Long-Term Maintenance of the Functional Changes Induced by Influenza A Virus and/or LPS in Human Endothelial ECV-304 Cell Sublines.

Influenza A virus and secondary bacterial infection may have remote effects in the form of cardiovascular complications or fibrosis in different organs. However, the mechanisms governing the development of complications remain poorly studied. The present work reports the comparative assessment of the functional changes which take place in human ECV-304 endothelial cell sublines obtained previously by the long-term culturing of cells after exposure to varying infectious doses (IDs) of influenza A virus, and/or bacterial lipopolysaccharide (LPS). It has been demonstrated that, in the course of long-term culturing (six passages) after exposure to pathogenic agents (influenza virus and/or LPS), endothelial cells maintain changes in their migratory activity, permeability, and expression of mRNA for cytokines TNFα and TGFß (along with the changes in their proliferation activity, which has been demonstrated earlier). The pattern of changes depended on the type of the agent (agents) to which the cells were exposed. The differences in migratory activity (which was at its maximum 4 h after wounding) between the cell sublines at the sixth passage correlated with the differences in their proliferation activity at the first passage (proliferation data were obtained previously). In particular, an increase in migration and proliferation was observed in the sublines exposed to low virus doses (ECV-1ID), as well as exposed to LPS (ECV-LPS), while the suppression of migration and proliferation was observed in the subline exposed to high virus doses (ECV-1000ID). In the ECV-1ID, ECV-LPS, and most notably in ECV-1ID + LPS sublines, we detected an increase in the expression of mRNA for cytokines TNFα and TGFß, which, however, didn't lead to the induction of apoptosis. We have also demonstrated an increase in cell permeability in the analyzed sublines, which was indicated by a decrease in the expression of the mRNAs for the genes encoding occludin and ZO-1, the tight junctions proteins . This paper also reports an evaluation of the effects of the antiviral preparations rimantadine and alpisarin on the functional state of cell sublines. As a result, it has been demonstrated that these drugs may be able to prevent the development of the pathological changes caused by influenza A virus and/or LPS in endothelial cells. The results obtained in the present work may be of use when studying the mechanisms of development of the influenza A virus and secondary bacterial infection complications.

[THE APPLICATION OF DOT-TECHNIQUE FOR DETECTING ANTIGENS OF ADENOVIRUS IN CLINICAL SAMPLES].

The article substantiates possibility of application of point enzyme-linked immunosorbent assay (dot-technique) for detecting viral antigens in samples from patients. To diagnose adenovirus infection conjugate of virus-specific monoclonal antibodies and peroxidase of horse-radish were used The chromatographic rectification of conjugate from free peroxidase permits diminishing background coloring of nitrocellulose membrane and therefore to increase sensitivity. The application of direct conjugates on the basis of virus-specific monoclonal antibodies increases specifcity of dot-technique and significantly shortens time period of analysis. As in case of application of direct conjugates on the basis of polyclonal serum, samples from patients require preliminary processing with detergent for preventing non-specific reactions. The dot-technique demonstrates good coincidence with data of polymerase chain reaction and after clinical trials it can be used in diagnostic of human viral infections.

Toxic and DNA damaging effects of a functionalized fullerene in human embryonic lung fibroblasts.

Water-soluble fullerenes have been studied as potential nanovectors and therapeutic agents, but their possible toxicity is of concern. We have studied the effects of F-828, a soluble fullerene [C60] derivative, on diploid human embryonic lung fibroblasts (HELFs) in vitro. F-828 causes complex time-dependent changes in ROS levels. Inhibition of Nox4 activity by plumbagin blocks F-828-dependent ROS elevation. F-828 induces DNA breaks, as measured by the comet assay and γH2AX expression, and the activities of the transcription factors NF-kB and p53 increase. F-828 concentrations>25µM are cytotoxic; cell death occurs by necrosis. Expression levels of TGF-ß, RHOA, RHOC, ROCK1, and SMAD2 increase following exposure to F-828. Our results raise the possibility that fullerene F-828 may induce pulmonary fibrosis in vivo.

INFLUENCE OF INFLUENZA A VIRUS AND BACTERIAL LIPOPOLYSACCHARIDE ON PROLIFERATION AND GENE EXPRESSION OF CYTOKINES AND OTHER CELLULAR FACTORS IN CELLS OF ESTABLISHED ENDOTHELIAL CELL LINE ECV-304.

