Current issue
1 . 2020
Hot points of immunology

Immune response against epidemic coronaviruses


In December 2019, an outbreak of severe acute respiratory infection COVID-19 (CoronaVirus Disease-19) caused by a novel coronavirus SARS-CoV-2 occurred in China. Since then, the virus has spread to nearly all countries and infected more than 1 million of people. World Health Organization has declared a pandemic because of the spread of the virus. The COVID-19 epidemic is a third one caused by coronaviruses, the first two being severe acute respiratory syndrome of 2002-2003 and Middle-East respiratory syndrome. In the present review, we briefly summarize information related to COVID-19. We review factors of innate and adaptive immune defense against epidemia-causing coronaviruses as well as mechanisms of viral evasion from the immune response. We also review potential mechanisms underlying the main feature of severe coronavirus infections, namely excudative pneumonia that, in a significant proportion of patients, develops into interstitial pneumonia and acute respiratory distress syndrome. Finally, we provide information concerning potential strategies of immunoprevention and immunotherapy of coronavirus infections.


Analysis of expression of immune system genes that are responsible for activation and inhibition of T-cell immune response in renal transplant recipients after extracorporeal photochemotherapy


Introduction. Due to the uprising need for kidney transplantation, there is a demand for new methods of preventing rejection of the transplant, based on the mechanisms of natural regulation of the immune response. One of the promising technologies in this area is extracorporeal photopheresis (ECP), based on electromagnetic influence on the recipient’s cells. The clinical efficacy of ECP has been confirmed by many studies, but the mechanism of therapeutic effect has not been sufficiently studied. Characterization of immune response during ECP is an important step towards formation of practical recommendations on clinical application of ECP and modification of immunosuppressive therapy.

The aim of this study was to analyze the changes in the expression level of the genes responsible for the activation and inhibition of the T-cell response in the recipients of the renal graft after ECP.

Material and methods. An open cohort randomized trial was conducted with 20 patients who underwent a single group cadaver kidney transplantation from an unrelated donor. The recipients of the compared groups received standard immunosuppressive therapy and the same therapy in combination with ECP procedures, according to the study protocol. The level of gene expression was analyzed by real-time RT-PCR. Comparison of the study groups was carried out in point 1 (1st-4th day) and point 2 (30th day).

Results. The study of organ function in recipients of paired kidney allograft who received standard immunosupressive therapy and therapy in combination with ECP was carried out. The analysis of clinical data did not show statistically significant clinical differences, but in the specific consideration of each pair of cases the tendency to some improvement of the transplant organ engraftment rate in the early terms after the transplant, better survival of the graft and the recipient was revealed. The level of expression of genes involved in immune response regulation (CD28, CTLA4, PDL1, FOXP3) and cytokine genes (TNFA, IL1, IL2, IL10, IFNG) in the studied groups was determined.

Conclusion. According to the results of comparative analysis the level of expression of pro-inflammatory cytokines' genes IL1, IL2 and IFNG decreased to 30th day after transplantation. A 30-day comparison between groups shows an increase in the expression level of the PDL1and FOXP3 genes (p = 0.0009 and 0.0013, correspondingly). Increased expression of these genes may be associated with the activation of regulatory T-cells and the development of peripheral tolerance.


Aerobic glycolysis is dispensable for pro-inflammatory cytokine production by dendritic cells


Introduction. Activation of innate immune cells is accompanied by an increase in glycolysis; however, the links between glycolysis and specific parameters of innate immune response have not been conclusively established.

Aims to study the relationship between glycolysis and proinflammatory cytokine production by dendritic cells (DCs) activated by lipopolysaccharide.

Material and methods. DCs were generated from blood monocytes of healthy donors and stimulated by LPS in the presence of different combinations of D-glucose, 2-deoxy-D-glucose (2-DG) and D-mannose. Cytokine production was analysed by enzyme-linked immunosorbent assay, cytokine gene expression by real-time PCR with reverse transcription, glycolysis by XFe96 real-time metabolism analyzer.

Results. We show that aerobic glycolysis is dispensable for pro-inflammatory cytokine production by human DCs in vitro. Upon activation with LPS, macrophages and DCs produce comparable amounts of the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6), despite the much lower rate of glycolysis in DCs. Inhibition of glycolysis by glucose starvation (culturing in glucose-free medium) does not significantly affect TNF, IL-6 and IL-12p70 production by LPS-activated DCs. At the same time, 2-deoxy-D-glucose (2-DG), a widely used glycolysis inhibitor, suppresses production of these cytokines by LPS-activated DCs and compromises their viability. Unlike glucose starvation, 2-DG induces an unfolded protein response (UPR). Cancellation of this UPR by an excess of D-mannose alleviates negative effects of 2-DG on DC cytokine production and viability.

Conclusion. Obtained data allow to partially revise current views on the role of glycolysis in the development of innate immune response.

