Vol 23, No 4 (2020)

Influenza is a highly contagious respiratory disease widespread throughout the world that causes disease in humans, birds and many mammalian species. Annually, around 20% of the global human gets sick with influenza so that more than 500,000 people die its various complications. Secondary bacterial pneumonia poses the peak threat during influenza infection, being most frequently caused by S. pneumoniae. Multiple studies in humans confirm the negative impact of influenza virus infection on subsequent outcome of bacterial pneumonia and provides insight into increased morbidity and mortality due to complicated influenza infection. In particular, the last 2009 influenza pandemic caused by H1N1 virus revealed that 25-56% cases of severe disease forms were associated with secondary pneumonia, among which 14-46% of them were fatal. Based on the aforementioned, it is of high priority to investigate a role of influenza virus proteins in developing of pathogen synergism in viral-bacterial pneumonia, particularly influenza virus non-structural protein NS1. The study objective was to examine effects of NS1-specific antibodies on course of influenza infection and secondary bacterial pneumonia in mice. For this, we used an experimental model of sublethal influenza infection followed by secondary Streptococcus pneumoniae bacterial pneumonia. Influenza A/Puerto Rico/8/34 (H1N1) virus and S. pneumoniae No. 3405 strain were used to simulate influenza infection. Rabbit serum containing antibodies against recombinant NS1 protein from A/Puerto Rico/8/34 virus and native rabbit serum (contain no specific antibodies) were used for vaccination. The study was carried out with female BALB/c mice, weighing 20-22 g. Protective activity of animal serum was assessed by using the three criteria: infection-related mortality, life expectancy and body weight change. The data obtained showed that passive transfer of antibodies specific to influenza virus NS1 protein did not lowered viral replication in sublethal murine model of influenza infection. Subsequent secondary bacterial pneumonia induced by S. pneumoniae revealed no protective effect of anti-NS1 protein antibodies assessed by measuring survival rate, lung viral and bacterial titers in treated vs. control mice.

Disruption of neutrophilic granulocyte (NG) functioning underlies one of the key causes for negative polymicrobial synergism during virus-microbial co-infections. In connection with this, it is important to develop experimental models for viral-bacterial co-infections in vitro, which might allow to uncover NG involvement in effector events and assess reorganization of inter-connected functionally relevant NG receptors in response to various immunotropic agents. Understanding molecular mechanisms related to any molecule including drug molecules provides its safe use allowing them to become drug of choice. NG functional activity is associated with surface receptors CD64, CD32, CD16, CD11b, which are assigned to several NG subsets exhibiting distinct phenotypes, and their activation leads to complex processes of pathogen elimination. Study objective: to elucidate types of phenotype transition for NG subsets CD64^-CD32⁺CD16⁺CD11b⁺ and СD64⁺CD32⁺CD16⁺CD11b⁺ and assess an opportunity for phenotype reprogramming exposed to hexapeptide arginyl-alpha-aspartyl-lysyl-valyl-tyrosyl-arginine (НP) in experimental in vitro model of viral-bacterial coinfection. Materials and methods. We examined 39 samples of peripheral blood (PВ) collected from healthy adult volunteers (7 women, 6 men) aged 21 to 32 years, subdivided into 3 groups: comparison group 1 (intact NG); comparison group 2 – model of viral-bacterial infection; Study group – to assess HP effects. Model of viral-bacterial co-infection was created by incubating PВ samples sequentially with dsRNA (10^-7M) for 60 min followed by fMLP (10^-7M) for 60 min, 37 °С. To assess HP effect, PC samples preincubated with dsRNA and fMLP were next exposed to HP (10^-6 g/L) for 60 min at 37 °С. We analyzed percentage of CD64^-CD32⁺CD16⁺CD11b⁺ and СD64⁺CD32⁺CD16⁺CD11b⁺NG subsets as well as receptor expression density (MFI) by flow cytometry (FC 500, Beckman Coulter, USA) using MAbs (Beckman Coulter International SA, France). Results. Transformation of CD64^-CD32⁺CD16⁺CD11b⁺ and СD64⁺CD32⁺CD16⁺CD11b⁺NG subset phenotype was experimentally revealed in virus-bacterial model manifested as upregulated expression of all receptors examined. Our data on HP effects ambiguously demonstrated phenotype transformation in CD64^-CD32⁺CD16⁺CD11b⁺NG, СD64⁺CD32⁺CD16⁺CD11b⁺NG in in vitro viral – bacterial coinfection model contributing to recovery of proper NG response.