Change of state of endothelial cells occurs under the action of viral infection and bacterial lipopolysaccharide (LPS) that leads to cell dysfunction. Therefore, the aim of the current study was to investigate the effect of LPS from Escherichia coli and influenza A virus on proliferative activity of human endothelial cells (ECV-304) and gene expression of several cytokines and cellular factors: TNFá, TGFâ, IFN-ã, MMP-9, NF-êB, Rho A, eNOS and iNOS. It was found that ECV-304 cells once infected with very low infectious doses of influenza virus acquire the ability to long-term active proliferation (over 8 passages). Addition of LPS E. coli reduced the virus-stimulated cell proliferation. It was shown that influenza virus and LPS can affect on gene expression of cytokine and other cellular factors. When endothelial cells had been infected with influenza A virus in the presence of LPS, there was a significant increase in the expression of several genes and replacement of some genes expression on the expression of other genes. Expression of MMP-9 gene was inhibited in the case of separate exposure to the virus and LPS, but it was significantly increased during the first day under the adding of the virus and LPS together, as well as the activity of the IFN-ã gene; gene of TNFá was active for only 1­3 days whereas genes expression of other factors (TGFâ, eNOS, iNOS, NF-êB and Rho A) increased significantly at the 5th day as in the case of adding only LPS. Thus, the change of physiological state of endothelial cells occurs in the presence of influenza A virus and LPS and it can be caused during different time periods (as well as by varying degrees of virus infection of cells) by different cellular factors and possibly with involvement of different signaling pathways.

[IMPACT OF VARIOUS MULTIPLICITY OF INFECTION OF INFLUENZA A VIRUS ON PROLIFERATION AND APOPTOSIS INDUCTION IN CULTURED CELL LINES OF LYMPHOCYTIC AND MONOCYTIC ORIGIN (JURKAT, NC-37, THP-1 AND U-937)].

The severity of disease caused by influenza A infection depends not only on biological characteristics of the virus but also on the number of viral particles than penetrate the body. T- and B-lymphocytes as well as monocytes (macrophages) play a key role in the development of cell-based and humoral immunity as well as influenza virus elimination from the body. The present study describes the effect of influenza A virus infection on cell proliferation and induction of apoptosis in human cultured cell lines of T-, B-lymphocytic and monocytic origin infected with various multiplicity of infection (moi). Low moi of the virus stimulated cell proliferation; maximal effect has been registered 3-4 days after infection. But the fate of T-cells, B-cells and monocytes after initial infection was different: Jurkat cells continued intense proliferation while proliferation of NC-37, THP-1 and U-937 cells lowered. Prolonged (for 3 passages) cultivation of Jurkat, NC-37 and U-937 cell lines has shown that infection of these cell lines not only with low but also with medium and high moi also leads to stimulation of proliferation. Using a variety of methods for the detection of viral reproduction has clearly shown that infection of non-permissive human T-, B-cells and monocytes with influenza A virus leads to latent infection. So, low moi interferes with normal formation of viral particles, which in turn might stimulate cell proliferation and then be followed by induction of apoptosis. Antiviral drags rimantadine and ribavirin suppressed virus-induced cell proliferation; at the same time, induction of apoptosis was suppressed only by rimantadine and was enhanced by ribavirin. The data obtained provide strong support for the role of influenza A virus in the observed effects.

Synthesis and antiviral activity of PB1 component of the influenza A RNA polymerase peptide fragments.

This study is devoted to the antiviral activity of peptide fragments from the PB1 protein - a component of the influenza A RNA polymerase. The antiviral activity of the peptides synthesized was studied in MDCK cell cultures against the pandemic influenza strain A/California/07/2009 (H1N1) pdm09. We found that peptide fragments 6-13, 6-14, 26-30, 395-400, and 531-540 of the PB1 protein were capable of suppressing viral replication in cell culture. Terminal modifications i.e. N-acetylation and C-amidation increased the antiviral properties of the peptides significantly. Peptide PB1 (6-14) with both termini modified showed maximum antiviral activity, its inhibitory activity manifesting itself during the early stages of viral replication. It was also shown that the fluorescent-labeled analog of this peptide was able to penetrate into the cell. The broad range of virus-inhibiting activity of PB1 (6-14) peptide was confirmed using a panel of influenza A viruses of H1, H3 and H5 subtypes including those resistant to oseltamivir, the leading drug in anti-influenza therapy. Thus, short peptide fragments of the PB1 protein could serve as leads for future development of influenza prevention and/or treatment agents.

