[EFFECT OF INFLUENZA VACCINES ON SUBPOPULATIONS OF BLOOD DENDRITIC CELLS].

AIM: Study the effect of Vaxigrip split, Influvac subunit and Grippol plus immune-adjuvanted vaccines on the content of myeloid (mDC) and plasmacytoid (pDC) dendritic cells (DC) in blood of vaccinated healthy women. Materials andmethods. Blood of 30 healthy women aged 18-50 years was studied at days 7 and 30 after the vaccination. pDC (CD14+CD16-/CD85k(ILT3)-PE/ CD123-PC5) and mDC (CD14+CD-16-/CD85k(ILT3)-PE/CD33 -PC5) immune phenotyping was carried out using mAbs (Beckman Coulter, France) and flow cytometer Cytomix FC-500 (Beckman Coulter, USA). RESULTS: Use of unadjuvanted vaccines Vaxigrip and Influvac resulted in an increase of the numbers of mDC and pDC (p<0.05) in blood of the vaccinated only at day 7 of the observation. Grippol resulted in a more significant (2.2 - 3.6 times, p<0.05) increase of DC subpopulations (compared with unadjuvanted vaccines) at both day 7 and a month after the vaccination. CONCLUSION: Influenza vaccination activated innate effectors - the first component on the way of infection penetration - dendritic cells of both myeloid and lymphoid origin. Wherein, a more pronounced and prolonged effect of such activation is observed when immune-adjuvanted vaccine is used compared with subunit and split vaccines.

[Cloning of alternative isoforms of the catalytic subunit of the human thelomerase (htert)].

Most human tumors, including cervical cancer, are characterized by telomerase activation (cell proliferation activation enzyme). Such activation is implemented in the elongation of the terminal segments (telomeres) of the telomerase chromosome. The gene of the enzyme is RNA-encoded, the RNA in tumors being observed in a few isoforms. The hTERT RNA role in cell activation and control was simulated using cervical cancer, as well as its pretumoral states (CIN), as a model object. The goal of this work was to clone of the human hTERT isoforms (normal, α-, ß-, and α+ß-splice-variants). The genetic constructions containing normal hTERT sequence, α- and ß-deletion variants based on the lentivirus vector pR780 were obtained. The α- and ß-deletion variants were not obtained in this variant because of methodological problems. In further research, we plan to implement splice-variants of hTERT in eukaryotic human cells.

[Features of dynamics of sera cytokines in patients with erythema multiforme during immunotherapy].

AIM: Study the features of cytokine profile in patients with exudative erythema multiforme (EME) and dynamics of basal level of pro-inflammatory and anti-inflammatory cytokines during immunotherapy. MATERIALS AND METHODS: 39 adult patients with erythema multiforme were examined. The patients were split into groups based on therapy variant. One group (14 individuals) received Immunovac-VP-4 against the background of basic therapy; the other (12 individuals)--cagocel against the background of basic therapy; comparison group (13 individuals) received only basic therapy; 15 individuals composed a group of healthy individuals. All the patients had the level of pro-, anti-inflammatory and regulatory cytokines determined in blood sera by solid-phase EIA method by using Biosource (Austria) test-systems at the beginning of the study and after the therapy. RESULTS: In patients with exudative erythema multiforme Immunovac-VP-4 therapy facilitated a significant (p < 0.05) increase of serum IFN-gamma level, insignificant (p > 0.05) increase of IL-1beta and decrease of IL-17. Whereas cagocel lead to an increase of IL-4 (p > 0.05), IL-2, IFN-gamma (p < 0.05) and decrease of TGF-beta and IL-12 (p < 0.05). At the same time basic therapy facilitated a significant increase of IL-5 and decrease of IL-6, IL-12, IFN-gamma. CONCLUSION: Immunovac-VP-4 facilitates the increase of secretion of IFN-gamma, IL-1beta against the background of TGF-beta that facilitates normalization of cooperation of cells in immune response including against viral infections, and thereby influencing the trigger factor in EME patients.

[Activating effect of a germanium-organic compound on immunocompetent cells during intranasal immunization of mice with a live influenza vaccine].

