[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.

[TYPING OF LEPTOSPIRA SPP. STRAINS BASED ON 16S rRNA].

AIM: Comparative typing of Leptospira spp. strain collection based on analysis of 16S RNA fragment. MATERIALS AND METHODS: 2 pairs of primers were used for PCR, that jointly flank 1423b.p. sized fragment. Sequences of Leptospira spp. strain 16S rRNA, presented in the international database, were used for phylogenetic analysis. RESULTS: A high similarity, including interspecies, of the 16S fragment in Leptospira spp. strains was shown independently of the source, serovar and serogroup. Heterogeneity of the primary matrix, spontaneous mutations of hotspots and erroneous nucleotide couplings, characteristic for 16S sequence of pathogenic Leptospira spp. strains, are discussed. Molecular-genetic characteristic of certain reference Leptospira spp. strains by 16S sequence is obtained. CONCLUSION: Results of the studies give evidence on expedience of introduction into clinical practice of identification of Leptospira spp. by 16S sequence directly from the clinical material, that would allow to significantly reduce identification time, dismiss complex type-specific sera and other labor-intensive methods.

[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.

Interspecific polymorphism of gene lipL32 restriction profiles of pathogenic leptospires.

We analyzed restriction fragment length polymorphism of lipL32 gene encoding outer membrane protein in three Leptospira genomic species (L. interrogans, L. kirschneri, and L. borgpetersenii) using Bsa29 I and Bam HI restriction endonucleases. It was found that restriction profiles of the studied gene were similar at both the intraspecific and serogroup levels; variability was revealed only at the interspecific level, which can be explained by relatively low variability of lipL32 gene.

Surface-plasmon-mediated single-molecule fluorescence through a thin metallic film.

We demonstrate that fluorescence of single molecules in the nanometric vicinity of a thin gold film can be effectively excited and detected through the film with an epi-illumination scanning confocal microscope. A full theoretical treatment of the fluorescence signal indicates that both excitation and emission are surface-plasmon mediated. Remarkably, the number of photons detectable from chromophores perpendicular to the interface is enhanced by the presence of the metal.

Altered phenotype and function of blood dendritic cells in multiple sclerosis are modulated by IFN-beta and IL-10.

Multiple sclerosis (MS) is assumed to result from autoaggressive T cell-mediated immune responses, in which T helper type 1 (Th1) cells producing cytokines, e.g. IFN-gamma and lymphotoxin promote damage of oligodendrocyte-myelin units. Dendritic cells (DCs) as potent antigen presenting cells initiate and orchestrate immune responses. Whether phenotype and function of DCs with respect to Th1 cell promotion are altered in MS, are not known. This study revealed that blood-derived DCs from MS patients expressed low levels of the costimulatory molecule CD86. In addition, production of IFN-gamma by blood mononuclear cells (MNCs) was strongly enhanced by DCs derived from MS patients. IFN-beta and IL-10 inhibited the costimulatory capacity of DCs in mixed lymphocyte reaction (MLR) and showed additive effects on suppression of IL-12 production by DCs. Correspondingly, DCs pretreated with IFN-beta and IL-10 significantly suppressed IFN-gamma production by MNCs. IFN-beta in vitro also upregulated CD80 and, in particular, CD86 expression on DCs. In vitro, anti-CD80 antibody remarkably increased, while anti-CD86 antibody inhibited DC-induced IL-4 production in MLR. We conclude that DC phenotype and function are altered in MS, implying Th1-biased responses with enhanced capacity to induce Th1 cytokine production. In vitro modification of MS patients' DCs by IFN-beta and IL-10 could represent a novel way of immunomodulation and of possible usefulness for future immunotherapy of MS.

Further studies on Nivalin P-induced changes in muscle fiber membrane processes.

Nivalin P, composed of Nivalin (galanthamine hydrobromide) and Pymadin (4-aminopyridine hydrochloride), was applied extracellularly to isolated skeletal muscle fibers during prolonged activity (fatiguing) to better understand the effects of the drug on membrane ionic processes. Changes in intracellular action potential (ICAP) and twitch (Tw) parameters were monitored from treated and untreated fibers during uninterrupted activity (endurance time, ET) produced by repetitive stimulation every 200 msec for 3 min. Nivalin P-induced a shortening of the ET, drastic changes in repolarization of the ICAP corresponding to changes in negative afterpotential and falling area and an initial increase of the Tw amplitude and duration. These results suggest that Nivalin P: (i) inhibits the Na+, K(+)-pump due to nonspecific reduction of Na+ influx, stimulates the Na(+)-Ca2+ exchanger and inhibits K+ conductance; (ii) increases Ca2+ release and delays Ca2+ uptake under sufficient depolarization. It was concluded that fatigue develops faster in the presence of Nivalin P.