Current issue
4 . 2020
Cellular immunology

Dynamics of T-lymphocytes regeneration after lymphopenia induction by cyclophosphane


Introduction. Chemotherapy drugs used in medicine, mainly for the treatment of cancer, belong to a group of factors that cause damage of the immune system. The study of the mechanisms of T-cell recovery after exposure to cytostatic agents is important both for understanding the pathogenesis of their damaging action and for finding the correct therapy.

Aim of the study - to investigate the role of thymus and the peripheral lymphoid organs in T-cell restoration after exposure to cyclophosphamide in C57BL/6 mice.

Material and methods. Cyclophosphamide was administered intraperitoneally to experimental animals once at a dose of 125 mg/kg. Mice were euthanized on days 0, 10, 20, 30, and 60 after cyclophosphamide administration. The total number of cells and the lymphocyte subpopulations in the thymus and spleen were determined by flow cytometry.

Results. We found that actively dividing thymus cells are more defenseless to the action of the drug than spleen lymphocytes. At the same time, recovery processes in the thymus begin earlier (on day 20), followed by slow recovery of spleen lymphocytes (by day 60). However, complete regeneration of cytotoxic T-cells (CTLs) in the spleen is not observed even by 60 days. The study of the age phenotype of T-cell subpopulations showed that cyclophosphamide intensifies the phenomenon of the conversion of the naive T-cells into central memory T-cells.

Conclusion. Cyclophosphamide has a heavy cytotoxic effect on the T cells of C57BL/6 mice, with predominant damage in highly dividing thymocyte populations. Thymopoie-sis is unable to compensate for the deficiency of peripheral T-cell populations, which leads to an increase in homeostatic proliferation of T-cells, conversion of naive T-cell phenotype and the accumulation of T-helpers and CTLs with the phenotype of central memory T-cells. These changes can narrow the repertoire of T-cell receptors, lead to impaired immune surveillance and the possible growth of oncological, autoimmune and infectious diseases in the future.

Effect of collagen matrikines on the functional state of human leukocytes


Introduction. Matrikines - biologically active compounds formed during partial proteolysis of proteins and glycosaminoglycans of the extracellular matrix, act as endogenous regulators of many physiological and pathological processes of the body. Matrikines obtained from collagen proteins (CM) enhance the proliferation of connective tissue cells, stimulate their adhesion, migration and synthesis of various components of the extracellular matrix, cytokines and growth factors. CM regulate vascular tone and permeability, participate in protein-protein interactions that change the functions of other molecules, and contribute to the regeneration of nervous tissue. However, the effects of CM on immune system cells have not been fully studied.

The aim of this study was a comprehensive assessment of the effect of low-molecular weight CM on the functional activity of human peripheral blood leukocytes.

Material and methods. The fraction of low-molecular weight CM (Mw <10 kDa) was obtained by hydrolysis of the collagen matrix with bacterial collagenase. Depending on the method of investigation, the study of the effect of CM was performed either in whole heparinized venous blood, or in the fraction of neutrophils isolated from the blood of healthy donors. The effect of CM on the absorption and digesting functions of white blood cells, as well as their production of reactive oxygen species, was been evaluated.

Results. There has been evidence that CM are able to change the effector functions of phagocytes: although peptides do not increase the absorption capacity of phagocytes, they stimulate their bactericidal and synthetic activity. The effectiveness of CM depends on their concentration: the maximum effect was observed at low and medium concentrations of CM (10-100 mcg/ml) and was leveled at the maximum concentration of CM (1000 mcg/ml). Conclusion. Thus, CM exhibit immunoregulatory activity against innate immune cells.

Innate immunity

A comparison of three types of cell-based test systems for the assessment of biological activity of NOD2 receptor agonists


Introduction. NOD2 receptor agonists are a promising class of activators of innate immunity. Test systems for qualitative and quantitative measurements of their biological activity are required.

Aim - to compare main characteristics of three reporter cell types devised to assess biological activity of NOD2 agonists: cells with endogenous NOD2 expression, with transient NOD2 overexpression, with stable NOD2 overexpression.

