Modern immunology education: traditions and actual trends
Currently, fundamental and clinical immunology is undergoing rapid development, and this science is becoming one of the leading disciplines of modern biology and medicine, interdisciplinary, the base of life sciences. Thus the growing interest in immunology, and accordingly, spread of teaching thereof, observed in various countries. This review presents the main stages of development of teaching immunology in our country: creation of the 1st Department of Immunology at the II N.I. Pirogov Moscow Medical Institute by acad. R.V. Petrov, publication of the 1st textbook on immunology, establishment of the 1st departments of immunology and allergology in medical universities, creation of a new specialty - «physician allergologist-immunologist», the recent publication of the 4th edition of textbook «Immunology» by R.M. Khaitov. The principle of immunology education in 2 semesters within a single department was formulated and proved its practicability. Considering the fact that immunology is one of the most rapidly progressing sciences and disciplines taught in higher education, important for future doctors of all specialties, biologists of many specialties, etc., there is a need to make additions to the basic university medical and biological education. Particularly, there is a need for the introduction of the discipline «Allergology and immunology» into the new generation of the Federal State Educational Standard, supplemented with an obligatory exam, which will greatly increase student motivation. To increase the efficiency of the education process, it is advisable to conduct practical classes using interactive forms of studying based on the active interaction between teachers and students: role-playing and business games, collective problem solving, work in small groups (teams), problem-based learning, etc. Cross-disciplinary education is encouraged, specifically the use of knowledge derived from different fields, their grouping and application in the context of the discussed subject. The importance of information and communication technologies in the process of immunology education is increasing as well. The establishment of a unified educational environment ensures effective interaction between teacher and student. These approaches are most relevant in the context of the current COVID-19 pandemic, when the significant part of education is on-line performed. This review also debates the issues regarding the difficulty of training highly qualified immunology teaching staff. Science is developing dynamically, soliciting the need to ensure the continuity and consistency of immunological education of students, medical residents and physicians of different specialties.
Relevance of the β-defensin family in reduction of male reproductive function
Introduction. Nowadays infertility became a global sociodemographic problem, which affects about 15 % of couples around the world. In the last few years more and more studies, which research an influence of antimicrobial peptides of the β-defensins family on reproductive processes, have been appearing.
The aim of this study was to estimate the role of β-defensins HBD1 and HBD26 in the pathogenesis of male infertility and the association of carriership of mutant HBD126 gene del allele (rs11468374) with its’ gene expression levels in sperm cells, and the level of its’ protein product in the seminal fluid of infertile patients.
Material and methods. The study included 88 male subjects. The participants were divided into a group of 65 men affected by idiopathic infertility, and a group of 23 healthy donors. Separation of the ejaculate into seminal fluid and spermatozoa was performed via centrifugation of the ejaculate in a density gradient. To assess the DEFB1, DEFB126 genes expression and the polymorphic marker (rs11468374) of DEFB126 gene, the techniques of DNA/RNA extraction, reverse transcription reaction, and real time PCR were used. The level of the HBD1, HBD26 peptides in the semen fluid was assessed by enzyme-linked immunosorbent assay.
Results. We demonstrated a decrease of expression of DEFB1 and DEFB126 genes in spermatozoa of patients with idiopathic infertility with severe asthenozoospermia by 4.5 times (HBD1) and 13.57 (HBD26) in comparison with heathy donors, with the concentration levels of these defensins in seminal plasma reduced by 2.7 and 13.84 times respectively. We also estimated a distribution of allele and genotype frequencies of the rs11468374 polymorphic marker of the DEFB126 gene among Moscow population. We demonstrated that the carriership of the mutant del allele is statistically associated with a higher risk of impairment of male fertility (χ2 = 14.67, р < 0.001, OR = 6.83; 95 % CI 2.409-19.366).
Conclusion. These data can be used for a more detailed study of β-defensins’ influence on male reproduction processes and establishment of infertility. Also the results of our work could be a foundation of an early diagnosis of susceptibility to decrease of fertility function.
