Peer-rewieved medical journal for medical immunology
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Gamma delta (γδ) T cells: origin, phenotype, functions
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. Over recent years, numerous data were presented on the diversity of γδT cell subpopulations and its role in physiological and pathological processes, sometimes being controversial, or even antagonistic. Hence, the purpose of this review was to systematize data on the biology of γδT cells, including their origin, phenotype, functions, and approaches to their clinical application. The article presents modern concepts regarding the origin of γδT cells, stages of their intrathymic differentiation, and extra-thymic potential for trans-differentiation of some subpopulations. The review presents a modern classification of human γδT cells based on expression of γ- and δ-chains of the T cell receptor, their phenotype and describes the properties of the most common populations of Vδ1, Vδ2, Vδ3 T cells. Classification of human γδT cells based on their cytokine production and expression of intracellular messengers is proposed, the properties and functions of the most studied subpopulations are described in details: γδT1, γδT17, γδNKT, γδTreg, γδTAPC, γδTfh. The review pays special attention to the phenotype of various populations, their ability for cytokine secretion, and provides data on expression of surface receptors of human γδT cells and their functions. In particular, the structural features and ligands of the γδT cell receptor, are addressed as well as specific 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, chemokines 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 human γδT cell subpopulations under various physiological and pathological conditions, and their role in the tumor surveillance. On this basis, possible prospects for usage of γδT cells in the treatment of distinct diseases are also discussed.
Single cell transcriptomics in prostate cancer research
The objective of our study was 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 (PCa). PubMed, Medline, Web of Science, and Embase scientific databases were analyzed. 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 for studying cellular heterogeneity in PCa. The methodology of the analysis is also presented in detail, cellular heterogeneity in PCa is characterized, current research in the field of single cell transcriptomics in PCa is described, and promising applications of the results in clinical practice are also 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 PCa diagnostics and therapy. 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 a personalized approach to prostate cancer treatment, which opens prospects for improving the efficiency of treatment.
Role of mechanical properties of T cells in shaping the immune response
Recent studies in immunology highlight the critical role of mechanical factors in shaping the immune response. Mechanoimmunology, being an emerging interdisciplinary field, concerns the influence of mechanical stimuli on immune cell behavior, in particularly, T lymphocytes. Microenvironment stiffness, mechanical interactions with the extracellular matrix, and changes in membrane tension are able to 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 may 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 expanding knowledge on their role in immune interactions suggests a high degree of interconnected modulation, resulting into a system of coordinated responses to mechanical stimuli. Mechanomodulation alters the intracellular environment, acting as a determinant of metabolic profile of T cell populations. Moreover, these studies presume that mechanosensitive signaling pathways may regulate intercellular interactions and adaptive immune responses, offering broad opportunities for modifying immune reactions. Understanding the mechanotransduction mechanisms provides new prospects for the development of novel therapeutic strategies. Mechanical signals may 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, 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 of immune responses via mechanical properties of the cells could provide a precise regulation of immune activity based on the specific characteristics of pathological processes.
Prospectives of novel therapeutic and prophylactic medications based on symbiotic bacterial strains for correction of immune disorders mediated by intestinal microbiota in COVID-19
Recent studies on the role of intestinal microbiota in animals and humans show that the microorganisms are an important determinant of host health, participating in the pathogenesis of various infectious and non-infectious diseases. Currently, the effects of functional gut-lung axis are being actively investigated in the new coronavirus (COVID-19) infection. Gastrointestinal tract may be the point of entry for infection, suggesting involvement of intestinal microbiota in the infectious process. On the one hand, the changes in gut microbiota (dysbiosis) in SARS-CoV-2 patients may be among the factors contributing to development of secondary bacterial infection, sepsis, systemic inflammation and multi-organ failure. On the other hand, impaired gut microbiota contributes to severe clinical course and mortality in patients due to bidirectional coupling of the gut microbiota with immune system via cytokine network. The studies have shown a link between the severity of COVID-19 in patients, cytokine levels, and presence of distinct types of pro- and anti-inflammatory bacteria in the intestinal biotope. Immune abnormalities in COVID-19 patients are also mediated by altered metabolome profile associated with dysbiotic microbiota. The relations between gut microbiota composition, cytokine levels and inflammatory markers suggests that the gut microbiome may influence the progression of coronavirus infection. Therefore, the “symbiotic potential” of normobiotic microbiota may be used to develop prevention and rehabilitation strategies for the patients. This approach may be facilitated by the studies of human-microbiota symbiosis. A number of key mechanisms for studying the integration of Bifidobacteria and Lactobacilli with the host mediated by the immune system, hormones and neurotransmitters are opening new perspectives for medicine, including development of differently targeted probiotic strains for therapeutic and preventative correction of impaired functions within the organism. The studies of microsymbiocenosis, being a vector of associative symbiosis have enabled a technical approach for intermicrobial “friend or foe identification”, where bifidobacteria are used as a diagnostic culture, since “friendly” strains are characterised by synergism (support). By contrary, encountering the “foreign cells” leads to antagonism. This fundamental mechanism may be used for choosing the “host-friendly” bacteria strains eligible for design of a probiotic compositions.
