Vol 90, No 3 (2013)

Aim. Determination of virulence of enterococci strains isolated from clinical material from humans on pheno- and genotype levels. Materials and methods. 30 strains of enterococci isolated from wound exudate, urine, newborn skin lavage were used in the study. Strain identification was carried out by multiplex PCR. Hemolytic activity was determined by dish method, gelatinase - by dissolution of gelatin column, proteolytic - by biuret method; genes coding virulence factor synthesis (gelE, sprE, cylM, cylB, cylA, cylLs, cylLl, ESP, HYL, ASA) - by using PCR. Results. Clinical isolates of enterococci were assigned to E. faecalis and E. faecium species. Virulence factors on phenotype and genotype levels were detected in both species. Conclusion. Genetic determinants of virulence are more widespread among clinical isolates of E. faecalis species. Set of genes coding virulence factors in E. faecalis depends on biotope. Gene coding hyaluronidase synthesis is characteristic for E. faecium. A correlation between phenotypic manifestation of features and enterococci genotype was detected.

Aim. Study structure of a genetic determinant of amylase activity (amy gene) in Corynebacterium diphtheriae biovar gravis and mitis strains. Materials and methods. 87 C. diphtheriae strains (31 gravis biovar strains and 56 mitis biovar strains) as well as C. diphtheriae PW8 strain were analyzed to detect structural features of C. diphtheriae strains of various biovars. 10 pairs of primers were used in PCR that flank mutually overlapping regions within DIP0357 locus as well as additional primers that flank DIP0353 - DIP0354, DIP0357 and DIP0358 loci. Results. All the C. diphtheriae biovar gravis strains were established to contain a full-size DIP0357 locus (amy gene) whereas in all the mitis biovar strains this genome fragment is absent. All the studied C. diphtheriae biovar gravis strains do not have significant changes within DIP0354 - DIP0357 loci (amy gene) whereas in genome of 57 studied C. diphtheriae biovar mitis strains the major part of this fragment including the complete nucleotide sequence of amy gene is absent. Conclusion. C. diphtheriae biovar gravis strains have a genetically determined ability to produce amylase that can be viewed as an additional pathogenicity factor giving microorganisms wider capabilities to colonize the mucous membrane of oropharynx.

Aim. Evaluation of CD3+ and CD3-/CD56+ proliferative response on hepatitis C virus antigens in healthy medical workers. Materials and methods. The study included 15 medical workers with length of service of 2 and more years without common risk factors (blood and blood products transfusion, abdominal operations, invasive procedures, use of intravenous drugs). Control group consisted of 9 healthy individuals without risk factors. Peripheral mononuclears were isolated from blood and then incubated 72 hours in the presence of mitogen/PHA, Core+NS4 1b genotype HCV, NS4 HCV 2a+3a genotypes or medium. Proliferative activity was registered by the presence of cell division marker ki67 by using FACS. Results. The initial immunogram of medical workers differed from the control group by a significantly lower quantity of CD3+ lymphocytes, in particular CD3+/CD8 + population. Incubation with PHA resulted in a decrease of quantity ofCD3+/ ki67+, CD4+/ki67+ and CD3-/CD56+/ki67+ in the medical workers group. Cultivation with HCV antigens resulted in a significant decrease of Treg (CD3+/CD25high/FoxP3+) and activated T-lymphocytes population in the case of stimulation by Core and NS4 1b genotype antigens. Analysis of cell response on virus antigens based on proliferative activity index detected significant differences only for CD8+/ki67+. Stimulation by Core and NS4 1b genotype antigens resulted in an increase of quantity of these cells whereas in the case of NS4 2a+3a genotypes their decrease was observed. Conclusion. The described changes may reflect exhaustion of cell reactivity due to extra antigen load in the group of medical workers, while differentiated immunologic shifts on hepatitis C viruses of various genotypes are noted.

Aim. Study safety, reactogenicity, immunogenicity and prophylactic effectiveness of polysaccharide pneumococcus vaccines during immunization of adult HIV-infected patients. Materials and methods. 200 HIV-infected patients at stages 3 to 4A of the disease aged 20 to 50 years with the quantity of CD 4+ T-lymphocytes in blood of no less than 500 μl -1 took part in the study. 100 individuals immunized with polysaccharide 23-valent pneumococcus vaccine (Pneumo 23, Sanofi Pasteur, France) constituted the observation group. 100 individuals not immunized against pneumococcus infection constituted the comparison group. The groups were standardized by sex, age and disease stage. Vaccine reactogenicity was evaluated by detection of general and local postvaccination reactions, degree of their intensity and duration. Vaccine safety was evaluated based on comparative evaluation of results of general clinical and biochemical studies of blood, general urine analysis, determination of IgE in blood sera, CD 4+ T-lymphocytes level, quantity of HIV RNA (viral load) before vaccination and 28 days after the immunization. Vaccine immunogenicity was evaluated by determination in blood sera ofIgG against a mixture of Streptococcus рneumoniaе polysaccharides comprising Pneumo 23. Prophylaxis effectiveness of the preparation was evaluated by juxtaposition of acute respiratory illness morbidity in observation and control groups. Results. During immunization of HIV-infected patients against pneumococcus infection postvaccination complications, severe local and general postvaccination reactions were not detected, laboratory studies carried out before and after the immunization gave evidence on the lack of progression of the main disease and activization of the infectious process. After the immunization the geometric mean antibody titer against S. рneumoniaе increased by 2 times and reached protective level. Index of prophylactic effectiveness of Pneumo 23 vaccines during immunization of HIV-positive patients was 5.6, and relative risk of the disease in the immunized group - 0.07, in the control group - 0.42. Conclusion. The data provided give evidence on the high prophylactic effect of vaccination of immune compromised HIV-positive patients with a lack of deterioration of the main disease course.

