Vol 13, No 5 (2023)

B-cell receptors can interact with antigen epitopes on various objects: macromolecules, microorganisms or on the surface of other cells, e.g., follicular dendritic cells. Accordingly, B cells, on the one hand, have the ability to evaluate the location of pathogen surface epitopes, and, on the other hand, they must adapt their receptor apparatus to different epitope locations and antigen-bearing surface properties. Indeed, B-cell receptors and antibodies better bind objects with regular and dense epitope arrangement characteristic of many pathogens. As a result, such epitope arrangement can be recognized as a pathogen-associated geometric pattern, but the conditions for such recognition depend on the isotype of membrane immunoglobulin and the degree of B cell maturity. Young B cells express membrane IgM, which is involved in B cell development and the selection of their repertoire. Receptors with IgM do not impose strict requirements on epitope location and can activate B cells even upon binding a monovalent antigen. Receptors with membrane IgD are expressed later and predominate on naive B cells before entering the immune response. These receptors are optimized for two-point antigen binding and strictly require this type of interaction to induce an activation signal. Before contact with antigen, B-cell receptors are grouped in discrete membrane zones — nanoclusters, due to close interactions with the actin cytoskeleton. Contact with the antigen leads to the detachment of receptors from the cytoskeleton, rise in their mobility and the combining nanoclusters into microclusters — large clusters enriched with signaling molecules. The most dynamic changes are observed upon contact with an antigen fixed on the membrane of adjacent cell. In this case, free actin moves to the periphery of the intercellular contact zone, where it forms the cytoskeleton of the processes carrying receptor clusters. The processes spread across the surface of the partner cell and then contract, moving the antigen-binding microclusters to the center of the contact zone. Finally, the microclusters combine into a central cluster of the immune synapse, the intensity of the activation signal drops, and the cell prepares for endocytosis of antigens grouped at the local site. Thus, the structure of B-cell receptors can contribute to the response of the B-lymphocyte to antigens with a characteristic spatial location, while the dynamic interaction between B-cell receptor apparatus and the cytoskeleton allows optimizing the binding of antigens presented on various carriers. Knowledge on spatial aspects of antigen recognition may be useful for the construction of vaccines based on virus-like particles or antigens on other artificial carriers.

A significant part of the complications of COVID-19 and manifestations of post-COVID syndrome is associated with autoimmune reactions caused by SARS-CoV-2. The key mechanism for enabling autoimmunity in COVID-19 results from molecular mimicry, which is involved in developing cytokine storm, systemic multiorgan hyperinflammation, endothelial dysfunction, also being a trigger for arising post-COVID-19 autoimmune diseases (autoimmune thrombocytopenia, autoimmune vasculitis, Guillain–Barré syndrome, Miller–Fisher syndrome, autoimmune neuropathy, autoimmune thyroiditis, rheumatoid arthritis, etc.). Overall, there have been identified 59 common immune determinants in 80 epitopes of the SARS-CoV-2 spike protein and 53 anti-inflammatory proteins, receptors regulating cell proliferation, differentiation and apoptosis as well as immune response. It was found that among the 37 viral proteins, only 8 of them bear no immunogenic regions identical to human proteins. Cross-reactivity results in emergence of more than 15 distinct types of autoantibodies including antiphospholipid antibodies against cardiolipin and beta-2-glycoprotein I, antibodies specific to transmembrane adenosine receptor A2b, adiponectin, phosphatidylserine-prothrombin, antinuclear antigens, mitochondrial M2, type I interferons, and other cytokines, chemokines, complement components and cell membrane proteins. Autoantibodies formed during COVID-19 react to antigens of cells located in the thyroid gland, cardiac and skeletal muscles, lung, joints, liver, kidneys, brain and bone marrow, peripheral nervous system, skin and adipose tissue, gastrointestinal tract, testicles, eyes as well as mitochondrial antigens, mediating development of severe disease-related complications and post-COVID syndrome. The presence of 24 homologous pentapeptides with those found in B. pertussis, C. diphtheriae, C. tetani, H. influenzae and N. meningitidis poses a risk of developing ineffective vaccination immune response paralleled with higher risk of autoimmune complications. It is imperative to take into account the phenomenon of molecular mimicry while proposing new approaches for rehabilitation and treatment of COVID-19 as well as in development and testing of vaccines against SARS-CoV-2.