Material and methods. Reporter cells with endogenous NOD2 expression (293Luc) were obtained by stably transfecting HEK-293T cells with luciferase reporter gene under an NF-кВ-dependent promoter. Cells with transient NOD2 overexpression (293Luc-tNOD2) were obtained by transiently transfecting 293Luc cells with a plasmid encoding NOD2 gene under a constitutive promoter. Cells with stable NOD2 overexpression (293Luc-stNOD2) were obtained by prolonged culture of NOD2-transfected cells with selective antibiotic. For each cell type, control cells were created enabling to confirm NOD2-dependent NF-кВ activation. Cells were stimulated for 24 h with a NOD2 agonist, muramyl dipeptide (MDP); as the readout, luciferin-dependent chemiluminescence of cell lysates was measured.

Results. Background signal in 293Luc-tNOD2 and 293Luc-stNOD2 was, respectively, 30 and 170 times higher than in 293Luc cells, pointing at NF-кВ activation in the absence of NOD2 agonists. At the same time, limit of quantitation of MDP by 293Luc-stNOD2 cells was an order of magnitude lower than by 293Luc cells (1.6 nM and 20 nM, respectively). Systems based on 293Luc and 293Luc-stNOD2 cells possessed acceptable accuracy and repeatability, 293Luc-tNOD2 cells showed unsatisfactory repeatability as well as increased time and reagent consumption.

Conclusion. 293Luc and 293Luc-stNOD2 cell lines can be used to measure biological activity of NOD2 receptor agonists.

Vaccines and vaccination

Immunogenic and protective properties of the recombinant human adenovirus serotype 5 expressing the glycoprotein G gene of the rabies virus vaccine strain RV-97


Introduction. Rabies is an acute infectious disease common to both humans and animals. To prevent the disease before contact with the pathogen, pre-exposure prophylactic vaccination (PrEP) of risk groups is used. The classic inactivated culture anti-rabies vaccines are used for immunization of risk groups in Russia. These vaccines are highly effective, but can cause severe adverse reactions and requires a multiple administration. Therefore, it is relevant to develop new safe rabies vaccines to achieve a protective effect with a single administration.

The aim of our study was to create replication-defective recombinant human adenovirus serotype 5, that carries the gene for glycoprotein G rabies virus, and to study its protective and immunogenic properties in immunizing laboratory mice.

Material and methods. Recombinant adenovirus was obtained using the kit AdEazy Adenoviral vector system (Stratagen, USA). To evaluate the protective properties and immunogenicity of Ad5-RV97 BALB/c mice were immunized once intramuscularly. The humoral immune response was studied using the fluorescent virus neutralization reaction, the T-cell response using the ELISPOT method.

Results. Recombinant adenovirus Ad5-RV97 was 100 % protection against rabies viruses. Within 14 days after immunization, high level of virus-neutralizing antibodies was reported, which continue to grow up until day 42 after immunization. Significant level of splenocytes (both CD4+- and CD8+-cells) producing IFN-γ in response to antigen stimulation was detected.

Conclusion. The data obtained allow us to consider Ad5-RV97 as the basis for creating a candidate rabies vaccine, which can be used to treat various risk groups.


Peripheral blood CD8+-lymphocytes populations and its value for direct and outlying treatment results in triple negative breast cancer patients


Introduction. Triple negative breast cancer (TN BC) is characterized by an aggressive course and a high likelihood of the disease recurrence. An important role in the regulation of the growth of malignant neoplasms, as well as in the effectiveness of various types of antitumor therapy, is played by the immune system. Establishing the relationship of some CD8-positive lymphocyte populations with the effectiveness of treating patients with breast cancer may help establish reliable immunological predictive and prognostic factors and increase the effectiveness of treatment for patients.

The aim of the study was to determine the value of the original number of CD8+ peripheral blood lymphocytes (CD8+, CD3+CD8+, CD8+CD28+, CD8+CD28, CD3CD8+) cells for immediate (degree of therapeutic tumor pathomorphosis) and distant (progression of disease and survival) of treatment results of patients with three-year negative breast cancer.