Immunological mechanisms of therapeutic efficacy of oxidized dextran in spermatogenesis disorders of infectious and inflammatory genesis
The article summarizes experimental and clinical data on the immunological properties of oxidized dextran. The study addresses pathophysiological effects of oxidized dextran, in particular, its ability to selectively activate tissue macrophages, including testicular macrophages, which are directly involved in spermatogenesis. In experimental part of the work, a rat model of spermatogenesis disorders was produced by injection of Escherichia coli endotoxin. We have shown that oxidized dextran significantly reduces pathomorphological changes in the testes. At the same time, such effect is associated with the activation of tissue macrophages and release of corresponding cytokines, which exert anti-inflammatory effects and normalize microcirculatory hemodynamics. The presented experimental data correlate with the sufficient results on the high clinical efficacy of oxidized dextran in spermatogenesis disorders and male infertility. Clinical part of this work concerned the effects 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 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 severity of intracellular infections, in particular, tuberculosis. Analysis of experimental and clinical data allows the authors to propose a theoretical model for the mechanism of therapeutic effectiveness of oxidized dextran in intracellular infections. The bulk of data presented by the authors allow us to consider oxidized dextran as a quite effective tool for the treatment of intracellular infections, including urogenital infections causing male infertility.
Targeting Group A Streptococcus with a Recombinant Chimeric Vaccine: Integrating ScpA and SpeA Fragments
Our objective was to develop a vaccine against group A streptococci (Streptococcus pyogenes, GAS), the causative agent of a broad spectrum of infections with varying severity. The expression vectors pET27 and pQE30 were used 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 5 × 107 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. 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 into 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. These findings emphasize 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.
Functional activity of anti-GD2 CAR-T cells with different antigen-recognition module
Neuroblastoma (NB) is the most common extracranial solid tumor in children comprising, 8 to 10% of all pediatric tumors, with 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. Among the main promising antigens for development of CAR-T therapy against NB is the disialoganglioside GD2, the expression of which is shown in 100% of NB cases. Most clinical variants of anti-GD2 CAR-T cells 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 based on 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 it is not detected 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. In all these cases, the anti-GD2 CAR contains insertion domains in extracellular portion of the molecule. Results of separate clinical trials have been published for CAR-T based on 14G2a and hu3F8. So far, however, there are no data on the usage of 8B6 antibody in CARs. The aim of the present study is to obtain 2nd generation chimeric antigenic receptors based on three antibodies with different lengths of extracellular domain, and to evaluate their functional activity against a number of cell lines to justify further clinical trials.
