Vol 96, No 1 (2019)

Aim. Estimation of activity of native human serum and its antimicrobial peptides fraction against clinically important yeasts and comparison with the activity of some mammals sera. Materials and methods. Pooled samples of human, bovine, rabbit and mouse sera and collection strains of yeasts Candida albicans, Rhodotorula mucilaginosa, Malassezia furfur, Cryptococcus neoformans, Geotrichum candidum, Trichosporon cutaneum, Saccharomyces cerevisiae were used in the study. Antimicrobial peptides fractions (AMP) were obtained by filtration through molecular filters with 100 kDa pores. Activity of sera and their AMP-fractions were estimated by spectrophotometric method. Results. Activity of native mammal sera varied in diapason 73÷89% independently from yeast genus, although AMP-fractions activity varied more significantly. The minimal sensitivity to AMP-fractions of sera demonstrated M. furfur (activity values were equal 0÷13,5%) and G. candidum (0÷6,5%), but the maximal — R. mucilaginosa (12,3÷56,4%), C. albicans (22,0÷32,9%), and C. neoformans (17,1÷29,9%). Activity values of AMP-fractions of human serum were correlated meaningfully with no of the values of other mammals (Pirson coefficient r=0,459÷0,527). Considerable correlation of the indexes took place between rabbit and bovine sera (r=0,827), as well as between rabbit and mouse sera (r = 0,753). Conclusion. The differences between AMP-fractions activity towards studied yeast genera/specia indicate the occurrence of its specificity probably related with structural organization of cytoplasmic membrane of yeast cells as well as with variations in AMP composition in different mammals.

Aim. Comparative study of immunobiological properties of cell wall surface antigens and extracellular protein-containing antigens of Staphylococcus aureus. Materials and methods. Preparations: surface antigens of the cell wall (peptidoglycan, teichoic acids, protein antigens) of the strains of S. aureus containing in the staphylococcal vaccine «Staphylovac» (SV) and extracellular protein-containing antigens of S. aureus (EPCA). The parameters of innate immunity were evaluated by the effect of preparations on the immunophenotype of mononuclear leukocytes (ML) of the spleen of mice, expression of Toll-like receptors (using flow cytometry), phagocytic activity of macrophages of peritoneal exudate of mice after the introduction of SV and EPCA; the protective activity of preparations was studied in experiments of active protection of BALB/c mice. Results. To isolate EPCA, we used the virulent strain of S. aureus №6, and for obtaining surface antigens of the cell wall, 4 strains were selected, the most immunogenic of which was the low virulent strain of S. aureus №1991. Both preparations increased the number of TLR2 and MHC II expressing cells; CV administration caused an increase in the number of cells with CD25 marker, reflecting the early activation of immunocompetent cells, and EPCA immunization — led to a shorter expression of this marker. On the other hand, an increased number of CD19 positive cells were detected for a longer period of time during EPCA immunization. The longest activation of phagocytosis under the action of SV was established. High protective activity of bo-th types of the studied preparations was noted. Conclusion. The antigens of the cell wall and extracellular protein-containing experimental drugs possessed comparable immunological  properties, however, the surface antigens to a greater extent activate innate immunity, and extracellular — adaptive immunity.

Aim. Some viruses can subvert host defense mechanism, autophagy, to their own benefit. We analysed the effect of Rubella virus (RV) infection on autophagy in human alveolar epithelial cells A549. Materials and methods. Cells were infected with the wild type and lab-attenuated strain, C-77w and C-77a, respectively, with a multiplicity of infection of 1.0, in parallel, the expression level of genes encoding Beclin1, Atg5, Rab7, and p62 (SQSTM1) proteins participating in different steps of autolysosome formation was measured. To investigate the role of autophagy on RV replication cycle, we measured the amount of infectious RV particles, together with the viral RNA in supernatants and cell lysates, after incubation of A549 cells with wild type or attenuated strain in the presence of the autophagy inhibitor, Bafilomycin A1, or inducer, Rapamycin. Results. The significant increase in Beclin1 and Atg5 gene expression at 24-48 (for the wild type) and 24-72 (for the attenuated type) hours after infection was observed, while significant induction of either Rab7 or SQSTM1 gene expression was not noticed. This effect was correlated with more delayed increase of IFNβ expression and IFNβ-mediated pro-apoptotic gene expression leading to apoptotic cell death 72-96 hours after infection. Moreover, Bafilomycin A1 diminished the RV infection non significantly, as evidenced by the RT-qPCR and plaque assay, while Rapamycin increased the amount of infectious RV particles released by the infected cells more dramatically with wild type comparing with attenuated strain. Conclusion. Thus, we hypothesized that RV can use an antiviral mechanism to prevent degradation and ensure its replication, differentially regulating the process of autophagy, by stimulating the initiation and suppression of later steps.

