[Production of mycobacterial antigenes merged with cellulose binding protein domain in order to produce subunit vaccines against tuberculosis].

Protein genes Ag85A, Esat-6, and Cfp10 of Mycobacterium tuberculosis were sequenced using the database GenBank to implement selection and synthesis of primer pairs of given genes. PCR was used to obtain target amplicons of the genes. Chromosome DNA of M. tuberculosis H37Rv was used as the DNA amplification matrix. The PCR products were obtained using the plasmid pQE6, cloned, and amplified in the Escherichia coli M15 strain. Chimere products containing mycobacterial genes and cellulose binding protein domain (CBD), were obtained using the plasmid treated with restriction endonucleases. CBD fragment obtained using similar treatment of the ptt10 plasmid. The plasmids containing merged sequences of mycobacterial genes-antigenes and CBD were selected. The 3 mycobacterial genes were expressed in the E. coli M15 cells resulting in biosynthesis of corresponding recombinant proteins of expected molecular weight. Concentration of CBD, Cfp10-CBD, Ag85A-CBD, and ESAT6-CBD was 20%, 15%, and 15% total protein, respectively. The resulting chimere proteins provide high affinity for cellulose and high stability. Immobilization of CBD-containing recombinant proteins proceeds as one-stage process providing target protein purification and adsorption on cellulose. The vaccines produced using this technology are inexpensive because of low cost of cellulose sorbents as well as simultaneous use of cellulose for purification and immobilization of protein. Many cellulose preparations are not toxic, biocompatible, and widely used in medicine.

Effects of immunization with group a streptococcal antigens on the transplantability of mouse bone marrow stromal stem cells, counts of stromal precursor cells, and their osteogenic characteristics.

Immunization of CBA mice with killed group A streptococcus (type 5) vaccine changed the counts of stromal precursor cells (CFC-F) in bone marrow transplants at different donor-recipient combinations (normal, N, or immune, I). CFC-F counts in bone marrow transplants from normal mice transplanted to immunized animals decreased 4-6-fold depending on the transplant age in comparison with similar transplants in normal recipients. The percentage of CFC-F colonies with alkaline phosphatase (osteogenesis marker) activity decreased more than 2-fold. Similarly, the count of CFC-F in the transplants was 2-fold lower during delayed (7 months) period after bone marrow transplantation from immunized donors (8-12 days after the end of immunization) to intact recipients, while 2 months after transplantation it was 3-fold lower. The mean optical density of the bone capsule in preparations stained for glycogen and alkaline phosphatase was 1.5-3 times lower in the N-->I and I-->N experiments in comparison with the control (N-->N). On the other hand, CFC-F count in the femoral bone marrow of immunized animals was significantly (3.5-2.5 times) higher during the period from 8 days to 8 months after the end of immunization compared to CFC-F count in the femoral bone marrow of intact mice. These results attest to a significant prolonged effect of streptococcal antigens on the bone marrow stromal tissue. These data also indicate that not all CFC-F, the counts of which increased in response to antigens, are responsible for transplantability of the stromal tissue in heterotopic transplantation. Immunization by streptococcal antigens seemed to suppress transplantability and osteogenic activity of stromal stem cells. The efficiency of CFC-F cloning in mouse bone marrow cultures increased significantly (2-3-fold) in the presence of sera from immune mice. The levels of TNF-α and IFN-γ were low in this serum (2.7 and 6 times lower, respectively) in comparison with normal serum. Presumably, the effects of streptococcal antigens on stromal tissue were mediated through serum cytokines.

[Production of the recombinant human bone morphogenetic protein-2 in Escherichia coli and testing of its biological activity in vitro and in vivo].

