Vol 91, No 6 (2014)

Aim. Analysis of occurrence of the third type secretory system (TTSS) effectors in clinical P. aeruginosa strains. Materials and methods. Intra-hospital (n=164) and extra-hospital (n=30) strains of P. aeruginosa were studied. Detection of exoS and exoU genes was carried out by PCR in DNA Engine Dyad Thermal Cycler («Bio-Rad», USA). Metallo-beta-lactamase (MBL) producers were detected by the presence of blaVIM-2 gene. Results. Screening of intra- and extra-hospital strains for the presence of genes coding ExoS and ExoU showed, that exoS is detected in genome of clinical isolates in 59.8% and exoU - 31.1% of cases. At the same time, strains with exoS-/exoU+ genotype predominated in ICU (ф=0.466; p=0.0000). A significant association between the presence of the respective effectors and material of strain isolation was not detected. exoU gene was more frequently detected in genome of MBL producers (ф=0.784; p=0.0004). Conclusion. A significant association between exoU and blaVIM-2 could be explained by clonal prevalence of P. aeruginosa ST235 VIM-2, circulation of those is noted on all the territory of Russia. As a rule, ExoU is produced by highly virulent poly-antibiotic resistant hospital isolates that determine unfavorable outcomes of pseudomonas infection.

Aim. Study the main surface parameters of mi11ed po1yacry1ic materia1s using atomic force microscopy and primary microbia1 adhesion of periodontopathogenic group bacteria and Candida albicans fungi taking into consideration the method of samp1e po1ishing. Materials and methods. Studied samp1es: mi11-treated without po1ishing (contro1); ergobox po1ished; po1ished in denta1 1aboratory conditions; po1ished by a rubber brush in dentists’ office. Microbia1 strains be1onging to periodontopathogenic species (c1inica1 iso1ates) that had been iso1ated from periodonta1 pockets of periodontitis patients: Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus sanguis, C. albicans fungi were used for mode11ing experiments of primary adhesion of microbes to the materia1 samp1es. Results. S. sanguis had the highest degree of adhesion to po1ymer after mi11ing, P. gingivalis, C. albicans - medium, F. nucleatum - 1ow. A significant reduction of adhesion is observed during po1ishing in denta1 1aboratory conditions or ergobox, 1ess significant - during po1ishing in denta1 office. Conclusion. The data obtained a11ow to make a conc1usion that the samp1es from po1ymer materia1s for preparation of prosthesis basis have varying degree of intensity of microbia1 adhesion of members of periodontopathogenic microf1ora and C. albicans fungi that depends on the po1ishing method, that according1y determined the differences in co1o-nization resistance against formation of microbia1 biofi1m during po1ymer use in c1inica1 conditions.

Aim. Deve1opment and testing of a rea1-time PCR method for detection of Neisseria meningitidis serogroup A, B, C and W DNA. Materials and methods. Reference strains and 187 samp1es of cerebrospina1 f1uid (CSF) from meningococci meningitis patients were used in the study. Mu1tip1ex PCR was carried out in an instrument with 5 channe1s of fluorescent detection. Results. Ana1ysis of specific serogroup 1oci of the genome and design of o1igonuc1eotides for the detection of DNA of a11 the capsu1e meningococci and 4 serogroups in particu1ar was carried out. PCR conditions were optimized; specificity was shown and ana1ytica1 sensitivity was eva1uated using reference strains. DNA of the fo11owing serogroups was detected during study of c1inica1 CSF samp1es: A - in 103 samp1es (55%), B - in 45 (24%), C - in 30 (16%), W - in 5 (3%). On1y DNA of meningococci capsu1e gene ctrA was found in 4 samp1es; presumab1y, they contained DNA of other serogroups. Mu1ti1ocus sequence-typing and detection of antigenic determinants of PorA and FetA genes for 27 DNA samp1es of group A menincococci as we11 as DNA of 5 group W meningococci and 4 ungroupab1e was carried out. Conclusion. The method proposed a11ows to carry out serogrouping of no less than 95% of strains or DNA samples isolated from CSF of meningococci infection patients. Combined with other recommended non-cultural methods of genotyping, it may be useful for complex characteristics of pathogenic meningococci.

Aim. Study the influence of staphylococcus vaccine on functional activity of antigen-presenting cells. Materials and methods. Mice intraperitoneally received 500 ^g of «Staphylovac» vaccine. Phagocytic activity of peritoneal macrophages against Staphylococcus aureus 1991 was determined in animals at various time intervals. Phagocytic index (PI) and phagocytic number (PN) in smears made at 30 and 60 minutes of incubation were calculated. Dendritic cells (DC) were obtained from bone marrow precursors during cultivation with 20 ng/ml GM-CSF and 20 ng/ml IL-4 (BioSource International Inc., Belgium). At day 6 of incubation staphylococcus vaccine (50 ^g/ml) was added to immature cells for induction of DC maturation. DC phenotype evaluation was carried out by flow cytometry using monoclonal antibodies against cell antigens (Beckman Culter, USA). Results. PI at 30 and 60 minutes of incubation increased by 0.12 - 1.4 times and 1.11 - 1.52 times, respectively, compared with control. PN at 30 minutes of incubation of cells with microbial suspension increased from 8.6 to 11.4% against 5.9% in control, at 60 minutes of incubation - from 7.7 to 8.1% against 5.1% in control. In DC culture during their incubation with the vaccine, content of cells with expression of intercellular adhesion marker CD38, antigen presenting marker MHCII and DC terminal differentiation marker CD83 increased. Expression of CD34 and CD14 was also noted, that may give evidence on partial direction of cell differentiation to macrophages. Conclusion. «Staphylovac» vaccine during intraperitoneal administration to mice had activating influence on functional activity of antigen-presenting cells and peritoneal macrophages.

