Search

The protection against tuberculosis infection is largely determined by the ability of host tissue macrophages to limit the growth and spread of mycobacteria. Able to multiply within the host macrophages, mycobacteria have developed a number of protective mechanisms preventing phagosome-lysosome fusion, thereby evading damaging effects of lysosomal enzymes. Saposins are small, acidic, thermostable, non-enzymatic glycoproteins that participate as co-fac-tors in degradation of short oligosaccharide head group glycosphingolipids. Saposins A, B, C and D are formed in acidic endosomes due to cleavage of initial prosaposin molecule. The effect of saposins on human immune response is mediated by their involvement in presenting mycobacterial antigens on CD1 molecules. Preliminary studies with electron microscopy allowed to uncover saposin D-bound damaging effect on Mycobacterium tuberculosis in acidic environment. These data allowed us to suggest that saposin D is an important protective component fighting against TB infection. The aim of the study was to explore how saposin D deficiency might affect formation of anti-tuberculosis immune response and ability of macrophages to inhibit M. tuberculosis growth. Materials and methods. Interstitial pulmonary macrophages and peritoneal macrophages were isolated from wild type C57BL/6 strain and saposin D deficient C57BL/6-SapD-/- mouse strains. Results. It was found that as compared to macrophages from mice, macrophages from wild type strain significantly better controlled mycobacteria growth in vitro. To study an opportunity of compensating for deficient saposin D in peritoneal macrophages from C57BL/6-SapD-/- mice, a saposin D gene-bearing lentiviral vector was created. Transfection of SAPD-deficient peritoneal macrophages with expression vector compensated for saposin D deficiency in such cells and restored bactericidal function. The mechanisms of action for current anti-TB drugs are mediated by various metabolic pathways in mycobacteria (inhibited biosynthesis of fatty acids, arabinogalactan, peptidoglycan and protein; inhibition of DNA-dependent processes, proton pumps and cytochrome P450-dependent monooxygenases). Conclusion. It was shown that saposin D deficiency affects activation of macrophage bactericidal function in vitro. Our study data may be a prerequisite for biologically substantiated potential of using a vector construct bearing natural human protein gene such as saposin D, as a new anti-tuberculosis drug.

Inhibition of inflammatory processes in the model organisms using non-steroidal anti-inflammatory drugs (NSAIDs) can be an effective geroprotective method. The mechanisms of NSAIDs action in insects have not been studied enough. It is assumed that they are similar to those in mammals and are based on the inhibition of cyclooxygenase 2, which leads to a decrease in the synthesis of prostaglandins. Prostaglandins are central signaling molecules for mediated coordinated cellular immunity of insects and control the imago eclosion, egg production and oogenesis of Drosophila. Obviously, signaling pathways exist where the role of prostaglandins has not yet been shown. In our work, the resistance to starvation of Drosophila melanogaster of wild type stock Canton-S under pharmacological inhibition of prostaglandin metabolism at different stages of ontogenesis was analyzed. In the experiments, nimesulide was used in three different concentrations – 0.1, 0.05, and 0.025 mg/ml. The results of the experiments have shown, that the development of larvae in the medium containing NSAID nimesulide leads to a decrease in resistance to stress factor – starvation on average by 19.2% in females and by 7.4% in males. Resistance to starvation of the most stress-resistant (10% of individuals with the longest life span) females decreases during the development of larvae in the medium containing nimesulide in concentrations 0.1 and 0.05 mg/ml. The consumption of nimesulide by imago at a concentration of 0.025 mg/ml during the first days of life increases stress resistance and life span at starvation in females by 12.4% and in males in all variants of the experiment on average by 17.8%. Under the same experimental conditions, life span during starvation increased in the most stress-resistant females Canton-S. Thus, pharmacological inhibition of prostaglandin metabolism leads to an increase of resistance to starvation if virgin adults are exposed to nimesulide during the first day after eclosion, and stress resistance decreases if nimesulide is consumed by larvae. This is obviously, due to that somatic tissues of the adult flies are almost entirely composed of postmitotic cells, while intensive processes of cell division are characteristic of growing larvae. The data obtained indicate that the action of nimesulide on postmitotic imago cells promotes transition of cells to increased stress tolerance, while the impact on actively dividing cells of larvae leads to a decrease in the resistance of the adults.

