, intermediate and non-classical) contributed to the decrease. Decrease in HLA-DR expression and increase in CD11b expression was observed in total PMC, caused by the fraction of classical monocytes, while the expression of CD162, CD86 and TREM-1 did not change significantly. Subpopulations of monocytes contributed differently to the changes in the expression levels of activation markers, associated with recurrent miscarriage, and these changes were not always manifested in the total monocyte population. The results obtained suggest that recurrent pregnancy loss is accompanied by a decrease in the content of free monocytes in the peripheral blood and changes in the antigenic phenotype of monocytes, characterizing a weakening of the proinflammatory properties and an increase in the adhesive properties of these cells. These changes may underlie the pathophysiological processes leading to premature termination of early pregnancy. Determination of the expression patterns ofactivation markers characteristic of a particular obstetric pathology contributes not only to the identification of pathophysiological mechanisms of reproductive disorders, but also to the improvement of methods for their diagnosis and the development of pathogenetically reasoned methods of therapy.

Introduction. Chronic obstructive pulmonary disease (COPD) is one of the most common diseases of the bronchopulmonary system. Determination of new inflammatory markers for early diagnosis, optimization and monitoring of therapy is a promising direction of modern research. Objective. To evaluate the levels of YKL-40 and NGAL in serum and induced sputum and to determine their significance as markers of inflammation in patients with COPD. Materials and Methods. The study included 50 patients with COPD, 60 patients with asthma. The control group consisted of 30 volunteers, comparable in age and sex, without allergic and bronchoobstructive diseases. Clinical and anamnestic data, indices of external respiratory function were evaluated. Induced sputum was collected with subsequent assessment of cellular composition. Immunophenotyping of lymphocytes by flow cytometry was performed. The concentration of YKL-40 and NGAL in serum was determined using an enzyme immunoassay test system (R&D Systems). The obtained data were processed using STATISTICA and SPSS Statistics software systems. Results. A significant increase in the absolute number of T-lymphocytes in patients of the studied groups compared to the control group was found. The highest number of T-cytotoxic lymphocytes and NK-cells was registered in the COPD group. Analysis of the cellular composition of induced sputum in patients with COPD revealed the predominance of neutrophilic pattern of inflammation. The maximum concentration of YKL-40 in serum was registered in COPD patients. The level of NGAL in serum of COPD patients was not significantly different from that of the asthma group. NGAL level in induced sputum was significantly higher in COPD group. Correlation analysis confirmed the correlation of YKL-40 and NGAL levels in serum with neutrophilic inflammation indices, smoking index, number of hospitalizations due to COPD exacerbation during a calendar year. It was found that YKL-40 and NGAL levels were significantly increased in patients with frequent COPD exacerbations compared to the group with infrequent exacerbations. Conclusions. YKL-40 and NGAL are promising markers of neutrophilic airway inflammation in COPD patients. The obtained data allow us to consider YKL-40 and NGAL as markers of non-T2-endotype COPD with neutrophilic inflammation pattern and high risk of exacerbations.

Abstract

Sarcoidosis is a systemic inflammatory disorder of unknown etiology characterized by tissue infiltration with macrophages and lymphocytes, including CD8+ T cells, and associated non-caseating granuloma formation. The aim of the study was to investigate various peripheral blood CD8+ T cells from patients with chronic respiratory sarcoidosis using markers of T cell maturation and ‘polarization’. Peripheral blood samples were collected from 34 patients with newly diagnosed chronic sarcoidosis of the respiratory organs with the background of a natural course of disease and without immunosuppressive therapy history. The diagnosis of pulmonary sarcoidosis was performed according to the standard criteria and was confirmed by histological examination for 94,12% of patients. Peripheral venous blood samples from healthy volunteers (n=40), matched by gender and age with patients with pulmonary sarcoidosis, was used as a control group. Multicolor flow cytometry revealed that patients with sarcoidosis had decreased levels of CD45RA+CD62L+ ‘naïve’ and CD45RA–CD62L+ central memory CD8+ T cells if compared with healthy controls, as well as the frequencies of ЕМ1 (CD45RA–CD62L–CD27+CD28+) и pre-effector type 1 (CD45RA+CD62L–CD27+CD28+) cells were also reduced. Next, to assess relevant ‘polarized’ CD8+ T cell subsets, we identified Tc1 (CCR6–CXCR3+), Tc2 (CCR6–CXCR3–), Tc17 (CCR6+CXCR3–), and double-positive Tc17.1 (CCR6+CXCR3+). We found that CXCR3-expressing CD8+ T cell subsets (Tc1 and Tc17.1) were significantly decreased both in relative and absolute numbers in patients with sarcoidosis if compared to healthy controls. Oppositely, Тс2 CD8+ T cell were significantly elevated. Furthermore, the relative numbers of Tc1 cells negatively correlated with serum ACE levels (r=–0,456 with р=0,010), while Тс2 levels positively correlated with serum ACE levels (r=0,623 with р<0,001). Thus, our results indicate that CD8+ T cells may play a role in the pathogenesis of sarcoidosis. More extensive clinical and immunological comparisons are required for further systematization of the obtained data.

Abstract

Hereditary angioedema (HAE) is a genetically determined disorder classified as a primary immunodeficiency involving complement system dysfunction. In most patients, the disease is characterized by a deficiency of C1 inhibitor (type I HAE) or impaired functional activity of the C1 inhibitor (type II HAE). In such cases, the diagnosis is based on laboratory findings. In HAE with normal C1 inhibitor levels and activity, the diagnosis can only be established based on family history and/or genetic testing. Among patients with HAE with normal C1 inhibitor, mutations in the F12 gene are most frequently observed, particularly in women. However, mutations with uncertain clinical significance are often identified. Given the limited number of HAE cases, it is not feasible to experimentally determine the clinical relevance of newly discovered polymorphic variants. A potential solution to this problem is the in silico analysis of each novel polymorphism.

The aim of our study was to evaluate the predictive potential of bioinformatic analysis methods in assessing polymorphic variants in the F12 gene.

The study focused on four polymorphic variants — NC_000005.9:g.176831285C>G, NC_000005.9:g.176831258C>G, NC_000005.9:g.176831232G>C, and NC_000005.9:g.176831232G>T — with varying clinical significance statuses. To predict the effect of these polymorphic variants on the F12 protein, various web-based tools employing different algorithms were used, including SIFT, PolyPhen-2, FATHMM-XF, MutationTaster2021, MutPred2, MUpro, I-Mutant 2, HOPE, and ChimeraX.

 

Results. In silico analysis demonstrated that the mutations NC_000005.9:g.176831232G>C (p.Thr328Arg) and NC_000005.9:g.176831232G>T (p.Thr328Lys) have a pathogenic effect, which is fully consistent with their previously established clinical status. At the same time, the polymorphic variants NC_000005.9:g.176831258C>G (p.Gln319His) and NC_000005.9:g.176831285C>G (p.Arg310Ser) do not appear to be independent causes of the disease, although their potential role in modifying the clinical phenotype cannot be excluded.

Bioinformatic analysis plays a key role in the preliminary assessment of the significance of newly identified mutations in the F12 gene and facilitates a more precise identification of pathogenic variants. The integration of bioinformatic tools into diagnostic workflows is essential for determining the cause of disease in patients with hereditary angioedema who present with normal levels and functional activity of C1 inhibitor.

Abstract

The aim of this study was to determine the mechanisms of action of {Mo₇₂Fe₃₀} on erythropoiesis and hematological parameters *in vitro* and *in vivo*. The obtained results showed that neither the nanocluster polyoxometalate {Mo₇₂Fe₃₀} nor its destruction products (low-molecular-weight iron and molybdenum-containing ions) affected the activity of non-specific esterase in the cytoplasm of central macrophages of bone marrow erythroblastic islands, thus indicating no disruption of endotoxin and xenobiotic detoxification processes carried out by these macrophages. However, administration of the studied substances resulted in a decrease in macrophage phagocytic capacity without altering cellular involvement in phagocytosis.

Analysis of erythroblastic island cultures revealed the following patterns: administration of {Mo₇₂Fe₃₀} and its destruction products (PD) accelerated erythroid cell maturation within the erythroblastic island corona. At 24 hours after their administration, a decrease in the number of class 3 and involutive erythroblastic islands was observed due to accelerated maturation and dissociation of erythroblastic islands. This process was confirmed by an increase in the number of erythroid cells in the myelogram at 24 hours after these substances administration. The simultaneous increase in the number of reconstructing erythroblastic islands in the groups treated with the polyoxometalate and its PD suggests the development of an additional wave of erythropoiesis within the island corona. On day 2 after administration of {Mo₇₂Fe₃₀}, there was an increase in the number of proliferating erythroblastic islands of the class 2 and 3, while the number of class 1 islands remained unchanged. This is attributed to the involvement of new macrophages into erythropoiesis and active erythroid cells proliferation. On day 3, a shift towards a more mature proliferating erythroblastic islands of the class 2 was observed. These data indicate the acceleration of erythrocyte maturation within erythroblastic islands after 3 days of the {Mo₇₂Fe₃₀} administration. A similar trend was observed with the PD administration, although the erythropoiesis-accelerating effect was less pronounced.

Analysis of rat bone marrow myelograms revealed an increase in bone marrow cellularity after seven days of administration of the studied substances ({Mo72Fe30} and PD). This increase was attributed, in part, to an increase in the number of erythroid cells and reticulocytes.

These data are consistent with peripheral blood parameters in rats. A statistically significant increase in erythrocyte count, hemoglobin levels, and hematocrit values was observed following administration of the studied substances ({Mo72Fe30} and PD).

The experimental results suggest a potential stimulatory effect on erythroid lineage development.