Bone morphogenetic protein-2 (rhBMP-2) represents the osteoinductive protein factor which plays a dominant role in growth and regeneration of a bone tissue. In clinical practice the bone grafting materials on the basis of rhBMP-2 are widely applied; the Russian analogues of similar materials are not produced. The fragment of the bmp2gene coding for a mature protein was cloned in Escherichia coli. The effective overproducing strain of rhBMP-2 was created on a basis of the E. coli BL21 (DE3). The rhBMP-2 production was about 25% of total cell protein. The biologically active dimeric form of rhBMP-2 was obtained by isolation and purification of protein from inclusion bodies with subsequent refolding. The rhBMP-2 sample with more than 80% of the dimeric form was obtained, which is able to interact with specific antibodies to BMP-2. Biological activity of the received rhBMP-2 samples was shown in the in vitro experiments by induction of alkaline phosphatase synthesis in C2C12 and C3H10T1/2 cell cultures. On model of the ectopic osteogenesis it was shown that received rhBMP-2 possesses biological activity in vivo, causing tissue calcification in the place of an injection. The protein activity in vivo depends on way of protein introduction and characteristics of protein sample: rhBMP-2 may be introduced in an acid or basic buffer solution, with or without the carrier. The offered method of rhBMP-2 isolation and purification results in increasing common protein yield as well as the maintenance of biologically active dimeric form in comparison with the analogues described in the literature.

[Immunization of mice by group A streptococci and its effect on bone marrow stromal cells and level of serum cytokines].

AIM: To assess effect of continued immunization of mice from CBA line with inactivated group A streptococcal vaccine on levels of serum cytokines and number of stromal bone marrow progenitor cells in immunized mice and in heterotopic transplants after different variants of transplantation. MATERIALS AND METHODS: CBA mice were immunized during 3 weeks with heat-killed vaccine prepared from group A streptococci type 5. Levels of pro- and antiinflammatory cytokines were measured with BioPlex device. Number of stromal progenitor cells was determined on quantity of colonies formed by cells explanted to monolayer cultures. RESULTS: Significant 2.3-fold increase of number of stromal progenitor cells in femoral bone marrow of immune mice was demonstrated. Experiments with heterotopic transplants showed that bone marrow in transplants of mice immunized with streptococci--variant Normal-->Immune (N-->1)-- is defective both on efficacy of cloning and number of stromal progenitor cells. Even short-term presence of stromal tissue in immune organism (variant I -->N) significantly changed these parameters, especially at late time after transplantation. In serum of immune mice changes of cytokines levels, especially TNFalpha, were observed. The level of the latter was decreased (mean--2.5-fold) in all immune serum samples compared to normal serum. CONCLUSION: Immunization of mice with group A streptococci leads to changes in stromal tissue and, possibly, to damage of microenvironment functions including hemo- and lymphopoiesis.

[Feasibility to use of recombinant domains of immunoglobulin-like protein LigA as a promising marker for serological testing of patients with leptospirosis].

AIM: To clone the DNA fragment encoding conservative domain of LigA protein of Leptospira interrogans into Escherichia coli and to investigate antigenic properties of constructed chimeric protein. MATERIALS AND METHODS: E. coli strain M15 [pREP4], recombinant plasmid pTT10 encoding cellulose-binding domain (CBD), restriction endonucleases BamHI, BglI, BglII, XbaI, T4 DNA-ligase, RNAse were used in the study. Molecular cloning of ligA gene fragment was performed using standard protocols, and expression of hybrid genes--according to "Qiagen company's protocols. Extraction and purification of proteins were performed using original method. RESULTS: DNA fragment encoding immunoglobulin-like domain 5 of LigA was cloned in E. coli. Effective strain-producer of chimeric domain D5-CBD consisting of the immunoglobulin-like domain 5 of LigA, Gly-Ser spacer, and cellulose-binding domain (CBD) was obtained. The high-purity D5-CBD preparation was obtained using one-stage purification on cellulose. Antigenic specificity of this chimeric protein was studied and it was shown that it could be used as a marker for the development of diagnostic ELISA kit. CONCLUSION: Recombinant domain of LigA in chimeric protein produced in E. coli retains antigenic properties of native LigA protein. Obtained results confirm the feasibility to use recombinant antigen D5-CBD as a marker for development of diagnostic kits on the basis of ELISA.

