THE INFLUENCE OF QUERCETIN ON THE HEPATIC DIFFERENTIATION OF HUMAN ADIPOSE-DERIVED MESENCHYMAL STEM CELLS.

The human adipose-derived mesenchymal stem cells was shown to be hepatic differentiated under influence of inductor factors. The differentiation of MSCs was induced by fibroblast growth factor-4, hepatocyte growth factor, oncostatin M and dexamethasone. The influence of quercetin on the hepatic differentiation of MSCs in culture was investigated. The adding quercetin into induction medium (1 or 10 mmol/l) enhanced the manifestation of signs of hepatic differentiation MSCs such as urea secretion, cytokeratin 19 and a-fetoprotein synthesis. Quercetin modulating action on the CYP1A ­ cytochrome P450 activity in differentiated cells was found. MSCs which was differentiated in the presence of quercetin had a higher viability and resistance to oxidative stress.

[The influence of fibroblast growth factor (FGF2) on cardiomyocytes differentiation of mesenchymal stem cells of bone marrow ex vivo].

The influence of FGF2 on the efficiency of cardiomyocytes differentiation of mesenchymal stem cells (MSC) of bone marrow induced by 5-azacetidine (5-aza) was studied. The effect of FGF2 developing by the 14th day after the combined action of a differentiating agent and growth factor was manifested in an increase in Mef2A, Mef2D and gene transcription and a rise of ionized Ca2+ concentration in cytoplasm keeping cell viability and proliferation activity. In the presence of FGF2 this approach provided cardiomyogenesis and the increase in the formation of early precursors of cardiomyocytes.

[Peculiarities of growth and development of cultured mucosal cells from the upper respiratory tract stimulated by growth factors].

Specific features of growth and development of cultured mucosal cells from the upper respiratory tract were studied during their in vitro stimulation by keratinocyte growth factor (KGF) and epidermal growth factor (EGF). Phenotypic composition and quantitative characteristics of cultured epithelial cells was investigated with the use of monoclonal CD49F antibodies and flow cytofluorometry. The culture technique makes it possible to obtain a large amount of cells for the evaluation of their pathological changes. Moreover, cell cultures can be used to restore lesioned mucosa of the upper respiratory tract both in experiment and under clinical conditions.

[Biological and immunogenic characteristics of Chlamydia trachomatis strain MT-2A (serovar D) and its use for development of experimental inactivated vaccine].

Biological characteristics of C. trachomatis author's strain MT-2A (serovar D) is presented. Stages of development on its basis the experimental formalin-inactivated vaccine against Chlamydia were described. Humoral and cellular immune response to the vaccine administered on 3-dose immunization schedule in conjunction with polyoxidonium as adjuvant was studied. Significant immunological efficacy of the vaccine was shown. T- and B-cell immune responses were characterized. Titer of IgG antibodies against Chlamydia in blood serum after 3rd dose of the vaccine was 10,880+/-1,817.76. Assessment of T-cell response showed that reaction of delayed hypersensitivity with formation of granuloma presented in 60% of animals. Proportion of immunoblasts in reaction of blast-transformation was 29.3+/-2.8%. Perspectives of further studies of the developed corpuscular vaccine against Chlamydia are discussed.

[Regulation of morphologicaland functional properties of astroglial cells by hydrogen peroxide].

Effects of hydrogen peroxide on morphological characteristics, proliferation index, menadione-dependent lucigenin-enhanced chemiluminescence of C6 glioma cells were studied. It was established that H2O2 at 1 x 10(-8) - 5 x 10(-7) M concentrations acts as a regulator of morphological and functional properties of astrocytes by inducing their reactivation that is manifested as a cell body hypertrophy and an increase of proliferative activity and of menadione-dependent production of superoxide (O2- ). Cytodestructive action of hydrogen peroxide at a concentration higher than 1 microM on C6 glioma cells shows itself as a decrease of their proliferation index and the ability to generate O2- under menadione action. Using lipopolysaccharide B as a functional stimulator it has been shown that H2O2 modifies signaling pathways leading to the increase of mitotic activity of C6 glioma cells and decreases the yield of lucigenin-enhanced chemiluminescence of astrocytes under menadione action to the level of control values.