Interrelationship of IFN-γ, IL-4, pituitary-thyroid and pituitary-adrenocortical systems in cold airway hyperresponsiveness in patients with asthma
Introduction. The mechanism of development of thyroid insufficiency in asthma is associated with the influence of Th1 cytokines on the peripheral part of the hypothalamic-pituitary-thyroid axis.
Aim of the study - to assess the profile of cytokines IFN-γ and IL-4, the activity of the pituitary-thyroid and pituitary-adrenocortical systems after exposure to the cold trigger on the respiratory tract of asthma patients.
Material and methods. 57 asthma patients with cold airway hyperresponsiveness (1st group) and 79 patients without hyperresponsiveness (2nd group) were examined. Asthma control was assessed using the Asthma Control Test questionnaire (ACT, Quality Metric Inc., 2002). We studied the lung function, the airway reaction to 3-minute isocapnic hyperventilation with cold (-20 °C) air (IHCA). In the exhaled breath condensate, the content of IFN-γ and IL-4 was determined before and after IHCA; in blood serum - thyroid stimulating (TSH) and adrenocorticotropic (ACTH) hormones, thyroid hormone fractions, cortisol; in leukocytes - cyclic adenosine monophosphate (cAMP).
Results. After the IHCA, in the persons of 1st group, a significant increase in the concentration in the blood serum of only free T3 was recorded, in 2nd group - TSH, free T3, T3, and free T4. Patients of 1st group had lower baseline concentrations of cortisol and cAMP than patients of 2nd group, and cold air bronchoprovocation promoted an active decrease in the content of cortisol in blood serum and cAMP in leukocytes, in contrast to patients who did not respond to the stimulus. When examining the IL-4 content in the exhaled air condensate, both before and after IHCA, no significant intergroup differences were found. In patients of 1st group, in response to IHCA, lower IFN-γ values were recorded than in patients of 2nd group: 12.9 [3.05; 24.85] and 34.5 [29.1; 51.8] pg/mL, respectively, (p = 0.001). Close correlations were found between the fall in FEV1 after IHCA, IFN-γ, IL-4, TSH, thyroid hormone fractions, cortisol, cAMP.
Conclusion. The cytokines IFN-γ and IL-4 are involved in the pituitary-thyroid and pituitary-adrenocortical regulation of the cold airway responsiveness in asthma patients. Thyroid insufficiency contributes to the disadaptation of the respiratory system to effects of cold air and is a factor influencing the imbalance in the cytokine system. Cold airway hyperresponsiveness is associated with low baseline levels of cortisol and leukocyte cAMP.
Combined immunotherapy of metastatic carcinoma by resection of the primary tumor and subsequent reprogramming of macrophages and dendritic cells using a TLR4 agonist in laboratory mice
Introduction. The successes of the last decade in the development of new methods of treating cancer patients are associated with the use of inhibitory receptor blockers and their ligands (PD-1, PD-L1, CTLA4); agonists of activation receptors of immune cells; bi- and trispecific ligands that contribute to the antitumor effect of cells of the immune system; as well as CAR-T, CAR-NK, TIL cells grown ex vivo for cell therapy. Despite the fact that many of the aforementioned variants of immune therapy for cancer patients have already been approved for use or are in the final stages of clinical trials, the achieved treatment efficacy is still far from the desired level. Consequently, the development of new methods of immune therapy for cancer patients is urgent.
The aim of the investigation was to study the effectiveness of immunotherapy of a solid metastatic tumor using a TLR4 agonist for reprogramming myeloid cells in the tumor microenvironment from protumor state to antitumor one.