Introduction. The SARS-CoV-2 coronavirus pandemic has been a major challenge for all areas of medical science, causing a surge of new developments in various fields ranging from diagnostic techniques to therapeutic and preventive approaches. Intranasal vaccination is an innovative approach to immunization against SARS-CoV-2, which has attracted the attention of many drug developers. Dynamics of blood virus-neutralizing antibodies (VNAs) in recovered COVID-19 patients or vaccinated healthy volunteers is one of the objective parameters for assessing vaccine immunological efficacy, which requires high standards of bioanalytical techniques within the framework of clinical trials. Immunogenicity data on the two-component Salnavak^® (intranasal) and Gam-COVID-Vac^® (intramuscular) vaccination obtained in randomized double-blind multicenter phase 3 clinical trial interim analysis are presented. The objective of the study was to assess immunogenicity of intranasal and intramuscular vaccination against COVID-19 using a neutralization reaction with pseudoviral particles and HEK293T-hACE2 cell culture. Materials and methods. A total of 137 healthy volunteers with a baseline anti-RBD IgG level not exceeding 100 BAU/ml received immunization by a two-component (Ad26 and Ad5 based) intranasal or intramuscular vaccine administered on day 1 and day 21. Immunogenicity level based on VNA quantitative analysis using a neutralization reaction with pseudoviral particles and a HEK293T-hACE2 cell culture as well as to SARS-CoV-2 S-protein receptor-binding domain (anti-RBD) IgG antibodies on days 21 and 42 after administration of component I was assessed. Results. The geometric mean VNA titer against SARS-CoV-2 on day 42 was 238.34±3.93 and 616.94±3.73 in the Salnavac^® and Gam-COVID-Vac^® groups, respectively. Trial data shows sufficient immunological efficacy of both intramuscular and intranasal vaccines based on a high protection level at VNA titer of more than 100 while using the pseudoviral neutralization method. The geometric mean of the anti-RBD IgG level by day 42 was 131.22±3.91 and 782.03±3.04 in the Salnavac^® and Gam-COVID-Vak^® groups, respectively. A direct moderate correlation was shown between VHA and anti-RBD IgG. Conclusion. Neutralization reaction using pseudoviral particles was successfully validated and used to determine the VNA titer during clinical trial. Trial interim data revealed that intranasal vaccine Salnavac^® vs intramuscular vaccine Gam-COVID-Vak^® resulted in lower but sufficient stringency of humoral immunity.

According to the WHO data, the number of infected people exceeded 765.2 million people during the COVID-19 pandemic. The severity of patient’s condition is determined by immune system hyperactivation. Activation of T- and B-lymphocyte subsets plays a prominent role in the control of infectious process. A content of small circular DNA molecules — T-cell receptor excision circles (TREC — T-cell receptor excision circles) and B-cell (“kappa”) excision rings (KREC — Kappa-deleting recombination excision circles) — in the peripheral blood can be used as a marker of the functionally active T and B cells maturation. The purpose of this work is to quantify peripheral blood TREC and KREC level in patients with the new coronavirus infection COVID-19 of varying severity. Materials and methods. The material consisted of 1028 blood samples from patients with a confirmed diagnosis of COVID-19, as well as 717 blood samples from apparently healthy volunteers. The content of TREC and KREC DNA fragments in the total DNA fraction was assessed by quantitative Real-time PCR using the “TREC/KREC-AMP PS” test system (St. Petersburg Pasteur Institute, Russia). Blood cell phenotyping was carried out using flow cytometry. Results. TREC/KREC levels were significantly reduced in COVID-19 patients (p < 0.0001 at 95% CI). A significant direct correlation was established between the levels of peripheral blood TREC molecules and level of CD45⁺CD3⁺CD19^– T cells (r = 0.59, p < 0.0001), as well as between KREC level and count of CD45⁺CD3^–CD19⁺ B cells (r = 0.66, p < 0.0001). The level of TREC molecules in patients with severe vs. moderate infection was significantly reduced in patients aged 30–39 years old (p = 0.0404) and 40–49 years old (p = 0.0356). The negative correlation between severity of COVID-19 clinical manifestations and TREC level in the blood of patients in 30–49 year age group indicates about an opportunity of using this analyte as a diagnostic and prognostic laboratory marker of patient’s condition. A simple PCR analysis algorithm makes it relevant to use the described method for assessing a state of immunity in coronavirus patients in the context of systemic negative impact of the SARS-CoV-2 virus on human organism.

