Vol 9, No 1 (2019)

Over the last two or three decades, a concept regarding functional capacities of neutrophilic granulocytes has been remarkably extended owing to new technologies. Neutrophil-related effector potential against infectious agents, including phagocytosis, production of reactive oxygen and nitrogen species, degranulation coupled with the released multiple enzymes and antimicrobial peptides, and the extracellular trap formation have been studied in detail. In particular, it was found that many of the factors used by neutrophils to directly destroy pathogens also exert regulatory effects on other immune cells as well as on neutrophils in an autocrine manner. In addition, activated neutrophils are able to de novo synthesize a range of biologically active molecules. Neutrophil-related immunoregulatory effects on macrophages, dendritic cells, T-lymphocytes and B-lymphocytes may be mediated both via direct intercellular contacts and indirectly through production of cytokines and other biologically active mediators. Ambiguous proactive and suppressive neutrophil-related effects on immune cells evidence about their important role played both in homeostasis and diverse pathologies, particularly in developing malignant tumors. Diverse, even opposing impact exhibited by neutrophilic granulocytes on immune cells and cells in non-lymphoid tissues, point at their functional plasticity and, probably, heterogeneity. Upon that, modality of effects elicited by neutrophils largely depends on surrounding microenvironment while they exit from the circulation. Widely considered as an inf lammatory response inducer, neutrophils are able to simultaneously trigger mechanisms facilitating restriction and resolution of inf lammatory reaction. Using intravital microscopy in animal models, it allowed to discover that neutrophils are able to re-enter circulation after exiting into the extravascular space, thereby challenging a classical concept about a unidirectional neutrophil migration from the blood vessels to body tissue. In addition, evidence that neutrophils can serve as antigen-presenting cells for T cells under certain conditions and are recruited from inf lammatory sites to draining lymph nodes were also obtained. Although a body of the data were obtained in vitro or in animal models, which therefore require to be further examined and verified, it may be unequivocally acknowledged that a neutrophil-related impact is not only limited to innate immunity.

Infectious diseases hold one of the most crucial places in healthcare. In Russia, annual prevalence of infectious diseases comprises around 50 million cases, wherein acute respiratory viral infections cover up to 90%. In non-influenza (non-peak) season, adenoviruses, respiratory syncytial virus, parainfluenza viruses etc. represent the main infectious cause of acute respiratory viral infections. Adenovirus-induced infections are characterized by heterogeneous manifestations poising them both as an interesting challenge, yet difficult in clinical diagnostics. Use of rapid, sensitive and specific tests is of high priority for routine clinical laboratory practice. In Russia, differential diagnostics of adenoviral infections includes a widely used ELISA and immunofluorescence analysis based on polyclonal specific sera. Importantly, a pattern and range of specific reactions depend on origin of animal-derived antibodies and their composition. Introduction of monoclonal antibodies specific to certain viral antigenic epitopes ensures high sensitivity and specificity allowing to reach an expected standardization level for such diagnostic products. Adenovirus hexon protein bears genus-specific antigens and relatively conserved amino acid sequence among diverse adenoviruses. Moreover, it is produced at high amount in infected cells and may be purified in a native form. Moreover, it is produced at high amount in infected cells and may be purified in a native form, thereby posing it as a promising antigen for producing monoclonal antibodies able to detect various adenoviruses types. A panel of adenovirus hexon-specific monoclonal antibodies was generated, which were further examined for biological and diagnostics properties. Western blotting data allowed to conclude that all of monoclonal antibodies generated by us were able to bind to adenovirus hexon oligomers. Specific activity of the new monoclonal antibodies against various adenovirus types was examined by ELISA and indirect immunofluorescence assay. In particular, monoclonal antibodies 4B7 and 6B12 were shown display top specific activity measured by ELISA (antibody titers comprised as high as 10^–6). Moreover, monoclonal antibody 6B12 exhibited peak hexon-specific activity assessed in indirect immunofluorescence assay (against various adenovirus types), which resulted in prominent granular nuclear fluorescence in cells infected with adenovirus types 3, 4, 6, and 19. Thus, the data obtained evidence that monoclonal antibodies developed 4B7 and 6B12 maybe potentially used for developing high-quality adenovirus-specific diagnostic assays.

