Immunologiya

Introduction. The COVID-19 pandemic, which began at the end of 2019 in Wuhan, China, has unfolded differently in different countries. These differences cannot always be explained by different healthcare organizations. Clinical variations, severity of infection, favorable or fatal outcome of COVID-19 may also be associated with immunogenetic differences in hosts. Classical HLA proteins, the main function of which is to present peptides of infectious pathogens on the surface of Ag-presenting cells to T cells, which initiates an adaptive immune response. Different HLA alleles present different peptide repertoires, which can potentially influence the immune response. It has been shown that different alleles of HLA genes have been involved in host susceptibility or resistance to diseases such as tuberculosis, malaria, hepatitis B, dengue, influenza, and MERS.

Aim – to search for markers of sensitivity and severity of COVID-19 disease among alleles of the HLA-A, HLA-B, HLA-DRB1 genes in different age groups of patients.

Material and methods. All subjects included in the study are residents of Moscow. Population control – 98 people. Patients with COVID-19 were in the Infectious Clinical Hospital No. 1 of the Moscow in 2020–2021. The presence of the SARS-CoV-2 was confirmed by PCR. Patients with mild/moderate infection and a favorable outcome 65 years and younger (n = 316): m/f = 160/156, age Me = 48 years (18–65 years), over 65 years (n = 102): m/f = 43/59, age Me = 72 years (66–96 years), patients with severe disease and a favorable outcome, 65 years and younger (n = 75): m/f = 43/32, age Me = 52 years (32–60 years), over 65 years (n = 139): m/f = 49/90, age Me = 72 years (66–90 years); patients with severe infection and death 65 years and younger (n = 23): m/f = 12/113, age Me = 54 years (29–60 years), over 65 years (n = 88): m/f = 23/65, age Me = 74 years (66–94 years). Alleles of genes HLA-A, HLA-B, HLA-DRB1 were determined by NGS.

Results. After the number of studied alleles correction, the studied age groups of patients (over 65 years old and younger than 65 years) did not differ from the population control in the frequency profile of HLA genes. Markers of severe disease, but with a favorable outcome in the age group 65 years and younger were B*08:01 and DRB1*11:03. A marker of a milder course of the disease in the over 65 years old group was HLA B*5701. The marker of lethal outcome of severe COVID-19 infection in the over 65 years old group was HLA B*15:01.

Conclusion. The studied HLA genes are more likely associated with the severity and outcome of infection than with the fact of infection itself.

Introduction. Currently, there is a high demand for new promising drugs for the treatment of respiratory viral infections. The great deal of these infections, including those leading to life-threatening complications, are caused by human respiratory syncytial virus (RSV). Synthetic peptides are being widely developed and tested as potential antiviral drugs that can be used in treatment of RSV infections as well. Development of new peptides with proposed antiviral activity strongly relies on rational design of their structures and research of their physicochemical properties to determine the correlation between structure and activity.

The aims – to create and experimentally determine structures of new synthetic antiviral peptides, to study their activity against RSV.

Material and methods. The structures of the peptides are based on structural fragments of several nucleolin ligands, the cellular target protein of RSV, as well as on previous results of the authors. The peptides were obtained by solid-phase peptide synthesis, identified with mass spectrometry, quantitated with CHNSO-analysis, their purity was determined by capillary zone electrophoresis. Antiviral activity was determined using a cultured RSV strain in vitro. Cytotoxicity was determined by MTT test.

Results. All synthesized peptides were found to have significant antiviral properties. We assume that the greatest effect is achieved by combining aromatic and cationic amino acid residues in the peptide structure, in particular Y, F, R, K, O. Antiviral effect was demonstrated for dendrimeric peptides as well as for linear. The positive net charge of the peptide and/or pronounced amphipathy contributed to the manifestation of antiviral properties. These particular structural properties are often found in antiviral, cell-penetrating, and antimicrobial peptides, including natural ones.

Conclusion. Five novel antiviral peptides were synthesized, characterized with physico-chemical methods and were found to have significant anti-RSV activity. These peptides can be used in further development of new antiviral peptide drugs. A possible explanation is provided for correlation between the structure of peptides and their activity.