The aim of the work is to assess biological properties of the enteroaggregative Escherichia coli (EAgEC) strain isolated from the colon mucosa biopsy material from a patient with ulcerative colitis, and to develop a new method for PCR identification of E. coli strains. 

Materials and methods. The study involved 46 patients with a verified ulcerative colitis. Isolation of 87 E. coli strains was carried out by using colon biopsy material obtained during a standard endoscopic procedure. The description of the biochemical bacterial properties was combined with the molecular genetic detection of virulence determinants and presence of antibiotic resistance mechanisms. Using the “AmpliSense^® Escherichiose — FL” reagent kit, a screening for the presence of diarrheagenic E. coli strains was performed, which revealed the presence of a single strain of EAgEC. The methodological approach to creating a new method for strain identification was based on the search for a unique genetic sequence within the glutamate decarboxylase (gad) gene.

 Results. E. coli 18-726 can be described biochemically typical to its species. The studied strain was characterized by a multiple resistance phenotype (MDR) to antibiotics, as well as a lack of sensitivity to five bacteriophages. The E. coli 18-726 strain had a unique sequence of the gene encoding the O-antigen, which differed from 188 known O-antigens (ONT) and, by the identity of the nucleotide sequence of the gene encoding the synthesis of the H-antigen, the strain belonged to the H30 serovar. Thus, the antigenic formula of strain EAgEC 18-726 was expressed as ONT:H30. The E. coli 18-726 strain contained a significant spectrum of genes that enable properties of virulence (iss, capU, aggA, aggB, aggC, aggD, aap, aar) and resistance to antibiotics (bla_CTX-M-15, bla_TEM-1B, aadA1, aadA5, mph(A), catB3). A new method for identifying EAgEC in chronic bowel disease is based on the polymerase chain reaction method using primers for a specific region of bacterial glutamate decarboxylase (gad) gene. Conclusion. 1) The biochemical, antigenic and molecular genetic properties of E. coli ONT:H30 18-726 strain (No. B-8857) isolated from a patient with histomorphologically diagnosed ulcerative colitis were analyzed. 2) A method for PCR identification of EAgEC strains based on the detection of a specific region within the bacterial genomic glutamate decarboxylase gene has been created and patented.

