[Experimental approach to the gene therapy of motor neuron disease with the use of genes hypoxia-inducible factors].

Motor neuron disease (MND), or amyotrophic lateral sclerosis, is a fatal neurodegenerative disorder characterized by a progressive loss of motor neurons in the spinal cord and the brain. Several angiogenic and neurogenic growth factors, such as the vascular endothelial growth factor (VEGF), angiogenin (ANG), insulin-like growth factor (IGF) and others, have been shown to promote survival of the spinal motor neurons during ischemia. We constructed recombinant vectors using human adenovirus 5 (Ad5) carrying the VEGF, ANG or IGF genes under the control of the cytomegalovirus promoter. As a model for MND, we employed a transgenic mice strain, B6SJL-Tg (SOD1*G93A)d11 Gur/J that develops a progressive degeneration of the spinal motor neurons caused by the expression of a mutated Cu/Zn superoxide dismutase gene SOD1. Delivery of the therapeutic genes to the spinal motor neurons was done using the effect of the retrograde axonal transport after multiple injections of the Ad5-VEGF, Ad5-ANG and Ad5-IGF vectors and their combinations into the limbs and back muscles of the SOD1(G93A) mice. Viral transgene expression in the spinal cord motor neurons was confirmed by immunocytochemistry and RT-RCR. We assessed the neurological status, motor activity and lifespan of experimental and control animal groups. We discovered that SOD1(G93A) mice injected with the Ad5-VEGF + Ad5-ANG combination showed a 2-3 week delay in manifestation of the disease, higher motor activity at the advanced stages of the disease, and at least a 10% increase in the lifespan compared to the control and other experimental groups. These results support the safety and therapeutic efficacy of the tested recombinant treatment. We propose that the developed experimental MND treatment based on viral delivery of VEGF + ANG can be used as a basis for gene therapy drug development and testing in the preclinical and clinical trials of the MND.

[Contribution of L,D-carboxypeptidases in virulence of facultative intracellular pathogenic bacteria Listeria monocytogenes].

AIM: Evaluate influence of mutation of Listeria monocytogenes genes coding murein-tetrapeptide L,D-carboxypeptidase Lmo0028 and Lmo1638 on dynamics of infectious process and interaction of purified muropeptides with NOD1 receptor. MATERIALS AND METHODS: Wild type EGDe strain and recombinant strains GIMins1638 H GIMins0028 obtained on its basis by site-specific mutagenesis were used. Infectious process dynamics was studied on the model of intravenous infection of BALB/c mice. Ligand-receptor interaction activity of muropeptides isolated from recombinant and parent strains were assayed on HEK293-hNOD1 cell line expressing NOD1 receptor and containing in their genome beta-galactosidase reporter gene under the control of NF-kappaB dependent promoter expression. RESULTS: Lack of Lmo0028 decelerates reproduction of listerias in animal liver starting from 24 hours and at later terms after the infection whereas lack of Lmo1638 leads to increase of microbial load 6 and 24 hours after the infection with no influence on further infection. Differences in activation of NOD1 receptor by muropeptides isolated from recombinant and parent strains were not detected. CONCLUSION: Despite high homology murein-tetrapeptide L,D-carboxypeptidase Lmo0028 and Lmo1638 make a different contribution to the development of infectious process caused by L. monocytogenes in BALB/c line mice. Lack of differences in NOD1 receptor activation may be associated with compensation of enzymatic functions in strains with mutation in each of the genes owing to the presence of homologous protein.

[New approaches to therapy of persistent infections: elimination of intracellular Chlamydai trachomatis by exposure to low temperature argon plasma].

AIM: Study microbicidal activity of low temperature argon plasma (LTP) that is a stream of partially ionized argon having macroscopic temperature of the environment against Chlamydia trachomatis obligate intracellular parasites. Study viability of host cells in parallel. MATERIALS AND METHODS: McCoy line cells infected with C. trachomatis (Bu-434/L2 strain) were exposed to LTP obtained by using atmospheric pressure plasma SHF generator. Intracellular localization of chlamydiae was visualized by luminescent microscopy. RESULTS: Exposure of infected McCoy line cells resulted in the destruction of chlamydia inclusions and practically complete elimination of intracellular bacteria. At the same time LTP exposure did not result in immediate death of host cells, an insignificant reduction of the number of cells was observed 24 hours after the exposure to LTP. CONCLUSION: The effect of LTP for elimination of intracellular chlamydia without significant changes in viability of eukaryotic host cells was demonstrated.

