Vol 89, No 6 (2012)

Aim. Study of tight junction state and ultrastructure changes of rat jejunum enterocytes and colon colonocytes under the effect of cholerogen and protamine. Materials and methods. Cholerogen (cholera toxin, Sigma-Aldrich, Germany) and protamine sulfate (Russia) were used in the study. The study was carried out in Wistar line rats. Effect of cholera toxin and protamine on rat intestine epitheliocytes was carried out by incubating intestine segments in the respective solutions. Ultrastructure changes caused by cholerogen and protamine in rat enterocytes and colonocytes were assessed based on ultrathin section analysis by transmission electron microscopy of the cells themselves and tight junctions between them compared with control. Results. Effect of cholerogen on intestine mucous membrane epitheliocytes manifested in changes of cell ultrastructure, the form of which transformed as a result of increase of intercellular space without the destruction of tight junctions. Disappearance of cell plasma membrane lateral area folding and decrease of number of microvilli was noted. Enlargement of nuclei was noted only in individual cells. Effect of protamine on epithelial cell layer ultrastructure differed significantly from the effect of cholerogen. Increase of cell plasma membrane lateral area folding and significant enlargement of nuclei that moved to the central part of cells reaching its apical end were characteristic effects for protamine. Surface of a part of epitheliocytes lost microvilli with simultaneous destruction of tight junction structure. Protamine induced increase of folding only in colon without affecting jejunum. At the same time both of these substances caused increase of intercellular space in jejenum and colon epithelium. Conclusion. Differences in ultrathin structure of rat small intestine and colon epitheliocyte tight junctions under the effect of cholerogen and protamine were revealed.

Aim. Study the interaction of vaginal corynebacteria and lactobacilli in realization of oxidative mechanism of antagonistic relations of bacteria. Materials and methods. Effect of supernatants of corynebacteria inhibiting catalase on antagonism of peroxide producing lactobacilli to Staphylococcus aureus was studied. Results. High frequency (55.5 - 72.7%) of potentiating of antagonism of lactobacilli with medium and high level of hydrogen peroxide production under the effect of supernatants of corynebacteria inhibiting catalase was established. The frequency of potentiation of antagonism of lactobacilli and corynebacteriae depended on the intensity of hydrogen peroxide production and on the ability of corynebacteria to suppress catalase of staphylococci. Conclusion. Potentiation of antagonism to S. aureus of peroxide producing lactobacilli and corynebacteria with catalase inhibitors gives evidence on realization of oxidative bacterial mechanism of colonization resistance in human organism.

Aim. Certification of V. cholerae strains stored at State collection of pathogenic microorganisms and cell cultures SCPM - Obolensk. Materials and methods. 50 V. cholerae strains were studied. Real-time PCR with primers to genes ctxA, ctxB, ace, zot, tсpА, toxR, hlyA, rtxC, rfbO1, ompU, ompW was used. Results. Membership of the studied strains in V. cholerae species was confirmed by molecular-biological methods. 46 strains belong to O1 serogroup, 42 of those - El Tor toxigenic, having all the virulence genes and 4 nontoxigenic strains. A strain had ace, zot, tсpA, toxR, rtxC, hlyA, ompU genes. 2 strains had ace, toxR, rtxC, hlyA genes, a strain had only toxR gene which is a global regulatory gene. 2 of the 4 serogroup O1 strains were non-toxigenic and had all the virulence genes (ctxA, ctxB, ace, zot, tсpA, toxR, rtxC, hlyA, ompU). 1 nontoxigenic strain has ace, zot, toxR, hlyA, ompU genes, and the other - toxR, hlyA genes. Conclusion. Genome certificates of all the V. cholerae strains stored in State collection of pathogenic microorganisms and cell cultures SCPM - Obolensk were created. Markers of epidemic threat - ctxA, ctxB, tcpA, toxR and additional virulence genes were determined.

