Characterization of the antiviral effects of interferon-alpha against a SARS-like coronoavirus infection in vitro.

Interferon (IFN)-alphas bind to and activate their cognate cell surface receptor to invoke an antiviral response in target cells. Well-described receptor-mediated signaling events result in transcriptional regulation of IFN sensitive genes, effectors of this antiviral response. Results from a pilot study to evaluate the clinical efficacy of IFN-alpha treatment of SARS patients provided evidence for IFN-inducible resolution of disease. In this report we examined the contribution of IFN-inducible phosphorylation-activation of specific signaling effectors to protection from infection by a SARS-related murine coronavirus, MHV-1. As anticipated, the earliest receptor-activation event, Jak1 phosphorylation, is critical for IFN-inducible protection from MHV-1 infection. Additionally, we provide evidence for the contribution of two kinases, the MAP kinase p38MAPK, and protein kinase C (PKC) delta to antiviral protection from MHV-1 infection. Notably, our data suggest that MHV-1 infection, as for the Urbani SARS coronoavirus, inhibits an IFN response, inferred from the lack of activation of pkr and 2'5'-oas, genes associated with mediating the antiviral activities of IFN-alphas. To identify potential target genes that are activated downstream of the IFN-inducible signaling effectors we identified, and that mediate protection from coronavirus infection, we examined the gene expression profiles in the peripheral blood mononuclear cells of SARS patients who received IFN treatment. A subset of differentially regulated genes were distinguished with functional properties associated with antimicrobial activities.

Redox-active metals in commercial preparations of lipopolysaccharide: implications for studies of cellular responses to bacterial products.

The mechanisms by which lipopolysaccharide (LPS) activates cells have been the subject of intense investigation for many years. Whereas much information on this process has been collected for mammalian species, little is known about the signalling path-ways operative in other animals. One general mode of cellular activation that has been recently pro-posed for pathways independent of the primary mammalian LPS receptor, CD14, involves reactive oxygen species (ROS) as intermediates in LPS-induced signalling pathways. Therefore, we used 2',7'-dichlorodihydrofluorescein, a fluorogenic probe of redox activity, to examine LPS-induced oxidative responses of a macrophage-like cell line from the rainbow trout, RTS11. Lipopolysaccharide dose-dependently increased oxidation of this probe by RTS11 cells, and a variety of other cell lines. This process was inhibited by catalase, superoxide dismutase and NG-methylarginine citrate, an inhibitor of nitric oxide synthases, suggesting the involvement of a diverse assortment of cellular ROS. More careful dissection of this phenomenon led us to conclude that the increase in oxidation was, in fact, due almost entirely to metals, particularly copper, in some LPS preparations, which is something to consider when experimenting with LPS.