To identify the features of the immune system functioning in patients with chronic opisthorchiasis bearing mutations in loci associated with predisposition to developing osteoporosis, comprehensive studies of cellular and humoral arms were carried out. The state of the phagocytic system was assessed by assessing absorption, metabolic activity and reactive oxygen species formation to restore nitrosine tetrazolium (spontaneous and stimulated NBT test). The phenotype of lymphocytes was determined by flow cytometry. The humoral immune arm was evaluated by the number of immunoglobulin classes M, G, A and E. Differences in the functional state of various arms of the immune system were revealed. In patients with chronic opisthorchiasis in the presence of rs1544410 polymorphism of the gene encoding the intracellular vitamin D receptor, the relative number of T-helper cells is significantly lower than in the group with the normal allele. In the presence of rs1800012 polymorphism of the gene encoding the α1-chain of type I collagen, the absolute lymphocyte count is significantly higher, spontaneous and stimulated NBT test were lower, the number of DN-T lymphocytes is significantly lower (both in relative and absolute values). In the presence of the rs3736228 mutation of the gene encoding the transmembrane low-density lipoprotein receptor, the level of myeloperoxidase and the neutrophil stimulation index are lower, the absorption activity of neutrophils is higher. The presence of the rs2234693 mutation for estrogen receptor gene leads to significantly increased level of stimulated NBT test and IgG concentration. Thus, patients with chronic opisthorchiasis bearing mutations in the COL1 A1, LRP5, ESR1(rs2234693) genes, have altered both nonspecific innate reactions and parameters of the adaptive immune arm; mutation of the VDR gene solely affects adaptive immunity. Analysis of the results suggests that the presence of mutations associated with the development of osteoporosis has a modulating effect on the immune response in chronic opisthorchiasis invasion. The identification of polymorphic genes associated with metabolic disorders of bone tissue and the study of the immunological profile in patients with chronic opisthorchiasis invasion will allow to implement an individual approach in the treatment of such patients.

The study aimed to assess the effect of inhaled bacteriophage therapy on oral mucosal immunity in children with acute tonsillitis.

 Materials and methods. We examined 212 patients aged 4 to 15 years old with acute tonsillitis and 110 age-matched apparently healthy children. Research methods: calculating the Neutrophil to lymphocyte ratio (NLR), saliva diagnostics — secretory immunoglobulin A (sIgA) and the pro-inflammatory cytokine (TNFα). Taking into account the scheme of the treatment, the patients were divided into mutually comparable groups: the first group included patients with acute tonsillitis who received the standard generally accepted treatment depending on the clinical form of the pathology, without using bacteriophage therapy — n = 107 (50.5%), the second group — patients receiving a course of bacteriophage therapy — n = 105 (49.95%), nebulizer bacteriophage therapy using liquid complex pyobacteriophage (PCL) (Microgen, Russia) from the first days of the disease along with standard treatment. Results. During bacteriophage therapy, on the 6th day of treatment, an increased sIgA level up to 97.2% was observed particularly in younger and adolescent patients up to 97.2% (p ≤ 0.05). At the same time, this parameter reached 75.8% and 81.6%, respectively (p ≤ 0.05), in patients who received only standard treatment. The following difference between the two study groups was observed: between patients in the younger age subgroup — 21.4%, in the older age subgroup -16.1% (p ≤ 0.05 relative to control group), which indicates a more effective drug-related effect in patients from the younger age group groups. Similar changes are observed while analyzing level of the pro-inflammatory cytokine (TNFα).

 Conclusion. The use of inhaled bacteriophage therapy in the combination treatment of children with acute tonsillitis helps to shorten the period of general and local clinical manifestations of the disease by 1.4-fold and improve mean local immunity from 5.7% up to 16.1% (p ≤ 0.05).

Background. Numerous hemodialysis patients (HD) suffer from severe, life-threatening inflammation that must be treated to prevent further complications. Early diagnosis of inflammation in HD is highly needed. The present study used matrix metalloproteinase-1 (MMP3) and tissue inhibitor of metalloproteinases-1 (TIMP1) to differentiate between patients with/without inflammation using the neural network analysis (NN).

Methods. The positive results of C-reactive protein were used as a criterion for the presence of inflammation in the patients (HD+CRP) versus the negative group (HD-CRP). The NN analysis was used to discriminate between groups using the measured biomarkers.