Avian influenza A (H1N1) in humans is characterized by severe clinical manifestation and high mortality. The main drawback of current human H5N1 vaccines is related to low immunogenicity. Prime-boost vaccination is considered as an effective approach to enhance vaccine immunogenicity. The aim of this study was to compare immune response and protective efficacy of diverse prime-boost immunization protocols: 1) prime and boost with live influenza vaccine (LAIV) А/17/Turkey/Turkey/05/133 (H5N2); 2) prime with LAIV А/17/Turkey/Turkey/05/133 (H5N2) followed by boost with inactivated influenza vaccine (IIV) “Orniflu” (H5N1). Both vaccination protocols were found to increase serum antibody level against homologous and heterologous influenza A virus strains. In particular, serum HAI antibodies were significantly elevated solely after LAIV/LAIV vaccination. A more sensitive sandwich ELISA assay revealed that serum virus-specific IgG antibody levels were significantly increased after both vaccination protocols as well as after a single LAIV or IIV vaccination. Both LAIV and IIV boost increased titers of serum IgG specific against unrelated influenza A (H5N1) strains: homologous A/NIBRG-23 (clade 2.2), A/Indonesia (clade 2.1) and, to a lesser extent, against clade 1 virus A/ Vietnam and even against heterologous А/New York (H1N1). Single LAIV vaccination was also able to induce antibody responses against all strains examined, though to a lesser degree as compared with either prime-boost protocols. However, amount of splenic CD8+  Тcells specific to homologous influenza A virus strain was solely observed after LAIV/IIV vaccination. Moreover, both LAIV and IIV boosting effect demonstrated high protection level against lethal challenge with А (H1N1) WT virus and significantly decreased lung viral titer compared to control group. Furthermore, both regimens resulted in lung virus clearance after non-lethal challenge with clade 1, 2.1 or 2.2 influenza А (H5N1). In conclusion, we demonstrated that both LAIV/LAIV and LAIV/IIV regimens were able to induce cross-clade A (H5N1) response and that prime-boost immunization was a promising approach to improve immunogenicity of influenza A (H5N1) virus vaccine.

Here we present the data comparing maturation of peripheral blood mononuclear cell-derived dendritic cells (DCs) isolated from healthy volunteers and Staphylococcus aureus-positive patients with chronic osteomyelitis. Dendritic cells were cultured in a standard maturation cell medium (RPMI-1640,  supplemented with antibiotics, L-glutamine, 15% calf embryonic serum) added with interleukin-4 and granulocyte-macrophage colony-stimulating factor, followed by adding a stimulating factor cocktail containing interleukin-1β, tumor necrosis factor-α, interleukin-6, and prostaglandin E2. Dendritic cell maturation was analyzed by estimating visual characteristics under Zeiss ODSERVER.Z1 inverted microscope using Axiovision Rel.4.8 imaging software as well as light and phase-contrast microscopy at magnification of ×40, ×100, ×200, ×400, ×630. Dendritic cell immunophenotyping was carried out by using a panel of anti-human monoclonal antibodies: anti-CD80 FITC-conjugated, anti-CD86 (B7–2) PE-conjugated, anti-HLA-DR PC7-conjugated (all from Beckman Coulter, USA), anti-CD14 PerCP-Cy5.5-conjugated, anti-CD83 APC-conjugated, anti-CD40 PE-Cy7-conjugated (Becton Dickinson, USA) as well as isotype-matched control antibodies on the FACS Canto II f low cytometer (Becton Dickinson, USA). It was shown that while maturation dendritic cells derived both from patients or volunteers increased in size and underwent dendrite formation. Moreover, expression of CD86, CD83, CD80, and CD40 markers on dendritic cells derived from patients vs. volunteers was lowered. However, DC stimulation resulted in significantly increased percentage of DCs positive for CD83 DCs co-stimulation molecules CD86, CD80, CD40 chronic osteomyelitis. However, such differences found in immature DCs in both groups disappeared upon maturation, so that expression of the key markers on day 10 was maintained at close level. In particular, expression of CD83 differentiation marker and the CD80 co-stimulation molecule on DCs from patients vs. volunteers was increased stronger. Thus, a maturation potential in DCs isolated from patients with Staphylococcus aureus-caused chronic osteomyelitis was not impaired in vitro. The data obtained open up an opportunity to use dendritic cells as a natural adjuvant-substituting component for development of individualized vaccines in treatment and prevention of recurrent chronic osteomyelitis.