Recombinant influenza vaccines.

This review covers the problems encountered in the construction and production of new recombinant influenza vaccines. New approaches to the development of influenza vaccines are investigated; they include reverse genetics methods, production of virus-like particles, and DNA- and viral vector-based vaccines. Such approaches as the delivery of foreign genes by DNA- and viral vector-based vaccines can preserve the native structure of antigens. Adenoviral vectors are a promising gene-delivery platform for a variety of genetic vaccines. Adenoviruses can efficiently penetrate the human organism through mucosal epithelium, thus providing long-term antigen persistence and induction of the innate immune response. This review provides an overview of the practicability of the production of new recombinant influenza cross-protective vaccines on the basis of adenoviral vectors expressing hemagglutinin genes of different influenza strains.

[Prospects for the use of low-temperature gas plasma as an antimicrobial agent].

Results of application of LTP at atmospheric pressure as an antibacterial agent during the last decade are considered with reference to physicochemical mechanisms of its bactericidal action. The principles of designing modern LTP sources are described in conjunction with the results of LTP application against pathogenic bacteria in vitro and in biofilms. The possibility to destroy biofilm matrix by LTP is estimated along with the results of its testing for the treatment of acute and chronic wound surfaces. Prospects for the development of "plasma medicine" in this country and abroad are discussed with special emphasis on its advantages, such as the absence of long-acting toxic compounds, small probability of spontaneous mutations accounting for resistance to LTP, relatively low cost of LTP sources, independence of LTP effect of the surface relief, painless application.

[Advantages and prospects of the use of genetic vaccines for the protection from dangerous and socially significant infections].

High frequency of epidemiological threats (H5N1 influenza, SARS, etc.) in modern world calls for the development of new flexible technologies for manufacturing efficacious vaccines and rapid reorientation of their production as appropriate. Genetic vaccination is one of such technologies aimed at prophylaxis of dangerous and socially significant infections. The technology is based on administration of one or several functionally active genes encoding for antigens of pathogens which induces formation of both cellular and humoral immunity against the respective microorganism. This property of genetic vaccines is used for the development of prophylactic schemes. New vaccines are currently being designed to prevent a variety of infections. The aim of the present review is to outline major trends in genetic vaccination leading to the improvement of its efficacy.

[The role of pattern-recognizing receptors in anti-infectious immunity].

Pattern-recognizing receptors (PRR) play a key role in the functioning of human immune system. They are the primary sensors of infection capable of distinguishing between various highly conservative molecular patterns (pathogen-associated molecular patterns (PAMPs)) contained in pathogenic organisms. Binding of these molecular patterns to PRR induces a variety of reactions of innate (secretion of proinflammatory cytokines and antimicrobial peptides, activation of phagocytosis, etc.) and adaptive (antibody processing and presentation, polarization of T-cell response, etc.) immunity. Great interest in the molecular mechanisms of pathogen recognition resulted in the discovery of numerous PRR. The aim of this review is to systematize the currently available data on PRR, their specificity, and role in the formation of anti-inflammatory immunity.

[Opportunities for effective skin regeneration by topical stimulation of immunity].

A murine model of incisional wound was used to evaluate effect of topical application of purified bacterial lipopolysaccharide on the wound healing process. Thirty five ICR mice were used in the study. It was shown that bacterial lipopolysaccharide is a strong promotor of wound healing. It increases tensile strength, accelerates completion of the inflammatory process, stimulates collagen deposition and early remodeling.

[Methods of identification and assessment of safety of genetically modified microorganisms in manufacture food production].

Methods of identification of genetically modified microorganisms (GMM), used in manufacture food on control probes are presented. Results of microbiological and molecular and genetic analyses of food products and their components important in microbiological and genetic expert examination of GMM in foods are considered. Examination of biosafety of GMM are indicated.

Effective Genetic Expression of Nanoantibodies by Recombinant Adenoviral Vector in vitro.