[Photodynamic inhibition of infection caused by herpes simplex virus type 1 in the cultured cells, by using 5-aminolevulinic acid-induced porphyrins].

On simulating infection caused by different herpes simplex virus type 1 (HSV-1) variants responsive and unresponsive to the drugs acyclovir and phosphonoacetic acid in the cultured Vero and C6 cells has revealed the higher ability of target cells to accumulate 5-aminolevulenic acid (ALA)-induced endogenous porphyrins, which determines the selectivity of their photo damages. Optimal conditions have been defined for all the studied HSV-1 variants to show a virus-inhibiting effect upon photodynamic exposure of infected and ALA-treated cell cultures.

[Specific clinical, epidemiological patterns and laboratory diagnostics of enterovirus infection in the Republic of Belarus].

The clinical and epidemiological patterns as well as the results of the laboratory verification of the outbreak of enterovirus infection (EVI) in Minsk during the period of summer-autumn, 2000, are presented. During this outbreak a variety of clinical forms were observed, the serous meningitis being prevalent (57.5%). Practically simultaneous occurrence of infection on the territory of all administrative districts of the city, the predominant involvement of children aged up to 14 years into the outbreak, a high proportion of simultaneous casualities in the multiple foci. A number of circulating enteroviruses (EV)--ECHO 30, ECHO 6 of three serotypes and Coxsackie B5--were simultaneously isolated from clinical material. EV of the same serotypes were isolated from tap drinking water, and neutralizing antibodies to these serotypes were often detected in the patients blood sera. Infectious EV were also present in samples of bottled water and in water reservoirs used for bathing. The routes of EV transmission and the improvement of EVI control are discussed.

Photodynamic inhibition of infection caused by drug-resistant variants of herpes simplex virus type I.

Membranotropic amphiphilic chromophore merocyanine 540 sensitized photodynamic inhibition of drug-resistant and sensitive variants of type I herpes simplex virus in cultured Vero cell. Optimal conditions of photodamage to virus particles and infected cells were determined (merocyanine 540 concentration 1 microM, illumination dose 32.5-65.0 kJ/m(2), exposure at early stages of infection). Infected cells actively bind the photosensitizer, which explains their selective photodamage.

[Experience in the use of chemical compounds for the primary identification and differentiation of enteroviruses]. / Opyt ispol'zovaniia khimicheskikh soedinenii dlia pervichnoi identifikatsii i differentsiatsii énterovirusov.

Data on the usage of chemical inhibitors nifan and belvtazid, which possess a selective and antienteroviral effect, in the primary identification of enteroviruses and their differentiation into polio- and non-poliomyelytic ones isolated from human clinical materials or the environment by using the cell culture are presented in the article. The method is recommended for the practical use by the virology service in the diagnostics of enteroviral infections and in the identification of cytopathic agents isolated from the enviroment.

Viral water contamination as the cause of aseptic meningitis outbreak in Belarus.

In the recent years Echovirus-30 associated outbreaks have taken place in different European countries. Aseptic meningitis caused by Echovirus-30 was the main diagnosis of a large outbreak in Belarus in Summer-Autumn, 1997, involving 460 patients. Echovirus-30 was detected in cerebrospinal fluid of the patients with aseptic meningitis. This serotype played the dominant role in the outbreak. Minor serotypes and mixtures of enteroviruses were detected in faeces and nasopharyngeal lavages. Investigation of environmental samples gave evidence of expressed viral contamination of drinking water and water sources (river and ground sources). River water sources were considerably contaminated with viruses. The incidence of virus isolation was 50%. After cleaning procedures, the incidence became two times lower, proving imperfect water purification and disinfection procedures. Sequence analysis of isolates from Belarus (isolates from water and patient's cerebrospinal fluid) showed the difference of 0.2%. The outbreak peculiarities such as high attack rate and wide-spread of the disease incidences, clinical form variability, isolation of outbreak strain from water and a good agreement between minor serotypes isolated from faeces and water samples as well as correlation in the dynamics of acute intestine infections, aseptic meningitis morbidity and bacterial water contamination can be considered as evidence of its water-borne. Echovirus-30 isolates from Belarus were very closely related to each other and to several European isolates. Sequence difference between isolates of 1994-1998 from European countries was found to be 4.3%. The data can point to the common primary source of enterovirus infection, connected to water and to the possibility of epidemic strain transmission from neighbouring states to the Republic of Belarus.