[Comparative study of MDCK and CaCo-2 cell lines for influenza virus isolation].

Study of effectiveness of CaCo-2 cell line for influenza virus isolation was carried out. It was shown that the properties of this cell line strongly depended on the source of its origin and cultivation conditions. The infectious activity of the influenza viruses on CaCo-2 cell line was virtually the same as in the MDCK cell line. The rate of the viral isolation was virtually identical for both cell lines tested, but viruses from post-mortem materials were isolated only in CaCo-2 cell line. In general, the CaCo-2 line is believed to be a valuable cell line for virological research, particularly for influenza virus isolation.

Oxidized extracellular DNA suppresses nitric oxide production by endothelial NO synthase (eNOS) in human endothelial cells (HUVEC).

Circulating DNA from patients with cardiovascular diseases reduce the synthesis of NO in endothelial cells, which is probably related to oxidative modification of DNA. To test this hypothesis, HUVEC cells were cultured in the presence of DNA containing ~1 (nonoxidized DNA), 700, or 2100 8-oxodG/10(6) nucleosides. Nonoxidized DNA stimulated the synthesis of NO, which was associated with an increase in the expression of endothelial NO synthase. Oxidized NO decreased the amount of mRNA and protein for endothelial NO synthase, but increased the relative content of its low active form. These changes were accompanied by reduction of NO production. These findings suggest that oxidative modification of circulating extracellular DNA contributes to endothelial dysfunction manifested in suppression of NO production.

[New monoclonal kits for the diagnosis of the adenoviral infection].

Diagnostic properties of new monoclonal antibodies (MAbs) to hexon adenovirus antigen (AB) monoclonal ELISA kit for early diagnosis of adenoviral infection were tested. Developed ELISA kit and FITC-conjugate of new monocional antibodies for immunofluorescent analysis were used for detection of different types of adenoviruses in clinical materials. The availability of their use in clinical and epidemiological practice was validated.

[Role of cellular cytoskeleton in influenza A infectious cycle].

Influenza remains a significant social threat especially regarding the emergence of new mutant or reassortant strains. Measures of prophylaxis do not provide complete and stable protection from infection and the use of antivirals results in high-level occurrence of resistant forms of viruses. Nowadays more and more attention is paid to find new targets for antiviral therapy that are not directly connected with virus proteins but can act indirectly through cellular mechanisms involved in viral replication. This approach requires complete understanding of various cellular pathways used by influenza virus. Here we present a brief overview of interactions between influenza A virus and the cell cytoskeleton. This interaction is initiated from the very beginning of influenza infection--adsorption--and continues with endocytosis, release of viral RNP and its entry into the nucleus. The role of cytoskeleton during the late stages of infection is also of great importance. It takes part in NP translocation from the nucleus to the cytoplasm, virus assembly and budding. The presence of cellular actin in certain influenza virions is therefore not accidental but reflects the peculiarities of interaction between a virus and a host cell.

[Karyotypic variability of human myeloma cell lines].

Cytogenetic analysis of human myeloma cell lines L363, Karpas 707, RPMI 8226 and U-266 has been performed. Chromosome numbers retained near-diploid (L363, Karpas 707 and U-266) or increased to hypotriploid (RPMI 8226) during many years of maintenance of the cell lines in vitro. Based on G-banding analysis, the complex karyotypes with abnormalities of virtually all chromosome pairs were found in these cell lines but common chromosome translocations were not observed. In addition, chromosome loci involved in structural rearrangements in these cell lines often overlapped with loci of DNA copy number imbalances revealed in myeloma cells in vivo. Besides, distinct types of karyotypic structure of cell populations were found in all of these cell lines which differed by combination of cells with main and additional structural variants of karyotype and cells with non-clonal chromosome aberrations. Taken together, it seems obvious that karyotypic variability of human myeloma cell lines corresponds to karyotypic progression of myeloma in vivo and, hence, has tumor specific pattern.

[Use of the most recent reagent (CuFL) for stimulation of NO synthesis by the medicinal leech salivary cell secretion in the cultures of human endothelium cells (HUVEC) and in rat cardiomiocytes].