Results. HD+CRP patients have a higher duration of disease, MMP3 and lower calcium than the HD-CRP level is significantly higher, while vitamin D is significantly lower in the HD+CRP group compared with both other groups (all p<0.05). TIMP1 is significantly correlated with inorganic phosphate and CRP. In NN#1, the model for the prediction of HD+CRP from HD-CRP has an area under the curve (AUC) of the receiver operating characteristic (ROC) of 0.907 with a sensitivity and specificity 89.2% and a specificity of 100.0%. The top predicting variable for the prediction of HD+CRP is MMP3 (100%), followed by creatinine (87.1%). MMP3 is linked to the pathophysiology of HD, at least through their correlation with the inflammation in HD. In NN#2, the AUC of the ROC for predicting the kidney disease and subsequent HD was 98.9%, with a sensitivity of 100.0% and a specificity of 97.1%. The top four predicting variables for the prediction of high risk of inflammation in HD patients are urea (100%), creatinine (100%), MMP3 (59.7%), and vitamin D (57.1%).

Conclusion. The NN analysis may differentiate between HD patients with inflammation from the HD without inflammation. Also, the measured parameters, especially MMP3, TIMP1, and vitamin D are useful as a diagnostic tools for the kidney diseases and inflammation linked with the disease.

The relevance of the research problem is justified by insufficient knowledge of the mechanisms for genetic regulation of inflammatory immune response during granuloma formation and arising inflammation in pulmonary sarcoidosis. It is believed that development of inflammatory process and formation of sarcoid granulomas occurs in subjects with genetically determined sensitivity to the effects of an unidentified etiological agent(s). The complexity of determining a causative factor(s) is accounted for by a variety of clinical forms and manifestations of the disease as well as a role for multifaceted immunological events in the pathogenesis of this disease. The process of inflammation, its intensity, may depend, among other issues, on host genetic background. Both enhanced and lowered production of pro-inflammatory factors can be observed in carriers of certain combinations of gene allelic variants. This, in turn, may determine human susceptibility to emergence of pulmonary sarcoidosis as well as alter clinical characteristics of the disease course and magnitude of developing immune inflammatory response. It should also be noted that the genetic background differs in various ethnic groups. Therefore, genetic background and environmental cues, ethnicity, may account for differed disease prevalence and phenotype. In this regard, it is relevant to search for allelic gene variations that could act as prognostic markers for development and progression of pulmonary sarcoidosis as well as characterize the features of its course. The data on the relationship between the carriage of allelic gene variations and susceptibility to pulmonary sarcoidosis as well as the contribution of IL1A rs1800857 polymorphic variant to development, progression, and therapy of the disease remain sparse and often contradictory. The current study assessed a risk of lung sarcoidosis in the subjects of Russian descent of the Republic of Karelia. According to the results of the studies, a significant association (p < 0.001) between the indicated IL1A gene allelic polymorphism and pulmonary sarcoidosis was established, with a risk of its development increasing by 3.47-fold (95% CI 2.41–5.01) in carriers of the T allele (p < 0.001). Thus, the single nucleotide polymorphism rs1800857 in the IL1A gene is associated with the risk of developing lung sarcoidosis in the subjects of Russian descent of the Republic of Karelia.

Introduction. In the Russian Federation, registration of individual COVID-19 cases started at the end of January 2020 that markedly increased in the second half of March. At the moment, the situation with COVID-19 is unstable. As of 2023, COVID-19 incidence in descending order is as follows: USA (104 958 987 people), India (44 684 775 people) and France (39 582 057 people). Russia ranked 10th regarding total COVID-19 incidence (22 137 084 people, including 395 727 fatalities). The infection is able to “overlap” with the underlying pathology, thereby worsening patient’s condition up to death. Lung multislice computed tomography (MSCT) is recommended for all patients with COVID-19. If unavailable, a chest X-ray is performed. The aim of the study was to identify the most frequent comorbid diseases and changes on radiological scan in patients with COVID-19. Materials and methods. The study was carried out at the Infectious Disease Department No. 1, Regional Public Health Institution “Clinical Hospital No. 1” Smolensk. For this, there were analyzed 69 hospital patient records with basic diagnosis — coronavirus infection. The data of anamnesis and additional instrumental investigations were taken into account. 