Abstract

The age-related dynamics of the main subsets of blood lymphocytes in children has been studied quite well, but the question of the limits of the normal ranges, especially at an early age, remains actual and important. The paper analyzes results of a direct study dataset of 624 blood samples of healthy children aged 1 week to 17 years and 11 months, residents of Moscow and the central regions of Russia, who underwent a clinical complete blood count during routine checkups. The main criterion for the inclusion of a child in the study was reliable information about the absence of acute and chronic diseases. The absolute number of lymphocytes was determined in a complex of indicators of automated blood analysis, using 5 Diff technology of flow hemocytometry with fluorescent staining of nucleic acids. The percentage and absolute values of T-lymphocytes, T-helper cells, T-cytotoxic cells, the ratio of helper/cytotoxic lymphocytes, as well as the percentage and absolute numbers of B-lymphocytes and natural killers were determined using the residual blood volume by flow cytometry with 3-4 color staining.  14 age groups were formed with the number of observations in each from 40 to 64. The Kolmogorov-Smirnov method confirmed the normal distribution of the obtained numerical indicators both in the general group and in all separate age groups. The article provides detailed results of processing the data obtained using nonparametric (median, interquartile range, range from 10 to 90 centiles) and parametric (mean and standard deviation) statistics methods. Based on an estimate of the range of 10-90 centiles, the normal limits for the percentage and absolute values of the main subpopulations of lymphocytes were calculated. The well-known dynamics of a gradual decrease in the absolute number of lymphocytes and absolute values for all major subpopulations, as well as relatively higher indicators of the helper/cytotoxic ratio in early age groups, have been confirmed. A higher percentage of B-lymphocytes was detected in children in the age groups from 2 months to 2 years, which, together with a high total number of lymphocytes, determined fairly high absolute B-cell counts in these age groups. The proposed normal ranges were validated on an independent group of 75 children aged 2 to 18 years. The individual indicators of the children in the validation group exceeded the norm in 4 cases (5.3% with an acceptable level of discrepancies up to 10%), which allows us to consider the norms validated. The most debatable results are about the relatively high absolute level В-lymphocytes in young children are in good agreement with the pooled data of the meta-analysis of the world literature. The proposed normal ranges are designed in a form that is convenient for practical use.

Abstract

Despite the availability of specific prophylaxis against viral hepatitis B (HBV), the problem of HBV morbidity among health care workers is not becoming less significant. A latent form of hepatitis (LHB) may play a role in maintaining the epidemic spread of HBV.  The aim of the study was to identify the dependence of the incidence of anti-HBc among employees of medical organizations on age, gender and professional categories.

Blood serum was tested for the presence of anti-HBc using the ELISA method among 1,643 employees of medical organizations (doctors, nurses, orderlies, other personnel). Blood serum samples were examined for the presence of anti-HBc by ELISA using the HepaBest anti-HBc-IgG enzyme-linked immunosorbent assay system manufactured by Vector-Best-Europe JSC. The results were statistically analyzed using Microsoft Office Excel 2010 and Prism9 (GraphPad, USA). For comparison groups of medical workers according to the frequency of occurrence of Anti-HBc, the chi-square criterion was used.

Anti-HBs was detected in 601 people, which amounted to 36.6% of the total study sample, in 1042 people or 63.4% anti-HBs was not detected. Almost half of the paramedical personnel from the study sample were IgG seropositive for the nuclear antigen of the hepatitis B virus (50.6%). There were no special differences in the proportion of anti-HBc detection in men (31.5%) and women (37.0%). In the structure of seropositive anti-HBc in a smaller percentage, anti-HBc is detected in the age category "young age" (24.0%), the largest share falls on the category of "middle age" (45–59 years) - 38.8%, which is associated with the length of service, i.e. with the risk of longer exposure to a biological factor, which is contact with HBV sources.

The frequency of detection of anti-HBc to HBV nuclear antigen along with anti-virus surface protein antibody (anti-HBs) is an indicator of the prevalence of HBV. We consider it possible to recommend conducting anti-HB tests among the personnel of medical organizations before immunization with additional tests (virus DNA) with a positive result in order to exclude medical workers as a source of infection and further monitoring them for timely provision of the necessary medical care, as well as adjusting the volume of immunization against HBV in this professional environment.

AbstractThe purpose of study was to evaluate the state of the enzymes of the purinergic system and the subpopulation composition of lymphocytes in patients with newly diagnosed infiltrative pulmonary tuberculosis, depending on the drug resistance of Mtb to anti-tuberculosis drugs. In 109 patients with drug-resistant and drug-sensitive Mtb who achieved significant or less pronounced improvement after the intensive phase of chemotherapy, the activity of adenosine deaminase (eADA-1, 2), the concentration of ecto-5'-nucleotidase (eNT5E), CD26 (DPPIV), and the subpopulation composition of lymphocytes were evaluated before treatment. In patients isolating drug-sensitive Mtb strains who achieved a "less pronounced improvement", the concentration and activity of ectoenzymes responsible for the formation of extracellular adenosine (eNT5E) and its transformation (eADA-1 and eADA-2), the proportion of cytotoxic T cells was higher compared with patients who achieved significant improvement. Patients isolating drug-resistant Mtb strains who achieved a "less pronounced improvement" had lower absolute counts of T-lymphocytes and helper T cells with an increase in the proportion of cytotoxic T cells and increased eADA-2 activity, compared with individuals who achieved significant improvement. Thus, prior to the initiation of tuberculosis chemotherapy, the activity of purine metabolism enzymes and the subpopulation composition of lymphocytes were not associated with the characteristics of Mtb drug resistance. The relationship between the parameters of purinergic regulation enzymes and the number/ proportion of lymphocytes was revealed in patients who achieved significant improvement, with less pronounced improvement, regardless of the drug resistance of Mtb, no such relationships were found. This indicates an imbalance of inflammatory factors and the immune response to Mtb in individuals who showed the worst results from the intensive phase of chemotherapy. Taking into account the contribution of each component of protective reactions is necessary for the appointment of adequate chemotherapy, pathogenetic therapy and immunocorrection aimed at stopping the progression of the disease.

AbstractPseudomonas aeruginosa, an opportunistic pathogen of considerable clinical import, presents a formidable challenge to susceptible individuals, particularly within the confines of nosocomial settings. Those with compromised immune systems, indwelling medical devices such as catheters, and extensive thermal injuries are especially vulnerable to its insidious and often devastating effects. This investigation sought to elucidate the roles of Toll-like receptor 4 (TLR-4), Toll-like receptor 5 (TLR-5), the chemokine CXCL8, and its cognate receptor CXCR1 in the context of P. aeruginosa infection. Our findings indicate a significant involvement of TLR-4 and CXCL8 in the host response to this pathogen. Notably, CXCR1 expression was observed to be downregulated both in cellular models and in whole blood samples obtained from patients afflicted with bacterial infections, specifically those caused by P. aeruginosa. Utilizing antibodies targeting these cell surface molecules, we further explored their influence on bacterial adhesion and the modulation of infection. The application of anti-CXCR1 antibodies resulted in a demonstrable increase in bacterial infection in both T24 and A549 cell lines. Conversely, the administration of anti-TLR-4 antibodies exerted an inhibitory effect on P. aeruginosa infection. Anti-CXCL8 antibodies, however, did not elicit a discernible impact on bacterial infection within the initial three hours of exposure. These observations suggest a dichotomous role for these molecules in the host response to P. aeruginosa: CXCR1 appears to function as a negative regulator, while TLR-4 appears to act as a positive regulator in the context of bacterial infection.

Abstract

Introduction. The issues of pathogenetic and restorative therapy of gestational pyelonephritis during pregnancy are relevant and debatable in modern evidence-based medicine. Treatment of these patients is associated with certain subtleties, since during pregnancy it is necessary not only to provide for the relief of the inflammatory process in the mother's urinary system, but also, using pharmacological agents, not to harm the health of the future child. This problem is characterized by an annual increase in morbidity and complications, which often have an extremely negative effect on the course of pregnancy and childbirth in 3-17% of women of childbearing age in Russia annually.

Purpose of the study: to determine the effectiveness of using Viferon in stabilizing immune disorders in gestational pyelonephritis.

Material and methods. The study included 80 women (mean age 32.4±3.7 years) divided into groups: control (consisting of healthy non-pregnant female volunteers), two groups with acute pyelonephritis in the second and third trimesters of pregnancy, receiving basic therapy, and two groups of pregnant women with a combination of basic treatment and the immunomodulator Viferon. The cytokine spectrum, the state of the complement system and the functional-metabolic activity of neutrophils were studied in the circulating peripheral blood. Blood was taken before the start of complex treatment, i.e. immediately upon admission and after the treatment, i.e. upon discharge from the hospital, with the achieved signs of clinical relief of the disease.

Results. More effective relief of immune inflammation of acute pyelonephritis was revealed when using Viferon in the 2nd and 3rd trimesters of pregnancy compared to traditional therapy. The proposed treatment method allows for faster normalization of altered immune status parameters and the fastest clinical recovery of patients. The use of this method in medical practice with traditional therapy is a relevant, safe and effective method for treating acute gestational pyelonephritis, which will reduce the incidence of obstetric and urological complications, reduce the number of hospital stays in a medical institution, reduce the cost of hospital stay, improve the quality of life of pregnant women and create a healthy environment for the formation of the fetus.