Antimicrobial activity of neutrophilic granulocytes (NG) is based on effective recognition and elimination of microbial pathogens, as well as on complex intracellular signal transduction pathways interconnecting these processes. NG dysfunction leads to emergence of atypical infectious and inflammatory diseases recalcitrant to standard interventions, which requires new vector platforms aimed at restoring normal NG functioning and overcoming antibiotic resistance. Moreover, we emphasize about special interest paid to the NOD1 and NOD2 agonist polymuramyl and NOD2 agonist glucosaminylmuramyldipeptide. Objective of the study was to compare effects triggered by NOD1 and NOD2 agonist polymuramyl and NOD2 agonist glucosaminylmuramyldipeptide on phenotype of neutrophilic granulocyte subsets CD64^- CD16⁺CD32⁺CD11b⁺, CD16⁺CD62L⁺CD63^-, CD16⁺CD62L⁺CD63⁺ in the in vitro system. 64 samples of peripheral blood (PC) collected from 8 apparently healthy children (4 boys and 4 girls) aged 3 to 9 years were examined by flow cytometry (FC 500, Beckman Coulter, USA) assessing NG surface receptors CD64, CD16, CD32, CD11b, CD62L, CD63 with MonAb (Beckman Coulter International S. A., France) by analyzing NG number (%) expressing receptors examined, density of receptor expression measured as mean fluorescence intensity (MFI). For this, there were assessed intact peripheral blood NG from apparently healthy children (comparison group) as well as those exposed to polymuramyl (PM) (at concentration of 10^-6 g/l) or glucosaminylmuramyl-dipeptide (GMDP) (at concentration of 10^-6 g/l) for 60 minutes at 37 °С temperature. Comparative analysis of surface receptor expression was performed on CD64^-CD16⁺CD32⁺CD11b⁺, CD16⁺CD62L⁺CD63^- and CD16⁺CD62L⁺CD63⁺ NG subsets that suggested about positive transformation of activation parameters in circulating NG exposed to NOD1 and NOD2 agonist polymuramyl as well as NOD2 agonist glucosaminylmuramyldipeptide. At the same time, similar effects of varying intensity were revealed manifested as increased count of NG subsets CD16⁺CD62L⁺CD63⁺ bearing increased level of CD16 and reduced CD62L expression, as well as differences uncovered as significantly increased expression of surface membrane molecules CD16 and CD11b in CD64^-CD16⁺CD32⁺CD11b⁺ NG subset from apparently healthy children exposed to polymuramyl as well as increased surface CD32 expression after incubation with GMDP.

Understanding changes in the cytokine-mediated mechanisms in immunopathogenesis of essential hypertension (EH) after COVID-19 poses a pressing scientific issue. SARS-CoV-2 exerts direct effects on macrophages with high probability altering regulatory M-CSF-VEGF-A-IL-34 axis, thereby accounting for change in cytokine-mediated patterns of hypertension progression. Immunopathogenesis of complications after SARS-CoV-2 infection and a role of M-CSF in EH pathogenesis justify study objective – to compare the serum M-CSF and VEGF-A, IL-34 levels in stage II EH patients prior to COVID-19 and one month after recovery to assess modality of altered M-CSF-mediated mechanisms behind hypertension progression. Four groups of patients were stratified depending on EH and clinical characteristics of COVID-19 (without/with pneumonia). Blood sampling was performed one month after COVID-19. The serum M-CSF and VEGF-A, IL-34 level was measured by using enzyme-linked immunosorbent assay. The data were statistically processed by using Stat Soft Statistica 13.5. Comparative analysis of serum M-CSF level in patients with stage II EH prior and after COVID-19 revealed that regardless of clinical course (with/without pneumonia) they were featured with higher levels of M-CSF one month after recovery (p < 0.001) vs baseline level. The serum VEGF-A level in patients with stage II EH did not change in papallel with increased M-CSF (458 pg/ml or more) one month after SARS CoV 2 infection. However, M-CSF stimulated rise in serum VEGF-A level and accounted for formation of marked coronary collateral network prior to infection. A relationship between the increased serum M-CSF level (higher than 392 pg/ml) and elevated percentage of COVID-19 with pneumonia in patients with stage II EH prior to the infection might be related to the hypothesis about “a role of dysregulated activation of mononuclear phagocytes in development of lung tissue damage”. The data presented prove scientific and clinical value of assessing a role for M-CSF with respect to altered cytokine-mediated patterns of EH progression after COVID-19 recovery.