[Isolation and characterization of collagen-binding domains from human von Willebrand factor].

DNA fragments encoding for two collagen binding decapeptides from the human von Willebrand factor (vWF-H1 and vWF-H2) were cloned in the Escherichia coil culture. Overproducing strains of the chimeric proteins vWF(H1)-CBD and vWF(H2)-CBD consisting of the corresponding decapeptide, Gly-Ser spacer and a cellulose binding domain (CBD) from Anaerocellum thermophilum were constructed. Using one-stage purification on cellulose, the highly purified samples of vWF(H1)-CBD and vWF(H2)-CBD proteins were obtained and the ability of these proteins to bind collagen was studied. These constructions are planned to be used for development of the recombinant collagen binding proteins with different biological activities, which, in its turn, will be used for development of the new generation products and materials for medicine, such as different kinds of implants, the coats, etc.

[Research into the immunogenic properties of the recombinant cellulose-binding domain of Anaerocellum thermophilum in vivo].

Development of new technology allows different antigens of a necessary degree of cleanliness to be obtained. This development is a major problem of modern medical biotechnology. A promising approach to this problem includes use of the affinity domains (tags) incorporated in structure of a recombinant antigen and capable to bind to corresponding sorbents. The method of preparation of ready-for-use injections containing complexes formed by soluble antigens on insoluble cellulose immunosorbent (not chemical conjugates) in one stage is based on the fusion protein technology. This approach includes preparation of two-component recombinant proteins containing an antigen of interest and the cellulose-binding domain (CBD), which spontaneously binds to cellulose containing sorbents with high binding constant. Research into the immunogenic properties of the CBD in the complex with cellulose and in the preparation of recombinant CBD in a rat model was performed. The titers of specific antibodies in rat serum induced by recombinant CBD and CBD in the complex with cellulose was evaluated. The CBD in the complex with cellulose was more immunogenic in comparison with CBD alone. The spectrum and levels of cytokines in collected rat serum induced by developed preparations was also measured using the microsphere-based Luminex Flowmetrix system (BioPlex). It was found that the amorphous cellulose was not an immunotolerant sorbent, because it induced the expression of the proinfammatory cytokines in vivo.

Effect of serum from mice immunized with group A streptococcus antigens on the efficiency of in vitro cloning of stromal precursor cells.

The efficiency of cloning of stromal precursor cell in mouse bone marrow culture increases significantly (2-3-fold) in the presence of serum from mice immunized with type 5 group A streptococcus antigens (5-20 microl serum/ml culture medium) in comparison with intact animal serum. The levels of TNF-alpha and IFN-gamma are significantly reduced (2.7 times and more than 6-fold, respectively) in the sera of immunized mice in comparison with normal serum. Serum levels of IL-2, -4, -5, -10, and -12 were about the same in both groups; no granulocyte-macrophage CSF was detected. These data attest to appreciable effect of immunization with streptococcal antigens on the bone marrow stromal tissue; this effect is presumably mediated through serum cytokines.

Genetic features of highly pathogenic avian influenza viruses A(H5N8), isolated from the European part of the Russian Federation.

