Humanized Mouse Model of HIV Infection.

The development of new drugs for the treatment of HIV infection requires testing of their efficacy in a relevant animal model, such as humanized mice, which, unfortunately, are not yet available in Russia. In the present study, we have developed conditions for the humanization of immunodeficient NSG mice with human hematopoietic stem cells. Humanized animals generated during the study showed a high degree of chimerism and harbored repopulation of the entire range of human lymphocytes required for HIV replication in the blood and organs. Inoculation of these mice with HIV-1 virus led to stable viremia, which was confirmed by the presence of viral RNA in blood plasma throughout the entire period of observation and proviral DNA in the organs of animals 4 weeks after HIV infection.

Generation of SARS-CoV-2 Mouse Model by Transient Expression of the Human ACE2 Gene Mediated by Intranasal Administration of AAV-hACE2.

One of the most important steps in the development of drugs and vaccines against a new coronavirus infection is their testing on a relevant animal model. The laboratory mouse, with well-studied immunology, is the preferred mammalian model in experimental medicine. However, mice are not susceptible to infection with SARS-CoV-2 due to the lack of human angiotensin-converting enzyme (hACE2), which is the cell receptor of SARS-CoV-2 and necessary for the entry of the virus into the cell. In present work, it was shown that intranasal administration of the adeno-associated vectors AAV9 and AAV-DJ encoding the hACE2 provided a high level of expression of ACE2 gene in the lungs of mice. In contrast, the introduction of the AAV6 vector led to a low level ACE2 expression. Infection with SARS-CoV-2 of mice expressing hACE2 in the lungs led to virus replication and development of bronchopneumonia on the 7th day after infection. Thus, a simple method for delivering the human ACE2 gene to mouse lungs by intranasal administration of the AAV vector has been proposed. This approach enabled rapid generation of mouse model for studying coronavirus infection.

[Generation of SARS-CoV-2 Mouse Model by Transient Expression of the Human ACE2 Gene Mediated by Intranasal Administration of AAV-hACE2].

One of the most important steps in the development of drugs and vaccines against a new coronavirus infection is their testing on a relevant animal model. The laboratory mouse, with well-studied immunology, is the preferred mammalian model in experimental medicine. However, mice are not susceptible to infection with SARS-CoV-2 due to the lack of human angiotensin-converting enzyme (hACE2), which is the cell receptor of SARS-CoV-2 and necessary for the entry of the virus into the cell. In present work, it was shown that intranasal administration of the adeno-associated vectors AAV9 and AAV-DJ encoding the hACE2 provided a high level of expression of ACE2 gene in the lungs of mice. In contrast, the introduction of the AAV6 vector led to a low level ACE2 expression. Infection with SARS-CoV-2 of mice expressing hACE2 in the lungs led to virus replication and development of bronchopneumonia on the 7th day after infection. Thus, a simple method for delivering the human ACE2 gene to mouse lungs by intranasal administration of the AAV vector has been proposed. This approach enabled rapid generation of mouse model for studying coronavirus infection.

Preclinical Study of Pharmacological Properties of Doxorubicin-NPh.

Preclinical study of therapeutic properties of an innovative drug Doxorubicin-NPh (doxorubicin in the form of ultrafine suspension of phospholipid liposomes) in comparison with free doxorubicin (Doxorubicin-Teva) and protected doxorubicin (Caelyx) was performed on transplanted murine tumor models. All these drugs were efficient in Ca755 breast carcinoma model (tumor growth inhibition ≈100%, increase in lifespan 90.6-114.3%). In P388 lymphocytic leukemia and LLC lung carcinoma, advantages of the protected doxorubicin by the benefit/risk ratio (width of therapeutic interval) were demonstrated: Caelyx>Doxorubicin-NPh>Doxorubicin-Teva. Doxorubicin-NPh and Caelyx exhibited similar therapeutic activity in the LLC model, especially when administered 3 times with 3-day intervals; for Doxorubicin-Teva, the optimal interval between the injections was 7 days.

The Antitumor Efficacy of a Complex Based on Two-Vector System for Coexpression of the Suicide Gene Fcu1 and Cre Recombinase.

In this study, we evaluated the antitumor activity of a gene therapy complex in which the tumor-specific control of the expression of the effector suicide gene FCU1 was performed using a two-vector system based on the site-specific Cre-LoxP recombinase system. The complex of interest showed a high therapeutic potential in a mouse colon adenocarcinoma model.