Aim. Study the effect of aluminium hydroxide on molecular-cell mechanisms of innate immunity activation and its adjuvant effect on immunogenicity of natural bacterial and synthetic pneumococci antigens. Materials and methods. Surface markers of dendritic cells (DC), mononuclear leukocytes (ML) and cytokine levels were determined by flow cytometry; IgG titers - by EIA. Protective activity was evaluated in experiments with active protection of mice from infection with virulent pneumococci strains. Results. Aluminium hydroxide increased the ML content of mice spleen expressing TLR2 and TLR4. Its addition into the culture of immature DC induced the appearance of a population of cells with mature DC markers - CD83, CD80, CD86, however, the level of undifferentiated cells (CD34) and cells with adhesion molecules (CD11c, CD38) did not change. DC produced IL-ф, IL-5, IL-10, IFNy into the cultivation medium. An increase of cytokine production took place 2 hours after the administration into mice and was retained for the observation period (24 hours). Th1 (IFNy, TNFa) and Th2 (IL-5, IL-10, GM-CSF) cytokine production gave evidence on immune response polarization by Th1/Th2 type. After 2 administrations of aluminium hydroxide into mice the number of ML with CD19+, CD5+, NK1.1+, CD25+, MHCII+ markers increased during decrease of CD3+, CD4+ and CD8+ T-lymphocytes. Adaptive immunity activation was characterized by high IgG titers to pneumococci capsule polysaccharide and protection of 90 - 100% of the mice against infection with lethal doses of S. pneumoniae strains, was detected during 2-fold immunization of mice with conjugates of synthetic pneumococci oligosaccharides with BSA, sorbed onto aluminium hydroxide, whereas natural bacterial antigens provided 90 - 100% survival of animals during immunization without the adjuvant. Conclusion. Data are provided on the effect of aluminium hydroxide on key effectors of innate immunity: DC, ML, TLRs and cytokine production. A reasonable administration of this adjuvant was shown to be in association with conjugates of pneumococci synthetic oligosaccharides with a carrier protein.

Aim. Determination of the content of various cytokines in coprofiltrates of individuals with bacteriologically confirmed intestine dysbacteriosis. Materials and methods. Qualitative and quantitative content of large intestine microbiocenosis was studied; IFNy, pro-inflammatory (IL-2, IL-6, IL-8, IL-10) and anti-inflammatory (IL-4, IL-10) content was determined using EIA in coprofiltrates of 139 individuals aged 18 - 60 years. All the indexes were juxtaposed with the cytokine index (CI). Results. A high content of IL-2, IL-4 and IL-10 with normal levels of IL-10, IL-6 and IL-8 was established. A comparable content ofbifidobacteria, lactobacilli and escherichia was detected in individuals with various CI index; in individuals with CI above 1 c.u. and above 10 c.u., against the background of proportionally intensifying IFNy induction, an increase of quantity of escherichia with decreased enzymatic activity and frequent detection of opportunistic en-terobacteria, staphylococci and Candida genus fungi is noted. Conclusion. The presence of opportunistic microflora at low content of IFNy with CI of less than 1 c.u. could be evaluated as a dysbiotic reaction, and the presence of opportunistic microflora against the background of high IFNy content with CI of above 10 c.u. - as a development of systemic inflammation due to translocation of dysbiotic microflora into the bloodflow.

Aim. Study apoptogenic activity of microbes-associants during Epstein-Barr virus infection (EBVI) on the model of mice peritoneal macrophages in vitro. Materials and methods. Evaluation of apoptosis induced by bacteria isolated from EBVI patients was carried out by characteristic morphological changes ofmacrophages in smears stained by May-Grunwald with additional staining by Romanowsky-Giemsa. Results. All the EBVI microbes-associants were established to have apoptogenic activity, however, the highest pathogenic potential was noted in Streptococcus pyogenes. Conclusion. The presence of apoptogenic activity in bacterial microflora accompanying EBVI against immune system cells could serve as means of their survival and be the pathogenetic basis for prolonged persistence in the organism.