Introduction. Excessive, poorly controlled inflammation in the lungs is one of the complications of the immune response during tuberculosis. On one hand, increased production of IL-6 in the lungs significantly correlates with the progression of tuberculosis in susceptible mice. On the other hand, a total deficiency of IL-6 on a tuberculosis-resistant genetic background leads to susceptibility to the infection. In this regard, the role of the cytokine IL-6 in the immune response during tuberculosis may differ between genetically sensitive and more resistant individuals.

Aim – to study the effect of IL-6 deficiency on the immune response and sensitivity to the infection in susceptible to tuberculosis MHC-II congenic mouse strains.

Material and methods. 2–3 months old females of IL-6-deficient B6.I.100.IL-6KO and B6.I.9.3.IL-6KO and wild-type B6.I.100 and B6.I.9.3 mice were used for aerosol infection with virulent strain M. tuberculosis H37Rv, 100 CFU/mouse. Determination of CFU numbers in the organs of infected animals was assessed by plating suspensions of lung and spleen cells on Dubos agar and macrocolonies counting after 21 days of cultivation. Cell surface phenotype and intracellular cytokine production after stimulation of lung cells with mycobacterial antigens in vitro were measured by flow cytometry. Lung cryosections were stained with hematoxylin and eosin.

Results. We found that IL-6 deficiency leads to a decreased lifespan after tuberculosis infection in the congenic strains B6.I.100.IL-6KO and B6.I.9.3.IL-6KO compared to the control animals B6.I.100 and B6.I.9.3, respectively. IL-6 deficiency also results in increased migration of neutrophils into the lungs of B6.I.9.3.IL-6KO mice at the beginning of the infection. Additionally, we observed lower numbers of specific IL-17-producing Th17 lymphocytes but a higher level of TNF-a production in the lungs of IL-6KO animals compared to wild-type controls. During the later stages of tuberculosis infection, we detected severe diffuse inflammation in the lungs of IL-6-deficient mice, whereas more concentrated areas of inflammation separated from uninfected lung tissue were observed in the lungs of wild-type animals.

Conclusion. IL-6 deficiency on the background of genetically determined high sensitivity to tuberculosis leads to an aggravation of the infection, which occurs due to mechanisms that differ from those realized in mice resistant to tuberculosis.

Introduction. Ovarian cancer is considered one of the most aggressive gynecological malignancies. Unfortunately, the methods that are currently considered the gold standard for the treatment of ovarian cancer, in most cases, lead to frequent relapses and progression of the disease after a limited period of time. Since the prognosis of this disease is often associated with the infiltration of the tumor by immune cells, immune checkpoint inhibitors can be used to activate the antitumor immune response in the treatment of tumors of this type. However, to date there is insufficient data to effectively select patients who can fully respond to this therapy.

The aim of the study is to study the level of soluble forms of PD-1 and PD-L1 (sPD-1 and sPD-L1) in ovarian tumors, their clinical and prognostic significance.

Material and methods. The study included 136 patients and 35 healthy women who were treated at the N.N. Blokhin NMRC of Oncology, MOH of Russia. The clinical diagnosis in all patients was confirmed by morphological examination of the tumor according to the International Histological Classification of Ovarian Tumors, adopted by WHO in 2014. The study included patients with epithelial ovarian cancer of three histological types: serous (77), endometrioid (13) and mucinous (8); benign neoplasms (22) and borderline tumors (10) of the ovaries, as well as non-epithelial ovarian tumors: dysgerminoma (3), sertolioma (1) and granulosa cell tumor (2). The levels of sPD-L1 and sPD-1 proteins was determined in blood plasma obtained according to standard methods before the start of specific treatment using an enzyme-linked immunosorbent assay. Statistical analysis of the obtained results was carried out using GraphPad Prizm v. 10. When comparing indicators and analyzing their relationships, nonparametric Mann–Whitney and Kruskal–Wallis tests and Spearman’s rank correlation coefficient were used. Overall survival analysis was performed by constructing survival curves using the Kaplan–Meier method. Differences were considered statistically significant at p < 0.05.