Abstract

Neuroblastoma (NB) is the most common extracranial solid tumor in children, 8-10% of all pediatric tumors and an incidence of about 1-1.3 cases per 100,000 children under 15 years of age. Despite the use of intensive treatment with surgery, high-dose chemotherapy and radiotherapy, the 5-year event-free survival rate is 25-50% and 10-40% after relapse. Over the past decade, a new type of cell therapy with chimeric antigen receptor (CAR) modification of lymphocytes has been rapidly developed. One of the main known antigens for the development of CAR-T therapy against neuroblastoma is the disialoganglioside GD2, the expression of which is characterized in 100% of cases of this disease. Most clinical anti-GD2 CAR variants are based on scFv 14.G2, originating from chimeric antibody 14.18 (denutuximab). In 2020, the FDA approved a new anti-GD2 humanized antibody, 3F8 (naxitamab) with a better safety profile.  Despite partial success, the results of anti-GD2 CAR-T therapy remain modest. One option to increase receptor specificity is targeting O-acetyl-GD2, a disialoganglioside derivative in which the external sialic acid residue is modified with an O-acetyl ester. Acetylation of GD2 occurs only in tumor cells and is not found in peripheral nerves. An 8B6 antibody is known to target O-acetyl-GD2. Thus, at least 3 therapeutic antibodies, 14G2a, hu3F8 and 8B6 compete with each other for targeting GD2 with CAR-T cells. And in all cases the anti-GD2 CAR contains insertion domains in the extracellular part of the molecule. Results of separate clinical trials have been published for CAR-T based on 14G2a and hu3F8, but there are no data on the use of the 8B6 antibody in CARs yet. The aim of the present study is to obtain chimeric antigenic receptors of the 2nd generation based on three antibodies, with different lengths of the extracellular domain and to evaluate their functional activity against a number of cell lines to justify further clinical trials.

Abstract

Adjuvants represent important components of vaccines, including medications for specific immunotherapy, which improve their efficacy and safety. Current scope of vaccines development includes approaches based on adjuvant inclusion into the immunogenic molecule. Such covalent adjuvants vary greatly in chemical structure, and particularly are found among peptides. Peptides have distinct advantages over substances of other chemical classes, such as high efficiency and biodegradation, while dendrimeric cationic peptides are often capable of enhanced transport ability when compared to linear peptides. In this work, the ability to enhance IgG induction was studied for two cationic dendrimeric cell-penetrating peptides with previously reported transmembrane activity: LTP and SA-40. The peptides were obtained by solid-phase synthesis and characterized by mass-spectrometry and zone capillary electrophoresis. To study adjuvant activity, peptides were conjugated with the recombinant protein Bet v 1, a major birch pollen allergen, produced biotechnologically using e. coli. Conjugation was carried out with Michael reaction after modification of the protein with maleimide function. Conjugates were purified by gel chromatography and dialysis, conjugation efficiency was estimated with SDS-PAGE. Conjugates and pure rBet v 1 were used to immunize BALB/c mice to compare IgG levels to rBet v 1 after four injections without using other adjuvants. Mouse blood sera were studied with ELISA for IgG levels against rBet v 1, which was target criteria of the study, as well as against the conjugates and free LTP and SA-40. It was found that both conjugates significantly increased the IgG level to the base protein, while one of them, LTP-rBet v 1, also induced serum reactivity both to itself and to free LTP. The increase in IgG level to rBet v 1 was approximately fourfold in the case of LTP-rBet v 1, and threefold in the case of SA-40-rBet v 1. Based on the results of the study, it was concluded that both LTP and SA-40 possess adjuvant activity. We believe that LTP and SA-40 structures can be used as references in the development of new peptide adjuvants, and SA-40 itself can be used as a covalent adjuvant. In turn, LTP may be seen perspective as a non-covalent adjuvant.

Abstract

Prostate cancer (PC); is the second leading cause of cancer mortality among men. Human papillomavirus (HPV) is the most common cause of cervical cancer, strongly associated with anal and vaginal cancers. Also, interleukin-12 (IL-12) induces antitumor immunity. This study aimed to investigate the role of HPV in PC; and determine its effects on serum IL-12.

 Between 2018 and 2022 in Ahvaz, researchers obtained 55 paraffin samples of malignant prostate lesions and 55 control samples of benign hyperplasia tissues from the prostate. Blood samples were collected from 24 diagnosed cancer patients to assess IL-12 levels before treatment initiation. Additionally, 24 patients with benign prostatic hyperplasia participated as controls. We performed DNA extraction using the phenol-chloroform method and examined the presence of papillomavirus DNA in tissues through Nested-PCR. Subsequently, IL-12 levels in serum were measured using ELISA. 

The findings did not show the relationship between HPV and PC; HPV infection was not correlated to the presence of IL-12 secretion. However, with the progression of cancer, the level of IL-12 decreased significantly in patients compared to the control group (P<0.05).

HPV infection can exist in prostate tissue, although this does not mean that it contributes to P.C. development. The most significant strains infecting prostate tissue are types 16 and 18. Compared to the control group and with different Gleason scores, prostate cancer patient's levels of interleukin-12 secretion are significantly lower. One can make effective measures to assess the prognosis, regulate the condition, or aid in treating individuals using this crucial cytokine.

Abstract

Malignant neoplasms often arise against the background of immunodeficiency. But anticancer chemotherapeutic drugs can also cause the development or aggravate immunodeficiency, affecting the bone marrow function. Previous studies have shown that the bioorganocomplex obtained from the chickens’ Fabricius bursa has an antioxidant and immunotropic effect. The aim of this research was to investigate the protective mechanism of the bioorganocomplex «Bursanatal» against cyclophosphamide-induced immunosuppression in the bone marrow, thymus, spleen of C57Bl/6 mice. Immunodeficiency was induced with single cyclophosphamide injection (200 mg/kg). Histological, morphometric, radiological methods were used, as well as immunohistochemical staining of the spleen and thymus with CD45 and CD3 antibodies. The studies showed that cyclophosphamide injection affects both peripheral blood and bone marrow. Moreover, neutrophilic, basophilic and erythroid precursor cells are less sensitive to the cyclophosphamide. So on the 8th day their compensatory hyperplasia is observed. The use of the bioorganocomplex is accompanied by the leukocytes, erythrocytes and hemoglobin increase in the peripheral blood. The cellularity of the bone marrow increases due to the dividing and maturation of erythrocyte, neutrophil, basophil and lymphocyte lineage cells. Bioorganocomplex administration causes a reliable decrease the area of the red pulp in spleen of mice with cyclophosphamide-induced immunodeficiency. The activation of extramedullary hematopoiesis are found in the spleen of treated mice – the number of colony-forming hematopoietic progenitors increases. Also it is noted that the area of the reactive center in the white pulp decreases with a significant increase the CD3+ cells in the red and white pulp under the «Bursanatal» treatment. At the same time, it does not affect thymus pathomorphology.

Abstract

Today, the clinical manifestations of COVID-19 patients range from mild to severe, especially in groups of patients susceptible to chronic diseases. The selection of the most informative indicators that provide a prognosis of the possible outcome or severe course of the disease in patients with COVID-19 is necessary at an early stage to determine effective treatment tactics. The earliest predictors of deterioration in patients with COVID-19 are elevated levels of interleukin-6, interleukin-10, C-reactive protein, procalcitonin, and other indicators of the innate immune system. One of the objectives of this study was to determine the most informative indicators of general clinical blood analysis and the main subpopulations of lymphocytes in patients with COVID-19, which can help in interpreting the severity of the disease and the development of a cytokine storm along with clinical manifestations and functional diagnostic methods. We conducted a retrospective cohort study of peripheral blood in 65 patients, of whom 57 were men and 8 were women, the age of the patients ranged from 32 to 82 years, the average age was 48.6 years. After examining the total blood count in 65 samples, cytometric analysis was performed using the Cytomics™ FC 500 series flow cytofluorometry system from Beckman Coulter (USA), CD45-ECD, CD3-FITC, CD4-PC7, CD8-PE, CD19-PC-5, CD16+56-PE monoclonal antibodies from "Beckman Coulter" (USA). Statistical processing of the research results was carried out in the IBM SPSS Statistics 26.0 program (IBM, USA). In the patients with COVID-19 examined by us, absolute lymphopenia is noted against the background of neutrophilosis at the initial medical treatment, which is expressed in an increase in the values of the neutrophil-leukocyte and leukocyte-T-lymphocyte indices. These indexes are quite informative and can be used in predicting disease risks. The determination of the most reliable indicators of cellular immunity, such as CD3+, CD3+CD4+, CD3+CD8+, CD3-CD16+CD56+ lymphocytes, in addition to a clinical blood test, can assess the features of the body's immune response to SARS–CoV-2 infection, help predict the severity of the disease, and make a decision to change treatment tactics if necessary.

Abstract

The ability of CIK cells to recognize and lyse tumor cells has been demonstrated in preclinical studies in vitro. The ClinicalTrials.gov portal has registered over 600 completed clinical trials devoted to the method of cellular immunotherapy, including over 30 on the use of CIK. There are many ways to obtain cells drugs based on lymphocytes. Their common drawback is the need for additional blood sampling to obtain a cell preparation in large volumes, so it remains relevant to develop new approaches that take into account the solution to this problem. The purpose of the study was to evaluate the possibility of using leukocyte filters as a source of lymphocytes to obtain large doses of CIK. Materials and methods. The article describes in detail the procedure for extracting lymphocytes from leukocyte filters (30 donors). Lymphocytes were cultured in a CO2 incubator for 10-14 days in a medium with IL-2 and IL-15. The level of cell activation was assessed by ELISA and flow cytometry. The subpopulation composition of T, NK, TNK, B lymphocytes (CD3, CD4, CD8, CD14, CD16, CD19, CD45, CD56) and activation markers on them (CD38 and HLA-DR) were assessed before and after activation. Results. The possibility of using leukocyte filters after separation of plasma with leukocytes remaining on them was assessed. The functional activity of extracted lymphocytes and the cytokine composition of supernatants after cultivation in a complete nutrient medium were studied. Peptide complex (supernatant) contained high doses of signaling proteins: IL-2, IL-6 IL-10, IFNγ, TNFα and retained its functional activity after long-term storage at low temperatures. The drug is sterile and consists predominantly of T-, TNK- and NK- cells with a high level of viability and contains activation markers: HLA-DR, CD38. Conclusion.  Thus, up to 350 million activated lymphocytes and aliquots with the supernatant were obtained from one filter. The drug can be used both for basic research and for administering AIT to cancer patients in order to restore the patients’ own antitumor immunity potential. This amount of cellular and acellular preparation is sufficient to conduct a larger number of AIT courses without additional blood sampling.

