Vol 93, No 2 (2016)

Aim. Comparative study of tight junctions and ultrastructure alterations of enterocytes of mucous membranes of jejunum of rats under the effect of lipopolysaccharides and cholera toxin. Materials and methods. Lipopolysaccharides (Sigma-Aldrich, Germany) and cholera toxin (Sigma-Aldrich, Germany) were used. The study was carried out in Wistar line rats. Effect of lipopolysaccharides and cholera toxin on epitheliocytes was carried out by a method of withdrawal of segments of rat jejunum and their incubation with the specified substances. Comparative analysis of ultrathin sections of enterocytes of jejunum of rats and tight junctions between them was carried out in control and under the effect of lipopolysaccharides and cholera toxin. Results. Effect of lipopoly-saccharides on ultrastructure of enterocytes of rat jejunum manifested in the change of cell form as a result of increase of intercellular space without destruction of tight junctions. Disappearance of desmosomes, increase of nuclei and more pronounced ER were noted in some epitheliocytes. Effect of cholerogen on epitheliocytes of mucous membrane of rat jejunum by a number of signs is similar to the effect of lipopolysaccharides, that manifested in an alteration of ultrastructure of cell, the form of those also transformed as a result of an increase of intercellular space, this process was not accompanied by destruction of tight junctions. Disappearance of folding of the lateral region of plasmatic membrane of cells and a reduction of a number of microvilli was observed under the effect of cholera toxin. Conclusion. A similar character of effect of lipopolysaccharides and cholera toxins on ultrastructure of cells and region of tight junctions of enterocytes of rat jejunum was detected, both substances caused an increase of intercellular space without the destruction of tight junctions.

Aim. Confirmation of taxonomic position of Lactobacillus fermentum 90 TC-4 strain using phenotypic (classic microbiological, MALDI TOF mass-spectrometry) and genetic (16S rRNA gene segment sequencing and full genome sequencing) methods. Materials and methods. Object of the study - Lactobacillus fermentum 90 TC-4 strains from various collections. Mass-spectrometric analysis was carried out using Autoflex MALDI TOF mass-spectrometer (Bruker Daltonics, Germany), study of biochemical properties of the strain was carried out using API 50 CHL strips (Biomerueux, France), “DNA-sorb B” kit was used for isolation ofgenome DNA (CRIE, Moscow). Sequencing of the accumulated fragments of 16S rRNA gene was carried out using GenomeLab GeXP sequencing (Beckman Coulter, USA), full genome sequencing was carried out in MiSeq platform (Illumina). Assembly ofgenome and bioinformation analysis was carried out using BLAST program (www.blast.ncbi.nlm.nih.gov/blast.cgi), «CLC Bio Assembly» and genome server RAST (rast.nmpdr.org). Results. L. fermentum 90 TC-4 strain was established to be contaminated by L. plantarum culture in a series of cases. As a result of identification of a pure culture of L. fermentum 90 TC-4 strain using a specter of high-technology methods, membership of the strain in L. fermentum species has been proven. Conclusion. Taxonomic status of L. fermentum 90 TC-4 strain was confirmed.

Aim. Study transformation of skin microflora during development of atopic dermatitis. Materials and methods. 45 patients with various forms of atopic dermatitis (AtD) were examined. Control group consisted of 26 healthy individuals. The strains were cultivated on elective nutrient media. Identification of the isolated strains was carried out by MALDI-TOF mass-spectrometry method. Results. A low frequency of occurrence of taxon Staphylococcus epidermidis on face skin and high frequency of occurrence of Staphylococcus aureus on upper and lower limb skin was established for AtD patients compared with healthy individuals. The frequency of occurrence of proteolytically active isolates of S. aureus in AtD patients was 3 times higher than in healthy carriers of this taxon. Taxons of microorganisms not inherent to healthy individuals such as Bacillus mycoides, Pseudomonas putida, Pseudomonas radiobacter were isolated on lower limb and neck skin of AtD patients. A high frequency of occurrence of Cryptococcus satoi, Candida albicans, Malassezia globosa fungi was noted. Conclusion. A decrease of barrier functions of skin during AtD facilitates contamination of patients’ skin with rare bacterial taxons and fungi. One of the possible mechanisms of suppression of immune competent cell functions could be proteolytic enzymes of S. aureus.

Aim. Study N-acetyl-β-D-glucosaminidase (chitobiase) (EC 3.2.1.30) in strains of Vibrio cholerae of O1/non-O1 serogroups of various origin, that is a component of chitinolytic complex taking into account object of isolation and epidemiologic significance of strains. Materials and methods. Cultures of V.cholerae O1/non-O1 serogroup strains were obtained from the museum of live culture of Rostov RIPC. Enzymatic activity analysis was carried out in Hitachi F-2500 fluorescent spectrophotometer using FL Solutions licensed software. NCBI databases were used during enzyme characteristics. Results. N-acetyl-β-D-glucosaminidase in V.cholerae O1/non-O1 serogroup strains was detected, purified by column chromatography, studied and characterized by a number of physical-chemical and biological properties. Comparative computer analysis of amino acid sequence of N-acetyl-β-D-glucosaminidases of V.cholerae (VC2217 gene), Serratia marcescens etc. has allowed to attribute the enzyme from V.cholerae to glycosyl-hydrolases (chi-tobiases) of family 20 and classify it according to enzyme nomenclature as EC 3.2.1.30. Conclusion. N-acetyl-β-D-glucosaminidase in V.cholerae of O1/non-O1 serogroups of various origin and epidemiologic significance, participating in chitin utilization was studied and characterized for the first time, and its possible role in biology of cholera causative agent was shown.