Currently, new insights into the general mechanisms of developing allergic diseases suggest that Th17 cells are involved in the pathogenesis of allergic rhinitis characterized by dominant production of cytokines IL-17A and IL-17F. Objective: to assess IL17A rs2275913 gene polymorphism in the Uzbek ethnic group and its linkage with development of allergic rhinitis. There were examined 83 patients with allergic rhinitis, among which 38 (46%) had intermittent form and 45 (54%) – with persistent disease form. In control group there were included 123 apparently healthy subjects. Genotyping of IL17A rs2275913 polymorphism was carried out by using real-time polymerase chain reaction (Real-Time) “SNP-EXPRESS”-RV.
While analyzing frequency distribution of the G and A alleles within the IL17A gene, it was found that the A allele dominated (38.7% vs 19.5%, respectively, χ² = 15.9; p < 0.05). The G/G genotype in the A/G IL17A gene polymorphism was much less abundant in AR patients compared to apparently healthy patients in control group. It was detected increased frequency of heterozygous G/A allele within the IL17A gene in AR patients vs. control group (56.9% vs 30.2%, respectively, χ² = 11.9; p < 0.05; OR = 3.1). While comparing the A/A genotype of the IL17A gene it was shown that its prevalence was significantly higher in AR patients compared to healthy subjects (14.1% vs 5.9%, respectively, χ2 = 4.6; p < 0.05).
Our findings demonstrated that the AA genotype in the IL17A rs2275913 gene is associated with developing AR in the Uzbek ethnic group. Such trait may be used as AR predictor, provide valuable information for design and implementation of pathogenetically justified methodological approaches to therapy and prevention of allergic rhinitis.

Molds Alternaria spp., Cladosporium spp., Aspergillus spp., and Penicillium spp. play a major role in populational sensitization. Fungal spores may become a predisposing factor in developing bronchial asthma (BA), allergic rhinitis (AR) and atopic dermatitis (AD). The aim of our study was to investigate prevalence and clinical manifestation of fungal sensitization in the City of Samara. 2016 – 2019 general clinical and allergological examination of 855 patients of different age suspected for respiratory allergy was performed. All patients were tested for measuring serum level of total IgE and IgE specific to most common respiratory and food allergens, including fungal allergens derived from Alternaria spp., Cladosporium spp., Aspergillus spp., Penicillium spp., (RIDA Allergyscreen, R-Biopharm, Germany). 26 patients previously diagnosed positive to native Alternaria extracts and 50 patients with symptoms of exacerbated allergic rhinitis during summer and autumn were tested for serum level of IgE specific to recombinant molecular Alternaria component (rAlt a l, Phadia ImmunoCAP). It was found that 28.4% pediatric and 18.2 % adult patients (р = 0.0322). 25% of patients appeared to be sensitized to fungal allergens. Sensitization to various mold-derived allergens was distributed as follows: Alternaria allergens – in 23% all patients examined, (27% children and 14% adults); Cladosporium – in 11% patients (10% children and 8% adults); Aspergillus – in 5% patients (6% children and 2% adults); Penicillum – in 2% patients (1% children and 3% adults). Moreover, sensitization to Alternaria allergens prevailed in more than 90% patients with fungal sensitization that was more common in young individuals (up to 18% in 7-17-year-old group), but profoundly declined in subjects over 50 years of age (p = 0.00001, χ² = 61.31). True sensitization to Alternaria (Alt a 1) was detected in more than half (63%) of patients with confirmed sensitization to native Alternaria extracts, whereas 66% and 25% patients with fungal sensitization suffered from allergic rhinitis and asthma, respectively. Sensitization to Alternaria is a risk factor of developing asthma in childhood (OR = 2.415). The data obtained evidence about prevalence of fungal sensitization and associated allergic diseases in the Samara region.