Cellular immunology

The role of the mitochondrial pore in the effector functions of human neutrophils


Introduction. Neutrophils release neutrophil extracellular traps (NETs) in response to numerous pathogenic microbes in the inflammatory foci. However, excessive NETosis or diminished NET clearance is engaged in the pathogenesis of numerous autoimmune and inflammatory disorders. The initiation of NETosis after recognition of pathogens by specific receptors is mediated by an increase in the intracellular Ca2+ concentration, therefore, the use of Ca2+ ionophore A23187 can be considered as semi-physiological model of NETosis. Induction of NETosis by various stimuli depends on reactive oxygen species (ROS) produced by enzymatic complex NADPH oxidase, however, NETosis induced by Ca2+ ionophores was suggested to be mediated by ROS produced in mitochondria (mtROS) or without any ROS in general.

Material and methods. A wide inhibitor analysis has been used in our work, as well as luminol-amplified chemiluminescence assay and fluorescence detection of neutrophil extracellular traps.

Results. Using mitochondria-targeted antioxidant SkQ1, we confirmed that mtROS are involved in the oxidative burst and NET formation activated by A23187, but not a powerful activator of protein kinase C PMA. At the same time, using specific inhibitors of NADPH oxidase, we showed that ROS produced by this enzymatic complex are also involved in NETosis induced by A23187. Application of specific inhibitors of mitochondrial pore (mPTP), allowed us to demonstrate for the first time that mPTP is involved in signal transduction from mitochondria to NADPH oxidase, in mtROS production, NETosis and the oxidative burst induced byA23187.

Conclusion. We assume that the interplay between two sources of ROS, NADPH oxidase and mitochondria, is triggered by Ca2+ influx, so that mtROS contribute to NETosis either directly or by stimulating NADPH oxidase. One of the possible physiological mechanisms mediating Ca2+-dependent increase in mtROS production may be associated with the opening of the mitochondrial permeability transition pore mPTP, a multicomponent protein complex located in the mitochondrial membrane. Excessive NETs formation or a decrease in the organism’s ability to eliminate them can cause the development of autoimmune diseases. NETs are also involved in the development of lung diseases. Therefore, understanding the signaling pathways of NET formation is extremely important for finding ways to combat these diseases.

Mechanisms of allergic reactions

Asthma associated with nasal polyps: clinical characteristics and analysis of local expression of IL37 gene


Introduction. Asthma associated with chronic rhinosinusitis with nasal polyps (CRSwNP) is considered to be one of the most complex phenotypes of asthma, not sufficiently characterized in terms of etiopathogenesis. Conflicting data from numerous studies create difficulties in making recommendations for the diagnosis and treatment of this condition.

The aim of our study was to analyze the level of local expression of the IL37 gene and Th1-, Th2-, Th17-cytokines in patients with asthma associated with CRSwNP.

Material and methods. 48 volunteers were included and divided into 4 groups: «Healthy volunteers», «Atopic asthma + CRSwNP», «Not-atopic asthma + CRSwNP», «CRSwNP without asthma». All participants in the study underwent clinical, laboratory, instrumental, histological examination, assessment of local expression of IL37 gene, as well as IFNG, IL1B, IL4, IL5, IL13, IL17F in the obtained biopsy material (polypous tissue) using RT-PCR.

Results. Our results confirmed the mutual influence of asthma and CRSwNP, and according to the SNOT-22 and ACQ-7 questionnaires, a combination of these diseases led to a more severe and uncontrolled course of both asthma and CRSwNP. These results correlated with an increase in the absolute and relative number of eosinophils in peripheral blood (p = 0.05) and by eosinophilic cell infiltration of nasal polyp stroma in the not-atopic asthma associated CRSwNP. We also found that local dysregulation of the Th1-, Th2-, Th17-immune response underly the development of CRSwNP in patients with asthma. Moreover, the high expression of IL37 gene in patients with asthma associated with CRSwNP, and especially in patients with not-atopic asthma associated with CRSwNP, probably indicates the inclusion of the compensatory mechanism.

Conclusion. IL37 can be considered as the future molecular biomarker of the severity of inflammation in asthma and CRSwNP. Further study of the pathogenesis of asthma and CRSwNP will help to understand the mechanisms that bind these diseases and consider possible target molecules for biological therapy.

Clinical immunology

Defects of immune system function in immunocompromised children with congenital cleft lip and palate and recurrent respiratory infections


Introduction. Problem of comprehensive rehabilitation in children with congenital cleft lip and palate (CCLP) is actual, because this pathology has high frequency of occurrence and often has no possibility to conduct stage-by-stage surgical treatment in time in connection with the development of recurrent infections of the respiratory tract and oral cavity.

The aim of the study was to identify the characteristics of the functioning of the immune system in immunocompromised children with CCLP compared to immunocompromised children without congenital malformations of the face with recurrent respiratory infections (10 or more episodes per year) and exacerbations of chronic diseases of the oropharynx and upper respiratory tract.