Results. 69 patients aged between 18 and 91 years were included in the study. Respiratory failure was not observed in 75.81% of cases. A group of patients (66.67%) with comorbidities of organs and systems was identified, among which most common were: arterial hypertension (95.65%), coronary heart disease (78.26%), diabetes mellitus (30.43%), grade 1–2 obesity (17.39%), chronic hepatitis (17.39%). Diseases of the gastrointestinal tract and respiratory system were less prevalent. The rate of reported complications is as follows: bilateral polysegmental pneumonia — 72.46%; unilateral polysegmental pneumonia — 2.8%; pleurisy — 4.35%; unilateral hydrothorax — 1.45%. Each of the inpatients underwent radiological examination during hospitalisation. X-ray examination accounted for a smaller proportion (39.13%) and multispiral chest computed tomography (MSCT) — for a bigger percentage (60.87%). Chest MSCT at the beginning of inpatient treatment visualized CT-2 stage (64.29%) in the vast majority, less — CT-1 (26.19%), least frequently — CT-3 (7.14%) and CT-4 (2.38%). Before patient discharge, an average of 12.16% improvement in the radiological picture was found in patients with initial CT-1 stage, 14% — in CT-2, 12% and 26% — in CT-3 and CT-4, respectively. Conclusion. The moderate disease course without respiratory depression prevailed among the patients. Co-morbidities were found mainly in the anamnesis of persons over 50 years old. The most frequent clinical complication of coronavirus infection was bilateral pneumonia. After patient’s hospitalization, clinical and instrumental picture gradually improved, which was confirmed by radiological data.

Introduction. In most cases, osteomyelitis is the monomicrobial disease caused by various Gram-positive bacteria. However, previous injury may contribute to formation of a microbial association. Presence of two or more pathogens in the infectious focus can markedly complicate the disease clinical picture. Kytococcus schroeteri is one of the rarest pathogens found in clinical samples. Slightly more than twenty described cases related to such infection have been recorded globally. The aim of the research was to analyse the first identified clinical case of post-traumatic osteomyelitis associated with Kytococcus schroeteri and Enterococcus faecalis. Materials and methods. The bacterial cultures were obtained from the wound discharge of the patient’s shin by the cultural method. Identification of the bacterial cultures was performed using a VITEK MS mass spectrometer followed by 16S rRNA gene sequencing. The morphological, tinctorial and biochemical features of the cultures obtained were established, and their antibacterial resistance was also determined. Results. The clinical case was associated with the diagnosis “Chronic post-traumatic osteomyelitis of the left shin”. Two types of microorganisms were isolated from the wound discharge, and identified as Kytococcus schroeteri and Enterococcus faecalis using the MALDI-ToF mass spectrometry method. 16S rRNA gene sequencing from the isolated bacterial cultures confirmed the MALDI-ToF mass spectrometry data. Antimicrobial susceptibility for this microorganism was determined according to the criteria for the staphylococcal group according to the clinical guide “Determination of sensitivity to antimicrobial drugs (rev. 2021)”. Conclusion. For the first time, the mixed infection caused by two Gram-positive bacteria K. schroeteri and E. faecalis has been described. Such associations can enhance the pathogenic effects of each other bacterium, which may contribute to transition of the infection to a chronic form with a low probability of positive cultural test. K. schroeteri is a representative of the normal skin microbiota, but this microorganism is able to cause various infections. The K. schroeteri species should be differentiated from other representatives of the order Micrococcales. At cultural examination, resistance to oxacillin is a suspicious sign indicating that the bacterial culture might be potentially assigned to K. schroeteri species. Due to the unavailability of current biochemical tests for differentiation K. schroeteri from related taxa, reliable identification of this species is recommended using MALDI-ToF mass spectrometry or 16S rRNA gene sequencing.