Academician R.M. Khaitov and development of postdiplomal education in immunology and allergology
Since the 1980s there has been an increase in interest of specialists in various fields of medicine in immunology as a science and clinical discipline. Neuroimmuneendocrine regulation of the processes of life support of the body is fundamental and recognized in the world. Education in immunology for students in our country began in 1971 under the guidance of Academician of RAS R.V. Petrov on the course of non-infectious immunology with the basics of immunogenetics. The first Immunology Chair of the II Moscow Medical Institute named after N.I. Pirogov (II MOLGMI) under the leadership of R.V. Petrov was founded in 1974. In 1982, the first textbook of immunology was published. New, modern textbooks on immunology and allergology were created under the guidance of Academician of RAS R.M. Khaitov, including his textbook «Immunology», which passed 4 editions. The logical continuation of the development of education in the field of immunology and allergology was the formation of a system of postgraduate education for medical professionals. One of the first chairs of clinical immunology and allergology for the training of physicians was established by R.M. Khaitov in 1990 at the Institute of Advanced Education of the FMBA of Russia. He headed this chair until 2017. From 2021 to the present, the corresponding member of RAS M.R. Khaitov is the head of the chair. Currently, the chair is a structural part of the Academy of Postgraduate Education of the Federal Scientific and Clinical Center for Specialized Medical Activities and Medical Technologies of the FMBA of Russia and is based in the «NRC Institute of Immunology» of the FMBA of Russia. Since the establishment of the chair of clinical immunology and allergology by R.M. Khaitov in 1990, a long way has been passed. Programs of training of specialists in residency, programs of professional retraining, advanced training in the specialty «Allergology and Immunology» have been improved, programs of primary and periodic accreditation have been developed.
Comparison of transcriptional profiles of human macrophages activated by agonists of NOD1 and TLR4 receptors
Introduction. Pattern-recognition receptor agonists are used to design immunostimulants and adjuvants that activate innate immunity to enforce desired protective effects. The choice of agonists most suitable in particular clinical situations should depend on knowledge of mechanisms of action of particular substances. To this end, detailed information about biological effects of particular agonists on immune cells is required.
Aim – to compare transcriptomes of human macrophages activated by NOD1 and TLR4 receptor agonists in vitro.
Material and methods. Macrophages were obtained by culturing healthy donor blood monocytes with granulocyte-macrophage colony-stimulating factor and stimulated by agonists of NOD1 or TLR4 for 1, 4 and 9 h. Transcriptomes were evaluated using next-generation RNA sequencing (RNA-seq) and real-time PCR (RT-PCR), followed by gene-set enrichment analysis and transcription factor binding sites analysis.
Results. Main difference between transcriptomes of macrophages activated through TLR4 and NOD1 was a stronger type I interferon response induced by the TLR4 agonist. NOD1 agonist activated several defense mechanisms in macrophages, including elevated expression of genes coding for antimicrobial proteins (aconitate decarboxylase 1, antimicrobial chemokines), inflammatory mediators and proteins required for Th17-differentiation.
Conclusion. Transcriptomic data allow recommendation of NOD1 agonists for prevention or treatment of infections caused by extracellular bacteria and fungi.
Dual effect of amino acid compositions upon antibacterial activity of human neutrophilic granulocytes
Introduction. It is known that amino acid compositions for parenteral nutrition exhibit an immunomodulatory effect on T and B lymphocytes, phagocytes.
The aim of the study was to evaluate the effect of amino acid compositions on the antibacterial functions of human peripheral blood neutrophilic granulocytes (neutrophils) under various experimental conditions.
Material and methods. Neutrophils were preincubated with amino acid compositions Amin or Vamin 14, then phorbol-myristate-acetate (PMA) was added and the respiratory burst of neutrophils was evaluated by flow cytometry. In another model neutrophils were incubated with bacteria (St. aureus) in a ratio of 1 : 10 or 10 : 1, washed, incubated with amino acid compositions, washed, lysed and sown on meat-peptone agar to account for colonies formed by surviving bacteria.
Results. Amin and Vamin 14 had a weak immunostimulating effect on the respiratory burst of neutrophils activated by PMA. The addition of amino acid compositions to neutrophils that phagocytized bacteria at a neutrophil/bacteria ratio of 1 : 10 led to an increase in the number of colonies formed by the surviving bacteria. With a neutrophil/bacteria ratio of 10 : 1, the studied amino acid compositions enhanced the bactericidal activity of neutrophils, causing a decrease in the number of surviving bacteria forming colonies. Direct addition of amino acid compositions enhanced colony formation by bacteria.
