Vol 95, No 5 (2018)

Aim. To study the subpopulational structure of blood cells of the experimental animals immunized with cellular walls (CW) of Francisella tularensis different subspecies. Materials and methods. Total 210 certified («Vector», Novosibirsk) outbred mice were immunized with CW preparations of F. tularensis different subspecies in 95 pg protein dose. After 3, 7, 10, 14 and 21 days of observation the phenotypes (CD45, CD3, CD4, CD8, CD19, CD69) of blood cells were detected. Results. Dynamics of leukocyte balance in experimental animals depended on F. tularensis subspecies. All studied preparations promoted development of cellular and humoral immunity that confirmed by the increase of monocytes and B-lymphocyte, and also by the expression of CD69 activation marker. CW of F. tularensis subsp. mediasiatica А-61 promoted higher activation of cellular immunity in experimental animals in comparison with other preparations. Probably, exactly this factor causes its high immunogenic activity (83 % survival rate). Conclusion. On the basis of the results there is a necessity to further detailed research of CW immunogenic properties of F. tularensis subsp. mediasiatica А-61 as a perspective component in design of a tularemia vaccine.

Aim. Of the study was to specify the factors responsible for outbreaks of domestic cluster HFRS morbidity during winter period in the Republic of Bashkortostan as the basis for preventive measures substantiation. Materials and methods. Analyzed have been the data from the Rospotrebnadzor Administration in the Republic of Bashkortostan (RB) on the HFRS incidence in 54 administrative districts of RB during winter season 2016-2017. Summarized have been the results of epidemic foci eradication with domestic HFRS morbidity during winter months of 2010-2017 in the territory of RB. Results. Winter sporadic HFRS incidence is registered annually among the rural and urban population residing in steppe, forest-steppe and forest zones of RB. It is outlined that epidemic foci with domestic cluster HFRS morbidity emerge more frequently in the territory of forest-steppe zone. It is established that HFRS infections of domestic type take place in residential areas adjacent to some forestry to one extent or another, as well as on visiting country, out-of-town objects situated in the territory of active natural HFRS foci. Thereat, the risks of domestic HFRS infection are entirely defined by the sanitary-hygienic state of accommodation and manufacturing facilities. Conclusion. The main tactical option for the prevention of epidemic foci emergence with domestic cluster HFRS morbidity among rural and urban population of RB is simultaneous barrier and semi-rural deratization and disinfection in areas of high risk of infection.

Aim. Production of monoclonal antibodies (mAb) to synthetic tetrasaccharide - repeating unit of the capsular polysaccharide (CP) of Streptococcus pneumoniae serotype 14 and their immunochemical characterization. Materials and methods. In order to generate the hybridoma producing mAb, mice were immunized with synthetic tetrasaccharide conjugated with bovine serum albumin (BSA) with following hybridization of B lymphocytes with mouse myeloma cells. Antibodies were obtained in vitro andin vivo. Immunochemical characterization of mAb to tetrasaccharide was carried out using a variety of ELISA options. Results. For the first time obtained mouse hybridoma, producing IgM to tetrasacchride. The IgM titer of anti-tetrasacharide antibodies in supernatants of clones and in the ascitic fluid of mice in ELISA detected by biotinylated tetrasaccharide and synthetic CP adsorbed on the solid phase was higher compared to the use of bacterial CP as well cover antigen. In the reaction of inhibition of the ELISA, the mAb recognized the corresponding carbohydrate epitopes of the bacterial CP of S. pneumoniae serotype 14 dissolved in the liquid phase better than tetrasaccharide ligand and synthetic CP. Conclusion. To detect mAb to tetrasaccharide in ELISA preferably to use synthetic analogues of the CP as solid phase antigens. The obtained mAb to tetrasaccharide can be used to determine the representation of the protective tetrasaccharide epitope of CP in the development of pneumococcal vaccines.

