[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.

[THE COMPARATIVE ANALYSIS OF EFFECTIVENESS OF QUICK TESTS IN DIAGNOSTIC OF INFLUENZA AND RESPIRATORY SYNCYTIAL VIRAL INFECTION IN CHILDREN].

The analysis was implemented concerning diagnostic parameters of commercial quick tests (immune chromatographic tests BinaxNOW Influenza A&B and BinaxNow RSV Alere, Scarborough Inc., USA) under detection of antigens of influenza virus A and respiratory syncytial virus in clinical materials. The polymerase chain reaction in real-time and isolation ofviruses in cell cultures. The analysis of naso-pharyngeal smears from 116 children demonstrated that sensitivity and specifcity of detection of influenza virus A using device mariPOC in comparison with polymerase chain reaction made up to 93.8% and 99.0% correspondingly at total concordance of results of both techniques as 98.3%. At diagnosing of respiratory syncytial virus using device mariPOC parameters made up to 77.3%, 98.9% and 862% as compared with polymerase chain reaction. The sensitivity, specificity and total concordance of results of immune chromatographic tests BinaxNOW in comparison ofpolymerase chain reaction made up to 86.7%, 100% and 96.2% correspondingly at detection of influenza virus A and 80.9%, 97.4% and 91.6% correspondingly at detection of respiratory syncytial virus. In comparison with isolation technique in cell cultures sensitivity of system mariPOC and immune chromatographic tests proved to be in 1.3-1.4 times higher at detection of influenza virus A and in 1.7-2 times higher in case of isolation of respiratory syncytial virus. There is no statistically significant differences between diagnostic parameters received for mariPOC and immune chromatographic tests at diagnosing influenza virus A and respiratory syncytial viral infection.

[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.

[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.

[Evolutionary variability of influenza B viruses in Russian Federation in 2005-2012].

Specific traits of influenza B viruses circulation in Russia and worldwide in 2005-2012 were studied and the amount of influenza B viruses in the whole population of influenza viruses isolated in Russia was estimated. The trend toward antigenic drift for both Victoria and Yamagata lineages was characterized. The genetic analysis revealed amino acid changes that influenced the antigenic properties of the viruses. The match of the epidemic isolates and vaccine strains was corroborated.

[Development of influenza surveillance in Russia in the system of the WHO national influenza center].

Analysis of development influenza activity season 2010-2011 is presented. Significant participation of influenza A(H1N1)pdm09 virus and influenza B of Victoria lineage virus in the epidemic morbidity structure with minor participation ofA(H3N2) virus was revealed. The influenza viruses isolated in Russia according to antigenic properties were similar to the strains included in the vaccine composition. Drift variants of influenza A(H1N1)pdm09 viruses isolated in Astrakhan and St.-Petersburg were recognized using WHO CC in London as representatives of three new genetic groups.

[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.

[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.

[The 2009 pandemic influenza in Russia. I. Diagnosis and molecular biological characteristics of the virus].

The analysis of 1558 clinical samples revealed influenza virus A(H1N1v) RNA in 339 patients with influenza and 163 fatal cases,which was made in May to December 2009. Data on the antigenic properties of more than 250 of pandemic virus strains isolated at the Research Institute of Influenza and the molecular genetic characteristics of 31 strains are presented. All the test isolates were found to have the S203 substitution in hemagglutinin, which was characteristic of one of 5 minor genome A(H1N1v) virus variants found in the United States and Mexico in 2009. All the test strains contain the S31N substitution in the M2 protein, which determines viral resistance to adamantine, and have no H275Y substitution in neuraminidase, which determines oseltamivir resistance. The substitution of amino acid residue of Asp to Gly at position 222 of HA was found in 8 (73%) of 11 isolates from postmortem lung and trachea samples and in 2 (10%) of 20 isolates from nasopharyngeal swabs. The determination of the pathogenic role of this substitution calls for further investigations.

[Analysis of pandemic influenza in Russia as a part of the global process based on data of an influenza monitoring reference center].