Material and methods. In patients with II–III stages of TN BC (n = 72, age median 50 years) before the start of neoadjuvant chemotherapy (NACT) (cisplatin + paclitaxel), the percentage of peripheral blood CD8+, CD3+CD8+, CD8+CD28+, CD8+CD28, CD3CD8+ lymphocytes was determined using a multi-parameter cytometric analysis. The relationship of the initial number of these cell populations with the degree of therapeutic pathomorphosis (TP), disease progression, and patient survival was studied. The observation period was 60 months.

Results. In patients with TP grade 4, in patients without disease progression and surviving patients, the percentage of CD8+CD28+-lymphocytes and the ratio of CD8+CD28/CD8+CD28 were statistically significantly higher before treatment than in patients with TP grade 1–3, recurrent patients and deceased patients. An increase of the initial number of CD8+CD28+-lymphocytes and the СD8+CD28/CD8+CD28 ratio was associated with an increase in relapse-free (RFS) and overall survival (OS) of patients compared with patients in whom these showings were reduced. In patients who died during the observation period, the number of CD3CD8+-lymphocytes was increased prior to chemotherapy. An increase in the number of these cells was also associated with a decrease in OS.

Conclusion. Populations of CD8+CD28+ peripheral blood lymphocytes and the ratio of CD8+CD28/CD8+CD28 cells could be possible predictive factors for patients with II–III stages of TNBC receiving NACT (cisplatin and paclitaxel).

Clinical immunology

The importance of serum amyloid A and C-reactive protein in predicting postoperative complications and exacerbation of tuberculosis after surgical intervention in children of older age


Introduction. The search for early laboratory markers of tuberculosis progression after surgical treatment in children and adolescents is an urgent problem.

Aim. Analysis of the level of production of markers of the acute phase of inflammation -serum amyloid A (SAA) and C-reactive protein (CRP) - before and after surgery in dynamics in order to predict postoperative complications and exacerbate the tuberculosis process.

Material and methods. The study was conducted on 28 patients who underwent various surgical interventions after different chemotherapy regimens. The 1st group included 22 patients who lacked complications and progression of tuberculosis. The 2nd group included 6 patients with postoperative signs of tuberculosis progression.

Results. SAA and CRP were examined before and one week and 1-6 months after surgery. In the 1st group, the SAA level before surgery was 13.11 ± 0.63 pg/ml, in the 2nd group - 14.86 ± 0.72 pg/ml and had no statistical differences. After 1 week after surgery in the 1st group patients, SAA level was significantly lower than in the 2nd group (12.3 ± 0.57 and 15.2 ± 0.6 pg/ml, respectively, p = 0.001747). Similar differences occurred after 1 month (13.57 ± 0.46 pg/ml and 18.5 ± 0.21 pg/ml, p = 0.0096). The CRP level had similar dynamics. One week and one month after the operation, the2nd group patients had a reliable rise of the CRP level (14.75 ± 1.4 mg/l and 18.8 ± 0.76 mg/l, p = 0.017581, and 12.8 ± 1.73 and 18.6 ± 1.1 mg/ml, p = 0.009067), respectively.

Conclusion. The simultaneous increase of CRP and SAA levels earlier after surgery can serve as prognostic criteria for postoperative complications (exudative pleurisy) or exacerbation of tuberculosis.

Clinical evaluation of B- and T-lymphocytes in patients with surgical sepsis


Introduction. Immune disorders in sepsis include a wide range of reactions from the «cytokine storm» to deep immunosuppression. The cellular reactions of the innate and adaptive immune systems are responsible for the changing scenario at different stages of the disease. There is very limited studies on aspects of the mechanisms of the immune response by adaptive immune response cells in sepsis. Important changes in the quantitative composition and the role of B- and T-lymphocytes in the regulation of inflammation at different stages of sepsis were noted. The question of the possibility of their use as biomarkers in septic conditions and criteria for the diagnosis of its phenotypes and endotypes remains open.

The aim. An expanded study of the cellular adaptive immunity in various clinical course options of sepsis in dynamics.