Abstract

The cellular composition of red bone marrow is composed of an extremely heterogeneous cell population, including stem cells, reticulum cells and cells of the five hematopoietic lineages. The current task for cell therapy and experimental studies is to obtain cell fractions of bone marrow enriched with a certain type of cells. In this paper we investigated the level of cytokine mRNA expression in bone marrow cell fractions isolated by counterflow centrifugation in an elutriator rotor. Cell fractionations were isolated at a rotor speed of 2500 rpm. Six cell fractions (F) were collected: F-1 at a buffer flow rate of 12 ml/min, F-2 – 15 ml/min, F-3 – 19 ml/min, F-4 – 23 ml/min, F-5 – 50 ml/min, F-6 – collected after stopping the rotor rotating. Cytomorphological analysis of the fractions showed that erythrocytes (80%) and lymphocytes (40%) are collected in the “light” fraction F-1, lymphocytes (44%), polychromatophilic (50%) and oxyphilic (51%) normocytes – in F-2, neutrophils (70%) and eosinophilic granulocytes (40%)  – in F-3 and F-4, macrophages (64%), megakaryocytes (95%), reticular (35%) and mast cells (62%) – in F-6. Blast cells of different hematopoietic lineages were detected mainly in F-5. Using RT-PCR, the maximum gene expression of the stem cell factor (Scf)  and granulocyte-macrophage colony-stimulating factor (Gm-csf) was detected in the “heavy” fraction F-6, gene expression of tumor necrosis factor-α (Tnf-α) and erythropoietin (Epo) – in F-4, F-5 and F-6, and gene expression of macrophage colony-stimulating factor (M-csf)  – in F-3 and F-4. Thus, this method allow to separate the "light" fractions of lymphocytes and erythrocytes from the bulk of bone marrow cells, which can be used in allogeneic bone marrow cell transplantation to reduce the risk of acute graft-versus-host disease. Another important advantage of the method is the ability to obtain fractions of "heavy" cells with high regenerative potential in order to use them in cell therapy to stimulate regenerative processes in organs and tissues.

Abstract

The main method of assessing male reproductive function is sperm analysis, which is not always indicative of the cause of infertility. In the literature, a special role in the violation of male fertility is attributed to oxidative stress, the result of which the male germ cells are damaged. Damage of spermatozoa membranes is accompanied by the release of their DNA into the extracellular space, which is an inducer of local inflammatory reaction. In this case, the maintenance of seminal fluid homeostasis will depend on the efficiency of degradation of extracellular DNA. In human beings, this function is performed by DNAase enzymes localized in all tissues and body fluids. Their role in the process of fertilization and fragmentation of genetic material of spermatozoa is also noted. Excess or deficiency of DNAases can lead to the development of a wide range of diseases. The aim of the present study was to establish reference intervals for ejaculate DNAase 1L3 and DNAase II and to search for relationships between the concentration of nucleases and sperm analysis parameters. The seminal fluid of 82 conditionally healthy men aged 18-49 years examined at the Research Institute of Immunology of SUSMU was used as a material for the study. Exclusion criteria were the presence of inflammatory diseases of the urogenital tract and STIs. Sperm analysis was performed according to the WHO laboratory manual. STI examination was performed by real-time PCR. DNAase concentration in the ejaculate was determined by ELISA method. Descriptive statistics, construction of histograms and reference intervals were performed in the program “R”. For the first time we have determined the levels of DNase 1L3 and DNase II in seminal fluid and established reference intervals for them. Shifts in DNAase 1L3 and DNAase II concentrations can most likely be considered as indicators of possible pathologic conditions. In particular, an increased concentration of DNase 1L3 is an indicator of a pathological process associated with low sperm motility, and high levels of DNase II can be considered as an indicator of a local inflammatory reaction.

Abstract

Introduction. In 2020 COVID-19 was declared to be a pandemic. Since then it was revealed that the severity of the disease pathology may depend not only on the viral strain, but also on the functioning of the immune system. Target. To study characteristics of expression and genetic factors of innate immunity in patients who have had COVID-19. Materials and methods. Material (scrapings from the mucous membranes and venous blood) from 148 patients was studied. Identification of the studied markers was carried out using reverse transcription and RT-PCR methods. Statistical analysis of the results was carried out using the Mann-Whitney test, Fisher's exact test, χ2 test, odds ratio and 95% confidence interval. Results. Our study demonstrated the prognostic role of polymorphic markers and haplotypes in the TLR9 (rs352140 and rs5743836) and TLR4 (rs11536889 and rs4986791) genes in relation to the risk of developing severe SARS-CoV-2 infection. As the result of studying the long-term effects of COVID-19 it was revealed that an imbalance in the expression of receptor and effector molecules at the mucosal level of the immune system remains in patients who have had the disease. There is a decrease in the expression level of both receptor molecules (TLR3, TLR7) and antiviral immune response factors (IL28) in the mucous membranes of the oropharynx with the background of a general increase of these markers in the epithelial cells of the nasopharyngeal mucosa. Conclusion. The expression and genetic factors of innate immunity leading to severe SARS-CoV-2 infection and, consequently, persistent changes in the immune system long after recovery, have been studied, which expands our knowledge of the molecular genetic mechanisms associated with the long-term course of COVID-19. The results obtained during the study can help assess the risks of developing severe infection caused by SARS-CoV-2 and developing complications in hospitalized patients.

Abstract

Objective: To develop a vaccine against group A streptococci (Streptococcus pyogenes, GAS), the causative agent of a broad spectrum of infections with varying severity.

Methods: The expression vectors pET27 and pQE30 were utilized to clone genes encoding recombinant proteins, which were subsequently affinity-purified. Female mice were immunized subcutaneously twice with the purified polypeptides (20 μg/mouse) formulated with Alum adjuvant (2:1) at three-week intervals. Immune sera were analyzed using ELISA to evaluate antigen-specific responses. Three weeks after the final immunization, mice were challenged intraperitoneally with GAS M1 serotype at a dose of 5x10^7 CFU/mouse.  Vaccination efficacy was determined by comparing bacterial clearance in vaccinated versus control animals, assessed by bacterial loads in the spleen at 3 and 15 hours post-infection.

Results: The vaccine candidate is a hybrid recombinant protein comprising fragments from two essential GAS virulence factors: C5a peptidase (ScpA) and SpeA exotoxin. T- and B-cell epitopes from conserved regions, common across multiple GAS serotypes, were identified and included in the construct using bioinformatics tools.

The integration of these epitopes is designed to confer broad-spectrum protection against GAS strains carrying exotoxin, particularly those linked to invasive infections. Immunogenicity studies in mice revealed a robust humoral immune response targeting both components of the hybrid protein. Further evaluation of protective efficacy demonstrated accelerated bacterial clearance in vaccinated animals, with the SpeA fragment playing a significant protective role.

Conclusion: These findings underscore the potential of this recombinant chimeric vaccine as a promising candidate for the prevention of group A streptococcal infections, addressing the critical need for effective prophylactic strategies against GAS-associated diseases.

Abstract

The article summarizes experimental and clinical data on the immunological properties of oxidized dextran. The main emphasis is on the pathophysiological mechanism of action of oxidized dextran, in particular on its ability to selectively activate tissue macrophages, including testicular macrophages, which are directly involved in spermatogenesis. In the experimental part of the work on the model of spermatogenesis disorders in rats induced by the introduction of Escherichia coli endotoxin, it was shown that oxidized dextran significantly reduces pathomorphological changes in the testes. At the same time, such an effect is associated with the activation of tissue macrophages and the release of corresponding cytokines, which are aimed at stopping the inflammatory process and normalizing microcirculatory hemodynamics. The presented experimental data correlate with the data on the high clinical efficacy of oxidized dextran in spermatogenesis disorders and male infertility. In the clinical part of the work on the effect of rectal administration of oxidized dextran in patients with severe spermatogenesis disorders, it was shown that oxidized dextran significantly increases the volume of ejaculate, the concentration of spermatozoa in the ejaculate, and the total number of spermatozoa. The authors link the data of experimental and clinical studies within the framework of a single concept of tissue macrophage activation and the ability of macrophages to change their phenotype depending on the microenvironment and the characteristics of the infectious and inflammatory process. This approach is also confirmed by clinical data on the effect of macrophage phenotypes on drug resistance and the severity of intracellular infections, in particular tuberculosis. The analysis of experimental and clinical data allows the authors to propose a theoretical model of the mechanism of therapeutic effectiveness of oxidized dextran in intracellular infections. The experimental and clinical data presented by the authors allow us to consider oxidized dextran as a very effective means for the treatment of intracellular infections, including urogenital infections causing male infertility.

Synthetic peptides are a good basis for HIV vaccine development.  The immune response are focused only on a specific epitope after their administration, they are able to activate both pathways (humoral and cellular) of the immune response, and they are safe and well tolerated. Having a low molecular weight, synthetic peptides possess a low immunogenicity, therefore it is necessary to use various immunoadjuvants in an immunogenic composition. V3 loops of the gp120 envelope  protein is one of the main protective epitope, and a number of monoclonal antibodies with broad neutralizing activity have been obtained to it. We conducted a study of the immunogenicity of peptides copying the V3 loop of the group M HIV1 virus consensus sequence and the Russian isolate RUA022a2. The impact of  the method of administration (subcutaneously and intraperitoneally) and the use of an immunoadjuvant was also assessed. A synthetic analogue of double-stranded RNA, poly (I:C) was used as an adjuvant. poly (I:C)  is a ligand of innate immunity receptors TLR3. The studies were conducted on balb/c mice. It has been shown that the method of administration does not affect an immune response development to the peptides. However, earlier production of specific IgG antibodies was observed in the groups where the immunoadjuvant was used. At the same time, the antibody titer was slightly higher in the groups where peptides were administered with the adjuvant after the third (last) administration. There were also no differences in the isotype of the induced antibodies. IgG1 antibodies were predominantly induced in all groups. Specific IgM antibodies were detected only after the third administration of the antigens. Their titer did not depend on the administration method and the antibody titer was slightly higher in the groups where peptides were administered with the poly (I:C).  The induced antibodies did not have neutralizing activity against isolate QF495.23.M.EnvA1. In the study of antigen-specific cellular activation the production of the marker of the Th1 response IFN γ is detected only in groups where poly(I:С) was used. In addition, a low level of anti-inflammatory cytokine IL10 was determined in groups where poly (I:С) was included in the immunogenic composition. In addition, the IL10 highest level was detected in groups with intraperitoneal administration. Studies have shown that the use of poly (I:С) adjuvant promotes the immune response development  to the synthetic peptides, that contributes to  earlier induction of specific antibodies, as well as switching to the Th1 pathway. The data obtained can be used in the design of HIV vaccines and other viral infections to increase their immunogenicity and the possibility of inducing a protective immune response.