AIM: Detailed characteristic of results of intranasal immunization of mice with one of two variants of vaccinating influenza virus, particularly in combination with a low molecular weight germanium-organic compound (LMW-GOC). An additional aim is evaluation of effect of LMW-GOC on the parameters of immune system in case of intranasal administration of the preparation without the addition of vaccinating virus. MATERIALS AND METHODS: The study was carried out in female CBA mice (18-20 g, 6 animals per group). Intranasal immunization was carried out by 2 different variants of B/Victoria influenza virus--once or twice with a 2 week interval. Cells for study were obtained from spleen and nasal- and bronchial-associated lymphoid tissue (NALT/ BALT) 24 hours and 7 days after intranasal administration of the preparations. The main method of the study--determination of the level of expression of various markers oflymphocytes in comparison with the level of the same markers in the cells of control group animals by using flow cytometry method. The mean parameters obtained were determined by using program package WinMDI 2.8. RESULTS: The main results were the increase of level of expression of various lymphocyte markers obtained from mice after intranasal administration of the vaccines and their combination with LMW-GOC or LMW-GOC only without the participation of the vaccines. A significant increase of the expression of TLR9 marker compared with other parameters was noted. Administration to mice of wild B/Victoria strain notably more frequently conditioned the decrease of expression of some parameters compared with administration of the cold adapted strain. Effect of LMW-GOC without the vaccine also conditioned the increase of levels of markers however a combination of the preparations with the vaccine was more effective. CONCLUSION: The increase of level of expression of a number of lymphocyte markers may serve as a sign of successful intranasal vaccination against influenza. LMW-GOC preparation increases immune stimulating effect of intranasally administered vaccines and in none of the cases weakens the stimulating result of effect of the vaccines, and in many cases increases it. LMW-GOC may be studied as a main or additional adjuvant for intranasal application of influenza vaccines.

[Effect of Immunovac-VP-4 and Kagocel immunotherapy on cytokine levels in patients with erythema nodosum].

Fixed erythema--a kind of clinical and histopathologic reaction, fixed drug eruption. The purpose of the study--the study of characteristics of the cytokine profile in patients with erythema and the dynamics of the basal levels of proinflammatory and antiinflammatory cytokines during immunotherapy. All 41 patients with fixed erythema at baseline and after treatment was carried out determination of levels of pro-, anti-inflammatory and regulatory cytokines in the serum by ELISA using test systems "Biosource" (Austria). In patients with fixed erythema Immunovac treatment increased serum IFN-gamma (p < 0.05), IL-1beta (p > 0.05), IL-6. While Kagocel led to an increase in IFN-gamma (p < 0.05), IL-1beta, IL-6 and reduction of TGF-beta (p < 0.05). At the same time in patients with fixed erythema basic therapy contributed to the significant increase in TGF-â and decrease in IL-10. Immunovac-VP-4 had the highest activity for the induction of IFN-gamma. Inclusion in the range of therapeutic and prophylactic measures in patients with fixed erythema immunomodulators promotes activation links innate and adaptive immunity triggers mechanisms, thus increasing the antiviral response in patients with erythema.

[Activation of lymphocytes under the influence of an influenza vaccine combined with a low molecular weight germanium organic compound].

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 CD3, CD4, CD5, CD8, CD19, CD25, Foxp3, NK1.1, gammadelta 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.

[Organization of metabolic pathways and molecular-genetic mechanisms of xenobiotic biodegradation in microorganisms: a review].

Contemporary data on the mechanism of biodegradation of aromatic hydrocarbons and biodegradation genes (genomic organization and pathways of evolution) in diverse groups of microorganisms have been reviewed. Studies of this problem are topical, in view of the need in identification and construction of new strains degrading xenobiotics, particularly those halogenated. For this reason, emphasis is placed on specific features of explored metabolic pathways that can be used for constructing new enzymatic systems not present in nature. Sections on the mechanisms of genomic rearrangements involving biodegradation determinants are presented from the same standpoint. Part of the review is devoted to analyzing methods used for studying the population dynamics of bacterial communities involved in xenobiotic degradation in natural biotopes or industrial waste disposal plants. Particular attention is given to methods of gene systematics.

[Cloning and expression of the IGA-specific endopeptidase genes from Neisseria meningitidis].

IgA1-specific proteinases (Igase) are acknowledged as a pivotal pathogenicity factor in meningococcus (Neisseria meningitidis) and in some related bacteria. These enzymes belong to trypsin-like clan of serine proteases. They exhibit high substrate selectivity being able to discriminate between IgA1 and IgA2. On the other hand, these enzymes are able to distinguish the human IgA1 from IgA1 of non-primate species of mammals. In addition to conventional IgA1-processing enzymes, alternative enzymes were recently reported to occur in meningococci. However, the substrate specificity of the conventional Igase, its role in pathogenesis, and ability to complement functionality remains obscure. Within the framework of the present project we studied the structure of the Igase genes and their products in two highly virulent N. meningitidis serogroup A strains M9 and A208. In particular, we succeeded to find both conventional and alternative Igase genes in each genome: nucleotide sequences of these genes were deposited in the NCBI Gene Bank under the access number AY770504, AY558158, AY558159. The DNA sequence of the conventional Igase was almost entirely conserved in the two strains, whereas the recently discovered alternative Igase (formerly known as meningococcal adhesine, type 1) exhibited occurrence of a variable region spanning about 900 bp in the 5'-terminal part of the gene. Conventional genes from both strains were expressed in E. coli rendering inclusion bodies. The recombinant products were used for immunization of rabbits and exhibited reaction with both recombinant and native antigen from the N. meningitidis cultural medium.