Material and methods. Experimental model of solid metastatic carcinoma 4T1 in BALB/c mice was used. 4T1 carcinoma cells were inoculated subcutaneously, after which solid tumors grew in 100 % of cases. All tumor-bearing animals died within 30-35 days from multiple metastases to the lymph nodes, spleen, lungs, liver and other organs. In this experimental model, we performed surgical removal of the primary tumor at its 2-3 mm size (11th day after inoculation of 15 thousand 4T1 cells). The next day after resection, immunotherapy against metastatic disease was started. The TLR4-agonist i.e. the acidic peptidoglycan (hereinafter APG) from the plant source that is an active component of a pharmaceutical drug Immunomax was used for immunotherapy. The drug was injected intraperitoneally every 4-5 days, in total - 7 injections per course of treatment. Groups of mice treated with a combination of surgical resection and immunotherapy were compared with groups of mice treated using either surgical resection of the primary tumor alone or immunotherapy with TLR4-agonist alone. The rate of tumor growth, the survival time of the animals, the frequency of relapses after resection, and the number of metastatic CFUs in the lungs were investigated.
Results of our experiments showed that surgical resection of the primary tumor does not save animals from death, only postpones the moment of death by about 30 days. Monotherapy with TLR4-agonist (without surgical resection of the primary tumor) also proved to be ineffective. The growth rate of tumors and the lifespan of the animals treated with APG only did not change. In contrast, the combination of surgical treatment and immunotherapy has shown high therapeutic efficacy in 4T1 carcinoma. Resection of the primary tumor followed by a course of immunotherapy with injections of a TLR4-agonist allows a complete elimination of the malignant neoplasm in 20 % and a significant prolongation of the life span of the remaining 80 % animals.
Discussion contains an analysis of the possible mechanisms of the TLR4-agonist therapeutic action, based on the reprogramming of macrophages and dendritic cells to their anti-tumor state. Experimental data are presented that prove the feasibility and effectiveness of the use of a TLR4 agonist, capable of this reprogramming, in the complex treatment of animals with absolutely lethal metastatic carcinoma.
Conclusions. 1. The TLR4 agonist (APG) stand-alone use for the treatment of metastatic carcinoma does not significantly affect the growth rate of the primary tumor, the process of metastasis and the survival of tumor-bearing mice.
2. After surgical resection of the primary carcinoma, the TLR4 agonist (APG) use is highly effective for treatment of the lethal metastatic disease.
The role of C-reactive protein, neopterin and melatonin in the diagnosis of Epstein-Barr virus infection
Introduction. The complexity of the diagnosis of Epstein-Barr virus (EBV) infection determines the relevance of the search for non-specific markers indicating the activity of the process.
The aim of the study is to evaluate the role of C-reactive protein (CRP), neopterin and melatonin in the diagnosis of EBV infection.
Material and methods. The blood levels of CRP, neopterin, and melatonin were studied in 90 people with chronic EBV infection, including 45 people with reactivation (24 - in the early and 21 - in the late phase of reactivation) and 45 people with latent infection.
Results. The average concentration of CRP in individuals at the late phase of reactivation (193 mg/l) is significantly higher than in the group with latent EBV infection (60.5 mg/l). The frequency of exceeding the reference values of CRP in the studied groups did not significantly differ. Elevated levels of melatonin were significantly more often detected in individuals in the late phase compared to the subgroup in early reactivation, and lowered - on the contrary. The average concentration of melatonin in individuals in late reactivation (40.2 pg/ml) is significantly higher than in the early phase (18.9 pg/ml). Reduced values of neopterin among persons with virus reactivation were detected significantly less often than in the latent form. The average concentration of neopterin in individuals in a late reactivation phase (5.5 nmol/l) is significantly lower than in those in an early one (9.0 nmol/l).
Conclusion. The study of the levels of CRP, neopterin and melatonin allows us to determine the presence of an infectious process. Further research will allow us to outline new prospects for the diagnosis, therapy and prevention of EBV infection.