 

Abstract

It has been established that exometabolites of paleobacteria Bacillus cereus strain 875 TS significantly activate the differentiation of monocytes in the subpopulation of intermediate (CD14⁺CD16⁺) and non-classical (CD14^loCD16⁺) monocytes, effector CD4⁺ and CD8⁺ T-lymphocytes with a change in markers of early (CD69), mid (CD25) and late (HLA- DR) activation, differentiation of Treg (CD3⁺CD4⁺CD25^hiCD127^-), and also stimulate the synthesis of cytokines IFNγ and IL-4 relative to control levels. The peculiarities of the influence of exometabolites of paleobacteria include the dependence of immunomodulatory activity on the method of their preparation - “cold” (obtained from bacteria when they are cultivated at 5ºC), “mid temperature” (22ºC) and “heat” (37ºC) metabolites.  “Cold” metabolites stimulate mainly the mechanisms of the immune response with pro-inflammatory activity, in particular, the differentiation of intermediate CD14⁺CD16⁺ monocytes, an increase in the activity of differentiation of CD8⁺ T-lymphocytes and the synthesis of IFNγ. “Heat” metabolites stimulate predominantly immune response mechanisms with anti-inflammatory activity, namely the differentiation of non-classical CD14^loCD16⁺ monocytes, increased differentiation activity of CD4⁺ T-lymphocytes and IL-4 secretion. Also a distinctive feature is the relationship between pro- and anti-inflammatory mechanisms, which do not depend on the type of exometabolites. Thus, during the first three days of cell culture, the differentiation activity of CD8⁺ T-lymphocytes prevails over the differentiation of CD4⁺ T-lymphocytes, and the level of IFNγ secretion exceeds the level of IL-4. On the third day, there is a significant increase in Treg levels, which is accompanied by a tendency to normalize the balance between IFNγ(Th1) and IL-4(Th2) by the seventh day. There is a clear influence of Tregs (CD3⁺CD4⁺CD25^hiCD127^-) on the strength and duration of the immune response. The increase in Treg levels occurs moderately and briefly, which, on the one hand, prevents the excessive development of pro-inflammatory mechanisms, and on the other hand, does not lead to the development of long-term immunosuppression. An increase in Treg levels on days 1-3 is accompanied by a decrease in the activity of monocyte differentiation in the subpopulation and the synthesis of the proinflammatory cytokine IFNγ.

Abstract

Staying in a combat zone is associated with a high risk of developing mental disorders. A significant amount of knowledge has been accumulated about the mechanisms of immune-mediated reactions in the development of neuropsychiatric pathology, in particular post-traumatic stress disorder (PTSD). Activated T-regulatory cells play an important role in the development of neuroinflammation under stress.

Purpose of the study: to study the indicators of the blood system, cytometric features of the cellular compartment of the immune system in participants in modern military conflicts with the presence of adaptation disorders associated with stress.

Materials and methods. We examined 97 veterans - participants in modern military conflicts, males from 35 to 55 years old, of which 35 were veterans of a special military operation in Ukraine (SVO) - the main group, 42 veterans of the second Chechen military campaign (comparison group), 20 people - healthy military personnel (control group. All patients underwent pathopsychological examination in accordance with clinical recommendations. 12% of Northern Military District veterans were diagnosed with PTSD, 77% had various types of neurotic, stress-related and somatoform disorders. In the comparison group, 2% of combatants were diagnosed with chronic personality change. A complete blood count was performed using a standardized method on a hematology analyzer. Immunophenotyping of lymphocytes using a Navios flow cytometer (Beckman Coulter, USA).

Results and discussion. In the group of SVO veterans, a decrease in the degree of dispersion of erythrocytes in volume, an indicator of heterogeneity and the average volume of platelets, lymphopenia, and monocytosis was noted, which reflects the multidirectional effect of hematopoietic cell regulators on individual lines of differentiation of mononuclear cells. Cytometric analysis of the composition of lymphocytes showed a decrease in T-helper cells and mature NK cells in the group of SVO veterans, which explains the presence of lymphopenia and may indicate a deficiency of the adaptive and innate compartments of immune defense in conditions of a prolonged response to stress. An increase in the number of TNK and T-regulatory lymphocytes, which prevent the transition of the immune response to an autoimmune reaction, has been established. Under stress, the transcription factor Foxp3 is involved in the upregulation of the glucocorticoid-induced TNF receptor in the T-regulatory cell line, potentiating the proliferative activity of the latter. A decrease in the number of T-helpers and T-regulatory cells with markers of early and late positive activation has been shown, limiting the development of both autoimmune reactions and the development of stress-induced neuroinflammation. There were practically no differences between the control group and the indicators of the comparison group, which indicates that the severity of stress-induced neuroimmune reactions leveled out over time.

Conclusion. Hematopoiesis induced by stress mediators and changes in the quantitative spectrum of lymphocyte subpopulations mediated by neuroimmune influences are a consequence of a complex multi-level neuropsychodynamic process of the central nervous system associated with clinical forms of adaptation disorders.

Abstract

Introduction: The COVID-19 pandemic has focused the attention of researchers around the world on the fight against this infection. A critical approach to combating COVID-19 has been the development of preventive vaccines based on a range of platforms, including DNA and RNA vaccines, vector and subunit vaccines. Subunit vaccines have become one of these platforms, primarily due to their unsurpassed safety profile. However, the safety of these vaccines is often associated with low effectiveness, so it is often necessary to use adjuvants, as well as use more complex immunization regimens. At the same time, an important advantage of subunit vaccines is scalability and relative ease of production, since there is no need to work with live virus or viral vectors during the production process. Our object of the work was to develop a candidate vaccine based on the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike S-protein of the Delta variant (B.1.617.2). Materials and Methods: The study used immunological methods and methods of genetic engineering and biotechnology. Results: In the course of work based on the mammalian cells CHO-K1 developed producer of recombinant RBD. To obtain a protein that meets the requirements of injectable drugs, a chromatographic purification scheme was developed, including affinity and ion exchange chromatography. A variant of the subunit vaccine «Delta-Vac» based on the obtained recombinant protein was proposed. Assessment of the immunogenicity of the Delta-Vac vaccine was carried out on the model of laboratory mice BALB/c. Animals were immunized twice with a dose of 50 μg of RBD in combination with Al(OH)₃ at a two-week interval. The ability of the candidate vaccine to induce the production of specific IgG and neutralizing antibodies in BALB/c mice was demonstrated. The specific antibody titers of immunized animals ranged from 1/10⁵ to 1/10⁶. At the same time, blood serums had neutralizing activity against SARS-CoV-2 (variant B.1.617.2 (Delta)) with a titer of up to 1/2000. Conclusion: The vaccine "Delta-Vak» developed is highly immunogenic and induces the production of neutralizing antibodies against homologous Delta variant and heterologous Wuhan and Omicron SARS-CoV-2 variants. Thus, «Delta-Vac» can act as a vaccine candidate and serve as a prototype for the development of subunit vaccines against COVID-19.

The increasing incidence of infertility determines relevance of studying the contribution of various cellular factors, including immune cells, to its pathogenesis. The changes are described in NK cell activation in decidua and peripheral blood during infertility, as well as in representation of NKT cells and T lymphocytes in uterus at miscarriage (MC). NK cells and NKT cells are characterized by expression of activating receptor NKG2D and receptor for immunoglobulin G Fc-binding CD16. Distinct populations of T lymphocytes express NKG2D. The aim was to analyze the expression of CD16 and NKG2D by NK cells, NKT cells and NKG2D by peripheral blood T lymphocytes in women with infertility. We examined 60 women with infertility. The control group consisted of healthy non-pregnant fertile women (n=17). Mononuclear cells were obtained from peripheral blood and treated with monoclonal antibodies to CD45, CD14, CD3, CD56, CD16, NKG2D. We showed that in patients with primary infertility, NKG2D receptor expression intensity by peripheral blood NKT cells was lower than in healthy non-pregnant fertile women. The established difference may be due to absence of an embryo implantation in medical history at primary infertility and accordingly absence of the influence of cytokine and cellular microenvironment specific for first trimester of pregnancy. In patients with secondary infertility and MC, relative number of CD16+ NK cells and CD16+NKG2D- NK cells in peripheral blood was reduced compared to this parameter in women with secondary infertility without a history of MC. Relative numbers of CD16+ NK cells and CD16+NKG2D- NK cells in peripheral blood were also reduced in patients with secondary infertility and MC compared to patients with primary infertility and to healthy non-pregnant fertile women. CD16 expression intensity of NK cells was shown to be reduced in subgroup of women with secondary infertility and a history of MC compared to control group and to women with secondary infertility without a history of MC. No differences in NKG2D expression by T lymphocytes associated with infertility were established. Reduced CD16 expression by NK cells of women with secondary infertility and MC may reflect the impaired functional differentiation of NK cells associated with this pathology.

