Fingolimod targeting protein phosphatase 2A differently affects IL-33 induced IL-2 and IFN-γ production in CD8(+) lymphocytes.

Multiple sclerosis patients are treated with fingolimod (FTY720), a prodrug that acts as an immune modulator. FTY720 is first phosphorylated to FTY720-P and then internalizes sphingosine-1-phosphate receptors, preventing lymphocyte sequestration. IL-33 is released from necrotic endothelial cells and contributes to MS severity by coactivating T cells. Herein we analyzed the influence of FTY720, FTY720-P, and S1P on IL-33 induced formation of IL-2 and IFN-γ, by using IL-33 receptor overexpressing EL4 cells, primary CD8(+) T cells, and splenocytes. EL4-ST2 cells released IL-2 after IL-33 stimulation that was inhibited dose-dependently by FTY720-P but not FTY720. In this system, S1P increased IL-2, and accordingly, inhibition of S1P producing sphingosine kinases diminished IL-2 release. In primary CD8(+) T cells and splenocytes IL-33/IL-12 stimulation induced IFN-γ, which was prevented by FTY720 but not FTY720-P, independently from intracellular phosphorylation. The inhibition of IFN-γ by nonphosphorylated FTY720 was mediated via the SET/protein phosphatase 2A (PP2A) pathway, since a SET peptide antagonist also prevented IFN-γ formation and the inhibition of IFN-γ by FTY720 was reversible by a PP2A inhibitor. While our findings directly improve the understanding of FTY720 therapy in MS, they could also contribute to side effects of FTY720 treatment, like progressive multifocal leukoencephalopathy, caused by an insufficient immune response to a viral infection.

Simultaneous and dose dependent melanoma cytotoxic and immune stimulatory activity of betulin.

Conventional cytostatic cancer treatments rarely result in the complete eradication of tumor cells. Therefore, new therapeutic strategies focus on antagonizing the immunosuppressive activity of established tumors. In particular, recent studies of antigen-loaded dendritic cells (DCs) eliciting a specific antitumor immune response has raised the hopes of achieving the complete elimination of tumor tissue. Genistein, fingolimod and betulin have already been described as active compounds in different types of cancer. Herein, we applied an integrated screening approach to characterize both their cytostatic and their immune-modulating properties side-by-side. As will be described in detail, our data confirmed that all three compounds exerted proapoptotic and antiproliferative activity in different B16 melanoma cell lines to a given extent, as revealed by an MTT assay, CFSE and DAPI staining. However, while genistein and fingolimod also affected the survival of primary bone marrow (BM) derived DCs of C57BL/6 mice, betulin exhibited a lower cytotoxicity for BMDCs in comparison to the melanoma cells. Moreover, we could show for the first time, that only betulin caused a simultaneous, highly specific immune-stimulating activity, as measured by the IL-12p70 release of Toll-like receptor 4-stimulated BMDCs by ELISA, which was due to increased IL-12p35 mRNA expression. Interestingly, the activation of DCs resulted in enhanced T lymphocyte stimulation, indicated by increased IL-2 and IFN-γ production of cytotoxic T cells in spleen cell co-culture assays which led to a decreased viability of B16 cells in an antigen specific model system. This may overcome the immunosuppressive environment of a tumor and destroy tumor cells more effectively in vivo if the immune response is specific targeted against the tumor tissue by antigen-loaded dendritic cells. In summary, cytostatic agents, such as betulin, that simultaneously exhibit immune stimulatory activity may serve as lead compounds and hold great promise as a novel approach for an integrated cancer therapy.

Neuraminidase inhibitor susceptibility of swine influenza A viruses isolated in Germany between 1981 and 2008.

European swine influenza A viruses donated the matrix protein 2 as well as the neuraminidase (NA) gene to pandemic influenza A (H1N1) viruses that emerged in 2009. As a result, the latter became amantadine resistant and neuraminidase inhibitor (NAI) susceptible. These recent developments reflecting the close connection between influenza A virus infection chains in humans and pigs urge an antiviral surveillance within swine influenza A viruses. Here, NAI susceptibility of 204 serologically typed swine influenza A viruses of subtypes H1N1, H1N2, and H3N2 circulating in Germany between 1981 and 2008 was analyzed in chemiluminescence-based NA inhibition assays. Mean 50% inhibitory concentrations of oseltamivir and zanamivir indicate a good drug susceptibility of tested viruses. As found for human isolates, the oseltamivir and zanamivir susceptibility was subtype-specific. So, swine influenza A (H1N1) viruses were just as susceptible to oseltamivir as to zanamivir. In contrast, swine H1N2 and H3N2 influenza A viruses were more sensitive to oseltamivir than to zanamivir. Furthermore, reduction in plaque size and virus spread by both drugs was tested with selected H1N1 and H1N2 isolates in MDCK cells expressing similar amounts of α2.3- and α2.6-linked sialic acid receptors. Data obtained in cell culture-based assays for H1N1 isolates correlated with that from enzyme inhibition assays. But, H1N2 isolates that are additionally glycosylated at Asn158 and Asn163 near the receptor-binding site of hemagglutinin (HA) were resistant to both NAI in MDCK cells. Possibly, these additional HA glycosylations cause a misbalance between HA and NA function that hampers or abolishes NAI activity in cells.

Antiviral potential and molecular insight into neuraminidase inhibiting diarylheptanoids from Alpinia katsumadai.

At present, neuraminidase (NA) inhibitors are the mainstay of pharmacological strategies to fight against global pandemic influenza. In the search for new antiviral drug leads from nature, the seed extract of Alpinia katsumadai has been phytochemically investigated. Among the six isolated constituents, four diarylheptanoids showed in vitro NA inhibitory activities in low micromolar ranges against human influenza virus A/PR/8/34 of subtype H1N1. The most promising constituent, katsumadain A (4; IC(50) = 1.05 +/- 0.42 microM), also inhibited the NA of four H1N1 swine influenza viruses, with IC(50) values between 0.9 and 1.64 muM, and showed antiviral effects in plaque reduction assays. Considering the flexible loop regions of NA, extensive molecular dynamics (MD) simulations were performed to study the putative binding mechanism of the T-shaped diarylheptanoid 4. Docking results showed well-established interactions between the protein and the core of this novel NA-inhibiting natural scaffold, excellent surface complementarity to the simulated binding pocket, and concordance with experimentally derived SAR data.