Aim. Study of mechanisms of attenuation of cold-adapted (ca) influenza virus strain A/ Krasnodar/101/35/59 (H2N2), associated with disruption of NS1 protein functions. Materials and methods. Study of interferonogenic activity of ca strain A/Krasnodar/101/35/59 (H2N2), its parent variant A/Krasnodar/101/59 (H2N2), virulent strain A/WSN/33 (H1N1) and a number of single gene and multiple gene reassortants between these strains, obtained using reverse genetics, was carried out. Study of dynamics of IFNp gene expression was carried out by using a methodical approach of RT-PCR in real time mode. Results. Inclusion of PB-1 gene of ca strain A/ Krasnodar/101/35/59 (H2N2) with reversion to wild type into genome composition of virulent strain А/WSN/33 (H1N1) does not result in a sharp change of interferonogenic activity of the reassortant. At the same time, similar inclusion of PB-1 gene of ca strain resulted in an incredible growth of interferonogenic activity of the reassortant. On the other hand, inclusion of NP-gene of wild type strain A/Krasnodar/101/59 (H2N2) into genome composition of the wild type strain А/WSN/33 did not differ by effect on interferonogenicity of the reassortant from inclusion of NP-gene of ca strain. Conclusion. Both constellations of genes of parent variants and mutations localized in these genes could affect formation of attenuation phenotype of reassortants. The data obtained allow to assume possible mechanisms of attenuation of ca strains, associated with disruption of NS gene function.

Aim: To develop a highly sensitive real-time polymerase chain reaction (PCR) system for detection of somatic mutations in 542 and 545 codons of exon 9 and 1047 codon of exon 20 of PIK3CA gene comprising more than 80% of all somatic mutations in this gene for application on histological material without macroand microdissection, and to analyze associations between these mutations and clinical and pathological characteristics of breast tumors.Materials and methods: The Allele-specific real-time PCR method with signal detection by TaqMan probes was used. For determination of its analytical sensitivity, plasmids carrying the mutations studied were constructed by standard genetic engineering methods using mutagenesis. DNA samples carrying various mutated/wild type DNA ratios (5.0; 2.0; 1.0; 0.5, 0.25%) were prepared. Results: Multiplex allele-specific real-time PCR method for detection of most common mutations in PIK3CA gene: p.E542K c.1624G>A, p.E545K c.1633G>A, p.H1047R c.3140A>G, p.H1047L c.3140A>T – was developed and optimized.Analytical sensitivity of mutation detection comprised 0.5% for p.E542K and 0.25% for p.E545K, p.H1047R and H1047L enzyme.Conclusion: The method developed for detection of somatic mutations in PIK3CA gene is sufficiently sensitive, specific and efficient, that allows to propose it for a routine screening in clinical diagnostic laboratories for evaluation of disease prognosis and monitoring of response to therapy and its modification.

CCR5del32 Homozygous deletion in the chemokine receptor R5 gene provides almost com- plete protection to individuals against HIV infection. However, data relating to the protective effect for CCR5del32 heterozygous individuals have been contradictory. The frequency of the CCR5del32 allele in population control cohorts was compared with that of a group of children (27 Kalmyks and 50 Russians) infected by G-subtype HIV-1 in a nosocomial outbreak. The frequency of the CCR5del32 allele was shown to be lower among the infected children in comparison with that of the control group; however, the differ- ence was small and statistically insignificant. Similar results were obtained in a number of earlier studies. The insignificance of the small differences could be a result of one of two reasons. (i) The fact that there is no protective effect of the heterozygous state, and that the phenomenon depends only on the fluctuation of allele frequencies. In this case, there would be no differences even if the infected cohort is enlarged. (ii)The protective effect of the heterozygous state is real; however, the size of the studied cohort is insufficient to demonstrate it. In order to discern between these two reasons, a meta-analysis of data from 25 published articles (a total of 5,963 HIV-infected individuals and 5,048 individuals in the control group, including the authors' own data) was undertaken. A conclusion was drawn from the meta-analysis that the CCR5del32 al- lele protects individuals against the HIV infection even in a heterozygous state (OR=1.22, 95%CI=1.10-1.36). The risk of HIV infection for CCR5 wt/del32 heterozygotes was lower by at least 13% as compared to that for wild type CCR5 wt/wt homozygotes. Prior to this study, no data of the type or any conclusions had been published for Caucasians. The mortality rate in the 15 years following the infection was found to be ap- proximately 40% lower for CCR5del32 heterozygotes in comparison with that for the wild type homozygotes in the studied group. The size of the studied group was insufficient to claim difference validity (OR=2.0; p= 0.705), even though the effect quantitatively matched the published data. The features of the meta-analysis influencing the threshold level and the statistical validity of the effects are being discussed. The level of the CCR5del32 protective effect on the chances to be infected with HIV and on the outcome of the HIV infection was assessed for various ethnic groups. KEYWORDS HIV; nosocomial infection; lethality risk; infection risk; chemokine receptor gene; allele CCR5del32; meta-analysis. ABBREVIATIONS HIV - human immunodeficiency virus; AIDS - acquired immune deficiency syndrome; PCR - polymerase chain reaction.