Vol 90, No 4 (2013)

Aim. Study of plant extracts that have the ability to neutralize cytotoxic activity of hemolysin. Materials and methods. Preparations of purified and recombinant V. cholerae eltor hemolysin as well as supernatants of V. cholerae strains were used. Determination of cytotoxic activity of hemolysin and neutralizing activity of plant extracts were carried out by using cell cultures СНО-К1 and CaCo2. Results. Out of 9 water extracts only 3 - extracts of Rhei rhizome, Limonium gmeli-nii and Quercus robur neutralized hemolysin in cell culture CHO-K1 and CaCo2, whereas the other extracts - Humulus lupulus, Ocimum basilicum, Chelidonium majus, Juglans regia, Achillea milefolium and Hypericum perforatum did not have anti-cytotoxic effect. Neutralizing properties of extracts are exhibited during their co-incubation with hemolysin preparations and supernatants of V. cholerae strains already within 10 minutes. Conclusion. Plant extracts that have anti-cytotoxic activity against hemolysin are perspective for development of therapeutic-prophylaxis preparations.

Hemorrhagic fever with renal syndrome (HFRS) remains an actual natural-focal non-transmissible infection comprising more than 90% in structure of all the hemorrhagic fevers registered on the territory of Russian Federation. On the whole in multiyear dynamics of HFRS morbidity in Russia a pronounced tendency of growth with an average increase of morbidity of 1.7% per year that is caused by both improvement of clinical and laboratory diagnostics and objective increase of morbidity is noted. Currently a regulatory methodical base for organization of epidemiologic surveillance and prophylaxis of HFRS in Russian Federation is created.

Aim. Detect features of microflora of upper respiratory tract on the example of flora of palatine tonsils and level of antibodies against intracellular parasites as markers of etiologic factors of respiratory infections in school-age children in remission period. Materials and methods. 466 children from frequently and episodically ill groups were examined. Bacteriologic study of smears from the surface of palatine tonsils was carried out in all the children. By using EIA with the corresponding commercial test systems IgG level against Herpes simplex virus, Cytomegalovirus, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Human respiratory syncytial virus was determined in blood sera according to instruction manual. Results. During remission period of infectious process in the structure of microflora of upper respiratory tract in frequently ill children characteristic differences from their episodically ill peers were detected. In children with frequent respiratory infections a higher occurrence of antibodies against intracellular causative agents of these diseases was also detected. In the group of frequently ill, a direct correlation between frequency of infectious diseases of respiratory tract and occurrence of carriage of pathogenic and opportunistic microorganisms as well as increase of antibodies against Herpesviridae, Cytomegalovirus, C. pneumoniaе and M. pneumoniae was detected. Conclusion. Higher occurrence of intra- and extra-cellular infectious agents as well as their associations may be considered as one of the reasons of insufficient effectiveness of prophylaxis measures in frequently ill children.

Aim. Study reaction of cells of APUD-system of lymphoid organs and intestine of guinea pigs at the stages of morpho- and immunogenesis in response to administration of Yersinia pestis EV Research Institute of Epidemiology and Hygiene (RIEH) line vaccine strain. Materials and methods. Vaccine process was modeled in guinea pigs during subcutaneous infection of animals with culture of Yersinia pestis EV RIEH line vaccine strain. Pieces of thymus, spleen, lymphatic nodes and an area of duodenum were taken for histological study. The preparations were stained by solution of hematoxylin and eosin, impregnated with silver by Grimelius and Masson in Hamperl modification. Results. Dose-dependent character of effect of anti-plague vaccination on reaction of apudocytes in immunocompetent organs and duodenum biomodels was established, that indirectly reflects the direction of processes of immunogenesis in central and peripheral organs of immune system. Changes in quantity and morphofunctional state of apudocytes in the intestine of immunized guinea pigs gives evidence of the interest of this segment of APUD-system in the processes of immunogenesis during plague. Conclusion. Range of changes of quantity and functional state of apudocytes in lymphoid organs and intestine of animals during anti-plague vaccination was established. Quantitative parameters of morphofunctional state of intestine adipocytes that may be used for characterization of the intensity of adaptation-compensatory process in the organism of biomodels during modeling of any experimental vaccine process as well as an additional indirect indicator during evaluation of reactogenicity of live anti-plague vaccines were determined.