Complex assessment of the cytokine profile, proteinograms and inflammation acute phase proteins in patients with acute brucellosis
Introduction. The pathogenesis of brucellosis is directly related to the induction of proinflammatory cytokines and activation of the Th1-type immune response. The role of inflammatory factors in acute brucellosis remains unexplored.
Aim of the study - to determine the level of proinflammatory cytokines (IL-1β, IL-6, IL-8, IL-12, IL-18 and IFN-γ), proteins of the acute phase of inflammation (neopterin and lipopoly-saccharide-binding protein) and features of the fractional composition of blood serum proteins in patients with acute brucellosis before and after complex antibiotic therapy and taking an immunomodulator Licopid®.
Material and methods. The object of the study were clinical blood samples from 65 patients with a laboratory-confirmed diagnosis of acute brucellosis, admitted to the State Medical Institution «City Clinical Hospital No. 2», Stavropol. The concentrations of cytokines (IL-1β, IL-8, IL-12, IL-18, IFN-γ) and acute phase proteins were determined in blood serum by enzyme-linked immunosorbent assay, IL-6 level - by immunochemiluminescence method, fractional composition of blood serum proteins - using electrophoresis.
Results. In the acute phase of brucellosis infection (before treatment), there is a high level of pro-inflammatory cytokines IL-1β, IL-6, IL-8, IL-18 and IFN-γ. After the course of antibiotic therapy (doxycycline and rifampicin, 6 weeks), high level of IL-8 and IL-18 remains in the blood serum of patients with moderate brucellosis, indicating active inflammation. The level of LPS protein and neopterin decreased statistically significant during therapy. Dysproteinemia was shown with an increase in the level of globulins, mainly due to α- and γ-globulin fractions, a moderate decrease in the concentration of total protein, hypoalbuminemia, and a decrease in the albumin-globulin coefficient.
Conclusion. The prospect of further study of the cytokine profile (IL-1β, IL-6, IL-8, IL-12, IL-18, INF-γ) to assess the intensity of the systemic response to infection and to predict the course of acute brucellosis is noted.
Ferritin level as a predictor of COVID-19 severe course
Introduction. The search for possible prognostic criteria for the severity of COVID-19 is very relevant, as it can provide significant assistance in choosing a more effective and personalized therapy. The degree of severity of the infectious process is reflected by acute-phase proteins, in particular ferritin. The degree of informative assessment of its level as a predictive criterion is debatable, which determined the purpose of this study.
Aim of the study - to reveal the clinical and immunological features of the course of COVID-19 in patients with a moderate variant of the course, depending on the level of ferritin.
Material and methods. The study included patients with moderate COVID-19 (43 patients), who were subsequently divided into two groups: with high ferritin levels - 1744 mcg/l [985.75; 2402.85] (22 patients) and with normal ferritin levels - 264.4 mcg/l [129.5; 375.6] (21 patients). The group of comparison consisted of practically healthy donors (31 persons). Blood samples was taken for 2-3 days from the moment of admission for general clinical and biochemical analysis. CD3+-, CD4+-, CD8+-, CD16+- and CD19+-lymphocytes were evaluated by flow cytofluorimetry, as well as the level of serum IgA, IgM and IgG - by radial gel immunodiffusion by Mancini. A computer tomography of the chest organs was performed for all study participants.
Results. Lymphopenia, increased levels of C-reactive protein, and active proliferation of CD19+-lymphocytes without increased antibody production were observed in the group of patients with high ferritin levels. Clinical manifestations in this group of patients are more severe than in the group with normal ferritin content.
Conclusion. An increase of the blood ferritin level in patients with moderate COVID-19 is a predictor of a more severe course and is accompanied by a violation of the maturation of the T-cell part of adaptive immunity and increased proliferation of B-lymphocytes without an adequate increase of antibody formation processes.