It is considered that Ambrosia trifida is not widespread in Russia, thereby avoiding its threatening effects to populational health. However, there are some areas in Russia where more than half of land area is covered by Ambrosia trifida. Many such foci are found in the Samara, Orenburg regions, Tatarstan and Bashkiria. Ambrosia trifida is distributed in Central Russia, the North Caucasus region and Siberia.
The objective of the study was to examine specific IgE-profile in ragweed sensitized patients in the Samara region. A clinical and allergy examination of 969 patients with allergic rhinitis was performed. Allergic diagnostics was performed by using skin testing (prick-test) with standard set of pollen, household, and epidermal allergens. The data of skin tests in patients co-sensitized with ragweed and Artemisia allowed to analyze IgE-antibodies specific to the major Ambrosia (Amb a 1, Аmb. trifida) and Artemisia (Art v 1) allergens. Patients with negative skin test for the Amdrosia artemisiifolia and clinical manifestations of seasonal allergy were assessed for level of sIgE-antibodies nAmb a 1 and Amb trif.
Skin test data demonstrated that seasonal and combined forms of allergic rhinitis dominated in patients examined. In particular, positive skin test data for pollen allergens dominated (50.6%) among all allergen groups so that more than half of them belonged to weed pollen allergens. Patients challenged with skin tests for Artemisia and ragweed allergens more likely displayed positive reaction to the Artemisia allergen (71.27%) and Ambrosia artemisiifolia (50.88%).
Co-sensitization (to Artemisia and ragweed) prevailed in this geographic region featured with equal rate of simultaneously detected sIgE specific to the two types of ragweed and artemisia (nAmb a 1 + nArt v 1 + Amb trif) as well as Ambrosia trifida and Artemisia (Amb trif + nArt v 1). Almost half of the patients with negative Ambrosia art. prick-test were found to bear sIgE specific to Amb trif. Further investigation will help to better understand this phenomenon and take a fresh look at diagnostics and treatment of ragweed allergy in relevant geographic area.

Ultrasound examination of the spleen is a promising approach allowing to accurately determine its normal size in various age groups as well as in pathological conditions. Objective – to evaluate spleen size changes in patients with febrile state and infectious mononucleosis. Ultrasound morphometric examination of the spleen was carried out in patients with febrile state (group “A”, n = 22) and infectious mononucleosis (group “B” n = 24) by assessing organ echo structure, contour clarity, and its uniformity. Morphometric data allowed to measure spleen weight and various coefficients by using the method proposed by Vozgoment et al. for calculating spleen weight (Wt) with formula: Wt = 0.34 * L2h, where L – the length of the spleen, h – the thickness of the spleen (cm); spleen weight coefficient (SWC) by using the formula: 1000 m / body weight (in grams), where m – the mass of the spleen; spleen weight-to-body height ratio (KI) by using the formula: spleen weight (g) / height (cm); spleen weight-to-body surface area ratio (Ks) by using the formula: spleen weight (g) / body surface area (m²). Statistical analysis was performed by using Statistica 10.0 software package. Febrile state in most cases is not accompanied by changes in spleen size. However, decreased spleen size was found in one (4.5%) patient aged 58 years, whereas splenomegaly – in 2 (9%) cases (SWC range 5.5- 5.9) in young patients aged 18 and 25 years. The course of infectious mononucleosis in 25% cases was not accompanied by change in spleen size. In other cases, both decreased (20.8%) and increased (54.2%) spleen size were observed. In particular, decreased spleen size was detected in patients with average age of 43.6±14.5 years, whereas increased spleen size (SWC range 4-8.9) was typical for young people aged 25±8 years. Febrile state in most cases is not paralleled altered spleen size. Infectious mononucleosis was associated with the three types of altered spleen size: no change, decreased spleen size characteristic to older patients, or increased spleen size observed in young subjects. Ultrasound examination of the spleen with measuring its morphometric parameters represent objective criteria allowing to fully assess the state of this immune organ and reveal degree of its involvement in the pathological process at the preclinical level.