The present study is devoted to the feasibility of expressing the single-domain mini-antibody (nanoantibody) selected from the library of sequences of the variable domains of special single-stranded antibodies derived from an immunized camel, a gene of which was introduced into eukaryotic cells within a recombinant adenoviral vector. A vector bearing the gene of a single-domain nanoantibody was obtained using the AdEasy Adenoviral Vector System (Stratagene). This method of delivering the nanoantibody gene facilitates efficient expression of this gene and functional activity of the nanoantibody. The results obtained can be used to produce passive immunizing tools against pathogens or new-generation immunobiological antitoxic medication.

[In vitro study of antimicrobial activity of lactoferrins from various sources].

Comparative antimicrobial activity of lactoferrins from various sources (native lactoferrin from Laprot, human hololactoferrin, recombinant human lactoferrin isolated from the cultural medium of permissive cell culture transfected using pseudoadenovirus nanostructure with the human lactoferrin gene, and native bovine lactoferrin) was studied to prove the possibility of their use for development of antimicrobial drugs. It was shown that all the substances were active against the Bacillus standard strains. The antibacterial activity was almost independent of the degree of saturation the lactoferrin molecules with Fe3+. The native human lactoferrin was more active than hololactoferrin against Candida when evaluated by the minimum inhibitory concentration (MIC). Fe(3+)-Non aturated recombinant human lactoferrin demonstrated the antimicrobial activity (by MIC) similar to that of the native human lactoferrin. The results showed that native and recombinant human lactoferrins might be used for the development of intravenous and intracavitary dosage forms, while the native bovine lactoferrin could be useful in development of oral drugs.

[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.

[An in vivo study of the biologic activity of the recombinant pseudoadenovirus nanostructure containing lactoferrin gene].

The Ad5-Lf recombinant pseudoadenovirus nanostructure (RPAN) based on adenovirus of the 5th serotype and containing lactoferrin (Lf) gene was constructed. The goal of this work was to develop a system for efficient production of human lactoferrin (Lf) in human body. It was shown using the model of cisplatin (DDP)-induced toxicosis that human Ad5-based RPAN with human Lf gene expressing cassette in its genome provides high rate of expression of Lf gene in animal body. In vivo recombinant human Lf demonstrates detoxification effect against acute DDP-induced toxic reactions similar to that of the native Lf. RPAN does not stimulate growth of primary and metastatic nodes of experimental tumors. Moreover, it inhibits the growth of Lewis lung carcinoma (LLC), Ehrlich carcinoma (ELD), and S37 sarcoma in early periods after tumor transplantation. The obtained experimental data are indicative of the good prospects of further biologic and medical study of RPAN and development of RPAN-based genetic engineering medicine of the new generation.

[Stem cells of intestine epithelium. Survival mechanisms and microbiota role].

The thanatogenetic mechanisms of stem cells (SC) of rapidly renewing system of intestinal epithelium still remain unclear. On the one hand, they are definitely involved into basic mechanisms of carcinogenesis in the gastrointestinal tract, because dysregulation of programs responsible for elimination of "unwanted" mutant cells (which are normally under immune and own intrinsic control) is one of the reasons of neoplastic expansion. On the other hand, elucidation and characterization of the regulatory machinery controlling SC survival are interrelated with problems of clinical medicine, including the increase of therapeutic efficiency of treatment of inflammatory and ulcer lesions of the gut, traumatic and surgical wounds, as well as restriction of side effects in normal tissues induced by application of intensive methods chemo- and radiotherapy of cancer. The latter is especially important for treatment of blood diseases and tumors of peritoneal cavity organs mainly due to bone marrow and intestinal epithelium damage. (These tissues are the most sensitive to these treatments.) The review considers data on exogenous and genetic modifiers of SC survival, and also the basic principles of mechanisms involved into renewal and regeneration of SC and the effects of microbiota on these processes.

[Assessment of protective effect of heat shock protein-lypopolysaccharide of Salmonella typhimurium recombinant construction in mice].