AIM: Characterization of features of influenza pandemic development in Russia in relation to global process. MATERIALS AND METHODS: Pandemic monitoring was performed by using results of integrative analysis of laboratory diagnostic and population morbidity data from 49 supporting bases of Federal center of influenza from various cities in Russian Federation. Isolation of influenza virus was carried out in MDCK cells and chicken embryos under BSL-3 conditions. Reference virus A/California/07/09 obtained from CDC (Atlanta, USA) and antisera against this strain contained in WHO kit were used for antigenic analysis; rat antisera, new monoclonal antibodies against pandemic influenza virus developed by Research institute of influenza were also used. RESULTS: Based on PCR monitoring during epidemic peak, rate of pandemic influenza identification reached 45-49% of examined patients. About 53% of lethal cases of respiratory infections were caused by pandemic influenza virus, while predominately young people died from pneumonia and acute respiratory distress syndrome. Russian isolates generally were antigenically and genetically similar to the parent pandemic strain--influenza virusA/California/07/09, but contained S203T substitution in hemagglutinin. A number of strains contained D222G mutation that is responsible for the expansion of substrate specificity, as well as strain specific substitutions in hemagglutinin and neuraminidase molecules. The investigated isolates were resistant to remantadin, but sensitive to oseltamivir. CONCLUSION: Due to the formation of population immunity after the end of the first pandemic wave new drift variants of the virus capable of overcoming this formed immunity should be expected that apparently will require the correction of vaccine composition for the 2011 - 2012 season.

[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.

Assessment of rat polyclonal antisera's suitability in hemagglutination inhibition assay for influenza surveillance and antigenic mapping.

This paper presents comparative hemagglutination inhibition (HI) assay data obtained using ferret or rat antisera to analyze influenza viruses. The results indicate that rat antisera can be successfully applied both for identification and for antigenic analysis of human influenza A and B viruses. Data gained with rat antisera were comparable to those obtained with ferret antisera. In-depth statistical analysis, based on Confusion Matrix analysis and Receiver Operating Characteristic (ROC) analysis, confirmed good coincidence between ferret antisera-based and rat antisera-based results. Two-dimensional antigenic mapping, based on HI assays using rat and ferret antisera, supported these findings. Both antisera types yielded identical antigenic attributions for the viruses analyzed, and both permitted visualization of contemporary human influenza virus evolutionary trends.

Stimulation of all epithelial elements during skin regeneration by keratinocyte growth factor.

Keratinocyte growth factor (KGF), a recently discovered 18.9 kD member of the fibroblast growth factor family has been shown to selectively induce keratinocyte proliferation and differentiation in tissue culture. To explore its potential stimulating keratinocyte growth and differentiation in vivo, we analyzed for the influence of KGF on epithelial derived elements within a wound created through the cartilage on the rabbit ear. KGF accelerated reepithelialization (p = 0.004) and increased the thickness of the epithelium (p = 0.0005) when 4-40 micrograms/cm2 recombinant KGF was added at the time of wounding. The regenerating epidermis showed normal differentiation as detected by cytokeratin immunostaining. Remarkably, however, KGF stimulated proliferation and differentiation of early progenitor cells within hair follicles and sebaceous glands in the wound bed and adjacent dermis. There was a transient but highly significant increase in specific labeling of cycling cells in both basal and suprabasal layers that extended into the spinous layer of the regenerating epidermis. As an indication of specificity, the inflammatory cells and fibroblasts within the wound were not influenced by KGF. The results indicate that KGF is unique in its ability to accelerate reepithelialization and dermal regeneration by targeting multiple epithelial elements within the skin. These results suggest that KGF may induce specific epithelial progenitor cell lineages within the skin to proliferate and differentiate, and thus may be a critical determinant of regeneration of skin. Furthermore, these findings illustrate the potential capacity of this system to analyze epithelial differentiation programs and disorders of epidermis, dermal glandular elements, and hair follicles.

[Etiological characteristics of the influenza epidemics of 2006-2009 in the Russian Federation (according to the data of the Research Institute of Influenza, North-Western Branch, Russian Academy of Medical Sciences)].

The basic trends in the evolution of influenza A and B in the Russian Federation during the epidemic seasons of 2006-2009 were studied on the basis of an antigenic analysis of 1774 Influenza isolated at the Research Institute of Influenza (RII), North-Western Branch, Russian Academy of Medical Sciences, and sent from resting bases (the regional centers of the Russian Inspectorate for the Protection of Consumer Rights and Human Welfare, which collaborate with the RII). Although the trends in the substitution of representative strains generally coincide with the world patterns, the authors revealed some specific features of the antigenic drift of influenza viruses in the Russian Federation and regional varieties. Data on some biological properties and those of the antigenic analysis of the first pandemic influenza A(H1NI)v strains isolated at the RII from Saint Petersburg patients in July-August 2009 are also given in the paper.

