Influenza A virus lacking the effector and C terminal domains of NS1 protein induces cytokines associated with high pathogenicity in mice.

Non-structural NS1 protein of influenza A virus counters host antiviral defences by antagonizing the interferon response. The C-terminal effector domain suppresses the host response and is associated with the pathogenicity of the virus.  To better understand the regulatory role of the C-terminal domain, we used reverse genetics system to generate NS1-truncated virus (NS80) and compared the cytokine profiles in the lungs of mice infected with the NS80 mutant and with the control virus A/WSN/33 (WSN). The NS80 virus was attenuated and the viral titer in the lungs was about 25 times lower than viral titer of control A/WSN/33. Mice infected with NS80 virus exhibited more severe clinical symptoms and 2 mice died 6 days post infection. NS80 virus activated retinoic-inducible gene (RIG)-1-like receptor signaling pathway more strongly than control WSN virus and mice infected with NS80 virus exhibited a greater abundance and more diverse cytokine profile.  Infection with NS80 virus induced the expression of the following factors: pro-inflammatory cytokines (IL-1α, IL-1ß, TNF-α, IL-16), interferons (IFN-α and IFN-ε), chemokines (CCL2, CCL11, CXCL1, CXCL5, CXCL10, CXCL11 and CXCL13), matrix metallopeptidase 9 (MMP-9), metallopeptidase inhibitor 1 (TIMP-1), macrophage colony-stimulating factor (M-CSF), and vascular cell adhesion protein 1 (VCAM-1). All these cytokines are associated with viral pathogenicity. Our data show that attenuation of the virus should not be directly linked with pathogenicity. Keywords: influenza virus; NS1 protein; cytokines; interferon; pathogenicity.

Genistein Induces Bcl-2 Expression in Human Dermal Microvascular Endothelial Cells: a Short Report.

It has been shown previously that oestradiol protects the vascular network, leading to increased skin flap viability associated with Bcl-2, VEGF and FGF-2 up-regulation. We have shown that genistein, a natural selective oestrogen receptor modulator, also increases skin flap viability in rats and induces Bcl-2 expression in human umbilical vein endothelial cells. In the present study we aimed to answer the question whether genistein increases expression of Bcl-2, a potent anti-apoptotic protein, in human dermal microvascular endothelial cells (HMVEC-d) as well. Our results showed that administration of genistein induces Bcl-2 expression in a concentration-dependent manner. Cell co-treatment with genistein and anti-ER compounds (MPP, PHTPP, ICI, G-15) diminished the observed positive effect of genistein on Bcl-2 expression. The decrease in Bcl-2 expression in HMVEC-d was most prominent after co-treatment with ICI (nuclear ER antagonist/ GPR30 agonist) and PHTPP (selective ER-ß antagonist). In conclusion, genistein increases Bcl-2 expression in HMVEC-d, contributing to its protective effect on the skin flap viability. However, the question whether the mechanism is ER-specific (via ER-ß) has to be answered in further studies using a model of gene silencing or genetically modified cells.

Genistein and Selected Phytoestrogen-Containing Extracts Differently Modulate Antioxidant Properties and Cell Differentiation: an in Vitro Study in NIH-3T3, HaCaT and MCF-7 Cells.

During the last decades, plant extracts containing phytoestrogens have increasingly been used as an alternative to oestradiol hormone replacement therapy. The aim of the present study was to compare the effects of genistein with those of different phytoestrogen-containing plant extracts (from red clover flowers and soybeans) on the proliferation and differentiation of NIH-3T3, HaCaT and MCF-7 cells. Our results showed poor correlations between direct anti/pro-oxidant effects and cytotoxicity of the tested samples. In contrast, genistein showed a direct correlation between significant pro-oxidative effects at cytotoxic concentrations and almost no pro-oxidative effects at non-cytotoxic concentrations. Moreover, the tested red clover extract and genistein induced keratin-8 (luminal and prognostic marker in breast cancer) expression only in MCF-7 cells, but this effect was not seen following treatment with the soybean extract. From this point of view, the effect of consumption of phytoestrogens in oestrogen-positive breast cancer remains to be elucidated. In conclusion, our study demonstrates that various phytoestrogen- containing plant extracts and genistein are able to specifically modulate antioxidant properties and differentiation of studied cells.

Comparison of transcriptional profiles of interferons, CXCL10 and RIG-1 in influenza infected A549 cells stimulated with exogenous interferons.

Type I and type III interferons (IFNs) are induced by viral infection. It was concluded that these IFN species are identical in regulation and biological functions. However, these two systems differ in the tissue expression of their receptors and their transcriptional regulation is fundamentally different as well as cellular signaling pathways that drive expression of each IFN. Here, we have investigated the transcriptional profile of endogenous IFNs after stimulation of cells with exogenous IFNs and subsequent infection of A549 cells with A/chicken/Germany/27 [H7N7] influenza virus. Both type I and type III IFNs exhibit high degree of the cross-induction. Our results show that type III IFNs (IFN-λ1, IFN-λ2 and IFN-λ3) are better inducers of CXCL10 than type I IFNs. The IFN-ß1a and IFN-λ2 were the most potent IFNs and they highly increased the level of IFN-α, IFN-ß, IFN-λ, and CXCL10 mRNAs. Since type I IFNs up regulated expression of retinoic acid-inducible gene 1 (RIG-1) mRNA, type III IFNs-λ down regulated expression of RIG-1 mRNA in influenza infected cells. IFN-α and IFN-ω induced similar amount of IFN-α, IFN-ß and IFN-λ mRNA but differ in induction of CXCL10 and RIG-1 mRNA.