Abstract

Monoclonal antibodies (mAbs) have the potential to trigger undesired humoral immune responses in the patients and the formation of ADA (anti-drug antibody) to a drug protein.

Neutralizing anti-drug antibodies are one of the main factors affecting the safety and effectiveness of the therapy. If it is impossible to use cell-based tests to determine neutralizing antibodies, competitive ligand binding assay it can be used as an alternative.

Pembrolizumab is a broad-spectrum antitumor drug and is a humanized IgG4 kappa antibody to the programmed cell death receptor-1 (PD-1) that blocks the interaction of the receptor with its ligands PD-L1 and PD-L2. Due to the complex analysis, the use of a cell culture test for pembrolizumab is impossible, as there is a high risk of obtaining unreliable results.

Aim. Development and validation of a method for determining neutralizing antibodies to pembrolizumab in human serum based on inhibition of binding of pembrolizumab to its PD-1 target.

Material and methods. The experimental drug pembrolizumab RPH-075 (R-Pharm) was used in the study. Anti-Pembrolizumab antibodies KRIBIOLISATM Anti-Pembrolizumab (KEYTRUDA®) ELISA, India) were used as a positive control sample for neutralizing antibodies. The determination of antibodies was carried out by the ELISA method with using acid dissociation of the immune complex and the ACE technique (Affinity capture elution).

Results. The ELISA method was validated according to the following characteristic: selectivity, sensitivity, specificity, "hook" effect, drug tolerance, precision. Due to the use of sample pretreatment approaches (acid dissociation, ACE technique) for the analysis of neutralizing antibodies, a sensitivity of 100 ng/ml was achieved in the presence of pembrolizumab 40 μg/ml.

In this paper, a method for calculating the cut-point, sensitivity, and selectivity based on ROC analysis and floating exclusion limit (PSCP) through the average values of OD NC and LPC in each individual analytical cycle was substantiated.

Conclusion. The developed method for determining neutralizing antibodies to pembrolizumab can be used to assess the undesirable immunogenicity of the pembrolizumab at the stage of clinical trials.

Abstract

The phenomenon of neutrophil extracellular trap (NETs) formation, or netosis, plays a key role in the pathogenesis of infectious and inflammatory diseases. However, the lack of standardized methods for assessing NETs makes it difficult to correctly interpret the results. The aim of the study was to develop a practical methodology to investigate the ability of neutrophils to form NETs, which would allow correct identification of netosis-associated phenomena.

Neutrophils were isolated from peripheral blood of 15 healthy donors by gradient centrifugation (Ficoll-Verographin, density gradient 1.077 - 1.105 g/mL). A mixture of bacteria (Lactobacillus reuteri, L. acidophilus, L. rhamnosus, Bifidobacterium longum) was used to stimulate netosis. Propidium iodide intercalating dye and mouse monoclonal antibodies to human CD15/FITC were used to visualize NETs and other luminescence-positive objects. Luminescence microscopy was performed at excitation wavelengths of 450-480 nm and emission wavelengths from 515 nm. Intact neutrophils (bright green objects without a stained nucleus), activated neutrophils (bright green with a red-orange, propidium iodide stained nucleus), early netosis cells (bright green cells with nuclear material in one or more locations outside the cell membrane), NETs: cloud-shaped (in the form of a «cloud» surrounding the neutrophil or its fragments on all sides and more than 1.5 times its size) and filamentous (in the form of filaments 2 or more times the size of the neutrophil) were recorded in the preparation. The proportion of each type of observed objects relative to all fluorescent-positive objects was determined. During the optimization of the method the key parameters were determined: the temperature of leukocytes incubation with the stimulant (37°C), its concentration (2.5x10⁹ bacteria/ml), stimulation time (30 min).

Evaluation of the reproducibility of the method showed an acceptable inter-series coefficient of variation: 13.8% for filamentous and 15.2% for cloud-shaped NETs. The study method was applied in different groups of patients with certain infectious (pulmonary tuberculosis) and non-infectious pathology (ulcerative colitis, aneurysmal bone cysts). The results of the assessment of neutrophils' ability to form NETs in the representatives of the examined cohorts allow us to conclude that the level of sensitivity and manifestability of the indicators registered with this technique is adequate.

The aim of this study - to investigate the associations of blood serum idiotypic and antiidiotypic antibodies specific to estradiol (IgA₁-E2 and IgG₂-E2) with genes polymorphisms of DNA-repairing enzymes in breast cancer patients (BCP) according to Ki-67 positive cells in tumor.  

Idiotypic and antiidiotypic antibodies in the blood serum of BCP (360 at the I stage and 376 at the II–IV stages) were measured by enzyme-linked immunosorbent assay. Prevalence of hOGG1 (rs1052133), XRCC1 (rs25489), XPD (rs13181), APEX1 (rs1130409) were determined by allele-specific polymerase chain reaction. High levels of Ki-67 positive cells in tumors (>30%) were revealed in 47.9% and 58.9% BCP II–IV stages with low and high IgA₁-E2 levels (p = 0.035); in 61.2% and 46.9% BCP II–IV stages with low and high IgG₂-E2 levels (p = 0.001). High Ki-67 tumor levels were found in 55.6% BCP with low levels both IgA₁-E2 and IgG₂-E2; in 67.6% BCP with high IgA₁-E2 levels combined with low IgG₂-E2 levels; in 43.2% BCP with low IgA₁-E2 levels combined with high IgG₂-E2 levels; in 52.2% BCP with high both IgA₁-E2 and IgG₂-E2 levels. So IgG₂-E2 inhibited the tumor proliferation but IgA₁-E2 blocked this effect. There were no revealed any associations of hOGG1, XRCC1, XPD, APEX1 genes polymorphisms with Ki-67 positive cells tumors levels. High IgA₁-E2 levels were found in 39.9% BCP with CC hOGG1 genotype and in 47.8% BCP with CG hOGG1 genotype (p = 0.049). High IgG₂-E2 levels were revealed in 65.3% and 53.7% correspondingly (p = 0.003). High IgG₂-E2 levels in combination with low IgA₁-E2 levels were revealed in 40.6% BCP with CC hOGG1 and in 27.2 % BCP with CG hOGG1 genotypes. The other combinations of low and high levels of IgA₁-E2 and IgG₂-E2 were found more frequent in CG hOGG1 BCP. The differences between CC and CG hOGG1 BCP according to prevalence of anti-proliferative and pro-proliferative immunological phenotypes were statistically significant (p = 0.003).

It was shown for the first time that the formation of antibodies that modulate the proliferative activity of a tumor may be associated with variants in the genes for DNA repair enzymes. In particular, the formation of idiotypic and antiidiotypic antibodies specific to estradiol were associated with hOGG1 gene polymorphisms in BCP. IgA₁-E2 and IgG₂-E2 immunoanalysis may be used in BCP I stage for tumor proliferation prediction.

The presence of FcᵧRIIIb (CD16) on the surface of neutrophil granulocytes (NG) in human blood endows these cells, through interaction with IgG, with a key property of adaptive immunity: an antigen-specific cellular  response. The purpose of the work was to compare the ex vivo reaction of human neutrophil FcᵧRIIIb in response to the addition in blood the live cells of  opportunistic (Escherichia coli, Staphylococcus aureus) and pathogenic (Yersinia pestis, Brucella abortus) bacterial species. Materials and methods. The density of CD16 expression on NG was determined by flow cytometry in arbitrary fluorescence intensity units (MFI) after staining leukocytes with CD45-FITC and CD16-PE reagents (Backman Coulter, USA) when immunophenotyping them in the blood according to the Lyse/No-Wash protocol. To model  bacteremia, we used attenuated (vaccine) strains B. abortus 19BA and Y. pestis EV NIIEG, as well as strains S. aureus ATCC 6538 (209-P) and E. coli ATCC 25922. Blood was obtained from healthy donors (n= 10), who were not vaccinated against plague and brucellosis. Bacteria of 4 species were added to the blood samples of each donor in the same dose of 10⁸ mc/ml and the results were taken into account according to the studied indicator after 30 minutes, 1, 2 and 6 hours of incubation. Results. Opportunistic bacteria, in contrast to Y. pestis and B. abortus, caused a sharp decrease in the density of FcᵧRIIIb expression on human blood NG after 2 hours of incubation, which was a marker for the development of ex vivo IgG-mediated anaphylaxis, caused by the presence in the blood of all healthy adults IgG to specific antigens of E. coli and S.aureus. Changes in the CD16 marker induced in NG by opportunistic bacteria preceded degranulation and lysis of these cells under ex vivo conditions, while in the presence of Y. pestis and B. abortus neutrophils did not undergo degranulation and cytolysis within 6 hours. Conclusion. The experimental data obtained in the work reflect the known fact of the participation in the neutralization of E. coli and S. aureus of the phenomenon of extracellular antibody-dependent cytotoxicity of NG, which is realized in the bloodstream using the NETosis mechanism and plays a decisive role in the prevention of sepsis. The absence of a reaction of the molecular trigger CD16 NETosis to Y. pestis and B. abortus indicates that in the human body not vaccinated against plague or brucellosis, this protective antibody-dependent bactericidal mechanism does not work. Evaluation of FcᵧRIIIb reactivity by flow cytometry in an ex vivo bacteremia model is promising from the point of assessing the intensity of post-vaccination immunity in humans. 

Summary

The ecologically disadvantaged Priaralye region is characterized by a high prevalence of chronic respiratory allergies in children, often combined with anemia. These conditions significantly impact children’s health and require comprehensive study of immune and physiological features.

Objective. To assess the characteristics of the cytokine profile in children with respiratory allergies on the background of anemia, depending on the presence of concomitant dermatoses, age groups, and the level of physical development.