Netrin-1 upregulates the its own gene expression and unc5b gene expression, but downregulates ccl19 gene expression in human macrophages THP-1 cell line
According to the WHO data of 2021, ischemic heart disease was the leading cause of human disability and mortality at a global level, with brain stroke being the third leading cause of death. These cardiovascular diseases, including myocardial infarction and aortic aneurysms, are largely caused by atherosclerosis, a systemic chronic inflammatory process. This disease develops over decades and is characterized by the disruption of the vascular endothelial layer integrity, excessive lipid accumulation in arterial walls, macrophage and smooth muscle cells dysfunction, and evolving inflammation of arterial walls. One of approaches to atherosclerosis regression may be emigration of cholesterol ester-laden macrophage from atherosclerotic plaque via lymphatic vessels to the regional lymph nodes, and then to liver for subsequent cholesterol metabolism. The protein netrin-1 is inhibitor of immune cells migration. Particular interest is the study of the autocrine/paracrine regulation of netrin-1 gene expression in human macrophages. The understanding of this process may provide a basis for discovery of the new approaches to atherosclerosis therapy. The aim of the present article was to study the influence of protein factor netrin-1 on the gene expression levels of ntn1, its receptor unc5b, and ccl19. The experiments were carried out on the macrophage-like cells derived from acute monocytic leukemia cell line THP-1. The expression of ntn1, unc5b, ccl19 genes was performed by reverse transcription and real-time quantitative PCR (mRNA level), and by flow cytometry (at the protein level). Netrin-1 at the concentration of 50 ng/mL induced ntn1 and unc5b gene expression, but suppressed ccl19 gene expression in human macrophages. However, netrin-1 applied at the concentration of 250 ng/mL caused a significant downregulation of the own ntn1 gene expression. Netrin-1 has autoregulatory effect on its own gene expression, and on the gene encoding its receptor (unc5b) in human macrophage-like cells. The downregulation of chemokine ccl19 expression in macrophages mediated by netrin-1 may be a potential way by which this effector inhibits macrophage emigration. The obtained results suggest a regulatory mechanism for expression of proteins responsible for macrophage migration, which in the future will enable application of these data for searching new targets in atherosclerosis therapy.
Characteristics of the antigenic phenotype of monocytes in peripheral blood of patients with recurrent pregnancy loss
Recurrent pregnancy loss is a significant clinical problem affecting 1 to 5% of the population. The cause of premature loss of pregnancy remains unknown in more than half of the cases. 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 pathogenesis of recurrent pregnancy loss has not been sufficiently studied. The aim of the present study was to determine the quantitative changes in contents and antigenic phenotype of platelet-free (not bound to platelets) monocytes within whole cell 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 physiological pregnancy (7-12 weeks). Monocyte content and expression of CD11b, CD86, CD162, HLA-DR, TREM-1 were determined by means of cytofluorimetric analysis in the total population and subpopulations of peripheral blood monocytes. We have found that the proportion of platelet-free monocytes in recurrent pregnancy loss was decreased (74.6%) compared to uncomplicated pregnancy (83.4%). All studied subpopulations of monocytes (classical, intermediate and non-classical) proved to contribute to these changes. Decrease in HLA-DR expression and increase in CD11b expression was observed in total cell population caused by a 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 expression levels of activation markers, being 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 decreased content of free monocytes in peripheral blood and changed antigenic phenotype of monocytes, reflecting a weakening of proinflammatory properties and increased adhesive properties of these cells. These changes may underlie the pathophysiological processes leading to premature termination of early pregnancy. Discerning the patterns of activation marker expression typical for particular obstetric disorders may contribute not only to deteciton of pathophysiological mechanisms of reproductive disorders, but also to the improvement of methods for their diagnosis, and development of pathogenetically justified methods of therapy
Studying adjuvant properties of cell-penetrating cationic dendrimeric peptides
The adjuvant agents 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 inclusion of adjuvant into the immunogenic molecule. Such covalent adjuvants vary greatly in chemical structure, and are found among particular peptides. Peptides have distinct advantages over substances of other chemical classes, e.g., high efficiency and biodegradability, whereas dendrimeric cationic peptides are often capable of enhanced translocation 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, i.e., LTP and SA-40. The peptides were obtained by solidphase synthesis and characterized by mass-spectrometry and zone capillary electrophoresis. To study adjuvant activity, the peptides were conjugated with recombinant protein Bet v 1, a major birch pollen allergen, produced as biotechnology product, using E. coli. Chemical conjugation was carried out with Michael reaction after maleimide functionalization of the protein. The conjugates were purified by gel chromatography and dialysis, followed by estimation of conjugation efficiency 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 4 injections without using other adjuvants. Mouse blood sera were studied with ELISA for IgG levels against rBet v 1, being the target criterion 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 initial 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 four-fold in the case of LTP-rBet v 1, and three-fold with SA-40-rBet v 1. Based on the results of this 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 reference agents in development of new peptide adjuvants, and SA-40 itself may be used as a covalent adjuvant. Moreover, LTP may be considered a prospective non-covalent adjuvant.
Evaluating the relationship between human papilloma virus infections, prostate cancer and interleukin-12 levels
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 IL-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.