Aim. Compare frequency of isolation of polioviruses in children living in closed-type facilities (orphanages) before and after the change in poliomyelitis vaccination scheme. Materials and methods. Feces samples of 207 children from 5 orphanages during immunization with oral poliomyelitis vaccine (OPV) and of 259 children from 4 orphanages during vaccination with inactivated poliomyelitis vaccine (IPV) were studied. Isolation and identification of polioviruses was carried out according to WHO recommendations. Results. In orphanages, where children were immunized with the oral vaccines, 21 polioviruses were isolated. In orphanages, where only inactivated vaccine was used, 10 polioviruses were isolated, the presence of polioviruses in these facilities is associated with their introduction from the outside. The percentage of poliovirus detection in children immunized with OPV was shown to be 16.9+3.4% and was significantly higher than in children vaccinated with IPV (6.1 + 1.9%). Polioviruses isolated from children immunized with OPV belonged to serotypes 1, 2 and 3 in 19.0, 14.3 and 66.7% of cases, respectively. Polioviruses detected in children immunized with IPV belonged to serotypes 1, 2 and 3 in 30, 40 and 30% of cases, respectively. All the isolated polioviruses turned out to be Sabin vaccine strains. Conclusion. Implementation of strict prophylaxis measures in orphanages is necessary in order to prevent the possibility of introduction, transmission and circulation of polioviruses. Improvement of control in children from closed-type facilities will ensure maintenance of Russian Federation status as the country free of poliomyelitis.

Aim. Evaluate immune response in mice against various L-asparaginases and determine their cross-immunogenicity. Materials and methods. The studies were carried out in C 57Bl 6j line mice. Immunogenicity of L-asparaginases was studied: Escherichia coli type II (recombinant) (Medak, Germany) (ЕсА); Erwinia carotovora type II (ErA); Yersinia pseudotuberculosis type II (YpA); Rhodospirillum rubrum type I (RrA); Wollinella succinogenes type II (WsA). Immune response against the administered antigens was determined in EIA. Results. Y. pseudotuberculosis L-asparaginase was the most immunogenic, E. coli - the least immunogenic. E. carotovora, R. rubrum, W. succinogenes asparaginases displayed intermediate immunogenicity. The results of cross-immunogenicity evaluation have established, that blood sera of mice, that had received YpA, showed cross-immunogenicity against all the other L-asparaginase preparations except E. carotovora. During immunization with E. coli L-asparaginase the developed antibodies also bound preparation from E. carotovora. Sera from mice immunized with W. succinogenes, E. carotovora and R. rubrum L-asparaginases had cross-reaction only with EcA and did not react with other preparations. Conclusion. Cross-immunogenicity of the studied L-asparaginases was determined. A sequence of administration of the studied preparation is proposed that allows to minimize L-asparaginase neutralization by cross-reacting antibodies.

Aim. Comparative evaluation of the effect of polyoxidonium and betaleukin on immunogenic and protective activity of a live plague vaccine in model animal experiments. Materials and methods. Plague vaccine EV, polyoxidonium, betaleukin, erythrocytic antigenic diagnosticum for determination of F1 antibodies and immune reagents for detection of lymphocytes with F1 receptors (LFR) in adhesive test developed by the authors were used. The experiments were carried out in 12 rabbits and 169 guinea pigs. Results. Immune modulation accelerated the appearance and disappearance of LFR (early phase) and ensured a more rapid and intensive antibody formation (effector phase). Activation by betaleukin is more pronounced than by polyoxidonium. The more rapid and intensive was the development of early phase, the more effective was antibody response to the vaccine. Immune modulation in the experiment with guinea pigs significantly increased protective activity of the vaccine. Conclusion. The use of immune modulators increased immunogenic (in both early and effector phases of antigen-specific response) and protective activity of the EV vaccine. A connection between the acceleration of the first phase of antigen-specific response and general intensity of effector phase of immune response to the EV vaccine was detected.

Aim. Use of a complex of methods for etiologic deciphering of an acute respiratory infection. Materials and methods. Clinical samples of blood sera, nasopharynx washes and sputum were obtained from 35 patients with acute respiratory disease (ARD). «Difco PPLO Broth» was used for M. pneumoniae cultivation. AHR, IFR, PCR, IFA were used in the study. Results. Results of the study have shown that M. pneumoniae antigens in blood sera samples were detected in AHR in 32 patients, and specific G and M class antibodies - in 21 and 18 cases, respectively. Simultaneous detection of IgG and IgM was registered in 14 patients. M. pneumoniae cell DNA was detected in 10 of 20 blood sera samples. Circulating immune complexes were isolated from blood sera of 8 patients (4 with pneumonia, 4 with ARD) and M. pneumoniae antigens were detected in them by using direct IFR. IFR study of sputum and nasopharynx smears has shown that M. pneumoniae antigens were detected in 29 of 35 samples. In 12 of 15 smear samples M. pneumoniae DNA was detected by PCR. In 10 cases results of antigen detection by IFR as well as DNA in PCR coincided. Results of analysis of all the clinical material have shown that in 33 of 35 patients positive results coincided for 2 or 3 and in some cases 4 of the laboratory study methods used. Conclusion. The use of diagnostic test complex significantly increases the accuracy of the study results, and detection of specific antibodies allows to determine disease period.