Materials and Methods. The study included 284 children aged 3 to 17 years, divided into three groups: 121 children with respiratory allergies (RA), 126 children with respiratory allergies and concomitant dermatoses (RA + D), and 37 practically healthy children (control group). Concentrations of key cytokines (IL-4, IL-6, IL-10, IL-18, TNF-α) and total IgE in blood serum were determined by enzyme-linked immunosorbent assay (ELISA). Physical development was assessed using the standardized WHO AnthroPlus methodology. Statistical analysis was performed using Welch’s t-test and ANOVA, with significance set at p < 0.05.

Results. Children with respiratory allergies, regardless of the presence of dermatoses, showed significantly increased levels of IL-4 and IgE compared to controls (p < 0.01), indicating the formation of a dominant Th2 response. The highest level of the anti-inflammatory cytokine IL-10 was found in the RA + D group (p < 0.01), likely reflecting a compensatory mechanism aimed at limiting inflammation. At the same time, pro-inflammatory cytokines IL-6 and TNF-α were significantly decreased in patients compared to healthy children (p = 0.001 and p < 0.001, respectively), possibly indicating hyporesponsiveness of the pro-inflammatory pathway in chronic allergic conditions.

Age-related analysis showed a tendency toward higher concentrations of IL-4 and IgE in children under 10 years old, though differences between age groups were not statistically significant. In children with delayed physical development, levels of IL-4 and IL-10 decreased while IL-18 relatively increased, consistent with literature data and suggesting possible mechanisms of immune dysregulation associated with impaired general health status.

Conclusion. The data confirm a predominant Th2 immune response in children from Priaralye with respiratory allergies, reflected by increased IL-4, IL-10, and IgE levels. The decreased levels of IL-6 and TNF-α warrant further study to understand inflammatory processes in chronic allergies under ecological stress. Accounting for cytokine profiles and physical development is important for developing personalized therapeutic approaches and improving disease prognosis.

 

Abstract

Using psychoactive substances can cause toxic encephalopathy, which contributes to the modulation of both innate and adaptive immune responses. The impact of the use of psychoactive substances, including those prohibited in the Russian Federation, on immune homeostasis remains insufficiently studied, especially in northern regions with extreme climatic factors. With regard to medical and social consequences, attention is drawn to the rapidly increasing social maladaptation of young patients, as well as the manifestation of organic damage to the central nervous system. This study is aimed at assessing the ratio of lymphoproliferation and lymphapoptosis processes in men with toxic encephalopathy caused by psychoactive substances in the conditions of the northern region. Materials and Methods: The work contains a comparative analysis of the immunological parameters of the peripheral blood of two groups of people living in the northern region of the Russian Federation: 20 men with toxic encephalopathy (mean age 33.8±1.8 years) on the first day after severe poisoning with psychoactive substances and 22 healthy volunteers (34.5±2.0 years; (control)). Phenotyping of peripheral blood lymphocytes was performed by the method of indirect immunoperoxidase reaction with monoclonal antibodies. Statistical data processing was performed using SPSS 25.0. The results revealed a statistically significant (p<0.01) increase in lymphocytes with the CD10⁺ marker in men with psychoactive substances abuse (0.54 (0.40-0.75) ×10⁹ cells/l) compared to the control (0.27 (0.16-0.51) ×10⁹ cells/l), which indicates hyperproliferation of lymphocytes due to CD10⁺. At the same time, a low number of lymphocytes with the CD95⁺ marker was recorded in men with psychoactive substances abuse compared to the control group, reflecting the suppression of apoptosis. A two-times increase in the CD10⁺/CD95⁺ ratio was established in men with psychoactive substances abuse, demonstrating an imbalance in proliferative-apoptotic processes. A distinctive feature for men with toxic encephalopathy is an increased content of CD10⁺ cells that stimulate immune responses via the classical pathway against the background of a low concentration of lymphocytes with the CD95⁺ marker, which indicates an increased expenditure of the reserve capacity of immune homeostasis and their reduction. A moderate decrease in the level of receptor expression (CD71⁺) on lymphocytes in men with psychoactive substances abuse was revealed. The data obtained indicate that in individuals with toxic encephalopathy in the conditions of the North, there is a disruption of both the long-term and short-term mechanisms for maintaining immune homeostasis due to an imbalance in the ratio of lymphoproliferation and apoptosis processes.

Abstract

Implants are currently widely used in dentistry and effectively provide lost teeth. However, in the presence of concomitant risk factors and low oral immunity, patients develop peri-implantitis, accompanied by inflammation and an immune response of the body, while the cytokine profile in saliva remains insufficiently studied. The purpose of this study was to study the cytokine response and evaluate the diagnostic information content of pro-inflammatory and anti-inflammatory cytokines in patients with peri-implantitis. 65 patients with reimplantations aged 50-65 years and 48 patients aged 50-65 years without reimplantations were examined. The diagnosis of reimplantations was carried out based on the results of a clinical examination, the presence of symptoms - bleeding during probing, suppuration during probing, bone loss on radiographs ≥ 3 mm and the depth of the probed pocket ≥ 6 mm. In saliva collected in the morning on an empty stomach, the content of a number of pro-inflammatory and anti-inflammatory cytokines was determined by enzyme immunoassay using Protein Contour kits (St. Petersburg). The cytokine profile of patients with peri-implantitis is characterized by the expression of both pro-inflammatory and anti-inflammatory cytokines. The saliva content of IL-1 increased particularly among patients with peri-implantitis to 205,9±4,2 pg/ml versus 67,5±3,1 pg/ml in participants without peri-implantitis, IL-6 to 29,4±1,3pg/ml versus 11,7±1,2 pg/ml, respectively, with a significant difference in all cases. In addition, the level of IL-19 increased to 54,9±2,3 pg/ml relative to the comparison group – 30,5±1,4pg/ml, TNF-α – to 202,4±3,8 pg/ml relative to 115,6±2,4 pg/ml, respectively (p<0,001). Along with this, the saliva concentration of patients with IL-4 and IL-10 peri-implantitis increased significantly to 18,2±1,4 pg/ml versus 3,8±0,6 pg/ml in the group without peri-implantations and to 66,5±2,7 pg/ml versus 15,3±0,9 pg/ml, respectively (p<0,001). The calculation of the Kullback information measure revealed the highest diagnostic information content for IL-4 (8,5), IL-10 (8,1), IL-1b (5,7), IL-6 (4,2). To objectify diagnostic criteria, it is recommended to use the levels of IL-4, IL-10, IL-1b and IL-6 established in saliva.

 

Abstract

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that predominantly affects small joints, causing persistent pain, functional impairment, and a marked reduction in patients’ quality of life. The pathological process is characterized by ongoing synovial inflammation, destruction of cartilage and subchondral bone, and extra-articular manifestations involving the cardiovascular, pulmonary, and nervous systems. A key pathogenetic element is the imbalance between pro- and anti-inflammatory mediators, among which tumour necrosis factor-α (TNFα), interleukins IL-17A and IL-17F, and osteoprotegerin (TNFRSF11B), a regulator of osteoclast differentiation, play central roles.

The present study aimed to assess the contribution of polymorphisms in the TNFA, IL-17A, IL-17F, and TNFRSF11B genes to individual susceptibility to RA in the Russian population of the Chelyabinsk Region. We hypothesised that the major genetic impact on disease development derives not from single nucleotide variations in isolation but from their combined multilocus configurations. Consequently, special attention was given to inter-genic interactions, which are often under-appreciated in conventional association studies.

Genotyping was performed by polymerase chain reaction (PCR). To analyse the data we applied the multifactor dimensionality reduction (MDR) algorithm, which constructs predictive case–control models and evaluates their robustness through ten-fold cross-validation and permutation testing.

The algorithm identified three most informative combinations comprising four to six SNPs each; every combination showed statistical significance and high predictive accuracy. Cross-validation consistency values exceeded 9/10, indicating excellent reproducibility of the models.

These findings confirm that a comprehensive multilocus genotype analysis is more informative than examining individual markers alone and can be used for patient risk stratification, early diagnosis, and the development of personalised preventive strategies based on targeted anti-cytokine therapies. Further studies in larger cohorts are needed to validate these results.

Abstract

Glioblastoma is the most frequent and aggressive brain tumor, with a low survival rate. One of the challenges in treating glioblastoma is the resistance of cells to temozolomide, the main chemical agent used to fight the disease. The STAT3 gene is known to play a significant role in the growth and metastasis of tumor cells. Inhibition of STAT3 or suppression of its expression reduces the resistance of glioblastoma cells to temozolomide. Enhancer RNAs (eRNAs) are non-coding RNAs that are transcribed from enhancer regions. eRNAs can affect the expression of key genes of various biological processes, including oncogenes. Previously, we conducted a search for an eRNAs that may potentially affect glioblastoma cell resistance to temozolomide. Knockdown of TMZR1-eRNA (ENSG00000289579) in DBTRG-05MG cell line resulted in decreased STAT3 gene expression and increased cell sensitivity to temozolomide. The effect of enhancer RNA knockdown on STAT3 gene promoter activity has been shown both alone and together with the enhancer from which this eRNA is transcribed. The current study was designed to investigate the potency of TMZR1-eRNA in a glioblastoma cell line U-251. Compared to the previously studied cell line DBTRG-05MG, the U-251 cell line is more aggressive, has a higher proliferation rate and was derived from patient tissue at a later stage of the disease. Knockdown of TMZR1-eRNA by small interfering RNAs resulted in a decrease in STAT3 gene expression. No effect on cell growth was observed both when temozolomide was added and in control conditions of culture medium with DMSO. To study the effect of constitutive suppression of eRNAs, U-251 cell cultures expressing short hairpin RNA (shRNA) targeting TMZR1-eRNA and control vector were transduced. A significant increase in STAT3 mRNA expression was observed in the obtained cell lines relative to the control. At the same time, viability analysis demonstrated a significant slowdown in the growth rate of the cell line expressing short hairpin RNAs to TMZR1-eRNA compared to the control. A significant effect was observed both when temozolomide was added and in culture medium with DMSO.