[Study of the antiviral activity of Russian anti-influenza agents in cell culture and animal models].

The study of the antiviral activity of Russian anti-influenza agents in the cultured MDCK cells demonstrated that arbidol and ribavirin inhibited the reproduction of various influenza A virus strains, including rimantadine- and ozeltamivir-resistant variants, as well as influenza B viruses (IC50 2-8.5 microg/ml). Rimantadine at concentrations of 1-5 microg/ml completely inhibited the reproduction of reference and ozeltamivir-resistant influenza A virus strains, and it had no effect on the reproduction of influenza B viruses and rimantadine-resistant influenza A viruses. Arbidol and ribavirin also inhibited the reproduction of pandemic influenza A/California/04/2009(H1N1), A/California/07/2009(H1N1), and A/Moscow/01/2009(H1N1)swl viruses in the cultured MDCK cells (IC50 = 1.5-4.0 microg/ml) while rimantadine had no effect on their reproduction. The cultured cells showed no significant antiviral activity of ingavirin at nontoxic concentrations (up to 200 microg/ml) against all study strains of influenza A and B viruses, including pandemic A(H1N1) influenza virus strains. The activity of rimantadine, arbidol, and ingavirin was found on a model of Influenza pneumonia in mice infected with their adopted influenza A/Aichi/2/69(H3N2) virus. The preventive efficacy of the three test agents was similar and most pronounced when they were used 96 hours before infection, by preventing 40-50% death in the animals and their body weight loss and by increasing their survival by 1.3-1.5 times. Arbidol and rimantadine were more effective when used for treatment and prophylaxis in doses of 30 and 10 mg/kg/day, respectively, by protecting the infected animals from 60-80% death, increasing their survival by 1.7-2 times, and preventing their body weight loss as compared with the control. The same experiments with ingavirin showed that this agent was less effective than arbidol and rimantadine. Thus, arbidol and rimantadine have a pronounced antiviral infection in both cell culture and a model of influenza pneumonia. The found efficacy of ingavirin on an integral model of murine influenza pneumonia without its activity in the cultured cells is likely to be due to other pharmacological properties of the drug rather than its direct virus-specific action.

PROBLEMS OF ISOLATION, IDENTIFICATION AND ANTIGENIC CHARACTERIZATION OF RECENT HUMAN A(H3N2) INFLUENZA VIRUSES.

Human A (H3N2) influenza viruses are distinguished by a high rate of evolution and regularly cause epidemics around the world. Their ability to adapt and to escape from the host's immune response and to change their receptor specificity is very high. Over the past 20 years, these viruses have lost the ability to agglutinate red blood cells of chickens and turkeys and have practically ceased to propagate in chicken embryos - the main source of influenza vaccines. Isolation of viruses in the MDCK cell culture led to the selection of strains that lose one of the potential glycosylation sites. Many of the A (H3N2) strains have acquired mutations in neuraminidase, which distort the results of antigenic analysis in the hemagglutination inhibition test - the cornerstone method for the analysis of the match between viral isolates circulating in human population to strains selected for the influenza vaccines. In this regard, the characteristics of the antigenic properties of influenza A (H3N2) viruses by traditional methods become poorly informative, and the selection of vaccine strains of this subtype is erroneous, which is reflected in the discrepancy between vaccine and circulating A (H3N2) viruses in recent years (2013-2014, 2014 -2015, 2015-2016). The search, development and implementation of new algorithms for the isolation and antigen analysis of influenza A (H3N2) viruses are extremely urgent.

Review article: nonclinical and clinical pharmacology, pharmacokinetics and pharmacodynamics of etrolizumab, an anti-ß7 integrin therapy for inflammatory bowel disease.