Human inborn immune-related errors comprise a heterogeneous group of rare genetically determined diseases of the immune system caused by loss or gain of function mutations altering relevant protein functions. The 2019 International Union of Immunological Societies recently proposed the classification for such pathologies now comprising 406 distinct disorders with 430 different gene defects. Predominantly antibody deficiencies represent most common group of human inborn immune-related errors, which diagnostics poses uneasy challenge for general practitioner due to a broad range of their clinical manifestations, such as infection, allergy, autoimmunity and malignancy. In addition, patients with human immune-related inborn errors may develop a vaccine-associated disease after administering live vaccines in accordance with the Russia-wide National Vaccine Schedule. Most common among vaccine-associated diseases are vaccine-associated paralytic poliomyelitis, vaccine-associated encephalitis (1 case per 1 000 000 doses of measles, rubella, varicella vaccine), vaccine-associated meningitis (1 case per 250 000 – 500 000 doses of mumps vaccine) as well as adverse effects related to BCG immunization: local (infiltration, cold abscess – 8.6 case per 100,000 vaccinated patients) and disseminated complications (BCG lymphadenitis – 15.5 case per 100 000 vaccinated patients, BCG osteitis – 3.5 case per 100 000 vaccinated patients). Vaccine-associated paralytic poliomyelitis in vaccinated patients occurs after the first, second and rarely third oral polio vaccine dose inoculation. Incidence rate for vaccineassociated paralytic polio after 1 and 3 oral vaccine inoculation ranges from 1 case per 700 000 vaccine doses to 1 case per 3 500 000, respectively. Vaccine-associated paralytic poliomyelitis mainly emerges due to inborn mutations related to humoral immunity after primary vaccination with oral polio vaccine or close contact of unvaccinated patients with subjects vaccinated with oral polio vaccine. Here, we describe a clinical case of vaccine-associated paralytic poliomyelitis in patient with primary immunodeficiency. Our is aimed at emphasizing importance of immunological alertness with regard to detecting primary immunodeficiencies and timely apply a replacement therapy prior to verifying type of immunodeficiency.

Acute kidney injury (AKI) developing after cardiac surgery remains unsolved issue despite the high level of surgical techniques as well as organ and tissue protection during artificial blood circulation. Various publications demonstrate that complications emerge in as many as 42% cases, whereas renal replacement therapy is required in up to 1-8% cases. Systemic inflammatory response syndrome and activation of the blood coagulation system largely underlie developing AKI finally resulting in acute renal failure. Release of large amounts of cytokines is associated with subclinical renal damage, primarily with blocking renal glomerular filtration. Measuring concentration of serum inflammatory markers that could reflect activity of inflammatory events is crucial for predicting and selecting treatment methods as well as for identifying predictors of severe course with opportunity of early onset renal replacement therapy after hospitalization. Objective of our study was to assess diagnostic and prognostic value of innate immunity parameters such as interleukin (IL) 6, 8, 10, 17, tumor necrosis factor alpha (TNFα), growth factor TGF-β1 in patients with acute kidney injury before and after CABG. Blood serum samples collected from 120 patients (males and females) with ischemic heart disease (IHD) were examined before and after CABG. It was found that patients with acute kidney injury had elevated serum level of cytokines IL-6, IL-8, IL-10, IL-17, TNFα, and TGF-β1 in all groups on day 1 and day 2 after surgery. Hence, we provided the data on more detailed investigation of immune alterations in cardiosurgical patients with AKI prepared for planned CABG.

Traumatic brain injury (TBI) is one of the most common type of injuries, so that its mild form prevails in overall injury pattern. Currently, it is known that brain injury triggers immune system response, but its role in translating into clinical manifestations, potential complications and sequelae remains poorly understood. It necessitates assessment of cellular immunity in patients with acute TBI of varying severity followed by investigating relationship between identified changes. It is now believed that immune system plays a lead role in brain functioning. It may be accounted for by interplay between peripheral immune cells and the brain, which may become augmented during developing immune response.
Here we quantitatively assessed composition of major peripheral blood helper T cell subsets in TBI patients by flow cytometry measuring percentage of central (CM, CD45RA^-CD62L⁺) and effector (EM, CD45RA^-CD62L-) memory Th cells. It was found that percentage of Th17 (CXCR5^-CXCR3^-CCR6⁺CCR4^-), DP Th17 (CXCR5^-CXCR3⁺CCR6⁺CCR4⁺) within CD3⁺CD4⁺T cell population were significantly increased (p < 0.05) compared to control group. Moreover, percentage of Th1/Th17 subset (CXCR5^-CXCR3⁺CCR6⁺CCR4^-) was significantly increased (p < 0.05) within EM and CM T cell subsets compared to control group. In addition, percentage of Th1 (CXCR5^-CXCR3⁺CCR6^-CCR4) was also significantly elevated in CD3⁺CD4⁺, EM and CM T cells compared to apparently healthy subjects. Hence, the data obtained allow to consider immune reactions among crucial arms in TBI pathogenesis related to concussion and its consequences. Thus, brain concussion affects cellular immune response triggering distortion in CD3⁺CD4⁺T cell composition as well as percentage of helper central and effector memory T cells.
Hence, the changes revealed in patients with acute brain concussion may predetermine disease course and developing long-term complications, which requires advancing therapeutic and rehabilitation protocols in such patients.