DNase 1L3 and DNase II as indicators of ejaculate quality
Sperm analysis is the main method of assessing male reproductive function, which is not always informative in cases of infertility. In the literature, a special role in the disturbed male fertility is attributed to oxidative stress which results into damage of male germ cells. 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 extracellular DNA degradation. In human beings, this function is performed by DNAse enzymes localized in all tissues and body fluids. Their role in the process of fertilization and fragmentation of spermatozoal genetic material was also noted. Excess or deficiency of DNases may lead to the development of different disorders. The aim of the present study was to establish reference intervals for ejaculate DNase 1L3 and DNase 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 South Ural State Medical University was used as a material for the study. Exclusion criteria were the presence of urogenital inflammatory disorders and sex-transmitted infections (STI). Sperm analysis was performed according to the WHO laboratory manual. Screening for STI was performed by real-time PCR. DNAse concentration in ejaculate was determined by ELISA method. Descriptive statistics, construction of histograms and reference intervals were performed in the “R” platform. Primarily, we have determined the levels of DNase 1L3 and DNase II in seminal fluid and established appropriate reference intervals. Shifts in DNase 1L3 and DNase II concentrations can most likely be considered as markers of possible pathologic conditions. In particular, an increased concentration of DNase 1L3 is an index of a pathological process associated with low sperm motility, whereas high levels of DNase II may be considered an indicator of a local inflammatory reaction.
Subpopulation profile of blood leukocytes in assessing severity of COVID-19 and in conditions associated with cytokine storm
Currently, the clinical manifestations of COVID-19 patients are classified from mild to severe, especially in groups of patients susceptible to chronic diseases. Selection of the most informative prognostic indices of possible outcome or severe course of the disease in COVID-19 patients is required at early stage in order to determine effective treatment strategy. The earliest predictors of clinical deterioration in COVID-19 are elevated levels of interleukin-6, interleukin-10, C-reactive protein, procalcitonin, and other indices of innate immune system. The objectives of this study included a search for the most informative parameters of general clinical blood analysis and the main lymphocyte subpopulations in patients with COVID-19, which may be helpful in interpreting the severity of the disease and 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 including 57 males and 8 females. The age of the patients ranged from 32 to 82 years (an average of 48.6 years old). After examining the total blood counts in 65 samples, cytometric analysis was performed using the Cytomics™ FC 500 series flow cytofluorometry system (Beckman Coulter, USA), with 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 group of patients with COVID-19, absolute lymphopenia is noted along with neutrophilia at the initial medical treatment, resulting into increased values of the neutrophil-leukocyte and leukocyte-T-lymphocyte indices. These indexes are quite informative and can be used in predicting disease risks. Determination of the most reliable indices of cellular immunity, such as CD3+, CD3+CD4+, CD3+CD8+, CD3- CD16+CD56+ lymphocytes, in addition to a clinical blood test, may assess the features of the patients’ immune response to SARS-CoV-2 infection, helping to predict the disease severity, and make a decision to change the treatment strategy if required.
Delta-Vac’ subunit vaccine induces strong humoral immune response against SARS-CoV-2
The COVID-19 pandemic has focused the worldwide attention on the fight against this infection. Development of preventive vaccines based on different platforms, including DNA and RNA vaccines, genetic vectors and subunit vaccines A has been the key approach to combating COVID-19. The subunit vaccines were among these platforms, primarily, due to their unique safety profile. However, the safety of these vaccines is often associated with low efficiency. Hence, their application often requires usage of immune adjuvants, along with more complex immunization regimens. Meanwhile, an important advantage of subunit vaccines is their scalability and relative ease of production, since there is no need to work with live virus or viral vectors during the production process. The purpose of our work was to develop a candidate vaccine based on the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike S-protein from the Delta variant (B.1.617.2). The study used immunological techniques employing methods of genetic engineering and biotechnology. In the course of this work, we have developed a producer of recombinant RBD based on the CHO-K1 mammalian cells. To obtain a protein meeting the requirements of injectable drugs, a chromatographic purification scheme was developed, including affinity and ion exchange chromatography. We have proposed a variant of the subunit vaccine “Delta-Vac” developed on this recombinant protein. Immunogenicity of “Delta-Vac” vaccine was tested in the BALB/c mice. The animals were immunized twice at a dose of 50 μg of RBD combined with Al(OH)3, at a two-week interval. The ability of the candidate vaccine was assessed by induced production of specific IgG and neutralizing antibodies in BALB/c mice. Specific antibody titers of immunized animals ranged from 1/105 to 1/106 . Moreover, the blood sera showed neutralizing activity against SARS-CoV-2 (variant Delta, B.1.617.2) at a titer of up to 1/2000. The developed vaccine “Delta-Vac” is highly immunogenic and induces production of neutralizing antibodies against homologous Delta variant as well as heterologous Wuhan and Omicron SARS-CoV-2 variants. Hence, “Delta-Vac” may act as a vaccine candidate and serve as a prototype for the development of subunit vaccines against COVID-19.