BACKGROUND: Novel treatments with superior benefit-risk profiles are needed to improve the long-term prognosis of patients with inflammatory bowel disease (IBD). Etrolizumab-a monoclonal antibody that specifically targets ß7 integrins-is currently under phase III clinical evaluation in IBD. AIM: This review summarises the available pharmacological and pharmacokinetic/pharmacodynamic data for etrolizumab to provide a comprehensive understanding of its mechanism of action (MOA) and pharmacological effects. METHODS: Published and internal unpublished data from nonclinical and clinical studies with etrolizumab are reviewed. RESULTS: Etrolizumab exerts its effect via a unique dual MOA that inhibits both leucocyte trafficking to the intestinal mucosa and retention within the intestinal epithelial layer. The gut-selectivity of etrolizumab results from its specific targeting of the ß7 subunit of α4ß7 and αEß7 integrins. Etrolizumab does not bind to α4ß1 integrin, which mediates lymphocyte trafficking to tissues including the central nervous system, a characteristic underlying its favourable safety with regard to progressive multifocal leucoencephalopathy. Phase I/II studies in patients with ulcerative colitis (UC) showed linear pharmacokinetics when etrolizumab was administered subcutaneously at 100 mg or higher once every 4 weeks. This dose was sufficient to enable full ß7 receptor occupancy in both blood and intestinal tissues of patients with moderate to severe UC. The phase II study results also suggested that patients with elevated intestinal expression of αE integrin may have an increased likelihood of clinical remission in response to etrolizumab treatment. CONCLUSION: Etrolizumab is a gut-selective, anti-ß7 integrin monoclonal antibody that may have therapeutic potential for the treatment of IBD.

Long-term impaired neutrophil migration in mice overexpressing human interleukin-8.

The proinflammatory chemokine interleukin-8 (IL-8/NAP-1) has been implicated in recruiting neutrophils to sites of acute and chronic tissue inflammation. In transgenic mice, elevated serum IL-8 levels ranging from 1 to 118 ng/ml were correlated with proportional increases in circulating neutrophils and proportional decreases in L-selectin expression on the surface of blood neutrophils. No change in the expression of the beta 2-integrins Mac-1 and LFA-1 was apparent on peripheral blood neutrophils of the IL-8 transgenic mice. Additionally, L-selectin expression on bone marrow neutrophils and neutrophil precursors was normal in all transgenic lines. IL-8 transgenic mice demonstrated an accumulation of neutrophils in the microcirculation of the lung, liver and spleen. Moreover, there was no evidence of neutrophil extravasation, plasma exudation or tissue damage in any IL-8 transgenic mice. Neutrophil migration into the inflamed peritoneal cavity was severely inhibited in IL-8 transgenic mice, but not in nontransgenic littermates. The IL-8 transgenic mice should serve as useful models for studying the putative role of neutrophils in mediating tissue damage in models of inflammation, such as hepatic ischemia and reperfusion injury, cecal puncture and ligation, and glomerulonephritis.

Neu differentiation factor upregulates epidermal migration and integrin expression in excisional wounds.

Neu differentiation factor (NDF) is a 44-kD glycoprotein which was isolated from ras-transformed rat fibroblasts and indirectly induces tyrosine phosphorylation of the HER-2/neu receptor via binding to either the HER-3 or HER-4 receptor. NDF contains a receptor binding epidermal growth factor (EGF)-like domain and is a member of the EGF family. There are multiple different isoforms of NDF which arise by alternative splicing of a single gene. To date, in vivo biologic activities have not been demonstrated for any NDF isoform. Since NDF, HER-2/neu, and HER-3 are present in skin, and other EGF family members can influence wound keratinocytes in vivo, we investigated whether NDF would stimulate epidermal migration and proliferation in a rabbit ear model of excisional wound repair. In this model, recombinant human NDF-alpha 2 (rhNDF-alpha 2), applied once at the time of wounding, induced a highly significant increase in both epidermal migration and epidermal thickness at doses ranging from 4 to 40 micrograms/cm2. In contrast, rhNDF-alpha 1, rhNDF-beta 1, and rhNDF-beta 2 had no apparent biologic effects in this model. rhNDF-alpha 2 also induced increased neoepidermal expression of alpha 5 and alpha 6 integrins, two of the earliest integrins to appear during epidermal migration. In addition, rhNDF-alpha 2-treated wounds exhibited increased neoepidermal expression of cytokeratin 10 and filaggrin, both epidermal differentiation markers. NDF alpha isoforms were expressed in dermal fibroblasts of wounded and unwounded skin, while both HER-2/neu and HER-3 were expressed in unwounded epidermis and dermal adnexa. In wounds, HER-2/neu expression was markedly decreased in the wound neoepidermis while neoepidermal HER-3 expression was markedly upregulated. Taken together, these results suggest that endogenous NDF-alpha 2 may function as a paracrine mediator directing initial epidermal migration during cutaneous tissue repair.