[IDENTIFICATION OF LEPTOSPIRA SEROVARS BY MALDI-TOF MASS-SPEC- TROMETRY].

AIM: An attempt to use MALDI-TOF mass-spectrometry method for identification of lep- tospiral isolates on the serovar level. MATERIALS AND METHODS: 8 reference Leptospira spp. and 11 leptospira strains isolated from leptospiral patients and infected animals in the North-Western region of Russia were included into the study. Mass-spectra of all the studied strains were obtained by direct profiling of cell extracts. The created main spectral profiles (MSP) of reference strains were used for identification of isolates. Evaluation of identification was carried out by calculating coefficients of matching rate of separate spectra of each isolate with MSP of all the reference strains. RESULTS: Results of identification have shown the similarity of spectra of isolates belonging to Pomona, Icterohaemorrhagiae and Canicola serogroups, with MSP of saprophyte strain L. biflexa Patoc I. It is assumed that spectra of the studied strains contained peaks of polysaccha- ride Ο-antigens. Wherein maximum mean values of matching rate coefficients between spectra of isolates and MSP of pathogenic reference strains of leptospira correctly matched serovar type of the isolate. CONCLUSION: Further extended studies may form the base of development of a simple and rapid method oftyping of leptospirosis causative agents on the level of serovars using MALDITOF mass-spectrometry.

[MOLECULAR-BIOLOGICAL MARKERS OF HEPATITIS B IN PATIENTS WITH LIVER FIBROSIS/CIRRHOSIS IN UZBEKISTAN].

AIM: Evaluate prevalence of genetic variants of hepatitis B, viruses in population of various regions of Uzbekistan with hepatitis of various genesis and different severity levels of liver fibrosis and cirrhosis. MATERIALS AND METHODS: Blood plasma and liverbiopsy from 39 patients with different severity levels of liver fibrosis and cirrhosis served as study material. Genotyping based on direct sequencing of Pre-S1/Pre-S2/S HBV DNAregion was applied. RESULTS: Hepatitis B virus was detected in 32 samples ofthe 39 provided, frequency of occurrence - 82%, respectively. Phylogenetic analysis has shown, that only genotype D was detected among the examined,patients, hepatitis B virus subtype D1 predominated (84.38%) compared with D2 (3.12%) and.D3 (12.5%) subtypes. CONCLUSION: Large-scale sequencing of HBV in, Central Asia will allow to evaluate routes of transmission and time of evolutionary separation or virus isolates. Understanding the epidemiology of the infectious process is important for development of programs for prophylaxis and therapy of the infection.

[HBV COVALENTLY CLOSED CIRCULAR DNA AS A MARKER OF PREVALENCE OF OCCULT HEPATITIS B IN PATIENTS WITH HBV, HDVAND HCV INFECTION IN UZBEKISTAN].

AIM: Evaluate significance of covalently closed circular DNA of hepatitis B virus as a marker for detection of occult viral hepatitis B in Uzbekistan population with hepatitis of various genesis. MATERIALS AND METHODS: Blood plasma and liver biopsy from 39 patients with different severity levels of liver fibrosis and cirrhosis served as study material. HBV covalently closed circular DNA detection was carried out according to Pollicino T et al. (2004). RESULTS: Covalently closed circu- lar DNA of hepatitis B virus was detected in 82% of samples, including in 54.5% of patients with chronic viral hepatitis C (CVHC) and in 100% of patients with hepatitis of unknown etiology. Quantitative evaluation of content of covalently closed circular DNA of hepatitis B virus in liver tissue in patients with CVHB has shown an average of 2.5 copies of HBV genome as ccc DNA per cell, in patients with CVHB + D an average of 0.7 copies/cell, in patients with co-infection by HCV and HBV - 0.5 copies/cell, in patients with CVHC an average of 0.12 copies/cell, and in patients with cryptogenic hepatitis - 0.2 copies/cell. CONCLUSION: Detection of HBV DNA is a complex problem for effective laboratory diagnostics of hepatitis. Detection of HBV ccc DNA as a marker of occult hepatitis B in patients with CVHC and patients with hepatitis of unclear etiol- ogy is an. important factor for diagnostics, selection of adequate therapy, prognosis of disease outcome and prevention of development of severe liver diseases.