Cytometric features of cellular immune compartment in participants of current military conflicts
Staying in a combat area is associated with a high risk of developing mental disorders. A significant knowledge has been accumulated concerning the mechanisms of immune-mediated mechanisms of neuropsychiatric pathologies, in particular, post-traumatic stress disorder (PTSD). Activated T regulatory cells play an important role in the development of neuroinflammation under stress conditions. Our purpose was to study the indices of blood system and cytometric features of immune cellular populations in the participants of current military conflicts in presence of stress-associated adaptation disorders. We examined 97 male veterans involved in modern military conflicts, 35 to 55 years old. The main group 35 included veterans of a special military operation in Ukraine (SVO), along with 42 veterans of the second Chechen military campaign (comparison group). Control group consisted of 20 healthy persons from military staff. All patients underwent pathopsychological examination in accordance with clinical recommendations. PTSD was diagnosed in 12% of SVO veterans, whereas 77% had various types of neurotic, stress-related and somatoform disorders. In the comparison group, 2% of combatants were diagnosed with chronic personality changes. A complete blood count was performed using a standardized technique with a hematological analyzer. Immunophenotyping of lymphocytes was performed by a Navios flow cytometer (Beckman Coulter, USA). In the group of SVO veterans, we have noted a decrease in RBC volume dispersion, reduced heterogeneity index and the average volume of platelets, as well as lymphopenia and monocytosis, thus reflecting the multidirectional effect of hematopoietic regulation factors on distinct differentiation lineages of mononuclear cells. Cytometric analysis of lymphocyte populations 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 adaptive and innate immune response upon prolonged response to stress. We have revealed an increased number of T NK and T regulatory lymphocytes which are suggested to prevent the development of autoimmune disorders. Under stress conditions, the FoxP3 transcription factor may upregulate the glucocorticoid-induced TNF receptor in the T regulatory cells thus potentiating their proliferative activity. We have also shown a decreased number of T helpers and T regulatory cells expressing markers of early and late positive activation, limiting the development of both autoimmune reactions and the development of stress-induced neuroinflammation. No significant differences were found between the indices control group and comparison group, thus indicating that the severity of stress-induced neuroimmune reactions leveled out over time. Hematopoiesis induction by stress mediators and indirect neuroimmune-mediated changes in the quantitative profile of lymphocyte subpopulations seem to be a consequence of a complex multi-level neuropsychodynamic process of the central nervous system associated with clinical forms of adaptation disorders.
VEGF deprivation affects endoglin expression in trophoblast cells and natural killers
Vascular Endothelial Growth Factors (VEGFs) are a group of proteins involved in differentiation of various cell types including endothelial cells, monocytes, macrophages, stem cells, tumor cells, vascular smooth muscle cells, trophoblast cells, and other cell populations that express VEGF receptors. Pathological conditions, such as abnormalities in placental development, can be caused by altered 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 on functional characteristics of trophoblast cells. 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 decidual tissue, it is important to evaluate their potential influence on phenotype of 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 expressed on the surface of various cell types. It plays a role in signal transmission from TGF-β family proteins. In particular, endoglin has been shown to regulate signaling from TGF-β by directing signals through the SMAD2/3 or SMAD1/5/8 pathways. According to the literature, endoglin inhibits the SMAD3- mediated signaling. However, similar effects of endoglin have not been confirmed for NK cells and trophoblasts. The studies of endoglin expression levels are of importance, since the signals from TGF-β are essential for differentiation of trophoblast cells. Disruption of TGF-β 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 was associated with reduced 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 reception of TGF-β family proteins by trophoblasts and natural killer cells.