[A new Escherichia coli strain producing human tumor necrosis factor].

An Escherichia coli strain producing human tumor necrosis factor (TNF-alpha) was obtained using a semisynthetic gene partially optimized in respect of codon composition and a phage T7 promoter. The expression product was accumulated in cells as inclusion bodies in a yield of 50-70 mg/l of culture medium. The recombinant TNF-alpha in the form of inclusion bodies was used for immunization of rats to give a polyclonal antiserum. The resulting antibodies were specific under the immunoblotting conditions to the antigen used for the immunization. A dilution-based refolding procedure was developed; it provided a yield of soluble protein exceeding 85%.

[Metabolic pathways responsible for consumption of aromatic hydrocarbons by microbial associations: molecular-genetic characterization].

Genes for catechol 1,2- and 2,3-dioxygenases were cloned. These enzymes hold important positions in the ortho and meta pathways of the metabolism of aromatic carbons by microbial associations that consume the following volatile organic compounds in pilot minireactors: toluene, styrene, ethyl benzene, o-xylene, m-xylene, and naphthalene. Genes of both pathways were found in an association consuming m-xylene; only genes of the ortho pathway were found in associations consuming o-xylene, styrene, and ethyl benzene, and only genes of the meta pathway were found in associations consuming naphthalene and toluene. Genes of the ortho pathway (C120) cloned from associations consuming o-xylene and ethyl benzene were similar to corresponding genes located on the pND6 plasmid of Pseudomonas putida. Genes of the ortho pathway from associations consuming o-xylene and m-xylene were similar to chromosomal genes of P. putida. Genes of the meta pathway (C230) from associations consuming toluene and naphthalene were similar to corresponding genes formerly found in plasmids pWWO and pTOL.

[Application of molecular systematics to study of bacterial cultures consuming volatile organic compounds].

A range of species of four mixed bacterial cultures was studied by molecular systematics methods with the use of 16S rRNA genes. The cultures had been developed for application in minireactors, to degrade volatile organic compounds (VOCs): ethyl benzene, m-xylene, styrene, and o-xylene. A sample of 30 plasmid rDNA clones was obtained for each of the mixed cultures. The clones were analyzed by RFLP according to two restriction sites. Major variants of the 16S-rDNA sequences, corresponding to the most abundant species, were determined for each association. Sequencing of four clones of predominant 16S-rDNAs showed that the culture consuming ethyl benzene was dominated by Pseudomonas fluorescens; o-xylene, by Achromobacter xylosoxydans; styrene, by Pseudomonas veronii; and m-xylene, by Delftia acidovorans. Minor components of all four cultures were generally similar. They included species of the genera Sphingobacter, Rhizobium, Mesorhizobium, Pedobacter, and Paenibacillus. Sampling sequencing of genes for 16S rRNA cloned from total genomic DNA allowed quantitative determination of the composition of actual bacterial associations consuming VOCs in minireactors.

[NP-detecting DNA technologies: solving problems of applied biochemistry].

Heterozygosity of CANP3, ACTN3, and GHR genes in specialized collections was studied using state-of-the-art DNA technologies for DNA analysis. A new dinucleotide deletion (AC) at the beginning of exon 21 was identified in five individuals with heterozygous CANP3 gene. Analysis of polymorphism (SNP1747 C-->T) of ACTN3 gene demonstrated a positive association of allele C with a high muscular performance. Real-time PCR assay of SNP1630 (A-->C) in GHR gene suggested a putative negative association of allele C of this SNP with a high muscular performance.

Isolation and oligomeric composition of cytochrome c nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens.

A new procedure for isolation of cytochrome c nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens increasing significantly the yield of the purified enzyme is presented. The enzyme is isolated from the soluble fraction of the cell extract as a hexamer, as shown by gel filtration chromatography and small angle X-ray scattering analysis. Thermostability of the hexameric form of the nitrite reductase is characterized in terms of thermoinactivation and thermodenaturation.