Aim. Optimization of the accelerated whooping cough method of isothermal amplification for DNA Bordetela pertussis. Materials and methods. The research was conducted on 35 standard collection strains and 169 strains of Bordetella allocated in bacteriological laboratories of territorial subjects of the Russian Federation. The research included 329 clinical samples received from patients with whooping cough and the persons, contact with them, hospitalized in IDCH No. 1 DZM. Chromosomal DNA was extracted with a standard method of boiling from strains, from clinical samples by means of commercial sets. Identification of causative agents of whooping cough were performed with use of the АмплиСенс® Bordetella multi-FL. Results. We performed optimization method of a diagnostics of whooping cough by LAMP with detection by means of an electrophoresis and with naked-eye inspection under normal light is developed. The developed method allows to detect a DNA of B.pertussis within 4 - 5 hours in clinical material. The analytical sensitivity was 10² GE/ml. Assessment of validity showed that the developed method possesses 99,6% sensitivity and 98,7% specificity; predictive value positiveness and negative result was 99,6% and 98,7%, respectively; the index of accuracy (diagnostic efficiency) - 99,4%; the likelihood ratio of positive and negative result - 76,6 and 0,004, respectively. Assessment of analytical reliability in 100% of cases showed convergence and reproducibility of a technique. Conclusion. Diagnostic test on DNA of B.pertussis identification by LAMP method will allow to increase efficiency of laboratory diagnosis of whooping cough.

Aim. We aimed to estimate the composition and the detection frequency of bacterial species in induced sputum samples from patients with bronchial asthma (BA), chronic obstructive lung disease (COPD) and its combined phenotype (ACOS). Materials and methods. Bacteriological examination of samples of induced sputum in patients with chronic obstructive pulmonary diseases (BA, COPD) was carried out. Results. Patients with asthma-COPD overlap syndrome exhibit more diverse bacterial species composition as represented both by gram-positive Streptococcus sрp., Staphylococcus spр., gram-negative Klebsiella pneumoniaе, Escherichia coli, Serratia marcescens, Pseudomonas aeruginosa, Haemophilus influenzae, Burkholderia cepacia and rodlike bacterium Corynebacterium spр., Actinomyces spр. и Tsukamurella рaurometabola as compared to patients with only one diagnosis of COPD or asthma. In addition, we revealed the differences between microbiological diversity and predominance of Streptococcus spр, Neisseria subflava with decrease of Enterococcus sрр. in samples from patients with complicated forms of obstructive lung diseases as COPD and ACOS, with pulmonary emphysema and/or pneumosclerosis. Conclusion. The biodiversity of lung microbiome could be one of the pathology risk factors in patients with chronic lung diseases, on the other hand reflecting the structural morphological changes in the lung tissue as a result of sustainable inflammation.

Aim. Study the functional properties of cationic antimicrobial peptides 0-defensins isolated from baboon Papio hamadryas blood leukocytes. Materials and methods. Baboon 0-defensins were extracted from leukocyte mass using ultrafiltration, preparative electrophoresis and reverse phase high performance liquid chromatography. The test microorganisms used were Gram-negative bacteria Escherichia coli, Gram-positive bacteria Listeria monocytogenes and Staphylococcus aureus, and fungi Candida albicans. Minimal inhibitory concentrations (MICs) were determined by testing serial dilutions of the test peptides by radial diffusion in agarose gel. Microbicidal action was evaluated by counting surviving colony forming units after incubation microorganisms with the peptides. The 0-defensins influence on E.coli membrane permeability was assessed using chromogenic markers o-nirofenil- β-D-galactopyranoside and nitrocefin. Results. The analysis of the 0-defensins antimicrobial properties showed that they produce antimicrobial activity against test microorganisms, exhibiting bactericidal and fungicidal effects at micromolar concentrations. We studied the influence of different environmental conditions (low and high ionic strength, blood serum in medium) for antimicrobial activity. It is shown that 0-defensins have the ability to increase the outer membran of E.coli permeability, however, in contrast to 0-defensins have no noticeable influence on the inner membrane permeability. Conclusion. Baboon 0-defensins isolated from blood leukocytes are effective antimicrobial agents with a broad spectrum of microbicidal action. 0-Defensins baboon unlike 0-defensins exhibit antimicrobial activity in environments with high ionic strength. 0-Defensins increase the outer membran of E.coli permeability but not the cytoplasmic membrane, suggesting that the mechanism of antimicrobial effect of these peptides other than 0-defensins.