AIM: To study protective activity of recombinant construction of heat-shock protein with lypopolysaccharide (rcHSP-LPS) as well as its variants (with destroyed protein or bounded LPS) against Salmonella typhimurium. It was also planned to study the ability of rcHSP-LPS to interact with toll-like receptors (TLRs) expressed on continuous cell lines. MATERIALS AND METHODS: One of the following preparations was administered to outbred mice: rcHSP-LPS; rcHSP-LPS treated by polymyxin B (PMB) for bounding of LPS - rc(HSP-LPS)PMB; rcHSP-LPS in which protein was treated by boiling during 30 min--rc (HSP-LPS)B; LPS (E. coli K-235); polymyxin B (PMB). Twenty-four hours after single or last administration of rcHSP-LPS, each mice was intraperitoneally inoculated with 63 LD50 of S. typhimurium 415 contained in 0.5 ml of physiologic solution. Antibody titer to LPS of Salmonella typhimurium was measured by immunoenzyme assay. RESULTS: It was demonstrated that rcHSP-LPS administered 24 hours before inoculation induced resistance to S. typhimurium infection. Protection formed after 3 injections of rcHSP-LPS with 10 mcg in each or single injection with 100 mcg/mouse. Forty to eighty percent of immunized mice survived after challenge while 90% of control animals died. Destroy of the HSP by boiling of the construction led to loss of protective effect. Bounding of LPS by PMB did not lead to loss of protective properties of the construction but they expressed only after its multiple administration with 10 mcg per mouse. LPS of E. coli in dose 0.0266 mcg per mouse as well as PMB did not influence the course of S. typhimurium infection in mice. CONCLUSION: It was shown that rcHSP-LPS effectively protects mice from S. typhimurium infection by activating innate immunity; one of the possible mechanisms for such protection determined by interaction with TLRs 2 and 4 was considered. Other studies are needed in order to elucidate other mechanisms of innate immunity, which can be activated by rcHSP-LPS.

[Lipid-associated membrane lipopeptides of M. arginini activate NF-kB by interacting with TLR2/1, TLR2/6, and TLR2/CD14].

Various strains of mycoplasmas cause activation of transcriptional factor NF-kB as a result of interaction with different combinations of Toll-like receptors (TLR). It is well known that the MALP-2 protein of M. fermentans activates the NF-kB through interaction with the TLR2/6, lipid-associated membrane lipopeptides (LAMPs) of M. penetrans through the TLR1/2, LAMPs of M. pneumoniae through combinations of Toll-like receptors (TLR2/6 and TLR1/2), and superantigene of M. arthritidis through the TLR2 and TLR4-dependent pathways. In this study, we defined specific Toll-like receptors for LAMPs of M. arginini. For carrying out the research we used cell lines 293-null, 293-hTLR2, 293-hTLR1/2, 293-hTLR2/CD14, 293-hTLR2/6, 293-hTLR4/ CD14-MD2 expressing certain combinations of TLR and their coreceptors. It was shown that LAMPs of M. arginini cause activation of NF-kB interacting with TLR2/1, TLR2/6 and TLR2/ CD14, but not with TLR2 alone or TLR4.

[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.

[Requirements to a medical and biologic assessment and the hygienic control of the food production received from recombinant-DNA microorganisms].

In work the characteristic of the created in the Russian Federation system of an estimation of safety of the foodstuff received from/or with use of genetically modified microorganisms (GMM) is given, at their admission to realization and the hygienic control of given production over a revolution. It is shown, that strategy of a safety at a stage of registration GMM, the established order and accepted control measures of the foodstuff received from/or with use GMM, in Russia their large-scale commercial use, and the normative-legal and methodical base based on the federal legislation on state regulation in the field of genetically engineering activity, about quality and effectively outstrip safety of foodstuff about protection of the rights of consumers, is harmonized with approaches of the international organizations.

[Influence of integrin-binding motif (RGD) on attachment and internalization of avian adenovirus CELO in mammalian cells].

Recombinant avian adenovirus CELO bearing sequence RGD in the structure of a HI-loop of long fiber was designed. Experiments in vitro revealed that introduction of RGD-motif into fiber of CELO increased the ability of the virus to be attached to a surface of CAR-negative cells, and raised efficiency of the process of internalization of the virus both in CAR-positive, and in CAR-negative cells.