Conclusion. The amino acid compositions Amin and Vamin 14 enhance the growth of bacteria, including phagocytized by neutrophils at a cell/bacteria ratio of 1 : 10, but stimulate the bactericidal activity of neutrophils phagocytized St. aureus at a cell/bacteria ratio of 10 : 1, as well as in the respiratory burst induction test in neutrophils activated by PMA.
Humoral immune response to linear and conformational epitopes of SARS-CoV-2 in patients with COVID-19
Introduction. Coronavirus disease 2019 (COVID-19) is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread all over the world and changed our daily lives. Although SARS-CoV-2 does not have a high lethality compared to other viruses such as virus of atypical pneumonia or Ebola virus, it has nevertheless led to the development of a pandemic and a very high numbers of deaths from the infection in the last 20 years. Antibodies against structural proteins, primarily to the spike protein of the SARS-CoV-2, play a main role in the elimination of the virus from the body.
Aim – analysis of the humoral immune response of patients with COVID-19 at the level of IgG antibodies to linear and conformational epitopes of the main structural proteins of the SARS-CoV-2 –nucleocapsid (N) and spike (S) proteins.
Material and methods. Using Western blot and enzyme immunoassay, IgG binding from the sera of 153 patients diagnosed with COVID-19 and conducted a correlation analysis with demographic and clinical indicators.
Results. Direct correlations were found between the values of the coefficient of positivity (CP) of antibodies to the N protein and RBD and the age of the patient, the patient’s need for oxygen and arterial hypertension. We have shown that the greater the CP value of IgG antibodies to the RBD domain of the S protein the faster the recovery occurs, which confirms the role of the RBD domain as a target for the protective immune response.
Conclusion. The study showed that in order to understand the course of the COVID-19 disease, predict the development of a prolonged form in a patient, and possible complications, such as switching to artificial lung ventilation, it is necessary not only to qualitatively but also quantitatively analyze the values of the CP of IgG antibodies to conformational and linear epitopes of the N and S proteins of the SARS-CoV-2.
Impact of SARS-CoV-2-specific cellular and humoral immunity on survival in patients with COVID-19 for the first time
Introduction. The COVID-19 epidemic is the largest pandemic in modern history. To date, according to WHO, more than 670 million cases of COVID-19 infection have been officially registered, but, according to experts, the real number of COVID-19 cases is much higher. The prevalence of the pandemic led the most massive vaccination in history, and even after the start of vaccination campaigns, the spread of the disease has not radically decreased. Wide-range immunization of the population was chosen as one of the strategies to combat the pandemic; at the same time, conflicting data appear in the scientific literature on the effect of the presence of specific immunity on the course and outcome of the disease.
The aim of this study is the search of correlation between the presence of specific T cell-mediated and humoral immune response in COVID-naive patients and the outcome of the disease.
Material and methods. Using the ELISpot it was determined the presence of cellular immunity to the most important targets (S-, N-, M-proteins) of SARS-CoV-2 (delta strain) in previously virus-naive patients (n = 97). Qualitative determination of IgG to various epitopes of SARS-CoV-2 was performed using a multiplex assay. A subgroup with more stringent inclusion criteria was identified to minimize the impact of comorbidity and patient age on disease outcome (n = 40).
Results. On the 8th day of illness, 100 % of patients did not have T-cell immunity. Its formation was completed by the 17th day of the disease. The highest survival rate of patients was in groups with the presence of both specific humoral and T-cell immunity and in patients without both of the types of specific immunity (statistically not significant). The absence of comorbidity (obesity, diabetes, neurodegenerative diseases, oncology) and the young age of the patient did not significantly affect the result.
Conclusion. Based on the results of the study, we did not find a significant correlation between the presence of humoral or cellular immunity and the outcome of the disease in previously unimmunized patients, both in the general patient group and in the group of patients with additional inclusion criteria.
Severe asthma and COVID-19 in a real clinical practice
Introduction. Existing data about the mutual influence of COVID-19 and severe asthma are controversial. Little is known about effect of asthma treatment on risk and severity of COVID-19.
Aim – to assess frequency and severity of COVID-19 in patients with severe asthma on different treatment as well as the influence of COVID-19 on severe asthma in a retrospective study.
Material and methods. We recruited 99 adult outpatients [men 35 (35 %)/women 64 (65 %)] aged 18–81 years with severe asthma. They were retrospectively assessed during 2020–2022. 49 patients were treated by conventional therapy (ICS/LABA ± tiotropium, montelukast, OCS) only and 50 patients additionally received biologicals (оmalizumab – 7 pts, мepolizumab – 14 pts, вenralizumab – 12 pts, dupilumab – 17 pts). Data concerning COVID-19 and appropriate vaccination were collected during on-site visits or by phone and different messengers. Asthma control was assessed by using Russian version of ACQ-5.