[PRODUCTION OF CERTAIN PRO- AND ANTI-INFLAMMATORY CYTOKINES DURING STIMULATION BY LIVE AND INACTIVATED LEPTOSPIRA PATHOGENS IN THE MODEL OF HUMAN WHOLE BLOOD].

AIM: Study of the ability of clinical isolates of leptospira to cause production of certain pro- and antiinflammatory cytokines in the model of human whole blood. MATERIALS AND METHODS: Leptospira interrogans strain was taken for the experiment. Cytokine content was determined by a method based on xMAP technology using a standard panel, composed of 9 analytes: TNF-α, MCP-1, IL-8, IL-4, IL-6, IL-10, IL-IRa, IL- 12 (p70), IFN-γ. RESULTS: An optimal concentration of L. interrogans was selected for stimulation of human whole blood--1 x 10(6) leptospirae/ml. For the first time in the model of human whole blood it was determined, that at early stages of incubation IFN-γ, IL-12(p70), IL-4 and IL-1Ra are more actively produced; at later stages (6 hour incubation)--IL-8 and TNF-α. CONCLUSION: A differential pattern of cytokine production stimulation was shown in the model of human whole blood by live and inactivated leptospirae.

[MALDI-TOF MASS-SPECTROMETRIC ANAIYSIS OF LEPTOSPIRA SPP. USED IN SERODIAGNOSTICS OF LEPTOSPIROSIS].

AIM: Creation of a classification model of Leptospira spp. serovar model using ClinProTools 3.0 software and evaluation of use of MALDI-TOF MS as a method of quality control of reference strains of leptospira. MATERIALS AND METHODS: 10 reference strains of Leptospira spp. were used in the study according to microscopic agglutination reaction from the collection of Pasteur RIEM. All the strains were cultivated for 10 days in Terskikh medium at 28 degrees C. Cell extracts were obtained by ethanol/formic acid method. α-cyano-4-hydroxycinnamic acid solution was used as a matrix. Mass-spectra were obtained in Microflex mass-spectrometer (Bruker Daltonics, Germany). External validation of the test-model was carried out using novel spectra of every reference strain during their repeated reseeding. RESULTS: Values of cross-validation and confirmatory ability of the optimal model, built on a genetic algorithm, was 99.14 and 100%, respectively. This model contained 11 biomarker peaks (m/z 2959, 3447, 3548, 3764, 3895, 5221, 5917, 6173, 6701, 7013, 8364) for serovar classification. Results of the external validation have shown a 100% correct classification in serovar classesin Sejroe, Ballum, Tarassovi; Copenhageni, Mozdoc, Grippotyphosa and Patoc, that indicates a high prognostic ability of the model in these classes. However, data from verification matrix have shown, that 50%.of the spectra from Canicola and Pomona serovars were classified as Patoc class, that could be associated with cross serological activity of Patoc serovar L. biflexa with pathogenic leptospirae. CONCLUSION: MALDI-TOF mass-spectrometry method combined with building and using the classification model could be a useful instrument for intra-laboratory control of leptospira reseeding.

Synthesis of some CC chemokines and their receptors in the synovium in rheumatoid arthritis.

We studied the expression of some CC chemokines and their receptors in the synovium of patients with rheumatoid arthritis, osteoarthrosis, and a history of joint injury. In patients with rheumatoid arthritis, the levels mRNA for some angiogenic and proinflammatory chemokines (CCL5/RANTES, CCL11/eotaxin, CCL24/eotaxin-2, and CCL26/eotaxin-3) and their receptors (CCR1, CCR2, CCR3, CCR4, and CCR5) was elevated. mRNA expression correlated with activity, stage, and serological status of rheumatoid arthritis. Obtained data confirm the importance of CC chemokines as mediators of angiogenesis and inflammation in the synovium in rheumatoid arthritis.