Aim. Selection of optimal conditions for the cultivation of Bordetella pertussis biofilms and comparative assessment of the ability of different strains to form biofilms. Materials and methods. Used a vaccine strain of B. pertussis № 475 and selected from this strain strain № 475а, characterized by high virulence. Cultures of strains grown on dense nutrient medium (DNM) and liquid nutrient medium (LNM) were used as inoculates for biofilms production. The intensity of biofilms formation in round-bottom 96-well polystyrene plates was estimated by staining with 0,1% gentian-violet solution. Results. Daily cultures of the strain № 475 with LNM formed moderate biofilms in the range of doses 5 — 1,25 IOU (international optical units) /ml, in the absence of growth at lower doses. The daily cultures of this strain with the DNM formed a dense biofilms when planting a doses in the range from 10 to 1,25 IOU/ml, moderate from 0,625 to 0,157 IOU/ml and weak biofilms at a dose of 0,079 IOU/ml. Strain № 475а with the DNM formed dense biofilms in the doses range of 10 to 0,04 IOU/ml and only at the dose of 0,02 IOU/ml were formed moderate biofilms. Conclusion. A simple and informative method has been developed to evaluate the ability of B. pertussis strains to form biofilms in polystyrene plates. Cultures obtained with the DNM formed a more significant biofilms than cultures with LNM. Identified high ability to biofilms formation by a selected strain №. 475a, compared to the original strain № 475.

The aim of this work was to study modern manifestations of seasonal brucellosis among the population of the Republic of Tajikistan. The materials and methods: of the study were the official statistics, as well as the results of previously performed studies on the risk assessment of the incidence in population. A retrospective epidemiological analysis of annual incidence rate for the period from 1997 to 2016 was conducted, depending on the most significant social and environmental risk factors. Results. The article shows the impact on the annual incidence rate of socio-economic transformations, including the privatization of collective livestock farms, accompanied by the massive movement of farm animals to private ownership, the loss of practice in animal breeding, and changes in environmental and climatic conditions. Against the background of the intensification of sheep breeding, there is a tendency in seasonality smoothing and shifting the maximum levels of population incidence of brucellosis to the spring season. Conclusion. The modern features of seasonal manifestation of brucellosis infection in the Republic of Tajikistan make it necessary to apply a differentiated approach to planning and conducting preventive measures in various areas of the country.

We aimed to investigate immunological patterns of inflammation and autoimmunity, in bronchial asthma (BA) associated with obesity. Materials and methods. 109 people aged from 17 to 58 years with various body weights have been examined in total, including 64 individuals with allergic diseases as bronchial asthma (BA) and allergic rhinitis (AR). We performed the measurement of the body mass index according to WHO criteria, and evaluation of the asthma severity and comorbid conditions. In the samples of peripheral blood we measured biochemical tests (cholesterol and its fractions), spontaneous and PHA-induced production of cytokines: IL-4, IL-10, IL-17, TNF-, and serum levels of C-reactive protein (CRP), leptin, total IgE and IgE-autoAT, specific for a number of tissue AH (epithelial keratin, collagen 3 and 6 types, elastin and myosin). Results. Our study showed that in both groups of adults, the obesity was associated with increasing of acute phase proteins, CRP, leptin and TNF-α in serum, being most enhanced in asthma group. Individuals with excess body weight are characterized by significantly an increased level of acute phase proteins (Westergren ESR, CRP) and pro-inflammatory cytokines (TNF-α) in serum, thereby confirming the involvement of systemic inflammation in the obesity pathogenesis. The phenotype of BA with obesity is characterized by overproduction of CRP and leptin, along with increased spontaneous production of IL-4 and TNF-α, and also revealed sIgE to self-antigen as keratin, that in total could indicate more prominent inflammatory pathways with the impairment of immune regulation in this endotype of patients. Conclusion. The revealed associations confirm the link between obesity, as a chronic inflammatory condition, with atopy and development of asthma with further immune-mediated inflammation of the conduction airways.