Aim. Genotyping of astroviruses isolated from children with acute enteric infection (AEI) on the territory of Nizhny Novgorod in 2006 - 2011. Materials and methods. Samples of feces from children hospitalized in intestine infection department of a Nizhny Novgorod infectious diseases hospitals served as study material. Astroviruses were detected by reverse transcription polymerase chain reaction method. Astrovirus genotypes were determined based on phylogenetic analysis of nucleotide sequence of genome fragment coding capsid protein. Results. During examination of 5759 children with AEI from July 2006 to June 2011 astroviruses were detected in 2.6% of cases, and in 2010 - 2011 the frequency of astrovirus detection (5.19%) was significantly higher than in the previous epidemic seasons and the average based on the whole observation period. Genotyping of the detected astroviruses showed that genotype 1, 2, 3, 4, 5 astroviruses circulated on the territory of Nizhny Novgorod with predomination of genotype 1. Genotype 1 astroviruses are presented by genetic lineages 1a, 1b, 1d with predomination of lineage 1a. From the start of 2010 all the detected isolates from genetic lineage 1a belonged to a single new sublineage - 1a-2010. The second by quantity of detected isolates were genotype 5 astroviruses identified for the first time in Nizhny Novgorod in July 2010. Genotype 2, 3 and 4 astroviruses were detected in isolated cases. Conclusion. Activation of astrovirus circulation in the population of Nizhny Novgorod that was shown by a significant increase of frequency of their detection in children with AEI in 2010 - 2011 epidemic season with the highest probability was caused by appearance of genotype 5 astroviruses, that had not previously been detected in this territory and other territories of Russian Federation.

Aim. Study phylogenetic interconnections of HIV-1 subtype A and B variants circulating in Novosibirsk region (NSR). Materials and methods. 268 HIV-1 variants isolated in 2007 - 2010 from blood samples of HIV infected patients in NSR, Samara, Congo and Moscow. HIV-1 variant genotyping was performed by analysis of 1.3 kb long pol gene nucleotide sequences. Phylogenetic analysis of nucleotide sequences was carried out by program Mega version 4.1 by constructing phylogenetic trees by nearest neighbor method. Nucleotide distances were calculated by Kimura method. Results. The studied HIV-1 subtype B variants form separate phylogenetic groups with a low HIV-1 nucleotide sequence homology level combined based on territorial principle and/or time of HIV infection in a territory but not possessing interconnection with a specific population risk group. Subtype A HIV-1 is a fairly homogenous monophyletic group. Phylogenetic differences during studies of HIV-1 isolated from risk group patients - injection drugs users and individuals infected through sexual contacts were not detected. HIV variants isolated from patients infected in Moscow and Samara generally grouped with HIV variants circulating in the European part of Russia. Conclusion. An independent circulation of genetically separate HIV-1 subtype B groups is observed on the territory of siberian region which is a result of multiple independent introductions of distant variants of the virus. The confirmed limited spread of this HIV-1 genetic variant with a subsequent territorial separateness creates a possibility of formation of genetically different virus populations. The studies of subtype A viruses performed confirm the high level of homogeneity detected earlier in other Russia territories of HIV-1 belonging to this genetic variant. Monophyly of subtype A HIV variants is explained by imposition of 2 factors - territorial mobility of the population inside the country and lack of specific transmission routes for HIV-1 subtype A.

Aim. Analyze the diversity and prevalence of mutations in human immunodeficiency virus type 1 (HIV-1) genome emerging in response to antiretroviral therapy isolated from HIV-infected individuals of Novosibirsk region in 2010, 2011. Materials and methods. Detection of mutations in HIV-1 genome responsible for the resistance to antiretroviral preparations (ARVP) was carried out by determination of pol gene nucleotide sequence and subsequent analysis of the data obtained by program HIVdb: Genotypic Resistance Interpretation Algorithm. Results. HIV-1 resistance mutations to antiretroviral preparations were detected in 23.6% of the total number of the studied samples. The most prevalent mutations are those conditioning resistance to nucleoside and non-nucleoside reverse transcriptase inhibitors (M184V, Y181C and K103N). In studies of HIV-1 isolated from 4 patients who had not received antiretroviral therapy (ARVT) transmission of HIV-1 resistant to various groups of preparations was detected. Conclusion. The detected facts of ARVP resistant HIV-1 circulation among patients who had not received ARVT and the data obtained on the mutations emerging in response to therapy underline the relevance of administration of HIV-1 resistance profile study during both decrease of ARVT effectiveness and primary administration of therapy to HIV infected patients.

