Ebola virion attachment and entry into human macrophages profoundly effects early cellular gene expression.

Zaire ebolavirus (ZEBOV) infections are associated with high lethality in primates. ZEBOV primarily targets mononuclear phagocytes, which are activated upon infection and secrete mediators believed to trigger initial stages of pathogenesis. The characterization of the responses of target cells to ZEBOV infection may therefore not only further understanding of pathogenesis but also suggest possible points of therapeutic intervention. Gene expression profiles of primary human macrophages exposed to ZEBOV were determined using DNA microarrays and quantitative PCR to gain insight into the cellular response immediately after cell entry. Significant changes in mRNA concentrations encoding for 88 cellular proteins were observed. Most of these proteins have not yet been implicated in ZEBOV infection. Some, however, are inflammatory mediators known to be elevated during the acute phase of disease in the blood of ZEBOV-infected humans. Interestingly, the cellular response occurred within the first hour of Ebola virion exposure, i.e. prior to virus gene expression. This observation supports the hypothesis that virion binding or entry mediated by the spike glycoprotein (GP(1,2)) is the primary stimulus for an initial response. Indeed, ZEBOV virions, LPS, and virus-like particles consisting of only the ZEBOV matrix protein VP40 and GP(1,2) (VLP(VP40-GP)) triggered comparable responses in macrophages, including pro-inflammatory and pro-apoptotic signals. In contrast, VLP(VP40) (particles lacking GP(1,2)) caused an aberrant response. This suggests that GP(1,2) binding to macrophages plays an important role in the immediate cellular response.

Thymocyte stimulation by anti-TCR-beta, but not by anti-TCR-alpha, leads to induction of developmental transcription program.

Anti-T cell receptor (aTCR) antibody (Ab) stimulation of T cells results in TCR down-modulation and T cell activation. Differences in the effect of anti-alpha-chain and beta-chain Ab have been reported on thymocytes. Anti-beta-chain Ab but not anti-alpha-chain reagents cause long-term TCR down-modulation. However, both types of Ab result in TCR cross-linking and activate early steps in signal transduction. In this study, we show that TCR internalization and calcium flux, hallmarks of T cell activation, are similar with aValpha and aVbeta treatment. Therefore, we have compared the gene expression profiles of preselection thymocytes stimulated with these reagents. We find that aValpha treatment does not cause any significant change in gene expression compared with control culture conditions. In contrast, aVbeta stimulation results in numerous changes in gene expression. The alterations of expression of genes known to be expressed in thymocytes are similar to changes caused by positive thymic selection, suggesting that the expression of some of the genes without known roles in thymocyte development and of novel genes whose expression is found to be altered may also be involved in this process.

Thymocyte stimulation by anti-TCR-ß, but not by anti-TCR-α, leads to induction of developmental transcription program.

Anti-T cell receptor (aTCR) antibody (Ab) stimulation of T cells results in TCR down-modulation and T cell activation. Differences in the effect of anti-α-chain and ß-chain Ab have been reported on thymocytes. Anti-ß-chain Ab but not anti-α-chain reagents cause long-term TCR down-modulation. However, both types of Ab result in TCR cross-linking and activate early steps in signal transduction. In this study, we show that TCR iternalization and calcium flux, hallmarks of T cell activation, are similar with aVα and aVß treatment. Therefore, we have compared the gene expression profiles of preselection thymocytes stimulated with these reagents. We find that aVα treatment does not cause any significant change in gene expression compared with control culture conditions. In contrast, aVß stimulation results in numerous changes in gene expression. The alterations of expression of genes known to be expressed in thymocytes are similar to changes caused by positive thymic selection, suggesting that the expression of some of the genes without known roles in thymocyte development and of novel genes whose expression is found to be altered may also be involved in this process.

Normalizing DNA microarray data.

DNA microarrays are a powerful tool to investigate differential gene expression for thousands of genes simultaneously. Although DNA microarrays have been widely used to understand the critical events underlying growth, development, homeostasis, behavior and the onset of disease, the management of the resulting data has received little attention. Presently, the fluorescent dyes Cy3 and Cy5 are most often used to prepare labeled cDNA for microarray hybridizations. Raw microarray data are image files that have to be transformed into gene expression formats--a process that requires data manipulation due to systematic variations which may be attributed to differences in the physical and chemical dye applications is to identify differences in transcript levels calculated from fluorescence ratios it is necessary to normalize fluorescence signals to compensate for systematic variations. Here, we will review current normalization strategies applied to cDNA microarrays and discuss their limits. We will show that experimental design determines normalization success.