Possible role of platelet-monocyte complexes in the pathogenesis of recurrent pregnancy loss
Recurrent pregnancy loss (RPL) is a significant clinical problem that affects 1-5% of population. Moreover, the cause of RPL remains unknown in more than half of cases. Possible reasons include imbalanced maternal hemostasis, thrombosis of uteroplacental vessels, decreased placental perfusion and hypoxia. Changes in morphofunctional features of monocytes and platelet-monocyte aggregates may be the factors causing pregnancy complications. However, role of platelet-monocyte complexes (PMC) in pathogenesis of RPL is unknown. The purpose of our study was to determine quantitative changes in contents and antigenic phenotype of PMCs in peripheral blood of the patients with RPL, and to assess the effect of platelets on the expression of monocyte surface proteins in normal and pathological pregnancy. The study groups consisted of 6 to12-week pregnant women diagnosed with RPL and females with uncomplicated pregnancy (7-12 weeks). PMC content and expression of CD62P, CD11b, CD86, CD162, HLA-DR, TREM-1 were determined in total cell population and subpopulations of peripheral blood monocytes using cytofluorimetry technique. It was found that PMC level was increased in patients with RPL (26.5%) compared to uncomplicated pregnancy (15.3%) with all monocyte subpopulations contributing to this increase. Decrease in HLA-DR expression and increase in CD11b expression was observed in total PMCs, while expression of CD62P, CD162, CD86 and TREM-1 did not change significantly. Monocyte subpopulations were differently involved into the RPLassociated expression of activation markers, while the changes detected in distinct subpopulations were not always evident in total monocyte populations in recurrent pregnancy loss. A comparison of PMC and free monocytes demonstrated that changes in surface phenotype of monocytes were caused by platelets and other exogenous factors. In patients with RPL, we observed platelet-induced increase in adhesive properties of monocytes which manifested as increased CD11b expression. In contrast, decrease in monocyte HLA-DR levels was not caused by platelets. The results obtained suggest that RPL is accompanied by increased level of peripheral blood PMC and changes in antigenic profile of platelet-associated and free monocytes, thus demonstrating the immunomodulatory effect of platelets, and also confirming the importance of determining expression patterns of surface antigenic markers of PMC for diagnostic and therapeutic purposes.
Increased incidence of abnormal TREC/ KREC levels is revealed in virtually healthy individuals over the post-pandemic period
The COVID-19 pandemic has posed an unprecedented challenge to global public health. Poor clinical outcomes in COVID-19 were associated with a pathogenic triad of interrelated mechanisms: immune dysregulation, coagulopathy, and secondary immunodeficiency, which collectively establish a selfperpetuating cycle of progressive multiorgan dysfunction. The failure of immune defense stems, in part, from T and B cell depletion due to immune resource exhaustion during acute infection. The extent of immune system impairment may be assessed by quantifying biomarkers of lymphocyte function: T cell receptor excision circles (TREC) and κ-deleting recombination excision circles (KREC) for T and B cells, respectively. The aim of our study was to evaluate the prevalence of abnormal TREC/KREC levels in peripheral blood among apparently healthy individuals during the post-pandemic period. We analyzed whole peripheral blood samples from 4,433 apparently healthy adults (≥ 18 years), residents of St. Petersburg and Leningrad Region. The samples were collected during two population studies: June 15-21, 2020 (n = 717) and September 4-29, 2023 (n = 3,716). TREC/KREC quantification was performed by means of real-time PCR with “TREC/KREC-AmpPS” commercial kit (St. Petersburg Pasteur Institute, St. Petersburg, Russia) following manufacturer’s protocol. The prevalence of reduced TREC/KREC levels increased significantly from pre-pandemic (14.2% and 5.7%, respectively) to post-pandemic periods (50.1% and 21.2%, respectively), thus representing a 3.6-fold increase. The mentioned differences in pathological biomarker levels were observed across all age groups. Characteristic immunological disturbances manifested as either isolated T cell deficiencies or combined T/B cell dysfunction, with no isolated B cell defects detected, thus suggesting the pivotal role of T cell impairment in observed immunopathological changes. Post-pandemic findings included decreased minimal population TREC values in older age groups and a paradoxical 50.5% increase in lower threshold KREC levels. Our findings demonstrate a substantially increased prevalence of abnormal TREC/KREC values, which seems to reflect long-term immune alterations following COVID-19. These changes underscore the critical need for the ongoing immunological surveillance in the post-pandemic era. Future research should focus on temporal dynamics of these disturbances and develop preventive strategies to mitigate long-term COVID-19 sequelae.
КОЗЛОВ ВЛАДИМИР АЛЕКСАНДРОВИЧ (К 85-ЛЕТИЮ СО ДНЯ РОЖДЕНИЯ)