Aim. The study of the mechanism of antiviral action and evaluation of the clinical efficacy, safety and tolerability of therapy with Fortepren^® in patients with chronic recurrent herpesvirus infection of genital localization (CRHVI). Materials and methods. Clinical studies were carried out of a drug Fortepren^® (0.4% sodium polyprenyl phosphate solution), which was administered to patients who underwent a basic therapeutic course of the drug Acyclovir-Acry® to relieve the acute phase of the disease. The study was performed on 80 male and female patients selected during the screening with a confirmed diagnosis of CRHVI. Two groups were formed. Patients of group 1 (experimental) were intramuscularly injected with Fortepren^® at a dose of 2 ml (8 mg) three times at intervals of 21 days by 3 ± 2, 24 ± 2 and 45 ± 2 days following the 10-day basic course of treatment of the acute phase of diseases with the use of the acyclovir tablets of 400 mg - 13 ± 2, 34 ± 2 and 55 ± 2 days from the beginning of the study. Patients of the 2nd group (control) were intramuscularly injected with placebo solution at a volume of 2 ml instead of Fortepren^®. To evaluate Fortepren^® efficacy, the following criteria were used: increase in the duration of the inter-recessive period, a decrease in the frequency of relapses over the entire observation period; decrease in the severity of relapses, estimated in points, changes in immunological parameters according to the dynamics of changes in the production of the main cytokines. Results. In patients treated with Fortepren^®, the inter-recurrence period for the entire study period increased statistically from 29.36 ± 2.16 to 42.98 ± 3.29 days, while in the control group this indicator have not changed. Accordingly, in patients treated with Fortepren^®, a statistically significant reduction in the incidence of recurrence of CRHVI from 3.03 ± 0.02 before treatment to 1.94 ± 0.19 was observed during treatment in the absence of a decrease in the frequency of relapses in the control. Evaluation of the severity of CRHVI relapses in patients treated with Fortepren^® indicates the efficacy of this protocol. The sum of the scores of the mean values of CRHVI symptoms signs was statistically significantly decreased in the group 1 from 7.36 ± 0.35 points at the 1 st visit before the start of treatment to 4.75 ± 0.35 points during the treatment. No changes were seen in the control group. The level of leukocyte virus-induced interferon (LVI-IFN) in the patients of the group 1 increased from 36% to 64% in the end of the clinical trial compared to the control group, in which the increase in LVI-IFN titers was not observed. To further justify the possibility of increasing the immune response of cells, establishing possible mechanisms that determine the efficacy of treatment for CRHVI with Fortepren®, evaluation of the production of IFNz, IFNy, IL-10, IL-12p40, IL-12p70, IL-15, IL-2, IL-4, MIF-Fz, TNFа was made. In the of the study levels of all these cytokines was increased in patients treated with Fortepren® compared with the control group. Conclusion. The efficacy of using Fortepren^® in a dose of 2 ml (8 mg) with intramuscular administration to patients with chronic recurrent herpesviral infection of genital localization at the stage of remission three times with an interval of 21 days by 3 ± 2, 24 ± 2 and 45 ± 2 days after the end of 10 day basic course of treatment of the acute phase of the disease with the use of the drug acyclovir.