Aim. Study the effect of metabolites of H 2O 2-producing lactobacilli on enzymatic and bactericidal activity of lysozyme. Materials and methods. 9 H 2O 2-producing vaginal lactobacilli, Micrococcus luteus NCTC 2665, Escherichia coli State Institute of Standardization and Control № 240367, Lactobacillus acidophilus Institute of Cellular and Intracellular Symbiosis № 37 were used. Ability of lactobacilli to produce H 2O 2 was evaluated by oxidation of tetramethylbenzidine by peroxidase. Lysozyme was modified by mixing with equal volumes of lactobacilli metabolites, metabolites of H 2O 2-producing vaginal lactobacilli previously treated with catalase were used in control. Lysozyme enzymatic activity was determined by speed ofM. luteus lysis, bactericidal - by survivability of E. coli in Endo medium and L. acidophilus - in MRS medium. Results. Decrease of enzymatic activity of lysozyme due to its contact with H 2O 2-producing lactobacilli metabolites was detected. This effect is accompanied by growth of bactericidal activity of lysozyme against E. coli and decrease against L. acidophilus. The degree of changes of enzymatic and bactericidal activity of lysozyme by lactobacilli metabolites depended on concentration of hydrogen peroxide in them. Conclusion. Modification of lysozyme by H 2O 2-producing lactobacilli metabolites resulting in opposite changes of its activity against autochthonous and allchthonous bacteria is one of the mechanisms of formation of stable microbial biocenosis in human organism.

Aim. Study species composition and biological properties of microorganisms isolated from lower respiratory tract of 34 patients of surgical departments during mechanical lung ventilation. Materials and methods. 59 strains of microorganisms were isolated and identified by established methods from tracheobronchial secretion during intubation and extubation. Hemolytic and anti-lysozyme activity was determined in isolates by methods described previously (O.V. Bukharin, 1999). Results. During extubation an increase of seeding of tracheobronchial secretion in patients, increase of microorganism species diversity and frequency of occurrence of highly virulent strains characterized by the ability to hemolyze erythrocytes and high values of anti-lysozyme activity were established. Conclusion. The data obtained may be used to prognose the risk of development of diseases of respiratory system.

Aim. Choice of informative biomarkers for diagnostics of Mycobacterium tuberculosis infection and differential diagnostics of active tuberculosis (TB) of lungs and latent tuberculosis infection (LTBI). Materials and methods. 54 tuberculosis patients, 47 contact by TB individuals and 43 healthy donors were examined. All the individuals included into the study had QuantiFERON-TB Gold In-Tube (Cellestis, Аustralia) carried out. Values of spontaneous (NIL) and antigen-induced (AG) production of10 cytokines (EGF, MIP-1p, VEGF, IL-2, IL-4, IL-6, IL-1a, IFN-a2, TGFa, TNFa) as well as sIL-2Ra and sCD40L using XMap technology were measured. IP-10 level was also determined in 48 individuals by using EIA. Results. 6 out of13 biomarkers distinguished active TB and LTBI. As a result of construction of a decision tree in JMP 9.0 program 3 most significant markers were selected. Use of combination of IFNy Ao -NIL, TGFa NIL and IL-6 AG cytokines allowed to divide TB patients and contact individuals with sensitivity of 96.3% and specificity of 80.7% (AUC=0.9). We also observed very high levels of IP-10 and IL-2 that correlated with IFNyag -nil (r=0.71 and 0.79, respectively). Conclusion. IL-2 and IP-10 as well as IFNy may be used as helpful biomarkers as a first stage for diagnostics of (M.tb.) infection. At the second stage determination of IL-6, IFNy and TGFa for differential diagnostics of active TB and LTBI is proposed.

Data on antimicrobial peptides (AMP) of lactobacilli, mechanism of their damaging effect, chemical nature and genetic control are presented. Regardless of the source of isolation AMP of lactobacilli except reuterin are peptides with low molecular weight (4-6 kDa), differ from each other by chemical structure, sensitivity to temperature, effect of various enzymes, active at neutral or more frequently low pH. Especially important are mechanisms ensuring fine regulation of phenotypic expression of bacteriocin synthesis and formation of immunity against their effect. Activity and most importantly the level of their production depend on the conditions in which these bacteria are present and are controlled by a three-component regulation system. This system includes signal peptide (pheromone), sensory histidine kinase, regulator protein activating transcription. Resistance of the producer to the effect of its own bacteriocin is ensured by the so called immunity protein. AMP of lactobacilli are able to influence septoformation, peptidoglycan and protein synthesis, affect cytoplasmic membranes causing their destabilization. Stages of this damaging effect are described: interaction of effector peptides with the membrane of the sensitive cell, positioning of the peptide in the region of connection with protein receptor, submerging into the core of the spiral structure membranes with the formation of a pore and exhaustion of ATP pool leading to cell death. Protection from AMP is determined by specific proteins blocking pore formation in the membrane by direct binding of damaging molecules or their receptors. Perspectives of further studies of the role of antimicrobial peptides of lactobacilli are discussed.