[Identification of Mycoplasma in patients with suspected prostate cancer].

AIM: Tests for Mycoplasma hominis, M. genitalium, Ureaplasma urealyticum in males with suspected prostate cancer. MATERIALS AND METHODS: Identification of mycoplasms was performed in prostate tissue samples using universal PCR as well as in serum samples of patients with suspected prostate cancer using ELISA for detection of IgG to M. hominis. Two hundred and fifty samples from each lobe of prostate were obtained from 125 patients with suspected prostate cancer by transrectal polyfocal biopsy. Blood samples were drawn from the same patients for ELISA. RESULTS: Out of 125 patients with suspected prostate cancer, 20.5% were positive for Mycoplasma by PCR. Between studied species, only M. hominis was found in big proportion of analyzed samples. Out of 118 serum samples, 30.5% were positive for IgG to M. hominis in ELISA. CONCLUSION: Fact of presence of Mycoplasma species in tissue of prostate was established in 20.5% pf patients with suspected prostate cancer. Obtained results show that M. hominis is frequently infects prostate tissue and that this infection was more common in patients with high grade prostatic interstitial neoplasia and prostate cancer than in patients with benign changes of prostate tissue or in persons without prostate disease. This allows to suggest that infection with M. hominis could play an important role in development of cancer.

[Protective properties of candidate genetically engineered vaccines against avian influenza viruses constructed on the basis of recombinant adenoviral vectors].

AIM: To design and study the properties of candidate vaccines against avian influenza based on recombinant adenoviral vectors expressing H5 hemagglutinin. MATERIALS AND METHODS: Recombinant adenoviral vectors were constructed as described in "Stratagene" (Ad Easy Adenoviral Vector System). For immunization of animals, recombinant candidate vaccines were administered intranasally twice. Titer of hemagglutinating antibodies were measured by hemaglutination inhibition assay. RESULTS: It was demonstrated that administration of vaccines to animals completely protects them from a lethal dose challenge with H5N2 influenza virus. Protective effect of vaccines remained for 6 months after immunization. Additionally, highly effective cross-protection of the immunized animals against heterologous strain of H5 influenza virus was demonstrated. CONCLUSION: Obtained results show good prospects for usage of recombinant adenoviral vectors as a basis for development of new generation effective vaccines against influenza.

[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.

[Recombinant pseudoadenovirus nanostructure with human lactoferrin gene: production and study of lactoferrin expression and properties in vivo usage of the construction].

The Ad5-Lf pseudoadenovirus nanostructure (RPAN) was produced using homologous recombination in E. coli cells. This construction provided efficient expression of the Lf gene in permissive cell culture with high production rate of recombinant protein similar to native human Lf in some physical, chemical, and biological properties. Single intravenous injection of the construction into mice and rats was effective for prolonged production and circulation of recombinant human Lf in blood of experimental animals without toxic effects. The produced construction is promising for providing prolonged production of recombinant human Lf in the human body.

[Production of human recombinant beta-interferon in the avian cell culture].

The avian recombinant adenovirus of serotype 1 (CELO) was obtained. The recombinant adenovirus of serotype 1 (CELO) induces expression of human beta-interferon (IB). The expression cassette containing IB gene was placed at the right end of the CELO genome under control of hybrid promoter hEF-1alpha/HTLV. The resulting recombinant adenovirus CELO-IB transduced the avian cell culture LMH. The level of production of the recombinant IB was 0.15 micro/ml. The IB protein yield after affine chromatography purification using Ni-NTA agarose was 50%. The biological activity of the purified IB was high (7.8 x 10(8) MU/microg protein). The purified IB inhibited replication of murine encephalomyocarditis virus (VMEC) in cell culture of human diploid fibroblasts (HDF). Thus, expression system based on avian cell culture is an effective system for producing biologically active protein of human interferon beta.

[Immunization with cellulose-immobilized antigens. The development of A. E. Gurvitch concept].