[Water-borne outbreak of serous meningitis caused by Echovirus-30 in Belarus]. / Vodnaia vspyshka seroznogo meningita, vyzvannaia virosom Echo-30, v Belarus.

In recent years outbreak of enterovirus infections caused by Echovirus-30 were rather frequently registered in different European countries. A major outbreak caused by this virus took place during the summer-autumn period of 1997 in the city of Gomel, Belarus. Sanitary epidemiological and molecular epidemiological studies made it possible to determine that the outbreak was water-borne. The sequence analysis of Echovirus-30 strains isolated from water and the cerebrospinal fluid of patients revealed a minor divergence between them (0.2%) indicative of their practical identity. The comparison of the Belorussian isolates with the strains isolated in Europe in 1994-1998 also showed a small percentage of differences in their genomes, which showed that the outbreak of Echovirus-30 infection was probably brought to Belarus from the territories of European countries.

[Use of rapid methods in detection of enteroviruses]. / Ispol'zovanie ékspress-metodov dlia detektsii énterovirusov.

Indirect immunofluorescence (IIF) and RT-PCR were used for rapid diagnosis of enterovirus infection during an outbreak of aseptic meningitis in Gomel. IgM to enterovirus were detected in 93.6% sera by IIF and in 71.4% cerebrospinal fluid specimens by RT-PCR. IIF takes only 1.5-2 h and RT-PCR 6-7 h. The methods are recommended for rapid diagnosis of enterovirus infection.

[Relationship between the oxidation-reduction system of astrocytes with production of active forms of oxygen]. / Sviaz' okislitel'no-vosstanovitel'noi sistemy astrotsitov s produtsirovaniem aktivnykh form kisloroda.

Cells of neuroglia--the astrocytes are of interest from the point of view of their participation in phagocytosis. Phagocyte ability to generate active oxygen forms (AOF) as used as the basic criterion of the estimation of their functional activity. For the purpose to clear up molecular and cellular mechanisms of phagocytosis a study of astrocyte redox-systems, participating in production of AOF, was undertaken. Registration of AOF in astrocytes was carried out using a method of luminol-dependent chemiluminescence. Primary culture of guinea pig astrocytes was used. Spontaneous chemiluminescence of low intensity was found for the astrocytes at the presence of luminol. The destruction of the cells was accompanied by a significant growth of the intensity of spontaneous chemiluminescence. Suspension of endocutosis inductors, particle of latex and phytohemagglutinin, added to astrocytes did not result in formation of AOF, characteristic for other cells, possessing phagocytosis. It was established, that addition of hydrogen peroxide destroys astrocytes at the presence of luminol and gives rise to the emission. Chemiluminescence was not observed in similar experiments with intact cells. A conclusion was made that inside astrocytes there are structures, which show peroxidase-like activity.

Swelling-induced activation of Na+,K+,2Cl- cotransport in C6 glioma cells: kinetic properties and intracellular signalling mechanisms.

Swelling of C6 glioma cells in hypotonic medium (180 mOsm) results in two- to three-fold activation of K+ (86Rb+) influx suppressed by 10 microM bumetanide. Bumetanide-sensitive transport of 86Rb+ is dependent on extracellular K+, Na+ and Cl- both in iso-osmotic conditions and under hypo-osmotic shock, supporting the notion that it is mediated by Na+,K+,2Cl- cotransport. Inhibitors of protein kinase C (10 microM polymyxin B and l microM staurosporine) had no significant effect on basal cotransport but reduced its hypotonic stimulation by 70-80%. Similar results were obtained with calmodulin antagonist R24571 (10 microM), indicating Ca2+/calmodulin-dependence of the process. Influence of polymyxin B and R24571 was not additive. Swelling-activated Na+,K+,2Cl- cotransport was also suppressed by protein kinase C activator PMA (l microM). By contrast, preincubation of cells with inhibitors of protein phosphatases (100 microM vanadate, 5 mM fluoride and 0.5 microM okadaic acid) activated greatly the bumetanide-sensitive 86Rb+ uptake in isotonic conditions, while a subsequent hypotonic swelling led to smaller or no increment. These results indicate the involvement of Ca2+/calmodulin-dependent staurosporine/polymyxin B-sensitive protein kinase other than protein kinase C in swelling-induced activation of Na+,K+,2Cl- cotransport in glial cells.