Aim. Confirmation of immunostimulating effect of an original low molecular weight germanium organic compound (LMW-GOC) during immunization of mice with Vaxigrip vaccine. Materials and methods. The experiments were carried out in CBA mice divided into 4 groups: control, those that received Vaxigrip influenza vaccine intraperitoneally, those that received LMW-GOC intraperitoneally and those that received both preparations at once. Effect of the preparations administered was evaluated by flow cytofluorometry based on changes of СD3, CD4, CD5, CD8, CD19, CD25, Foxp3, NK1.1, ƒƒ T, MHC II, TLR2, TLR4, TLR9 expressing cell content in mice spleens. The content of the colored cells was determined at normal, 24 hours and 7 days after the administration of the preparations. Statistical treatment of the data was carried out by using Win MD 128 program package. Results. LMW-GOC can enhance the effect of Vaxigrip vaccine that is expressed by an increase of content in spleen of some lymphocyte subpopulations 24 hours and 7 days after the intraperitoneal administration. In some cases LMW-GOC increases the content of some lymphocyte subpopulations in mice spleen after administration as a monopreparation, i.e. without the vaccine. LMW-GOC suppressed stimulating effect of the vaccine on the spleen content of various lymphocyte subpopulations in none of the observations. Conclusion. By using cytofluorometry method it is possible to form an understanding of an elevated role of various types of cells in the development of immune response to the vaccine as well as regarding additional enhancement of this response during administration of LMW-GOC to mice. The effect of the preparation is manifested for a few days after its administration. The preparation manifests adjuvant properties and after further studies may be suggested for use as an adjuvant.

Aim. Evaluate informativity of simultaneous determination of antibodies (AB) against extracellular (AB against streptolysin-O - ASL-O) and cellular (IgM against A-polysaccharide - A-PSC) antigens in patients with angina and soft tissue infections caused by serogroup A streptococci (SGA) and identify features of humoral immune response to SGA infection according to infectious process localization. Materials and methods. 2 groups of patients with bacteriologically confirmed SGA infection (50 cases of angina - group 1 and 51 case of soft tissue infection - group 2) were examined for the presence of ASL-O by using Architect ci8200 analyzer (Abbott, USA) and IgM against SGA A-PSC by EIA. Results. In group 1, 23 (46%) individuals were recognized as positive by ASL-O level, and in group 2 - 20 (39%; p>0.05); conditionally significant exceeding of normal values (more than 1.5 times) was detected in 25% of patients of each group. Increased level of antibodies against SGA A-PSC was detected in 43 (86%) patients of group 1, and in 30 (59%) of patients of group 2 (p<0.05). In group 1 exceeding of normal values of anti-A-PSC IgM was noted mostly by 1.50.5 times (74%). In group 2 in 43% of patients the level of anti-A-PSC IgM was above normal more than 2 times and in most cases in uncomplicated variants of disease course. In 45% of patients with severe form of soft tissue infection this parameter did not exceed normal values (p<0.05). Conclusion. In acute period of disease with simultaneous determination of ASL-O and IgM against A-PSC sensitivity of serologic diagnostics of SGA etiology angina and SGA infection of soft tissues was established to reach 92% and 72%, respectively, and humoral immune response to cellular AG in each form of SGA has its features.

Aim. Study the state of intestine microecology in patients with obstructive jaundice after decompression of bile ducts and administration of lactulose. Materials and methods. 58 patients of different gender and age who were under treatment in the 13th surgical department of City Clinical Hospital No. 7 due to obstructive jaundice were examined. Evaluation of lactulose administration was carried out in a blinded randomized study. The patients were divided into 2 groups of 29 individuals each: (1) patients who had undergone surgery without administration of lactulose (control) and (2) patients who had received immediately after decompression of bile ducts 30 ml of lactulose for 1 week (comparison group). Feces samples were obtained with a weekly interval for bacteriological study for dysbacteriosis. Results. Based on the results of bacteriological analysis of feces in all the 58 patients with mechanical jaundice disorders of intestine microecology of various severity degrees were detected. In the patient group who had received lactulose for 7 days after the decompression of bile ducts a tendency for an increase of population level of bifidobacteria and lactobacilli and a decrease of quantity of opportunistic microorganisms of various taxonomical groups was noted. Conclusion. Administration of lactulose to patients at 30 ml dose per day for 7 days positively affected the state of microbiocenosis of colon towards its normalization.