This article presents the results of a study of the epidemiology of hepatitis E in the Russian Federation. Obtained data explaining the phenomenon, known as the Balayan paradox — the wide distribution of anamnestic antibodies to the hepatitis E virus in the absence of a registered incidence. It was shown that imported cases of infection are not able to support the epidemiological process of hepatitis E in Russia. The most cases of HEV infection are autochthonous in nature and are associated with zoonotic transmission of genotype 3 virus from pigs.

The aim of the study was to evaluate the diagnostic value of the method of loop-mediated isothermal amplification of DNA with real-time fluorescent detection (real-time LAMP, or RT-LAMP) on the model of carnivore parvoviruses. Materials and methods. Samples of feces, blood and swabs from the rectum of different species of predatory animals with parvovirus enteritis (n = 39) and healthy animals (n = 31), as well as laboratory strains of mink enteritis virus, were analyzed by RT-LAMP using SYTO-9 and SYTO-82 dyes. Real-time PCR was used as a reference method. Results. In our study, the LAMP method with real-time fluorescence detection (RT-LAMP) in the carnivore parvovirus enteritis model provides high analytical sensitivity (1.5×103 copies of DNA/ml), diagnostic sensitivity and specificity (up to 100% under optimal conditions). Comparison of the two intercalating dyes showed that the SYTO-82 dye provides a higher signal-to-background ratio (22.6 ± 2.1) than the SYTO-9 dye (6.3 ± 1.5) (p <0.0000001 ). At the same time, SYTO-9 dye at the sensitivity limit (10 copies of DNA) provides an increase in fluorescence in the reaction mixture 13 minutes earlier than for SYTO-82 (23 and 36 minutes, respectively). Conclusion. RT-LAMP is a promising method for rapid and highly sensitive «point-of-care» diagnosis of infectious diseases, as well as in conditions of livestock farms or in field conditions.

Аim. A study of CXCL12 effect on the migration of mononuclear cells isolated from healthy patients, from patients with myelomonoblastic leukemia before and after chemotherapy and the study of CCR4, EGFR and CXCL12 genes expression after exposure to CXCL12. Materials and methods. The chemotaxis of mononuclear cells (MNCs) of healthy donors and patients with myelomonoblastic leukemia was studied in a Boyden chamber, followed by isolation of RNA, reverse transcription and PCR-RV. Results. A significant increase in myelomonoblasic cell chemotaxis towards CXCL12 after chemotherapy was demonstrated, as well as a decrease in the expression of this chemokine in tumor cells before chemotherapy after exposure to CXCL12. Сonclusion. Presumably, the tumor cells themselves produce CXCL12 in large amounts, which is necessary for the disturbance of intercellular interactions and further intravasation, whose production may decrease with external stimulation by the same chemokine. CXCL12 also helps to increase the expression level of EGFR and CCR4, which leads to increased tumor proliferation and migration of tumor cells.

Aim. To study the immunobiological activity of the complex preparation for Haemophilus influenzae infections prevention. Materials and methods. We used the complex preparation, containing 1 mcg of lipooligosaccharide and 10 mcg of protein-containing fraction, lipooligosaccharide and protein-containing fraction monopreparations, and capsular polysaccharide preparation. For studying cross-protective activity of the complex preparation white outbread mouses (weight 14-16 g) were intramuscularly immunized with a dose 0,5 ml. 10 days later the animals were inoculated with H. influenzae encapsulated and non-typed strains culture inoculum in a dose 5*109 microbial cells/mouse. The immunized animals specific antibodies level was determined by means of indirect enzyme-linked immunosorbent assay (ELISA). For studying complex preparation’s influence on the development of the infectious process immunized mouses were intranasally inoculated with H. influenzae encapsulated and non-encapsulated strains. The mouses dissections were performed in 3, 24 and 72 hours after inoculation. Sterile samples (lung tissue) were homogenized, titrated and plated onto 5% horse blood agar; then after 18-20 growth hours we counted the number of colonies and recalculated its number for one mouse. Results. Our investigations have shown that the complex preparation provided 90-100% animals’ defence from used in this experiment infectious agent strains. Mouses immunization with the preparation induced significant increase in the level of antibodies, revealed by means of indirect ELISA. Beginning with the third day after intranasal inoculation there was a pathogen multiplica112 tion in control group mouses lungs. In the same time immunized mouses had almost indetectable number of bacteria. Almost all animals from control group contained pathogen in lymph nodes and mesentery; though pathogen’s presence in immune mouses viscera was rather occasional. Conclusion. The complex preparation protected animals from all H. influenzae strains, used in our experiment. The dynamic of the infectious process directly depended on the development of immune response on the complex preparation injection.