[Some features of measles virus morphogenesis in highly differentiated human astrocyte cultures]. / Nekotorye osobennosti morfogeneza virusa kori v kul'turakh vysoko- differentsirovannykh astrotsitov cheloveka.

Astrocyte cultures of adult human brain were used to study the morphogenesis of measles virus at different multiplicity of infection. High-dose (1 to 10 TCD50/ml) inoculation of the culture was found to give rise to an acute infection with cytolysis on days 10-12 postinfection and formation of extracellular mature virions. Low multiplicity of infection (0.01-0.1 TCD50/ml) caused the persistence of virus. During a persistent infection the virus in titers 1.7 to 2.4 lg TCD50/ml was detected as long as up to the 3rd passage. Ultrastructural studies of such cultures revealed some features which were not observed in an acute infection: (1) the diameter of intracytoplasmic RNP increased by 1.5 times vs. that in other cell cultures because of coarse thickening of the envelope; (2) numerous immature virions in which nucleocapsids were completely or partially absent; and (3) uncommonly low number of mature virions. Moreover, alterations in the spatial orientation of cytoskeleton components, such as intermediate and thin (actin) filaments were observed. A conclusion is made that astrocytes as a reservoir for measles virus accumulation serve as the source of immature envelope virus production, which may be one of the possible causes of sclerosing panencephalitis. The causes of disturbances in RNP transport from the site of assembly to cell membrane are discussed.

[Morphological characteristics of the course of HIV infection in laboratory animals]. / Morfologicheskaia kharakteristika techeniia VICh-infektsii u laboratornykh zhivotnykh.

The integrative form of infection was obtained in cotton rats intraperitoneally or retrobulbarly infected with the highly-productive strain Zmb HIV-1. Clinically, it appeared as reduced animal weight gain and high mortality rates in the disease terminal stage. The proviral DNA was detected by the polymerase chain reaction in the genomes of cerebral and splenic cells in most animals. A comprehensive study of the time course of morphological changes within 6 months showed impairments in some CNS cells, giving rise to glial nodules and neurone dystrophy. It should be noted that the most profound changes were observed when the virus had been intraperitoneally injected. It was found that some pathomorphological changes were similar to those seen in human neuro-AIDS. In the spleen, HIV-1 first stimulated an immune response, caused an increase in the extent of white bulb follicles and an active formation of germinative centers, then involution of lymphoid formations took place.

[The use of a primary astrocyte culture for modelling HIV neurological infection]. / Ispol'zovanie pervichnoi kul'tury astrotsitov dlia modelirovaniia VICh-neiroinfektsii.

The article deals with the results of the experiment substantiating the in vitro model of HIV neuro-infection. In this work primary glial (astrocytic) tissue cultures obtained from normal human and animal (guinea pig) brain tissue were used. As revealed in this investigation, the following phenomena could be observed in human brain tissue monolayer culture, infected with HIV and subsequently subcultured: (a) the stimulation of tissue-cell growth; (b) the formation of multinuclear glial cells; (c) the presence of virus-specific proteins in astrocyte cytoplasm, detected by immunofluorescent and electrophoretic techniques; (d) the presence of HIV-1 DNA provirus in infected astrocytes. Cyto-destruction was not observed, reverse transcriptase activity was absent.

[The use of electron microscopy for diagnosing spongiform encephalopathies]. / Ispol'zovanie élektronnoi mikroskopii dlia diagnostiki spongioformnykh éntsefalopatii.

The authors suggest an unsophisticated approach to laboratory diagnosis of spongiform encephalopathies induced by unconventional prion viruses, consisting in electron microscopic detection of abnormal helical Scrapie-associated fibrils. These fibrils are detectable in human and animal brain tissue samples and in cell cultures.