Material and methods. The data of a prospective study are presented in which the results of the state of cells of adaptive immunity of 43 patients were analyzed. Given the severity of the condition and the severity of organ failure, they were divided into groups on a scale of SOFA (Sepsis-related Organ Failure Assessments Score Sequential Organ Failure Assessment). Blood sampling was performed in patients in the first 48 hours after admission to the intensive care unit and on days 21-28 of the disease. Determination of the level of B- and T-lymphocytes (CD3+CD19+, CD3+CD19-, CD3+CD4+, CD3+CD8+), as well as subpopulations of T1 lymphocytes Th1 (CD3+CD4+IFNγ+), Th2 (CD3+CD4+IL-4+), Treg (CD3+CD4+CD25brightCD127negCD45+) was carried out by flow cytometry.

Results. Lymphopenia was noted on days 21-28 of the disease in the group with a more severe course of sepsis (p < 0.05), both in comparison with the comparison group and relative to the initial stage of observation. The level of CD19+-lymphocytes was reduced in the group with a more pronounced severity of organ dysfunction compared with the comparison group (p < 0.05) on days 21-28. In groups with varying degrees of organ dysfunction at the initial stage of the disease (the first 48 hours) there was no imbalance in the ratio of Th1 and Th2. At later stages, days 21-28, Th2 subpopulations of T-lymphocytes predominate (p < 0.05), and in the group with severe organ dysfunction, increased content of Treg (p < 0.01) was recorded.

Conclusion. Minimal changes were revealed on the part of adaptive immunity cells at the initial stages of sepsis, regardless of the severity of the course and the degree of organ dysfunction. At a later date (21-28 days) in patients of both groups (depending on the severity of organ dysfunction), a decrease in CD19+-lymphocytes, a predominance of Th2-subpopulation of T-lymphocytes over Th1 (p < 0.05), an increased content of Treg compared with the comparison group was revealed. One of the markers that can determine the onset of the immunosuppressive phase of sepsis may be the quantification of Treg.

Interconditionality of the effects of types I and II of inflammation on the processes of lipid peroxidation and antioxidant protection in patients with obstructive jaundice of gallstone origin in the postoperative period


Introduction. The pathogenesis of obstructive jaundice is complex, characterized by the development of local and systemic inflammatory reactions, endotoxemia and associated organ and histodestructive changes. Inflammatory activation of immune cells stimulates the transcription of genes responsible for oxidative stress, reactive oxygen species destroy the membranes of immunocompetent cells, prolonging local inflammation.

Aim of the study was to assess the contribution of immuno-inflammatory changes to lipid peroxidation processes and the functioning of antioxidant defense in the formation of postoperative complications in patients with obstructive jaundice of gallstone origin.

Material and methods. 70 patients with a clinical picture of obstructive jaundice were examined. The concentration of 6 cytokines was studied: type I inflammation (IL-2, IL-8, TNFα, IFN-γ); type II (IL-4, IL-10) by enzyme immunoassay. The content of malondialdehyde and the activity of antioxidant enzymes were studied in red blood cells by spectrophotometric methods. Analysis of statistical data was carried out using the Statistica 10 application programs.

Results. The predominant role in the pathogenesis of obstructive jaundice with a complicated postoperative period of type I inflammatory process has been demonstrated, and in the pathogenesis of obstructive jaundice with an uncomplicated postoperative period of type I and type II inflammation. A significant increase in the content of malondialdehyde in red blood cells was found in patients of both compared groups, which confirms the strengthening of lipid peroxidation in the early postoperative period. At the same time, the results revealed in our work, indicating an increase in the activity of antioxidant defense, an increase in the activity of superoxide dismutase and catalase enzymes, which contribute to a decrease in the manifestations of oxidative stress and are more likely indicative of the protective role of type II inflammation in OJ patients with uncomplicated postoperative period.

Conclusion. In patients with OJ with a complicated postoperative period, an increase in the processes of lipid peroxidation, a decrease in the activity of antioxidant enzymes and a predominant type I inflammation are revealed. In patients with OJ with an uncomplicated postoperative period, the manifestations of oxidative stress are compensated by an increase in the activity of antioxidant enzymes, types I and II of inflammation are detected.