Abstract

The study of cellular metabolism in the formation of immunological memory is still relevant, as it can help to deepen our knowledge about the pathogenesis of immune-inflammatory diseases and provide a basis for improving their prevention and treatment.

The aim of this study is to investigate the activity of mitochondria in thymocytes and splenocytes after immunization of rats with human albumin.

Materials and methods. Male Wistar rats were immunized with human albumin for 4 days. The total amount of protein injected was 135 mg. Twenty-four hours after the last injection, we evaluated hematological parameters (general blood test) and mitochondrial activity of thymocytes and splenocytes using rhodamine 6 G fluorescence on the Evos M7000 imaging system in RFP mode. We also calculated the mass coefficients of the thymus and spleen relative to the body weight of the animals. Statistical analysis included calculation of the median, upper and lower quartiles, and comparisons of hypotheses using the Mann-Whitney U-test and correlation analysis according to Pearson's method.

Results. Hypertrophy of the thymus was detected in animals of the experimental group by 56.9%, p=0.032, and no changes in the mass of the spleen were observed. A decrease in mitochondrial fluorescence in thymocytes by 23.5% (p=0.037) and splenocytes by 13.7% (p=0.548), may be associated with the immunosuppressive effect of transforming growth factor beta during repeated administration of a foreign protein. At the same time, monocytosis exceeding the control by 59.4% (p=0.030), and thrombocytopenia by 20.8% (p=0.045), were observed in experimental rats, reflecting reactive changes occurring during immunization. Conclusion. Immunization of animals at the early stages is accompanied by inhibition of energy exchange in cells of the thymus and spleen. This may reveal features of the effect of inflammatory mediators on mitochondria of lymphocytes and identify promising targets for treatment of immuno-inflammatory pathologies.

AbstractSome patients with inflammatory bowel disease (IBD) may not have traditional criteria of inflammation: elevated CRP, ESR and leukocytosis. The aim of this study was to identify additional immunological and biochemical criteria for the risk of IBG in patients with no non-specific laboratory criteria of inflammation. The study involved 150 patients, of whom 2 groups were formed: an observation group (100 patients with a verified diagnosis of ulcerative colitis or Crohn's disease) and a control group (50 clinically healthy individuals). All subjects underwent a complete blood count, a biochemical blood test and determination of IL-1β, TNF-α, and IL-4 concentrations. Based on the results of general and biochemical blood tests, patients from the observation group were divided into two subgroups - with and without classic laboratory signs of inflammation. Three main laboratory blood parameters were assessed for diagnosing the inflammatory process: ESR, white blood cell count and CRP level. The presence of laboratory signs of inflammation was considered to be an increase in two or more of the above blood parameters. It was noted that 40% of patients with IBD had no non-specific laboratory criteria of inflammation: in ulcerative colitis in 37% of cases, in Crohn's disease in 46% of cases (p < 0.001). Further, a comparative analysis of the levels of cytokines and biochemical markers in the blood serum of patients from the control group and patients with IBD without laboratory signs of inflammation was carried out. Based on the obtained data, a prognostic model of the probability of the presence of IBD in patients with no non-specific laboratory criteria for inflammation, depending on biochemical and immunological blood parameters was developed. The model included such serum parameters as glucose, sodium and IL-4 concentrations. The predictive ability of the model was assessed using ROC analysis (AUC 0.970±0.018 95% CI: 0.936–1.000; p<0.001). An algorithm for predicting the risk of IBD in patients without non-specific laboratory criteria of inflammation was proposed. The obtained data made it possible to identify additional criteria for the risk of IBD in patients with the absence of non-specific metabolic criteria of inflammation.

Abstract

Introduction. The incidence of measles has increased significantly worldwide in recent years. Measles virus is transmitted by aerosol and is highly contagious. Health care workers infected with measles are particularly at risk, as they may contribute to the hospital-acquired spread of infection. At the same time, medical personnel are at risk for contact with patients and biological material that may be infected with measles.

The study objective - to study the dynamics of the content of anti-measles antibodies in health care workers over a five-year period.

Materials and methods. Statistical processing of the results of enzyme-linked immunosorbent assay of blood serum of 272 health care workers of Clinics of Samara State Medical University aged from 22 to 62 years using StatTech v. 4.1.2. program was performed. The health care workers were divided into two age groups: from 22 to 44 years, from 45 to 62 years (average age 43 years). At the first stage of the study in 2018, the baseline level of specific anti-measles IgG was assessed. Individuals who demonstrated negative and questionable results were subjected to cleansing immunization. In the second stage of the study, specific antibody levels were re-assessed in 2023.

Results. In both age groups, the average IgG concentration increased over the five-year period as a result of the 2018 revaccination (0.49 IU in 2023 vs. 0.20 IU in 2018). No statistically significant differences between age groups could be detected in 2018. By 2023, antibody concentrations were higher among those over 45 years of age. In the group without cleansing immunization, there was a decreasing trend in antibody values over the 5 years. The group with cleansing immunization showed an increase in positive results and a decrease in the proportion of negative and questionable results.

Conclusion. Despite double injection of the anti-measles vaccine in childhood, not everyone is immune by adulthood. The 2018 cleansing immunization was generally quite effective. Individual characteristics of the organism, as well as a new coronavirus infection, may influence the formation of measles humoral immunity. Regular seromonitoring is necessary to monitor the maintenance of anti-measles humoral immunity, especially for those at risk, including health care workers.

Abstract

Excessive alcohol consumption has a negative effect on hematopoiesis, which is expressed in a significant suppression of both the production of blood cells and structural changes in precursors, namely in the suppression of their maturation, up to pancytopenia. A distinction is made between the direct effect of alcohol (toxic effect on bone marrow, hematopoietic precursors and mature blood cells) and the indirect effect due to a deficiency of trophic factors. Alcoholics often exhibit anemia, as a consequence of the destruction of erythroid cells before they mature, and thrombocytopenia, which causes the appearance of petechiae and spontaneous bleeding. Chronic alcohol consumption also has a suppressive effect on the production and function of white blood cells, resulting in a poor ability to resist bacterial infection. We have previously identified the immunomodulatory properties of the innovative anticonvulsant meta-chlorobenzohydrylurea and demonstrated the positive psychoneuromodulatory effect of splenic lymphocytes modulated in vitro by the indicated anticonvulsant during chronic ethanol intoxication. In this study the influence of meta-chlorobenzhydrylurea-modulated spleen lymphocytes on bone marrow hematopoiesis and peripheral blood cells long-term alcoholized mice was studied. In the bone marrow of long-term alcoholized mice a decrease in the colony-forming activity of hematopoietic precursors was observed: the population of erythroid precursors was significantly reduced, and a decrease in the population of granulocyte-macrophage precursors was also recorded at a trend level. In peripheral blood, a decrease in the number of lymphocytes, platelets, erythrocytes and leukocytes was observed with an increase in the population of segmented neutrophils, indicating peripheral inflammation.     Lymphocytes precultured with meta-chlorobenzhydryl urea, after intravenous administration to syngeneic long-term alcoholized recipients, had a corrective effect on a number of hematopoietic parameters, which was manifested in the restoration of the colony-forming activity of bone marrow hematopoietic precursors to indicators comparable to those in intact mice of the corresponding age, in a decrease of segmented neutrophils and restoration of erythrocytes and lymphocytes populations with tendency to increase the number of platelets in the peripheral blood. The data obtained may indicate the effectiveness of meta-chlorobenzhydrylurea-modulated lymphocytes in correcting a number of changes in hematopoiesis provoked by long-term ethanol intoxication.

Abstract

Pruritus is a rare chronic recurrent polyethological skin disease characterized by severe itching, resulting in the formation of skin elements such as papulo-vesicles, papules, nodules and plaques. The disease is often difficult to treat with standard therapy, and patients have been suffering debilitating symptoms for years. The article presents a clinical case of a patient who suffered from nodular pruritus for more than 20 years, which worsened in the last 4 years after suffering from a mild covid-19 infection, when the disease became continuously recurrent. The means of traditional therapy were practically ineffective. Upon admission to the allergological department of the Clinic of Immunopathology of the Federal State Budgetary Budgetary Institution "Scientific Research Institute of Fundamental and Clinical Immunology" in Novosibirsk in April 2024, rashes were common, severe itching disrupted sleep, and significantly reduced the patient's quality of life. On 04/16/2024, GIBT was initiated with dupilumab at an initial dose of 600 mg, according to the instructions, followed by 300 mg every 2 weeks. After the first administration of the drug, significant positive dynamics was noted in the form of a decrease in itching, rapid regression of rashes, and the patient's skin was almost completely cleansed by the 4th injection. By the time of writing, the skin remains clean, there is no itching, and sleep has returned to normal. The patient continues to use emollients and take 2nd generation antihistamines. The effectiveness of dupilumab therapy, which blocks IL-4 and IL-13 signaling by specifically binding to IL-4Ra, a subunit common to IL-4 and IL-13 receptor complexes, is due to the fact that neuroimmune regulation in the skin is leading to the pathogenesis of chronic pruritus with nodular pruritus. Interleukins 4 and 13 are considered important itchogenic cytokines of Th2 cells, which increase the expression of IL-31R and other pruritus-related receptors, enhance neuronal activation in inflamed skin, transmitting signals to nearby efferent neurons and the central nervous system, initiating itching sensation, forming a vicious cycle of itch-scratching, enhance sensitization of histamine-independent sensory neurons to pruritogens, stimulate fibroblast proliferation, migration, and production of profibrous factors. Since the main goal of treating nodular pruritus is to block the vicious cycle of itching and scratching, which promotes nodular healing, dupilumab therapy leads to remission of the disease, normalizing the interaction of the immune and nervous systems in the skin. In the treatment of adult patients with nodular pruritus, dupilumab was approved in Russia not so long ago - just over a year ago and has already shown its high effectiveness. The presented case demonstrates the possibilities of using modern immunotherapy methods in the treatment of dermatological diseases with torpid course.