Here, we discuss development of f low cytometry technique for determining level of rabies antibodies in immune sera and immunoglobulin preparations, which is based on measuring fluorescence level in Vero cell line added with a mix of serially diluted anti-rabies serum or immunoglobulin preparation together with Moscow 3253 attenuated rabies virus strain, adapted for reproduction in cell lines. For this, rabies sera and immunoglobulin preparation were diluted 1:20 and 1:200 with PBS, respectively, whereas rabies virus was recommended for use at dose of 0.1 ID50/cell. Cell membrane fixation and permeabilization were performed by using a Cytoperm/Cytofix reagent (BD Pharmingen, USA) containing formaldehyde able to inactivate rabies virus, thus complying with biological safety regulations. Anti-rabies FITC-conjugated immunoglobulin (FGBI ARRIAH, Russia) was recommended for staining, that does not limit using similar reagents. Then, a f low cytometer equipped with a 20 mW argon laser (488 nm emission wavelength, throughput — 500 cells per second) was used for analysis. Vero cells displaying a fluorescence intensity exceeding 10 arbitrary units were considered infected. Flow cytometry allows to precisely measure amount of experimental infected cells as well as degree of infection by evaluating cell f luorescence intensity. Amount of antibodies in the samples examined by us was calculated by building a calibration curve based on depicting data for a standard rabies immunoglobulin with activity of 30 IU/ml (NIBSC, Potters Bar, United Kingdom). A high correlation between the data obtained by us and results from other detection methods used to assess activity of anti-rabies preparations such as biological neutralization of rabies virus in white mice (0.92) and modified FAVN test (0.98) was demonstrated. The comparison of the results of the analysis of the level of rabies antibodies obtained using f low cytometry, and the results obtained in tests in vivo, was carried out for the first time. Moreover, it is worth noting that for the first time the level of anti-rabies antibodies assessed by f low cytometry and in vivo tests was compared. An opportunity to perform rapid and easy-to-do analysis on assessing amount of infected cells by measuring fluorescence intensity in f low cytometry assay would allow to apply this approach for quality control while developing and manufacturing immunobiological anti-rabies preparations.

Oral mucosal lesions hold one of the lead places in the structure of dental diseases. Oral lichen planus (OLP) considered as a multifactorial disease is of top priority among dermatoses of oral mucosa and red lip border. Diverse putative concepts behind developing lichen planus pathogenesis are discussed including immunoallergic, viral, genetic and membrane-destructive theories. However, an immune theory is thought to play a crucial role in developing lichen planus. Despite documented induction of immune mechanisms, complex interaction between pathological process and normal defense response as well as stirred interest to it, multiple aspects of immunological conflict in lichen planus remain unclear. Few data describing altered immune parameters depending on clinical picture of lichen planus are currently available that suggested to perform the study aimed at examining immune status in patients with various forms of oral lichen planus. There were enrolled 286 oral lichen planus patients (248 females and 38 males), aged 27–84 years. Based on clinical picture, all OLP patients were divided into 6 groups: a typical type, exudative-congestive, erosive-ulcerous, hyperkeratotic, atypical, bullous type. An immune status in peripheral blood samples was evaluated by analyzing innate defense mechanisms as well as humoral and cellular immunity. Multiple altered immune parameters characterized by impaired phagocytic capture and metabolic activity, disimmunoglobulinemia, altered ratio of major immunocompetent cell types and subsets were documented during the study. Moreover, OLP patients with typical, hyperkeratotic, exudative hyperemic, atypical and erosive ulcerous forms were found to have increased amount of CD4+ helper T cells associated with a self-sustained immune response due to suppressed elimination of pathogenic agents consequently resulting in developing autoimmune process. While analyzing immune status in OLP patients, it allowed to find a relationship between dysphagocytosis signs, impaired humoral and cellular immunity as well as various clinical forms of the disease. Thus, it suggests imbalanced mechanisms responsible for pathogen elimination might play a role in OLP pathogenesis, including infectious agents being involved in its development.