Comparative analysis of modern methods for diagnostics of immediate allergic reactions


The effectiveness of treatment of allergic diseases (AD), most of which are associated with the development of immediate allergic reactions in patients (immediate-type allergy), is directly related to the diagnosis. For specific diagnostics of AD caused by the development of immediate-type allergy, both in vivo and in vitro tests are used, which are associated with the formation of an allergen-antibody (IgE) complex. Skin tests are easy to set up, the result of the study is known in 20 minutes. However, for skin tests, there are a number of contraindications, failure to comply with can provoke undesirable complications, including anaphylactic shock. Modern in vitro tests (REAST and allergochip) do not have contraindications for their conduct and allow to have more accurately determine the cause-significant allergen. They belong to the methods of molecular diagnostics (MD) because they use separate components of a natural allergenic complex, which includes several different antigenic determinants, which are divided into major and minor. The main (major) components of the allergen complex cause sensitization of 50 % or more patients, minor components of the allergen complex - in less than 10 % of patients. Using MD methods the mechanism of cross-allergic reactions to allergens to which the patient did not have primary sensitization was studied and cross-immunoreactivity maps were developed. MD methods are more expensive than skin tests, but their results can be used to evaluate the effectiveness of allergen specific immunotherapy (ASIT), which leads to a significant reduction in the cost of treatment. If patients are found to be sensitized to the main components of the allergenic complex, then ASIT will be effective, since the allergen preparations used for its implementation are water-salt extracts of only natural allergens, which contain significant amounts of the main allergenic components.


Periodontal diseases and atherosclerosis: microecological, metabolic and immunological mechanisms of interconnection


The review provides an analysis of the literature of recent decades on microecological, metabolic and immunological mechanisms of the relationship between periodontal diseases and atherosclerotic vascular lesion. The role of lipid metabolism and immune mechanisms, confirmed by modern ideas about the pathogenesis of atherosclerosis, is shown. The concept of the potential significance of infectious agents as components of the dental sulcus biofilm in the pathogenesis of atherosclerosis is discussed from the standpoint of recent data on the properties of persistent bacteria. Evidence of the participation of periodontal pathogenic bacteria in the formation of atherosclerotic plaques is presented and the mechanisms of such participation are considered. The analysis of modern literature which is devoted to the significance of periodontal diseases in the development of systemic pathology on the example of atherosclerosis has shown that one of the main binding components in this case is an etiological factor, including the ability of periodontopathogenic bacteria to influence lipid metabolism in tissues, enter into macrophages, persist in the bloodstream, participate in the formation of foam cells and atherosclerotic plaques, and influence the progression of the atherosclerotic process.

Epstein-Barr virus: vaccine development


Introduction. Vaccination is one of the most effective ways to reduce morbidity. Infection caused by the Epstein-Barr virus (EBV), in the absence of vaccination, is a serious health problem.

The aim of the systematic review was to study the problem of developing means of specific immunoprophylaxis of EBV infection.

Material and methods. The search was carried out in the databases eLibrary, Cyberleninka, The Cochrane Library, PubMed, and on the Researchgate website in accordance with the developed strategy, taking into account the criteria for inclusion and non-inclusion. The search depth was 20 years. The search period was 04.05.2020-19.05.2020. The final list of publications included 10 studies.

Results. Of the 10 selected articles, 7 displayed the results of candidate vaccines trials aimed at preventing EBV infection, and 3 described candidate vaccines for immunizing EBV-infected individuals for emergency prevention of infection or treatment of EBV-associated tumors. Clinical trials (phase 1) were conducted for two candidate vaccines, one of which subsequently passed phase 2. However, the results obtained did not allow further trials of this candidate vaccine.

Conclusion. None of the candidate vaccines had yet passed all phases of clinical trials.


Vladimir Alexandrovich KOZLOV

Musa R. Khaitov

Corresponding member of Russian Academy of Sciences, MD, Professor, Director of the NRC Institute of Immunology FMBA of Russia

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