The medicinal leech salivary cell secretion (SCS) may stimulate NO-production in cultures of human endothelium cells (HUVEC) and rat cardiomiocytes (RCM). This effect was detected using a NO specific reagent, - the complex Cu2+ with a fluorescein derivative (Cu-Fl). NO had also been detected in the cells by fluorescent electronic microscopy and determined quantitatively in the cells and in culture fluid by the fluorescence method. SCS stimulated NO synthesis in HUVEC cells (but not in RCM) is accompanied by NO release into intercellular space. Localization of NO synthesis centers is presented and it is shown that the increase in NO levels during the SCS action on HUVEC and RCM is associated with the increase in the activity of eNOS/nNOS, but not iNOS. In endothelial cells SCS activates nitrosylation processes, assessed by the increase of nitrite-ions in the culture medium. It is therefore important to use Cu-Fl, other than Griss-reagent, during the first hour of analysis of NO synthesis. The NO-depended mechanism of SCS action on endothelial cells might be a factor in providing of its positive action in hirudotheraphy.

[Cell-free DNA fragments increase transcription in human mesenchymal stem cells, activate TLR-dependent signal pathway and supress apoptosis].

Human mesenchymal stem cells (MSCs) are now widely adopted in regenerative medicine. However, many questions on the role of different signaling pathways in the regulation of stem cell (SC) functional activity within the organism remain unaswered. In damaged regions the level of cell death increases and DNA fragments from dead cells (cell-free DNA, cfDNA) are accumulated in blood. We showed that in adipose-derived MSCs exposed in vitro to cfDNA fragments the transcription level increased (the total amount of cellular RNA and the rRNA amount rose). GC-rich CfDNA fragments (GC-DNA) activated the TLR9-dependent signal pathway: the expression of TLR9 and of TLR9-signaling pathway adapter--MyD88--was up-regulated. AT-rich DNA fragments did not increase the TLR9 expression, though, the MyD88 expression level rose. So we suggest that AT-DNA acts via some other receptors that nevertheless activate MyD88-dependent signalling in MSCs. We also showed that cfDNA fragments decreased the activity of caspase, an apoptotic enzyme. So, ctDNA can significantly influence the functional activity ofMSC by activating TLR9- and MyD88-dependent signal pathways and lowering the apoptosis level.

[Dynamics of interferon status in patients with atopic dermatitis during standard therapy combined with cycloferon].

Interferon status at different stages of atopic dermatitis was evaluated in 28 patients. Its dynamics and disturbances during standard therapy combined with cycloferon were clarified. Positive laboratory and clinical results of the combined treatment give reason to recommend it for the management of patients with atopic dermatitis.

[Pandemic influenza 2009 in Russia. Characteristics of the isolation and biological properties of viruses].

Research Institute of Influenza, Ministry of Health and Social Development of Russia, Saint Petersburg The characteristics of the isolation of pandemic influenza A(H1N1)v viruses were studied on chick embryos (CE) and MDCK cell culture. The materials (nasal swabs and autopsies) were collected in different regions Russia in the period from 20 July to 30 December 2009. The paper gives the data of the antigenic analysis of isolates, their capacity to multiply in different species-specific and tissue cell cultures. The viruses isolated on CE were shown to have higher hemagglutination titers and to be more stable. Isolation from the autopsies was effective only on CE. All the test cell lines other than MDCK were insensitive to the isolated pandemic influenza strains. The antigenic analysis showed no significant antigenic drift of the viruses isolated during the first wave of the pandemic in the Russian Federation.

[Comparative study of the differential susceptibility of different cell lines to pandemic H1N1v influenza viruses and avian influenza, swine influenza, and human influenza viruses].

The proliferation characteristics of influenza viruses of different origin were tested in various human and animal cell cultures. Pandemic H1N1v influenza and swine influenza viruses were shown to have a low infectious activity in virtually all the test lines. In spite of this, the replication of this group of viruses may be detected by de novo NP synthesis. These viruses are able to activate programmed cell death. Moreover, a low inoculative virus dose exerts a stimulating effect on cell proliferation in both suspension and monolayer cell lines.

[Influence of rimantadine, ribavirine and triazavirine on influenza A virus replication in human monolayer and lymphoblastoid cell lines].

The influence of antivirals, such as rimantadine, ribavirine and triazavirine on influenza virus replication in human cell cultures was evaluated. All the antivirals inhibited viral nucleoprotein NP synthesis. The strongest effect was shown for ribavirine in lung carcinoma A-549 cells and endothelial ECV-304 cells. Hoechst-33258 staining revealed induction of apoptosis in all the cell lines. Rimantadine and ribavirine inhibited virus-induced apoptosis while ribavirine enhanced it. The effect was registered in monolayer cell cultures as well as in suspension cell cultures. The influence of the antiviral drugs on the virus-induced cell proliferation in the suspension cell cultures is also described.