Results. The use of biologicals in patients with severe asthma did not lead to more frequent incidence or more severe course of COVID-19. Hospitalization rate was lower in patients who received biologicals (24 %) compared to patients treated by conventional therapy (46 %, p < 0.05). COVID-19 led to worsening of asthma control in a half of patients with conventional therapy and in a third of patients with biologicals.
Conclusion. The use of biologicals added to conventional therapy in patients with severe asthma in a real clinical practice did not lead to more frequent incidence or more severe course of COVID-19. New coronavirus infection was associated with worsening of asthma control in some patients.
Changes in the expression of genes encoding ACE2 and innate immunity molecules (TLR7, IFN-α, IL-1β, TNF) in elderly patients with COVID-19
Introduction. The SARS-CoV-2 infection has placed a significant weight on the healthcare systems around the world. In this regard, the study of possible predictors of an unfavorable course and outcome of the disease, as well as the development of thrombotic complications, is an urgent and important task in the context of the ongoing circulation of SARS-CoV-2.
Aim. The aim of the work was to study the expression of genes encoding the ACE2, the innate immunity receptor TLR7, the cytokines IFN-α, IL-1β and TNF in peripheral blood leukocytes in elderly patients with COVID-19, as well as the association of the expression of these genes with the risk of thrombotic complications development, assessed by the level of D-dimer.
Material and methods. The study included 50 elderly patients (65–77 years old) with a confirmed diagnosis of COVID-19, the comparison group consisted of 15 persons (65–69 years old) without COVID-19. Gene expression was assessed by real-time PCR with reverse transcription in leukocytes obtained from peripheral blood.
Results. A significant decrease in ACE2 gene expression was shown in the main group compared to the group of healthy persons. Evaluation of innate immunity molecules showed an increase of the expression of the TLR7 and IFNA genes, as well as the TNF and IL1B genes, compared to the group of healthy persons. A special task of the study was to compare the tested parameters, taking into account procoagulant activity, assessed by the level of D-dimer.
The study showed that a high level of D-dimer (> 1 mg/l) in patients with COVID-19 is associated with a lower level of expression of the IFNA gene and an increase of the level of expression of pro-inflammatory cytokines genes TNF and IL1B compared with a subgroup with a low level of D-dimer (< 1 mg/l). In the subgroup with a low level of D-dimer, the expression of TNF and IL1B genes had no difference from the group of healthy persons.
Conclusion. An increase of the level of TLR7 gene expression combined with a decrease of IFNA expression in the group with a high level of D-dimer may indicate an imbalance in innate immunity factors. Elevated expression levels of the pro-inflammatory cytokines genes TNF and IL1B are associated with a high risk of thrombosis (as measured by D-dimer) and may be one of the markers of an unfavorable outcome of COVID-19.
Immunomodulating antitumor therapy causing false-positive results of newborn screening for a primary immunodeficiency
Introduction. Extended newborn screening will be enrolled in Russia in 2023 including screening for primary immunodeficiencies. The screening will elaborate excision recombinant rings (TREC/KREC) determination. Noteworthy, the TREC/KREC levels may be reduced to critical values not only due to the primary immunodeficiencies of genetic origin but also due to secondary conditions caused by external reasons.
Aim – analysis of the newborn’ TREC/KREC levels reduced to critical values cases revealed in course of an extended newborn screening pilot study. Search for the reasons of the reducing besides the primary immunodeficiencies.
Material and methods. The TREC/KREC levels were determined by real time PCR. The newborn dried blood samples were used obtained during newborn screening routine.
Results. Over 10 000 newborns underwent the screening form March 2021 to September 2022. No cases of true primary immunodeficiencies were observed. Three newborns were revealed showing subthreshold KREC levels. Mothers of all three newborns had undergone chemotherapy using rituximab. Rituximab is suspected to penetrate the placental barrier causing the fetal B cells number reduction. Cases similar to presented had been described. The newborns revealed in the present study are presumed to develop the B cell number reduction by the same mechanism.
Conclusion. Patients showing subthreshold KREC levels should be observed by a pediatrician and immunologist as well as comprehensive examination for serum immunoglobulins and WBC subpopulations. The patients’ TREC/KREC levels must be controlled dynamically.
Expression of NKG2D by cytotoxic T lymphocytes as a possible mechanism of hemorrhagic fever with renal syndrome immunopathogenesis
Introduction. Hemorrhagic fever with renal syndrome (HFRS) is an acute zoonotic viral infection, widespread in the Russian Federation. The disease is caused by hantaviruses, which have the ability to largely modulate immune response.
Aim – determination of the relationship between the phenotypes of the innate and adaptive immunity lymphocytes with their cytotoxic and immunoregulatory properties in HFRS at different stages and severity.