Material and methods. 62 children of both sexes (aged 1-3 years) were monitored. 3 groups of children were formed. The 1st study group included 20 children (8 girls, 12 boys) with CCLP, suffering from recurrent acute viral infections of the respiratory tract, herpes-viral infection and different infection of oral cavity. All patients were at the stage of preparation for surgery to correct a soft palate defect. The study group 2 (without CCLP) included 22 children (11 girls and 11 boys) suffering from recurrent acute respiratory infections and herpes-viral infection. The comparison group consisted of 20 conditionally healthy children of the corresponding gender and age. A clinical, anamnestic and immunological study was conducted. The state of CD3+-, CD3+CD4+-, CD3+CD8+-T-lymphocytes subpopulations, CD3+CD4+/CD3+CD8+, CD3-CD19+-B-lymphocytes, CD3-CD16+CD56+-natural killer cells (NK) was studied by flow cytometry. Serum IgA, IgM, and IgG levels were determined using ELISA. The phagocytic and microbicidal functions of neutrophilic granulocytes (NG), as well as their spontaneous and induced oxidase activity, were studied.

Results. All patients in the 1st study group (with CCLP) were immunocompromised. All of them had clinical signs of immunocompromising: the frequency of recurrent acute respiratory viral infections, herpes-viral infections and oral infections were more than 10 times a year, they were complicated up to 6-8 times a year by bacterial infections, and therefore children received up to 6-8 courses of antibacterial therapy per year. It was revealed that children with CCLP have combined defects of immune system: deficiency of NK, reduced level of serum IgG and disorders of the NG system: neutropenia, deficiency of actively phagocytic cells, impaired production of reactive oxygen species. In study group 2 (children without CCLP), clinical signs of immunocompromising were also detected: the frequency of repeated acute respiratory viral and herpes-viral infections reached 10 or more times a year, and the frequency of bacterial complications was observed 2-3 times a year. Other disorders of the immune system were detected in this group of patients: the inability of the immune system to respond to infectious pathogens prevailed due to an inadequate response of innate and adaptive mechanisms of antimicrobial protection in combination with various defects in the functioning of the NG.

Conclusion. Combined secondary immune system defects have been identified in immunocompromised children with CCLP. At the same time in immunocompromised children without CCLP we observed another change in the immune system: the defective response of antimicrobial immune defense mechanisms - an inadequate immune response to infectious pathogens in combination with various disorders of the functioning of NG. The obtained data justify the need to develop new immunotherapy approaches aimed at correcting the detected defects in the functioning of the immune system in children with CCLP.


Development and validation of method for glucosaminylmuramyl dipeptide determining in aqueous solutions by high-performance liquid chromatography


Introduction. For the determination of peptide-based drugs the reverse phase HPLC method with a gradient elution mode is widely used. Among the published works no validated methods suitable for the study of new dosage forms of glucosaminyl muramyl dipeptide (GMDP) were found. Due to the significant drawbacks of gradient elution, it is recommended to avoid the use of a gradient when developing methods for HPLC analysis, preferring isocratic elution to it.

The aim of the study is the development and validation of a method for the quantitative determination of glucosaminyl muramyl dipeptide in aqueous solutions using the isocratic elution regimen, which can be used in the development of new dosage forms of GMDP.

Material and methods. Quantitative determination of GMDP in solutions was carried out by reverse phase HPLC in isocratic mode on a Mediterranea Sea18 15 x 0.4 cm, 5 pm column using an eluent of the composition acetonitrile: 25 mM phosphate buffer solution (3:97), pH 7.3; a flow rate of 0.7 ml/min; a detector wavelength of 200 nm; and a column thermostat temperature of 25 °C.

Results. The developed technique was validated by the following characteristics: specificity, linearity, correctness, repeatability (convergence), intermediate (laboratory) precision, analytical area, suitability of the chromatographic system. The technique has been successfully tested on laboratory samples of transdermal therapeutic systems containing GMDP.

Conclusion. A method has been developed for the quantitative determination of GMDP in aqueous solutions by reverse phase HPLC with isocratic elution. The suitability of the method is confirmed by the compliance of the main validation characteristics with the acceptance criteria. The confirmed analytical range of the method was 0.5-50 pg/ml. The technique can be used in the analytical part of preclinical studies of new dosage forms of GMDP.


Immunopathogenic properties of SARS-CoV-2 as a basis for the choice of pathogenetic therapy


In the context of the pandemic caused by the new SARS-CoV-2 coronavirus, due to the lack of population immunity and vaccines, strict epidemic measures are the only way to slow the spread of infection, but in the case of spread of COVID-19 in the absence of effective ethiotropic therapy - an adequate, full-fledged response of the immune system. Analysis of scientific data shows that the outcome of COVID-19 depends on the activity of non-specific immune response in the infectious-inflammatory process. It is assumed that with the disclosure of the immunopathogenesis of the disease, the tactics of treating patients, especially old patients with physiological aging of the immune system, may change. The possible use of a potential immunotropic drug for complex treatment of patients with COVID-19 is theoretically substantiated.


Vitaliy Yakovlevich ARION (1933–2020)

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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