A highly purified TUL4-CBD chimeric protein was obtained by one stage purification method. TUL4-CBD protein consists of TUL4 Francisella tularensis mature peptide sequence, Gly-Ser spacer and cellulose binding domain (CBD) of Anaerocellum thermophilum. The TUL4-CBD protein was shown to induce production of specific antibodies to TUL4 protein in laboratory animals.

[The CELO-ANG recombinant avian adenovirus with human angiogenine gene inducing neovascularization in the anterior tibial muscle of rat]. / Recombinantnyi adenovirus ptits CELO-ANG, nesushchii gen angiogenina cheloveka, indutsiruet neovaskuliarizatsiiu v perednei bol'shebertsovoi myshtse krysy.

The CELO recombinant avian adenovirus carrying the gene coding the human angiogenine (ANG) synthesis was obtained. Expression of the angiogenine gene was shown in the LMH cell culture after infection with the CELO-ANG virus. The ability of CELO recombinant adenoviruses to carry out the delivery and expression of alien genes in muscle cells was demonstrated in experiments with laboratory animals (Wistar line rats). The induced neovascularization in rat muscles after the animals were administered the CELO-ANG viruses was shown.

[Eukaryotic vectors of Celo avian adenovirus genome, carrying GFP and human IL-2 genes]. / Eukarioticheskie vektory na osnove genoma adenovirusa ptits Celo, nesushchie geny GFP i IL-2 cheloveka.

Recombinant CELO avian adenoviruses carrying green fluorescent protein (GFP) and and human interleukin-2 (IL-2) genes were obtained by homologous recombination in cell culture. The resultant recombinant CELO viruses are reproduced in chick embryos in the renal tubular and chorionic allantoic membrane cells. The ability of CELO vectors to transduce human and animal cells was studied in vitro (in cell cultures) and in vivo (in laboratory animals). GFP gene delivery and expression in recombinant CELO virus in tumors in C57BL/6 mice were for the first time demonstrated for B16 melanoma. Human IL-2 gene expression and protein accumulation in allantoic fluid of chick embryos infected with CELO-IL-2 vector were detected for the first time.

[Delivery of secreted placental alkaline phosphatase (SEAP) gene in vitro and in vivo as a component of recombinant avian adenovirus (CELO)]. / Dostavka in vitro i in vivo gena sekretiruemoi platsentarnoi shchelochnoi fosfatazy (SEAP) v sostave rekombinantnogo adenovirusa ptits (CELO).

Recombinant adenoviruses capable of expressing the gene of secreted placentary alkaline phosphatase (SEAP) under control of CMV-promoter was obtained on the basis of CELO avian adenovirus and human adenovirus-5 (Ad5) genomes. The efficiency of the CELO vector was determined in experiments with transduction of human (293, A549, and H1299), mouse (B16), and avian (LMH) cell cultures. It was shown in C57BL/6 mice in vivo that SEAP gene is expressed under conditions of intravenous, intranasal, and intratumoral application of recombinant adenovirus CELO-SEAP. The duration of expression of the alkaline phosphatase CELO = SEAP gene in immunocompetent mouse body was 21 days. The level of SEAP gene expression was measured in the allantois fluid of chicken embryo infected with recombinant adenovirus CELO-SEAP.

[Purification and properties of the hexon antigen of canine adenovirus serotype CAV-1]. / Ochistka i svoistva geksonobogo antigena adenovirusa sobak serotipa CAV-1.

The main antigen and immunogen of canine adenovirus type 1 (CAV-1) has been purified to near homogeneity from cultural fluid of a CAV-1-infected primary cell culture by hydrophobic and anion-exchange chromatography. The hexon native form (trimer) was shown to be resistant against denaturation by SDS under conditions of SDS-PAGE performed without heating the samples. The monomer chain of the CAV-1 hexon was apparently identical in terms of electrophoretic mobility with that of the previously sequenced BAV-3 hexon polypeptide (103 kDA). In blot enzyme immunoassay only native trimers of CAV-1 hexon were detected by cross-specific polyclonal and monoclonal anti-hexon antibodies.