Currently, it is believed that TTV and HGV display multifaceted activities in developing diverse acute and chronic processes (disorders affecting the liver, respiratory tract, hematopoiesis oncology diseases etc.). Transfusion of contaminated blood and its components contribute in transmission of HGV and TTV infections. In connection with this, examining a role for HGV and TTV in etiological structure of pediatric liver damage in acute lymphoblastic leukemia (ALL) as well as their relation to developing toxic liver damage against during ongoing therapy is of special interest. Our study was aimed at assessing hospital-acquired HGV and TTV infection and its potential effect on the incidence rate and liver damage intensity in children with acute lymphoblastic leukemia. A rationale for the current study was previously verified hepatitis of unknown etiology excluding viral hepatitis B and C in such patients. In the study, there were examined 99 patients stayed at the Department of Hematology as well as 286 samples from diverse donor bloodderived products. The data obtained were examined by using epidemiological methods (retrospective and near real-time epidemiological analysis, prospective observation), microbiological monitoring, ELISA, PCR followed by analyzing with variation statistics methods. It was found that 51 children, mainly aged 2–4 years (47.1%), were diagnosed with new onset acute lymphoblastic leukemia during a six-year follow-up study. Treatment phase-specific dynamic investigation demonstrated a progressive increase in frequency of HGV-infected ALL patients from 9.8% (day 15 of therapy) up to 45.1% (maintenance therapy). Moreover, a therapeutic intervention was associated with TTV infection detected in 100% cases in baseline TTV DNA-negative patients 100%, and its rate was significantly increased. Thus, our study allowed to demonstrate that 48% and 77% ALL pediatric patients were infected HGV and TTV, respectively, at initial treatment phase (remission induction), that was paralleled with administering the maximum-dose parenteral therapy, including transfusion therapy. Finally, assessing blood donor-derived preparations allowed to detect HGV RNA and TTV DNA in 6.6±1.46% and 19.3±2.9% cases, respectively.

Subject of study. Examining an impact of the contemporary climatic changes on the natural CCHF focus as well as CCHF incidence in the South of the European part of the Russian Federation. Materials and methods. Annual Epidemiological Report on CCHF Incidence provided by Departments of the Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing in the subjects of the Southern and the North-Caucasian Federal Districts, CCHF Epizootologic Monitoring Report on the territory of the Volgograd Region, Rostov Region and Stavropol Region were used in the study. Hydrometeorological data were obtained from the database of the All-Russian Research Institute of Hydrometeorological Information — World Data Center, weather station archives as well as The 2011—2016 National Report on the State of Environment in the Stavropol Region. Results. For the first time, climatic factors affecting each phase of the life cycle of ixodid tick Hyalomma marginatum which is the main CCHF virus vector in the South of the European part of the Russian Federation were identified. A direct correlation between seasonal air temperature fluctuations determining ixodid tick count and CCHF incidence was demonstrated. It was found that the average air temperature and accumulated precipitation collectively affect H. marginatum population (e.g., in Stavropol Region). Peak CCHF incidence was demonstrated to correlate with temperature conditions favorable to developing ixodid ticks. In contrast, air temperature fluctuations were noted to negatively impact in the years preceding decreased CCHF incidence. A relation between CCHF incidence and degree of wetting during preceding spring-and-summer as well as autumn seasons affecting vital activity, metamorphosis in preimaginal stages and count of the next-generation H. marginatum was revealed. Moreover, CCHF virus tended to expand geographic range northwards due to an effects of climatic changes. Virus-carrying Ixodidae found on the territory of the administrative districts of the Volgograd Region adjacent to the Volga Federal District evidence about a risk of CCHF virus spreading outside the Southern Regions of the Russian Federation.