Antitumor efficacy of combined gene and radiotherapy in animals.

Antitumor efficacy of the combined suicide gene therapy and radiotherapy was studied on the model of CT26 murine colon adenocarcinoma. CMV-FCU1-IRES-mGM-CSF-pGL3 construct with PEG-PEI-TAT (FCU1-mGM/5-FC) block copolymer as a vector was used for intratumoral administration. Tumors were irradiated with a single 5 Gy dose. The efficacy was evaluated according to the grade of tumor growth inhibition (T/C) and lifespan of the animals. Pronounced antitumor activity of the combined use of FCU1-mGM/5-FC system with radiotherapy on the background of prolonged lifespan and the synergism of the applied methods was revealed.

[Polyelectrolyte complexes of lactoferrin and pH-sensitive microparticles on their basis].

Suspensions of insoluble polyelectrolyte complexes of dextran sulfate? (DS) of different molecular masses with lactoferrin (LF) have been fabricated and characterized. The encapsulation efficiency of LF and DS in a complex at pH 3.0 and 4.0 was assessed, and particles were characterized by their sizes and zeta-potential. The complexes formed at pH 3.0 differed by a higher stability level. The interaction with DS resulted in a twofold decrease in the antioxidant activity of LF, although the formation of complexes was not accompanied by conformational changes in LF molecules according to IR-spectrometry data. Microencapsulation was carried out by treating the suspensions with negatively charged LF-DS complexes with protamine and chitosane solutions with different molecular masses. The composition, size, and the zeta-potential of interaction products were assessed which allowed us to select the conditions for the preparation of pH-sensitive polyelectrolyte microparticles loaded with LF which would be able to gradually release glycoprotein under conditions that model the passage through the gastrointestinal tract of humans. These data indicate that this approach is promising for the creation of pH-sensitive biopolyelectrolytes suitable for oral administration of LF to target cells.

[Comparative analysis of the methods for the estimation of the biological age].

The present article was designed to report the results of comparative analysis of the methods for the estimation of the biological age based on the measurement of a number of anthropometric and functional characteristics in the relatively healthy persons at the age varying from 30 years to 75 years. The study involved 277 individuals (143 women and 134 men). All the patients underwent clinical, biochemical, hormonal and instrumental examination. The following two methods were used to determine the biological age of the participants of the study: the ontogenetic anthropometrical method (L.M. Belozerova, 1999) and the Kiev method (V.P. Voytenko, 1984). These mathematical models are based on a specific set of calculation techniques for each anthropometric and instrumental parameter and approximately equally reflect the mismatch between the calendar and biological age; however, they are different in the degree of correlation with the disturbances of metabolism, enzymatic maintenance of metabolic processes, and other parameters. By way of example, the method of L.M. Belozerova for the calculation of the anthropometric biological age largely correlates with the insulin resistance index, blood concentrations of aldosterone, triglycerides and glucose, whereas the results obtained by the method of V.P. Voytenko show stronger correlation with the activity of alkaline phosphatase, the ceruloplasmin level and self-estimation of the patients' health.

[Antioxidative and detoxifying effects of analogues of delta-sleep inducing peptide (DSIP)].

16 DSIP analogues with substitutions of 1-2 amino acid residues were synthesized in order to investigate their potential use in medicine. Antioxidative properties of these peptides were studied in vitro and their detoxifying activity was examined in vivo on a model of toxicosis that was induced by the cisplatin cytostatic, which has been widely used in the cancer treatment. Practically all the studied DSIP analogues were shown to exhibit considerable direct antioxidative activity (AOA), and that of the ID-6 analogue was higher than AOA of DSIP and comparable with AOA of vitamin C and ß-carotine. This analogue also demonstrated the most pronounced detoxifying effect towards cisplatin action, resulting in a decrease in the animal death from the acute cisplatin toxicity to 17% (in comparison with 50-67% for the control animals) and restoration of a number of cisplatin-sensitive biochemical blood parameters: decrease in the activity of aspartate aminotransferase and alanine aminotransferase and downregulation of the concentration of the final products of nitrogen exchange (creatinine and urea). Thus, the DSIP-relative peptides could be promising agents for the decrease in the toxic effects of cytostatics that are used in oncology.

[Experimental study of remaxol as a drug for supportive therapy in traditional and high-dose treatment of tumors].