Material and methods. 65 patients with HFRS of moderate and severe course were under observation. Flow cytofluorimetry of the blood cells was performed during each period of the disease with the determination of the phenotypic characteristics of the innate and adaptive immunity lymphocytes. 15 healthy people made up the comparison group.
Results. It was found that the most informative deviations in the febrile period of HFRS from the indicators in the comparison group are subjected to the dynamics of two categories of cells. Cytotoxic T lymphocytes (CTLs) expressing such activation marker as the C-type lectin receptor NKG2D (CD3+CD8+CD314+), as well as regulatory T cells (Treg) of mainly CD3+CD8+FoxP3+ phenotype. In the oligouric period of HFRS, the predominance of the NKG2D-dependent rather than CD25-dependent activation mechanism in the CTL was noted. In the polyuric period, a significant increase of NKT number was recorded as well as a decrease of T cells number (mainly T helpers) and NKG2D+ NK cells.
Conclusion. The obtained results, in addition to applied value, allow us to propose a hypothesis on the pathogenetic value of the expression of NKG2D receptors by CTLs and the possibility of interrelation of this mechanism with the peculiarities of the processes of reprogramming of the CTL to NKT, the formation of Tregs, the development of immunological memory in HFRS.
Severe course of HAE and a new variant of the SERPING1 gene in a large Caucasian family
Introduction. Hereditary angioedema (HAE) is a primary immunodeficiency with a defect in the complement system, which can be accompanied by severe life-threatening clinical symptoms in the form of abdominal attacks and angioedema. This article presents details of the study, follow-up, and treatment of a large Karachai family in which 10 people had suspected type I HAE.
Aim – identification of a mutation in the SERPING1 gene encoding C1-INH in family members with identical symptoms.
Material and methods. Eight people with angioedema and abdominal attacks were included in the study. Direct Sanger sequencing was performed based on the complaints, examination, and tests of the family members’ blood samples (changes in C4 level, C1-INH concentration and functional activity of C1-INH). At the same time objective assessment of HAE symptoms severity was performed using validated questionnaires.
Results. For the first time a new variant c.1127_1130dup [p.(Val378Phefs*48)] in the heterozygous state was detected in exon 7 of the SERPING1 gene in all patients. It is cosegregating with deficient levels and activity of C1-INH.
Conclusion. The obtained data extend the understanding of new mutation variants in the gene encoding C1-INH (SERPING1) out of more than 700 already known mutations that can lead to a decrease in protein synthesis or its functional insufficiency. Identification of mutations in the SERPING1 gene can reduce the waiting period for a patient to be diagnosed with HAE.
Methodology for evaluation of the level of IgG antibodies against different SARS-CoV-2 proteins using multiplex immunofluorescence analysis
Introduction. The definition of patients’ serological status for SARS-CoV-2 is required for a clinical COVID-19 history evaluation and evaluation of epidemiological situation in general and also for development of population revaccination strategy. Though there are many different test systems for SARS-CoV-2 antibodies identifying, their sensitivity and specificity vary greatly. Test systems differ depending on used platform and antigen kit which make difficulties in the interpretation and comparison of results.
Aim – define diagnostic characteristics of MILLIPLEX® SARS-CoV-2 Antigen Panel 1 IgG test system for multiplex immunofluorescence assay of SARS-CoV-2 antibodies level on FlexMap 3D device (Luminex, USA) in blood serum samples of persons who have recovered from COVID-19 and those who were vaccinated against COVID-19.
Material and methods. The study includes 3 groups of participants: persons with history of COVID-19; persons without history of COVID-19 and unvaccinated; vaccinated ones. Identifying of the level of IgG antibodies against SARS-CoV-2 and virus neutralizing antibodies level was made for all samples using ELISA and multiplex immunofluorescence assay.
Results. In the course of this study a threshold value of IgG antibodies to SARS-CoV-2 S1 subunit of S protein (spike) was determined using multiplex immunofluorescence assay and it was 3267 MFI, also the sensitivity and accuracy of this method were assessed, which are 0.78 and 0.92 respectively. A linear regression model was developed for converting MFI values to BAU/ml values. Seroprotective level of antibodies to S1 subunit of SARS-CoV-2 S protein was determined and it was 5192 MFI.
Conclusion. Identifying of IgG antibodies using multiplex immunofluorescence assay has a high diagnostic sensitivity and specificity. This method allows to evaluate seroprotectiveness by level of antibodies to S1 subunit of SARS-CoV-2 S protein. Besides that, results could be compared to international units BAU/ml using formula:
m – 492,770 + 31,320 × b ⇔ b = – 15,733 + 0,032 × m,
where b is a BAU/ml value, m is an MFI value.