Current study was aimed at investigating prevalence of overt and occult hepatitis B infection in HIV-positive individuals as well as molecular-epidemiological characteristics of the circulating hepatitis B virus (HBV) strains in the Far Eastern Federal District (FEFD). A total number of 297 blood serum/plasma samples obtained from HIV-positive patients residing in the FEFD were enrolled in the study. The first control group included 351 blood serum/plasma samples of general population without indication on HIV and HBV-infection that underwent laboratory check up at the Centers for AIDS Prevention and Control. After evaluating the group of HIV-positive patients 20 HIV-HBV positive samples were selected for further detailed analysis. The second control group included 43 patients with chronic hepatitis B. All groups were age and gender-matched. The research included serological and molecular-genetic (real-time PCR, positive for HBV DNA samples underwent clonal sequencing of PCR-amplified HBV P/S gene) assessment of the biological material followed by a phylogenetic analysis of the HBV sequences. Our research revealed that HIV-positive patients are exposed to a higher risk of HBV infection compared to general population enrolled in the study, which is evident from the prevalence of anti-HBcAg antibodies in the groups examined. HIV-positive vs. first control group was positive for anti-HBcAg antibodies at higher rate (35.02%, CI 95: 29.59–40.45% versus 22.22%, CI 95: 17.87–26.57%, p = 0.0003). Abundance of ongoing HBV-infection markers was also higher in HIV-positive individuals compared to general population (6.73%, CI 95: 3.88–9.58% versus 0.85%, CI 95: 0–1.81%, p = 0.0001, respectively). Should be noted, that HBsAg-negative HBV infection in HIV-positive patient cohort comprised 1.01% (CI 95: 0–2.15%) whereas in general population this index was as low as 0.28% (CI 95: 0–0.84%). Virus hepatitis С and D were revealed at higher rate in HIV-positive individuals compared to patients with HIV-negative chronic HBV infection (p = 5.84 × 10–7  and p = 0.000001 respectively). HCV and HDV prevalence rates comprised 50.0% (CI 95: 27.46–77.46%) and 40.0% (CI 95: 17.97–62.03%) in HIV-positive patients. Similar indices in control group were 4.65% (CI 95: 0–10.94%) and 4.65% (CI 95: 0–10.94%), respectively. The phylogenetic analysis of the six isolated HBV sequences showed that the five samples belonged to genotype D, with dominant subtype D2 (verified in 4 cases). Further, HBV genotype С was detected only in one case. The obtained data indicate a necessity for further in-depth diagnostic examination of viral hepatitis in HIV-positive patients to lower a risk of developing life-threatening complications as well as preventing hepatitis spread in human population.

A risk of introducing into the Russian Federation exotic infections including laboratory-confirmed melioidosis regularly recorded worldwide necessitates development and improvement of express diagnostics tools. Cross reactivity between phylogenetically related species of the genus Burkholderia complicates melioidosis diagnostics by express test methods based on using monoclonal antibodies against pathogen exopolysaccharide epitopes. Searching for target antigens to create the next generation group- and species-specific immunodiagnostic reagents for identifying Burkholderia pseudomallei is still of high priority. The study was aimed at cloning complete coding sequences for cell surface proteins differentiating  Burkholderia pseudomallei and optimizing recombinant antigens purification protocol. In silico comparative study allowed to select highly immunogenic B. pseudomallei outer membrane proteins Omp38 and OmpA/МotB as target biomolecules. For cloning, omp38 and ompA/motB gene-specific amplicons were obtained by PCR and ligated with the linear expression vector RIC-Ready pPAL7. Competent E. coli C-Max5α cells were transformed by a ligation mixture for producing recombinant plasmids, which were further purified to transform E. coli BL21 (DE3) cells for robust recombinant protein expression. Due to a potential multimeric protein structure, a standard protein purification protocol from native cell lysate was inefficient, which was modified to increase recombinant protein yield. However, by adding denaturing conditions at intermediate purification steps caused hydrolysis of peptide bonds in the target proteins, presumably between proline and asparagine residues. As a result, N-terminal fragments connecting recombinant proteins to the stationary phase of chromatographic column were eluted and evaluated for linear epitope detection according to their molecular weights. In silico analysis data identified highly antigenic motifs within the polypeptides studied. Thus, strains of E. coli BL21(DE3) BpsOmp39 and E. coli BL21(DE3) BpsOmpА engineered by us produce cell surface proteins Omp38 and ОmpA/МotB derived from melioidosis pathogen, which can be useful for developing diagnostic test systems.

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