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发布于:2018-7-7 19:03:46  访问:23 次 回复:0 篇
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Re counted in triplicate on three different occasions by light microscopy.
Re counted in triplicate on three MGCD516 biological activity different occasions by light microscopy. Retrovirology 2010, 7:21 http://www.retrovirology.com/content/7/1/Page 4 ofintermediate characteristics between those obtained from SIVmac251- and HIV-1-infected cultures. Apart from being phylogenetically closer to SIVmac251 than to HIV-1, the HIV-2 strain that we used killed the majority of the infected cells in eight days following infection, thus showing viral cytopathogenicity kinetics slower than HIV-1 and more rapid than SIVmac251. To assess whether the difference in the EC 50 values for SIVmac251 and HIV-1 IIIB cytopathic effects were attributable to the different kinetics of viral cytopathogenicity, we measured, by antigen-capture ELISA assays, the viral core antigen in supernatants collected at five days post-infection from both the SIVmac251 and HIV1 infected cell cultures. In this case, the ranges of the EC50 values for SIVmac251 and HIV-1 obtained in the different experimental set-ups were overlapping (Fig. 1B). We concluded that raltegravir inhibits SIVmac251 replication in human T-cell lines with similar potency as shown against HIV-1. As different types of kits had to be used to compare inhibition of SIVmac251 p27 and HIV-1 p24 production, we decided to confirm the results using another method allowing simultaneous and homogeneous measurements of antiviral efficacy against SIVmac251, HIV-1, and HIV2. We used syncytia counts in CEMx174 cells as a measure of lentiviral replication. SIVmac251 replication induces syncytia at an earlier time point as compared to the cytopathic effect induced in MT-4 cells, in which lentiviral replication mostly induces apoptotic and necrotic cell death [29]. The effectiveness of syncytia counts as a parameter for detection of the antiretroviral effects was confirmed by correlation analyses of syncytium formation and viral core antigen production in the presence of antiretroviral drugs (an example using raltegravir is given in the additional material [see Additional file 2]). CEMx174 cells were infected with SIVmac251, HIV-1, and HIV-2 viral stocks at the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28404814 same multiplicity of infection (MOI), and syncytia were counted by optical microscopy at 4-5 days post-infection. Results confirmed that raltegravir exerted potent and reproducible anti-SIVmac251 activity (Fig 1C). To assess the anti-SIVmac251 effects of raltegravir under conditions more closely resembling those occurring in vivo, 3 day-old PHA-stimulated peripheral blood mononuclear cells (PBMCs) from uninfected rhesus macaques (Macaca mulatta) were infected with SIVmac251, and viral replication was quantified in supernatants by ELISA at five days post-infection, in order to allow comparison with the results reported in the previous paragraph. Also in this PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27735993 case, raltegravir displayed an EC50 in the low nanomolar range (Fig 1D). To assess the effect of raltegravir in the rhesus CD4+ T cell population, i.e., the main target of SIVmac251 in vivo, we separated the CD4 + T cells from freshunstimulated PBMCs using magnetic beads. Flow cytometric analysis of the enriched CD4 + T cell fraction showed that 94 to 100 of cells expressed the CD4 antigen (data not shown). Cells were PHA-stimulated for three days, infected with SIVmac251, and, again, viral replication was quantitated in supernatants by ELISA at five days post-infection.
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