Aim. To study the qualitative composition, the quantitative content of microorganisms, to assess the viability and functional status of neutrophils in the oral fluid of patients using removable and non-removable DOC. Materials and methods. The present study used a free oral fluid of 100 patients aged 21 to 79 years. The qualitative and quantitative composition of the microflora was determined using the PCR method by means of the Femoflor-16 system (NPO DNA-technology, Russia). Determination of the total number of leukocytes was carried out in Goryaev's chamber, analysis of their viability was carried out with Trypan blue staining. Acridine orange was used to study the indices of lysosomal activity of neutrophils. Oxygendependent metabolism was studied using the Nitro Blue-Tetrazolium (NBT) test, and the phagocytic function was determined by the absorption of latex particles. Results. In persons using dentures, the qualitative and quantitative composition of the microflora of the oral cavity changes. The method of hygienic care of the DOC significantly does not affect the composition of the microflora of the oral fluid of patients. In persons using DOC, a decrease in the total number of leukocytes and their viable forms in the free oral fluid was established. However we registered the stability of functioning of viable neutrophils in patients with removable DOC and a significant decrease in the phagocytic ability and microbicidal oxygen-dependent potential of neutrophils when using non-removable DOC. Conclusion. Foreign body in the form of a removable or non-removable DOC changes the functioning of the factors of congenital anti-infective protection of the oral mucosa, which can lead to the development of infectious complications during dental prosthetics.

Aim.To find out microbial landscape characteristics of the palatine tonsils in healthy students, depending on the season. Materials and methods. It were examined 40 students without any oropharyngeal pathology aged 17-30 (23 ± 3.5). There were no signs of chronic tonsillitis at pha-ryngoscopy, no signs of acute respiratory infection; they did not take antibiotics for one last month prior to the study and had no tonsilitis for the last 5 years. Examination was made three times, once in a season: autumn from October 16 till November 2; winter from January 27 till March 6 ; spring from April 2 till April 19. Results. The normal microlandscape in healthy individuals aged 17-30 years permanently had Streptococcus viridans, Streptococcus pyogenes, Staphylococcus aureus, Staphylococcus epidermidis, Candida albicans, Neisseria, Micrococcus, Bacillus, and Pseudomonas aeruginosa. Presence of S. pyogenes andP. aeruginosa in the structure of microlandscape of the palatine tonsils did not cause any clinical symptoms in their carriers. We also revealed a seasonal dynamics expressed by the frequency of the increase of conditionally pathogenic flora on palatine tonsils in spring and the decrease in all microorganisms growth in winter.

A mandatory requirement for cell cultures used in scientific researches and biomanufacturing is the absence of their contamination by viruses. We have described the case of adventitious isolation of mammalian orthoreovirus in the rhesus macaque embryo kidney cells MA-104. PCR analysis for the presence of reovirus RNA of all probable sources of reovirus (trypsin, fetal bovine serum, clinical samples, cell culture) revealed no viral RNA in any of the samples. An important condition for the activation of the reovirus reproduction in the MA-104 cells was the presence of trypsin in the culture medium. The obtained results underscore the urgency of control for the reovirus contamination of chemicals of animal origin and cell cultures. Since reoviruses are associated with human diseases, such control in pharmaceutical production is mandatory.

The effectiveness of regeneration is provided by normal over exchange and regulatory responses, so the regenerative therapy should be directed at identifying and addressing the causes of dismetabolk influences. Mikrobiocenosis is an integral part of holistic human organism takes part in realization of all metabolic reactions of the media. Correction of disbiosis helps restore regenerative processes, for the development of this direction is necessary to develop a new generation of probiotic preparations.

Last decades a sepsis problem attracts the increased interest in the world community. In spite of definite achievements of modern fundamental and clinical medicine, sepsis as before is characterized by significant dissemination and high lethality. The problems of sepsis spread in various countries are discussed. It is shown that annually million cases of generalized purulent-septic infections are diagnosed. Steady sepsis increase is registered in industrially developed countries. Share of severe sepsis in pathology structure varies from 2 to 43% in different territories. Sepsis still remains among leading causes of human death being characterized by essential hospital lethality (from 30,6 to 80,4%). The patient categories belonging to high risk groups of sepsis development are shown. Special attention is directed to epidemiological manifestations of this pathological syndrome in pediatrics. Features of etiologic spectrum of the sepsis causative agents, increasing etiological importance of multi-resistant bacteria (Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter spp., MRSA, VRE, etc.) are demonstrated. In consideration of clinical-epidemiological, social and economic significance of sepsis, studying of its epidemiological aspects is the major direction of activities for Public Health services.