Aim. Determine subpopulation composition of blood lymphocytes and the level of expression of TLR2 and TLR9 by epithelial cells of cervical canal mucous membrane in women of reproductive age with inflammatory disease of small pelvis organs (IDSPO) at exacerbation stage and remission period. Materials and methods. Clinical-laboratory and gynecological examination of 105 women was carried out and 3 groups were formed based on the results: patients at IDSPO exacerbation stage; patients at remission stage; clinically healthy women. By using real time PCR, TLR2, TLR9 gene expression levels were determined in epithelial cells of cervical canal mucous membrane in women of all the 3 groups. Subpopulation composition of blood lymphocytes was determined by flow cytofluorimetry by using monoclonal antibodies with CD45+ CD3+ -T-cell, CD45+ CD3+ CD4+ -Т-helper, CD45+, CD3 + , CD8+ -Т-suppressors-cytotoxic killers, CD45 + , CD3-, CD16+, CD56+ natural killers, CD45+, CD3-, CD19+ -В-lymphocytes. Immune fluorescence reaction evaluation was carried out in flow cytofluorimeter Cytomics FC 500 (Becton Coulter, USA). Results. The level of expression of TLR2 gene in the studied groups of patients was established not to differ significantly from parameters in the comparison groups, however it should be noted that this parameter in women with IDSPO at exacerbation stage (causative agents of the infectious process - ureaplasma, staphylococcus, candida) was somewhat higher than in the comparison group. Significantly high level of TLR9 gene expression in cervical canal epithelial cells was detected to correlate with the presence of infectious causative agents. In the group of women with exacerbation of the infectious process the expression of TLR9 was 14.5 times higher compared with the group of women without IDSPO. Among groups of women with IDSPO significant differences in relation to control group in relative and absolute levels of CD3 + T-lymphocytes; CD4+ T-helpers; CD8+ cytotoxic killer T-suppressors, B-lymphocytes compared with the same parameters in clinically healthy women were not detected. Conclusion. The increase of TLR9 gene expression level in cervical canal cells of women with IDSPO may serve as an additional diagnostic feature of the presence and degree of severity of the disease.

Aim. Evaluation of quality indicators of constructed cholera antigen polymer diagnosticums by using a complex of specific anti-cholera sera. Materials and methods. Cell lysates of cholera vibrio strains Vibrio cholerae cholerae 1395, V. eltor Ogawa 2044, V. eltor Inaba 13020, V. cholerae О139 16064 were sensitins for experimental preparations. 3 sera from cholera patients, normal human sera, cholera O1 (Ogawa, Inaba) commercial horse, cholera O139 commercial rabbit and heterologic sera against shigella, salmonella, escherichia and yersinia as well as experimental cholera rabbit sera against O1 and O139 were used as control. Results. The study established that diagnosticums based on V. cholerae cholerae 1395 and V. cholerae О139 16064 strain sensitins by quality indicators may be used in the future for construction of these diagnosticums. Conclusion. Antibody containing preparations - commercial horse O1 sera, rabbit experimental and commercial sera and MCA O139 demonstrating titers not lower than 1/5120-1/10240 may serve as a control of experimental diagnosticums in the absence of human sera from cholera patients.

Aim. Study of adhesive activity and growth speed of fungi Fusarium genus isolated from the surface of cutaneous covering in patients with atopic dermatitis (AD). Materials and methods. Clinical and museum F. oxysporum, F. solani strains were used in the study. Seeding was carried out into agarized Czapek and Sabouraud medium. The cultures were studied for 9 days at 30+2°C. adhesive properties of fungi were determined by using model based on nitrocellulose film (S.A.Li-sovskaya et al., 2006). Results. The studies carried out showed statistically significant differences in adhesive properties and intergrowth intensity of various Fusarium spp. Adhesion level of F. solani microconidia was almost 2.5 times higher compared with F. oxysporum. Formation of the first germ tube by F. solani microconidia was noted during the first 10 hours whereas by F. oxysporum - only at day 2. Conclusion. The data obtained confirm species differences of Fusarium spp. pathogenic properties that emphasize the importance of species identification of fungi.

Various macroorganism factors influence spontaneous and therapy induced elimination of hepatitis C virus. Standard therapy is application of a combination of pegylated interfernos and ribavirin. However such tactics results in attainment of a resistant virological response in approximately half the cases during infection by genotype 1 virus. The future of viral hepatitis C therapy lies most probably in the sphere of application of specific antiviral preparations such as protease and/or polymerase inhibitors as an addition to standard therapy. The aim of this review was to describe mechanisms of resistance to therapy in light of reaction of innate and adaptive immune response as well as an attempt to determine factors capable of prognosing response to therapy.

System of innate immunity is a set of effector molecules and cells counteracting invasion of pathogens and their products. AIpha-2-macroglobulin (a2-MG) and lactoferrin (LF) play a significant role in primary protection of organism. A wide spectrum of transport and regulatory functions, high affinity to endocytosis receptor as well as structural features of these proteins allow them not only to effectively protect the organism during direct contact with pathogen but also to have an immunomodulatory impact on immunocompetent cells of adaptive immunity. However despite a common receptor and a number of ligands, mechanisms of realization of protective functions of a2-MG and LF differ significantly. The aim of this review is to system-ize knowledge on means and mechanisms of protection of a2-MG and LF against invasion.