Aim. Evaluate significance of epithelial-bacterial interactions for the formation and/or support of microbiocenoses. Materials and methods. Effect of vaginal epitheliocytes on growth properties of lactobacilli strains obtained from the same biotope from 16 women with vaginal normocenosis was studied. During intravaginal probiotic therapy in 24 women with vaginal dysbiosis probability of colonization of vagina by probiotic strain was evaluated depending on the result of effect of vaginal epitheliocytes on its growth properties. Results. Exometabolites of epitheliocytes in normocenosis were shown to render stimulating effect on the growth of autostrain of dominant microflora, that probably is the basis of formation and/or support of microbiocenosis. Stimulating effect of exometabolites of vaginal epitheliocytes of patients on growth properties of probiotic strain was revealed to be the prerequisite for successful probiotic therapy. Conclusion. The data obtained allow to examine epithelial-bacterial interactions as a basis of formation of microbiocenoses. Evaluation of these interactions may be used for individual selection of probiotic strains.

Aim. Evaluate safety of prophylaxis of viral hemorrhagic fevers by specific heterologous immunoglobulins. Materials and methods. Clinical-laboratory examination of 24 individuals after intramuscular administration of heterologous Ebola immunoglobulin was carried out. Anaphylactogenicity of the immunoglobulins was studied by WD 42-28-8-89 in guinea pigs compared with commercial preparations. Results. Immediate type reactions were not observed. In individuals with normal anamnesis the number of local reactions was 31%, general in the form of lung serum disease - 13%. In individuals with unfavorable anamnesis against the background of desensitization therapy there were almost no reactions; without it local reactions were present in 50%, mild severity serum lung disease - in 17%, medium - in 33%. Immunoglobulins against especially dangerous viral agents by anaphylactogenic properties did not differ from commercial heterologous preparations. Conclusion. Application of specific immunoglobulins from horse blood sera (the main means of protection from dangerous and especially dangerous exotic viral infections) with compliance by desensitization principles is relatively safe. Safe level of sensitization properties is characterized by anaphylaxis index up to 3.7 for guinea pigs.

Aim. Determination of cytokine content in various areas of dentition in patients with peri-implantitis associated with parodontopathogenic bacteria species of I and II order. Materials and methods. 32 patients with complications that developed in 3 months to 14 years after installation of intraosteal dental implants were examined. Content of cytokines in various areas of dentition was determined by using solid phase enzyme immunoassay in patients with developed peri-implantitis associated with parodontopathogenic bacteria species of I and II order. Multiplex polymerase chain reaction was used for determination of parodontopathogenic bacteria marker DNA. Results. Marker DNA of I order parodontopathogenic bacteria - Aggregatibacter (Actinobacillus) actinomycetemcomitans, Tannerella forsythia (Baсteroides forsythus), Porphyromonas gingivalis in peri-implantation tissues during implant rejection was detected with 34.4 - 75% frequency while II order (Prevotella intermedia, Treponema denticola, Parvimonas micros (Peptostreptococcus micros), Fusobacterium nucleatum/periodonticum etc.) - with significantly lower frequency. Total concentration of IL-1ƒ, IL-4, IL-6, IL-8, TNFƒ, IL-17А and INFγ in contents of pathological pocket in the area of implants and the levels of each of them were significantly higher than in the contents of parodontal pockets, areas with stable implants and gingival fluid from areas with healthy teeth. Total interleukin content in the contents of pathological pockets in the area of rejected implants was significantly higher than in other studied areas. In the exudate of parodontal pockets of the preserved teeth it was 2.4 times lower, in the area of stable implants - 4.6 times lower, and in the areas with healthy teeth - 4.8 times lower than with rejected implants (p<0.05). Conclusion. The results of the study conducted allow to make a conclusion regard ing reasonability of monitoring during dental implantation of parodontopathogenic microorganism strains and local cytokine response of the host organism with the objective of rational prophylaxis and therapy of inflammatory complications.