Age-related changes in the content of αβ-, γδ-T-lymphocytes and some its minor subpopulations in healthy children
Introduction. Determination of the qualitative and quantitative content of cells of the immune system in healthy children is important for the formation of normal cell’s parameters, which is necessary both for research purposes and for clinical use. Data on the quantitative content of αβ-, γδ-T-lymphocytes and, especially, its minor subpopulations in children of different age groups are limited and extremely contradictory, so there is a need for such studies.
Aim of the study – to search the relative and absolute content of lymphocytes, Т-lymphocytes, αβ- and γδ-Т-lymphocytes, minor subpopulations: double negative and double positive T cells (DNT- and DPT-cells) in the peripheral blood of healthy children of different ages and to assess the correlation between the studied parameters and the age of children.
Material and methods. The venous blood samples of 83 healthy children (boys and girls), aged from 7 months to 14 years, was carried out. Monoclonal antibodies conjugated with various fluorochromes were used to detect the content of lymphocytes and its subpopulations. Determination of the number and percentage of lymphocytes and its subpopulations was carried out by flow cytometry.
Results. A decrease in the absolute number of γδ-T-killers in the blood of older children was found. The content of γδ-Т-lymphocytes, DNT- and DPT-cells remained stable in all age groups.
Conclusion. The decrease in the percentage of lymphocytes in healthy children depends on age. The absolute number of lymphocytes, T-lymphocytes, αβ-T cells and γδ-T-killers progressively decreases with age, while the number of γδ-T-lymphocytes, γδ-T-helpers, DPT- and DNT-cells remains unchanged.
Possibilities of the Enzyme-Linked Immunospot Assay (Elispot) method in carrying out anti-tuberculosis measures in patients with HIV infection
Introduction. The problem of tuberculosis (TB), combined with HIV infection, currently occupies a central place in phthisiology. HIV infection significantly increases the risk of reactivation of latent tuberculosis infection to the clinical form of the disease, which requires a new approach to early detection of tuberculosis at the stage of development of latent tuberculosis infection (LTBI) in the most vulnerable group of patients. A key factor in the disruption of the immune defense mechanisms against tuberculosis, leading to the activation of LTBI, is a decrease in the number of CD4+-cells. For early detection of tuberculosis infection, including LTBI, immunodiagnostic tests based on the determination of the immune response to ESAT6-CFP10 proteins specific to the virulent MBT strain are currently available. According to many authors, the most effective method for early detection of TB in HIV-infected patients is the method of determining the number of mononuclear blood cells (spots) that release interferon γ (IFN-γ) in response to stimulation of ESAT-6 and CFP-10 and obtaining a cell suspension a certain concentration (ELISPOT) Assessment of the significance of the method in the diagnosis of tuberculosis infection in screening patients with different levels of immunosuppression seems relevant.
Aim of the study – to evaluate the results of T-SPOT.TB in patients with HIV infection with different levels of CD4+-cells, comparing them with other methods of detecting TB using the example of the Samara region.
Material and methods. In 2019–2020 an analysis of the T-SPOT.TB results was carried out in 398 patients with HIV infection who are under dispensary observation at the Center for the Prevention and Control of AIDS and Infectious Diseases. Inclusion criteria: age 18 years and older, diagnosis of HIV infection, CT scan of the lungs, the presence of a blood test for T-SPOT.TB, the presence of a test result with ATP.
Results. It was found that with changes on CT, characteristic of tuberculosis, out of 117 cases of verified tuberculosis in patients with CD4+-cells level 230.05 ± 15.3 CI [190.91–256.2], positive T-SPOT.TB was registered in 103 (88 %) patients. When comparing the results of the test with ATP and T-SPOT.TB at a CD4+-cells level of 500–350 cells/mcl, there was no statistically significant difference in the effectiveness of the tests (χ² = 1.28, p = 0.2579, OR = 2.28), with a decrease in the level of CD4+-cells less than 350 cells/μl, the role of T-SPOT.TB increases significantly. With a positive T-SPOT.TB result and no changes on CT, persons with suspected tuberculosis were diagnosed with LTBI in 17.6 %.