Aim. Determination of values of coefficients of thermal stability of TEOVac for prognosis of conservation of the vaccine (specific biological activity) during the process of warranty period storage. Materials and methods. TEOVac (masticatory tablets) in primary packaging was kept at increased temperature (accelerated and stress-tests) and at the conditions established by PAP for the preparation (long-term tests). Biological activity of the vaccine was determined by titration on 12-day chicken embryos. Results. A correlation between the value of coefficients of thermal stability and conservation of the prepared series of the condition preparation at the final date of storage was experimentally established. Conclusion. Coefficients of thermal stability could be used as a prognostic indicator of quality of the produced pelleted formulation of the preparation for evaluation of conservation of the vaccine during warranty period storage.

Etiologic diagnostics of sepsis is one of the most difficult problems of contemporary medicine due to a wide variety of sepsis causative agents, many of which are components of normal human microflora. Disadvantages of contemporary «golden standard» of microbiologic diagnostics of sepsis etiology by seeding of blood for sterility are duration of cultivation, limitation in detection of non-cultivable forms of microorganisms, significant effect of preliminary empiric antibiotics therapy on results of the analysis. Methods of molecular diagnostics that are being actively developed and integrated during the last decade are deprived of these disadvantages. Main contemporary methods of molecular-biological diagnostics are examined in the review, actual data on their diagnostic characteristic are provided. Special attention is given to methods of PCR-diagnostics, including novel Russian developments. Methods of nucleic acid hybridization and proteomic analysis are examined in comparative aspect. Evaluation of application and perspectives of development of methods of molecular diagnostics of sepsis is given.

Purine receptors are located on immune and somatic cells of animal and human organisms. Summation of signals from purine and TOLL-like receptors takes place on the level of inflammasome formation and results in summation of the first and second signals of innate immunity. The first signal - from PAMPs (pathogen associated molecular patterns), the second - from DAMPs (danger associated molecular patterns). Adenosine triphosphate (ATP) is the most studied DAMP. ATP connects with purine receptors, which include P2 (P2X7 receptors are the best described), that results in opening of channels of these receptors and transit of ATP into the cell. In parallel exit of K+ from cells and entrance of Ca2+ and Na+ into the cells is observed, that is associated with activation of the immune competent cell. Damaged cells dying via necrosis or apoptosis are the source of extracellular ATP, as well as activated immunocytes. Signals from P2 and TOLL-like receptors are summarized in effectors of immune response, and activation of P2 receptors in lymphocytes makes a contribution into activation of cells, mediated by T-cell receptor. Negative side of purine receptor activation is a stimulating effect on proliferation and metastasis of malignant cells. The practical output of knowledge on functioning of purine receptors for clinical immunology is the application of agonists and antagonists of purine receptors, as well as explanation of effect of immune modulators from the position of launch of K+/Na+-pump, resulting in prolonged activation of immune competent cells.

Aim. Analysis of epidemiologic features of a norovirus outbreak in Alagir city of the Republic of North Ossetia-Alania and effectiveness of measures of its liquidation. Materials and methods. Data from maps-schemes of water supply system of Alagir city and statistical documentation of Centre of Hygiene and Epidemiology in the Republic of North Ossetia-Alania were used in the study. Indication of norovirus in clinical material and water samples was carried out by polymerase chain reaction method. Results. Etiological agent of outbreak disease was established - genotype II norovirus. Realization of fecal-oral mechanisms of water transmission pathway of the causative agent of norovirus infection was detected. Conditions facilitating emergence and development of the indicated outbreak were determined - non-satisfactory sanitary-technical condition of water supply system of the city. Conclusion. The studied water outbreak of norovirus infection was caused by GII.17 genotype virus, that currently gradually displaces GII.IV genotype, and was characterized by an intensive start, involvement of all population age groups into the epidemic process (with primary infection of adults), low family focality, predominance of average severity disease forms in the clinical presentation. The counter-epidemic measures carried out ensured rapid localization and liquidation of the norovirus infection outbreak.

Aim. Evaluation of dysbiotic condition of intestine based on determination of local antioxidant index (LAI). Materials and methods. 155 patients with bacteriologically confirmed intestine dysbacteriosis were examined. 20 individuals with normobiocenosis of the intestine served as control. Feces dilution 10^-2 was used for biochemical study. Catalase, superoxide dismutase activity and level of malonic dialdehyde was evaluated photometrically by the value of optical density of the samples. Microbial landscape of feces was studied by a bacteriologic method with parallel determination in samples of coprofiltrates of activity of the indicated enzymes and content of malonic dialdehyde. Evaluation of disturbances of microecology of the intestine was carried out by a biochemical method by calculating local antioxidant index in juxtaposition with microbial map data. Results. LAI parameter was significantly higher than 20 in coprofiltrates of people with intestine normobiocenosis. In patients with detected intestine dysbacteriosis, depending on its degree, LAI parameter changes were registered in the range from negative to 20. Dysbacteriosis in a part of patients had a prolonged character, LAI was lower than 14, that reflected the presence of a persistent inflammation. A tendency of normalization of microbial ecology and growth of LAI was observed during administration ofa probiotic preparation. Conclusion. A screening criteria is proposed, that differentiates the degree of severity of intestine dysbacteriosis based on calculation of local antioxidant index.