Results. The study showed that the median level of sPD-1 in the blood plasma of healthy women in the group of comparison was 43.8 (33.6–54.6) pg/ml, and in the group of patients with ovarian cancer – 47.1 (35.1–61. 6) pg/ml. The median level of sPD-L1 in blood plasma in the group of comparison was 60.8 (26.2–91.7) pg/ml and was not significantly higher than in patients with ovarian cancer 40.5 (15.7–78) pg/ml (p = 0.075). The level of sPD-L1 in blood plasma was significantly reduced in benign and non-epithelial malignant ovarian tumors (p = 0.022 and p = 0.006, respectively) compared to the group of comparison. It should be noted that the level of sPD-L1 in patients with nonepithelial ovarian tumors was significantly lower compared to epithelial tumors (p = 0.02). For sPD-L1 level, there is a significant association with tumor progression factors. Thus, the level of sPD-L1 is significantly higher in the later stages of the disease, in the case of larger tumors, in the presence of ascites and regional metastases. The analysis of prognostic significance showed that a high level of sPD-L1 protein in the plasma of patients with malignant ovarian tumors is a significant unfavorable prognostic factor (HR = 2.28; p = 0.023).

Conclusion. The study showed that the level of sPD-L1 in the blood plasma of patients with ovarian tumors reflects the extent of the tumor process and can potentially be an effective tool for monitoring the course of this disease. Also, our results and literature data indicate the prospects of using sPD-L1 level as a marker of the effectiveness of OC therapy, and therefore the prognosis of the disease.

Introduction. Hepatocellular carcinoma (HCC) is the most common type of liver cancer with low survival rate due to late detection. The cytokine IL-27 plays a key role by activating STAT1 and STAT3 factors, influencing the development of HCC. Recent studies indicate the importance of the IL-27R signaling pathway in the control of innate cytotoxic cells, which may be relevant for the diagnosis and treatment of early-stage HCC.

Aim – to study the role of IL-27R signaling in the early stages of HCC development.

Material and methods. The study used laboratory mice with genetic modifications in the IL-27 receptor gene (Il27ra^–/– and Il27ra^+/–) intraperitoneally injected with diethylnitrosamine (DEN) to analyze hepatocyte apoptosis and proliferation. Quantitative analysis of neutrophils was carried out using flow cytometry. The level of proliferation was determined by staining samples for the Ki67 marker in liver sections, and TUNEL analysis of lysates was used to detect apoptosis. Analysis of phosphorylation of the transcription factor STAT3 in the liver of mice was carried out using the Western Blot. The expression level of Ifng and genes Il10, Retnla, Chil3l1, Cd44, associated with repair in mouse liver, was analyzed by real-time PCR.

Results. After 48 hours following DEN administration, Il27ra^–/– mice exhibited decreased levels of proliferation and apoptosis in the liver, as well as decreased neutrophil numbers. Within 24 hours after DEN administration, there was an increase in the expression of genes associated with tissue repair in the liver in Il27ra^–/– mice.

Conclusion. The results of the study indicate the influence of the IL-27 signaling pathway on inflammation, apoptosis and proliferation of hepatocytes, and liver repair processes, providing the opportunity to assess the prognosis of HCC development by measuring the level of IL-27 after a damaging effect on the liver.

X-linked lymphoproliferative syndrome type 2 (XLP2) is a rare primary immunodeficiency, characterized by high incidence of severe complications such as hemophagocytic lymphohistiocytosis and inflammatory bowel disease. The disease is caused by pathogenic variants in the XIAP (X-linked inhibitor of apoptosis) gene encoding XIAP protein that is involved in NOD2 signaling. Development of new methods for an early diagnosis of XLP2 is a matter of current interest.

The aim of the study was to evaluate tumor necrosis factor (TNF) expression in monocytes of XLP2 patients in response to stimulation by NOD2 ligand muramyldipeptide (MDP).

Material and methods. The study included 4 XLP2 patients, 3 mothers with hemizygous pathogenic variants in the XIAP gene, as well as 21 healthy donors over the age of 18. Fraction of mononuclear cells was isolated from peripheral blood samples, NOD2 receptor stimulation was carried out by incubation of cells in the presence of MDP. Lipopolysaccharide was used as a positive control, and a complete culture medium was used as a negative control. TNF expression in monocytes was evaluated by flow cytometry.

Results. A threshold value for evaluating the response to MDP stimulation was calculated. All samples of healthy donors showed significant increase in the proportion of TNF-producing monocytes in response to stimulation. Mothers with hemizygous pathogenic variants in the XIAP gene demonstrated response that was comparable to that of healthy donors. All patients included in the study, despite the presence of residual XIAP protein expression, showed no response to MDP stimulation. Additionally, the possibility of using cryopreserved mononuclear cells as a material for the assay was demonstrated on samples of 5 healthy donors and one patient.