Conclusion. Immunodiagnostic tests can be used in a diagnostic complex for monitoring patients with HIV infections to detect tuberculosis infection, which will allow timely anti-tuberculosis measures.
The proteins of γ-globulin fraction binding metal ions, in physiological immune regulation. Mutual action of copper and zinc
The data are summarized in the paper concerning possibility of chelation by proteins of human y-globulin fraction of slow molar excess of metal ions from their periglobular space during tissue damages or inflammation. Copper and zinc ions bound by γ-globulin fraction proteins and localized within antibodies molecules by common groups of their binding sites cause similar changes of effector properties of γ-globulins that similarly shift cell functions in response to Fc receptor activation by the proteins transformed with metal binding. During tissue damages or inflammation the proteins of γ-globulin fraction chelating the slow molar excess of copper and zinc ions exert mutual effector properties, support induction of cellular immunity, and simultaneously exclude pathological reactions towards own normal human cells by enhancing production of functionally opposite cytokines.
Results of the interaction of gastric epithelium with Helicobacter pylori: cell damage, participation of epithelial cells in the immune response, carcinogenesis
H. pylori causes chronic infection in about half of the world’s population. In most people infected, this infection is asymptomatic, but in some it leads to the development of gastritis, peptic ulcer disease, lymphoma or gastric adenocarcinoma. H. pylori possesses a large arsenal of virulence factors that damage or actively manipulate epithelial cells and cells of the immune system for the long-term survival of the pathogen in the host organism. The chronic influence of the microorganism on signaling and gene expression in the epithelium, as well as the action of pro-inflammatory mediators formed during a poorly effective antimicrobial cellular immune response, can lead to malignant transformation of epithelial cells and accelerate tumor growth. The review provides data on the molecular mechanisms of the cytotoxic and carcinogenic effects of H. pylori virulence factors, describes the protective reactions triggered by epithelial cells in response to infection.
Complex regulatory networks: relationships between metabolism, intracellular signaling pathways and epigenetic regulators in the control of Th1 functions
The regulation of the immune response is one of the most important issues in immunology. There is still no complete understanding of how the immune system works. An ever-growing amount of research on this topic demonstrates that the problem is becoming interdisciplinary. Thus, it has been shown that both intracellular and extracellular metabolism control the functions of immune cells. In this review, we collected and systematized data on the relationship between metabolism, intracellular signaling pathways, and epigenetic regulation of the expression of genes that mediate cell functions in the group of lymphocytes - type 1 T-helpers (Th1). Using the example of this population of T-lymphocytes, which determines the cellular type of the immune response and performs a number of other functions, the review shows how individual metabolic pathways can affect the course of a certain type of immune response - reactions of cellular immunity. The principles of metabolic regulation of T-lymphocytes and potential strategies for therapeutic interventions targeting the Th1 cell population were also highlighted.
Tissue-resident natural killer cells: features of functioning in the uterus and decidual membrane
Natural killers are a heterogeneous population of innate immune lymphocytes, among which in recent years 2 categories of cells have been distinguished - circulating (cNK) and tissue-resident (trNK). The circulating pool of these cells is now fairly well characterized: these are cells of bone marrow origin with predominantly cytolytic activity, predominanly CD56dimCD16+ phenotype and a high level of perforin and granzymes expression. The tissue-resident pool has not yet been sufficiently studied, but a number of its features have been established: the ability to differentiate in tissues, the predominance of the CD56brightCD16- phenotype, the ability to produce high levels of certain regulatory cytokines (IFN-γ, TNFα, GM-CSF), the nature of the expression of receptors specific to class I histocompatibility molecules, etc. One of the most important features of trNKs is that their properties correspond to the specific organ where they are localized. This review is devoted to the characteristics of the natural killer cells of the uterus (uNK) and the decidual membrane (dNK) during pregnancy, the features of their origin, phenotype, cytokine production and the set of implemented functions.