We have experimentally investigated the detoxifying and modifying effects of remaxol in the framework of traditional and high-dose chemotherapy in mice with transplanted tumors. The influence of remaxol in comparison with heptral was studied on the toxic and therapeutic action of cytostatic drugs gemzar, lastet (etoposide), and methotrexate during their traditional and high-dose administration in mice with transplanted P388 lympholeukosis. Remaxol demonstrated a detoxifying action with respect to these cytostatic agents, which decreased in the following series: gemzar lastet methotrexate (according to the results of lethality evaluation). Remaxol significantly increased the therapeutic efficacy of gemzar used in both traditional and high-dose regimes. This was manifested by slowing down tumor growth and increasing animal lifetime during combined administration of the drugs. This may be due to the remaxol potentiation of the antitumor effect of gemzar. In combination with lastet and methotrexate, remaxol does not alter their therapeutic efficacy. The detoxifying action of remaxol with respect to these cytostatics is more pronounced than that of heptral.

[The test system to identify mucin MUC1 in human blood serum using the technique of immune-enzyme analysis based on monoclonal antibody ICO25].

On the basis of genuine mouse monoclonal antibody ICO25 the test system IEA ICO25 was developed and standardized to quantitative detect tumor-associated antigen, mucin1 in human blood serum in format of inhibitory immune-enzyme analysis. The analytic characteristics of test-system correspond to the standards applied to immune-enzyme diagnostic kits. The results of identification of MUC1 in blood serum of healthy donors and female patients with breast pathology using IEA ICO25 fully correlate with the data concerning the detection of antigen CA15-3 using certified commercial kits. The test system IEA ICO25 can be used to detect MUC1 in human blood serum for research purpose.

[Study of detoxifying efficacy of remaxol in experimental model of cisplatin-induced toxicosis].

The detoxifying efficacy of remaxol in the experimental model of cisplatin-induced toxicosis has been studied and the possibility of using this drug in cancer patients therapy is evaluated. Remaxol exhibited pronounced dose-dependent detoxifying effect in the model of toxicosis induced by cisplatin in a toxic dose (LD50). It reduced the death rate in test animals about three times when used at a 130 ml/kg dose, and prevented lethal outcome at a 500 ml/kg dose. Remaxol also normalized biochemical blood indices that characterized liver and kidney functions in survived animals. Remaxol did not stimulate growth of experimental carcinoma, sarcoma, melanoma, and lympholeukosis. The drug did not interfere with the anti-tumor efficacy of cisplatin in the schemes with combined treatment of animals bearing tumors of various histogenesis. Remaxol can be recommended for clinical study as a detoxifying preparation for cancer patients with various malignancies.

[Effect of polysaccharides on biological activity of human lactoferrin].

The influence of neutral and ionic polysaccharides on the antioxidant (AOA) and detoxifying activities of lactoferrin (LF) and the duration of its circulation in the body was studied. In addition to natural polymers, we studied artificial chitosan derivatives with different functional groups. On the basis ofAOA test, five polysaccharides were selected. The study of the detoxifying effect of LF in two models of induced toxicity revealed polysaccharides that maintained or increased the detoxifying activity of LF. We established that the formation of a complex of lactoferrin with two galactomannans and succinyl chitosan caused positive changes in LF properties: the detoxifying activity of the protein remained unchanged or increased, whereas its elimination from the body was decelerated.

[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.

[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.

[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.

[Incorporation of labeled formate in rat liver mitochondrial proteins and initiation of protein synthesis]. / Vkliuchenie mechenogo formiata v belki mitokhondrii pecheni krysy i initsiatsiia belkovogo sinteza.

During incubation of rat liver mitochondria in vitro labeled formate is incorporated into TCA-insoluble substance during mitochondrial translation. Data from hydrolysis with CNBr (after methionine residues) or with 0.5 N HCl (deformylation of amino acid N-formyl derivatives) suggest that about half of the total protein radioactivity is incorporated in formate groups of N-terminal methionine. Labeling of growing polypeptides with formate (but not with phenylalanine or methionine) oscillates with a period of about 13 min. The potential initiation capacity is unchangeable and exceeds that observed experimentally by one order of magnitude. The data obtained are consistent with the previously proposed hypothesis no synchronization of mitochondrial protein synthesis which cannot be induced by the steps preceding the formation of the first peptide bound.