Highly pathogenic avian influenza viruses (HPAIV) A(H5N8) of group B (Gochang1-like) have emerged in the Tyva Republic of eastern Russia in May 2016. Since November 2016, HPAIV A(H5N8) has spread throughout the European part of Russia. Thirty-one outbreaks were reported in domestic, wild and zoo birds in 2017. The present study aimed to perform a comparative analysis of new HPAIV A(H5N8) strains. Phylogenetic analysis revealed four genetically distinct subgroups in HPAIV A(H5N8) from the 2016-2017 season. Russian strains consisted of three subgroups with differences between isolates from Tyva, Siberia (Chany Lake), and the European part of Russia. Strains from the European part of Russia showed the beginnings of divergent evolution. Slight differences of the Voronezh strains were suggested by sensitivity to antiviral compounds. Testing for host-specific mutations in sequenced strains revealed the absence of mutations associated with possible increased tropism/virulence in mammalian species, including humans. Only one residue of polymerase basic-1, 13P, is discussed, because the L13P mutation increased complementary RNA synthesis in mammalian cells. We concluded that the evolution of HPAIV A(H5N8) is continuous. Surveillance in Russia revealed new cases of HPAIV A(H5N8) and led to the elaboration of prevention strategies, which should be implemented.

[Method of comparative evaluation of cytotoxicity of low-soluble dust by lowered activity of 5-nucleotidase in macrophage culture]. / Sposob sravnitelnoi otsenki tsitotoksichnosti malorastvorimykh pylei po snizheniiu aktivnosti 5-nukleotidazy v kulture makrofagov.

The authors suggest a new method to evaluate cytotoxicity of silicosis-dangerous dust through lowered activity of 5'-nucleotidase, a marker of cytolemma, in macrophageal culture. The scientists used the new method to compare cytotoxicity of 4 groups of industrial dust and correlated the results with those obtained in the test of lowered viability of macrophages (by incorporation of trypan blue). Both tests appeared to have correlation of the dust ranks according to cytotoxicity grade. Damage and activation of macrophages by products of their destruction change the cytotoxicity grade in the same direction, so the results could be considered more easily than for the tests where the changes are in opposite directions.

[Hygienic regulation of dust of magnesium-silicate minerals and fireproof materials]. / O gruppovoi gigienicheskoi reglamentatsii pyli magnezialno-silikatnykh mineralov i ogneupornykh materialov iz nikh.

Chronic experiments on rats showed that the dust of silicomagnesian refractory and dunite (a raw material for refractory production) extracted in Kytlym differ insignificantly in fibrogenic activity, but rank below the quartzite extracted in Pervouralsk (MAC-1 mg/cu m). The short study analyzing the cytologic changes of bronchoalveolar lavage determined the cytotoxicity of silicomagnesian refractory and 4 other types of dunite. The estimated cytotoxicity also ranked below that of quartzite from Pervouralsk. The MAC equal to 4 mg/cu m was suggested for the group of aerosols produced by dunite and silicomagnesian refractories. These aerosols are assigned to the 3rd jeopardy class and possess basically fibrogenic activity.

[Experimental study on risk factors of pneumoconiosis caused by dust in the industry of new construction materials containing glass and coal waste]. / Eksperimentalnoe izuchenie pnevmokoniozoopasnosti pylevogo faktora v proizvodstve novogo tipa stroitelnykh materialov iz stekla i ugolnogo shlaka.

The study compared cytotoxicity and fibrogenic ability of quartzitic dust to that appearing at 3 sites of light building filler production based on ground glass or clinker. Obtained results prove possible pneumoconiosis development caused by exposure to such dust types, especially in the production using ground glass. The MAC in this type of production is 2 mg/cu m, for the dust in production using clinker the MAC is 4 mg/cu m.

[Significance of short-term cytotoxicity tests for hygienic assessment of industrial aerosols]. / O znachenii sistemhy kratkovremennykh testov na tsitotoksichnost dlia gigienicheskoi kharakteristiki promyshlennykh aérozolei.

The evaluation of low-soluble particles toxicity for human phagocytes (primarily, for macrophages) was proved to have advantages in predicting their effects on the human body and in rapid toxico-hygienic regulation. The preference is given to tests "in vitro". If comparative toxicities of substances in group are discordant according to different "in vitro" tests the decision must be made after the "in vivo" evaluation of cytotoxicity: in 24 hours after the intratracheal administration of small doses of these low-soluble particles the bronchoalveolar lavage is examined cytologically. Reliable neutrophilia in lavage is of great value.