[Development of competitive enzyme immunoassay for the detection of influenza A(H5) virus antibodies].

An indirect enzyme immunoassay (EIA) using the purified fraction of surface viral glycoproteins (GP) as an antigen for solid phase sensitization was not shown to be a specific method for the differential detection of influenza A(HS) (HS-Ab) virus antibodies (Abs) due to total conservative epitopes in the structure of GPs of influenza A(H5) and A(H1NI) viruses. The cross activity of some monoclonal Abs (MAbs) to influenza A(H5) and A(HIN1) viruses, which had been obtained at the Research Institute of Influenza, was proof of the presence of total immunodominant determinants in the structure of influenza H1 and H5 virus hemagglutinin (HA). In this connection, an EIA, which was based on the competition of influenza A(H5) H5-Ab virus HA-specific MAbs in the test sera for an association with influenza A(H5) virus, was proposed for the subtype-specific detection of H5 Ab. Comparison of the results of competitive EIA (cEIA), microneutralization (MN) test and HA inhibition test (HAIT) (using equine red blood cells) in the examination of sera obtained from 44 volunteers immunized with inactivated vaccine containing influenza A/Indonesia/5/2005 (H5N1) virus showed the high sensitivity and specificity of cEIA in detecting H5-specific Abs. The effectiveness of cEIA for the sera strictly positive for the content of H5 Abs was close to that of MN test and was 9-34% higher than HAIT (depending on those used in the analysis of H5 virus antigens). cEIA may be proposed to assess new influenza vaccines as an additional laboratory test. Since the infectious virus is not used during cEIA, it may be recommended for the serodiagnosis of influenza A(H5) at practical virological laboratories.

Enhanced expression of iNOS in human endothelial cells during long-term culturing with extracellular DNA fragments.

NO synthesis by endothelial cells plays an important role in normal function of the cardiovascular system. This work was designed to evaluate the role of variations in properties of extracellular DNA in the regulation of NO synthesis. We studied the effect of four DNA samples with various base sequences (50 ng/ml) on functional activity of endothelial cells HUVEC during 24-h culturing. Human DNA fragments with high content of CG repeats increased intracellular content of NO and its metabolites (nitrites and nitrates) and accelerated oxidation of nitrites to nitrates. Changes in the content of NO metabolites after 24-h culturing was shown to depend on the expression of gene for inducible, but not for endothelial NO synthase. Increased expression of inducible NO synthase positively correlated with an increase in the content of mRNA for the adapter protein MyD88, but did not depend on TLR9 gene expression that encodes protein receptor for CG-DNA recognition. The intracellular concentration of MyD88 mRNA did not depend on the content of TLR9 mRNA. The existence of a variety of DNA-binding receptors apart from TLR9 receptor on the surface of endothelial cells was hypothesized. Activation of these receptors by extracellular DNA fragments stimulates expression of the adapter protein MyD88.

Extracellular DNA affects NO content in human endothelial cells.

Fragments of extracellular DNA are permanently released into the blood flow due to cell apoptosis and possible de novo DNA synthesis. To find out whether extracellular DNA can affect the synthesis of nitric oxide (NO), one of key vascular tone regulators, we studied in vitro effects of three artificial DNA probes with different sequences and 10 samples of extracellular DNA (obtained from healthy people and patients with hypertension and atherosclerosis) on NO synthesis in endothelial cell culture (HUVEC). For detection of NO in live cells and culture medium, we used a NO-specific agent CuFL penetrating into the cells and forming a fluorescent product FL-NO upon interaction with NO. Human genome DNA fragments affected the content of NO in endothelial cells; this effect depended on both the base sequence and concentration of DNA fragments. Addition of artificial DNA and extracellular DNA from healthy people into the cell culture in a low concentration (5 ng/ml) increased the detected NO concentration by 4-fold at most. Cytosine-guanine (CG)-rich fragment of the transcribed sequence of ribosomal repeat was the most powerful NO-inductor. The effect of DNA fragments on NO synthesis was comparable with that of low doses of oxidizing agents, H(2)O(2) and 17ß-estradiol. Extracellular DNA samples obtained from patients with hypertension and atherosclerosis decreased NO content in cells and medium by 1.3-28 times compared to the control; the effect correlated with the content of CG-rich sequences.