Conclusion. Diagnostic method described in this study in combination with the assessment of XIAP protein expression allows for an early diagnosis of XLP2.

Introduction. The study of COVID-19 and its consequences remains an urgent task, as the risk of emergence of new dangerous strains of SARS-CoV-2 and other infectious agents because a similar mechanism of action is still relevant. The present study contributes to the understanding of the immune system function during COVID-19 and its results may be useful for practical public health care.

Аim – to determine the prognostic potential of sCD40L, MDC, FKN, IP-10 and VEGF in relation to the severity and outcome in COVID-19.

Material and methods. Serum samples were collected from 1614 patients with various severity of COVID-19, including 302 fatal patients. Levels of sCD40L, MDC, FKN, IP-10 and VEGF were determined in the samples by multiplex immunofluorescence analysis.

Results. In severe and fatal disease cases, MDC and sCD40L concentrations were significantly lower and IP-10 concentrations were higher than in cases with milder forms of COVID-19. The significance of differences in sCD40L levels disappears when analysing data from patients without comorbid load (Charlson index ≤ 2). FKN concentration on admission to hospital was significantly lower in those cases that developed a cytokine storm.

Cellular senescence is a fundamental biological process which consists of an irreversible blocking of cell division induced by extrinsic or intrinsic factors. Cellular senescence is required for development and maintenance of normal structure of tissues and organs as well as for prevention of malignant transformation. However, in an aged organism, accumulation of senescent cells in tissues becomes an important factor contributing to tissue damage and aging-associated diseases. Negative effects of senescent cells are mediated by secretion of pro-inflammatory cytokines in the context of the so-called senescence-associated secretory phenotype (SASP). In immune system, increased numbers of senescent cells contribute to generalization of tissue damage and compromises defense against pathogens. In experimental models, elimination of senescent cells results in delayed development of aging-associated pathologies, increases life span of animals. Senotherapeutic drugs, designed to treat age-associated diseases by eliminating senescent cells or modulating SASP, are a subject of intense pre-clinical and clinical research.

Pulmonary fibrosis is a pathological process that is characterized by the proliferation of scar tissue in the lung parenchyma, leading to the destruction of alveolar structures and the development of respiratory failure. The growing number of patients with pulmonary fibrosis, including after the COVID-19 pandemic, and the lack of effective treatment methods dictate the need for a deeper understanding of the pathogenesis of this pathology. At the same time, increasing attention is being paid to research into the immune mechanisms of fibrosis development. Macrophages in the lung tissue are heterogeneous population of cells that differ in location, origin and function. They play a key role in the pathogenesis of pulmonary fibrosis. The review presents current data on various subtypes of pulmonary macrophages, their role in the fibrotic process, as well as some mechanisms regulating the process of fibrogenesis.

Allergic rhinitis (AR) is an inflammatory disease of the nasal mucosa, which affects up to 30 % in Europe and 20 % in Russian Federation. Despite the fact that AR is not a severe pathology it causes significant economic issue (1–1.5 billion euros is annual direct costs of AR treatment in European Union). Moreover, 40 % of patients with AR subsequently develop more severe disabling pathology – bronchial asthma (annual mortality reaches up to 300,000 people worldwide). Approaches to AR therapy include pharmacotherapy with corticosteroids, histamine H1 receptor blockers and leukotriene antagonists, and allergen-specific immunotherapy. However, current treatments are insufficient, as evidenced by the continuing rise in morbidity. The search for new ways to prevent and control of this disease is an urgent task.

According to the current knowledge, the main role in AR pathogenesis play immune cells: Th2-lymphocytes, B-cells and eosinophils, which secrete proinflammatory cytokines (mainly IL-4, IL-5 and IL-13) and other inflammatory factors that induce manifestations of the pathology. However, with the development of new molecular methods a lot of evidence has been accumulated for the participation of epithelial cells of the respiratory tract and proinflammatory cytokines derived from them (IL-25, IL-33 and TSLP) in the pathogenesis of AR. In the current review, we summarize the data on the role of the triad of epithelial cell-derived cytokines in the pathogenesis of AR.