Genomic DNA functions as a universal external standard in quantitative real-time PCR

Real-time quantitative PCR (qPCR) is a powerful tool for quantifying specific DNA target sequences. Although determination of relative quantity is widely accepted as a reliable means of measuring differences between samples, there are advantages to being able to determine the absolute copy numbers of a given target. One approach to absolute quantification relies on construction of an accurate standard curve using appropriate external standards of known concentration. We have validated the use of tissue genomic DNA as a universal external standard to facilitate quantification of any target sequence contained in the genome of a given species, addressing several key technical issues regarding its use. This approach was applied to validate mRNA expression of gene candidates identified from microarray data and to determine gene copies in transgenic mice. A simple method that can assist achieving absolute quantification of gene expression would broadly enhance the uses of real-time qPCR and in particular, augment the evaluation of global gene expression studies

Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection

Progressive loss of T cell functionality is a hallmark of chronic infection with human immunodeficiency virus 1 (HIV-1). We have identified a novel population of dysfunctional T cells marked by surface expression of the glycoprotein Tim-3. The frequency of this population was increased in HIV-1–infected individuals to a mean of 49.4 ± SD 12.9% of CD8+ T cells expressing Tim-3 in HIV-1–infected chronic progressors versus 28.5 ± 6.8% in HIV-1–uninfected individuals. Levels of Tim-3 expression on T cells from HIV-1–infected inviduals correlated positively with HIV-1 viral load and CD38 expression and inversely with CD4+ T cell count. In progressive HIV-1 infection, Tim-3 expression was up-regulated on HIV-1–specific CD8+ T cells. Tim-3–expressing T cells failed to produce cytokine or proliferate in response to antigen and exhibited impaired Stat5, Erk1/2, and p38 signaling. Blocking the Tim-3 signaling pathway restored proliferation and enhanced cytokine production in HIV-1–specific T cells. Thus, Tim-3 represents a novel target for the therapeutic reversal of HIV-1–associated T cell dysfunction

Host Genetics and HIV-1: The Final Phase?

This is a crucial transition time for human genetics in general, and for HIV host genetics in particular. After years of equivocal results from candidate gene analyses, several genome-wide association studies have been published that looked at plasma viral load or disease progression. Results from other studies that used various large-scale approaches (siRNA screens, transcriptome or proteome analysis, comparative genomics) have also shed new light on retroviral pathogenesis. However, most of the inter-individual variability in response to HIV-1 infection remains to be explained: genome resequencing and systems biology approaches are now required to progress toward a better understanding of the complex interactions between HIV-1 and its human host

Differential expression of type I interferon genes in plasmacytoid dendritic cells from HIV-infected patientss

Background: Plasmacytoid dendritic cells (pDCs) are the most potent producers of type I interferons (IFN). Human genome contains thirteen IFN alpha genes and one IFN beta gene. Research in mice suggests that different IFNs are induced by different stimuli, but whether this is true in human cells is unknown. Patients with HIV-1 infection show chronic interferon response in peripheral T cells, which caused us to analyze the induction of the IFN alpha genes in their dendritic cells. Methods: Uninfected, acutely infected and long-term non-progressive donors were leukopheresed, following which pDCs were isolated by negative depletion. The dendritic cells were then treated for with one of the following: influenza virus, sendai virus, HIV virus, CpG DNA, imiquimod, or media alone, and the cells’ RNA was analysed by real time qPCR for changes in the RNA levels of four IFN alpha genes: α1, α2, α7, α8, as well as IFN beta. Results: Final results were not available at the time of abstract deposition

Distinct Transcriptional Profiles in Ex Vivo CD4(+) and CD8(+) T Cells Are Established Early in Human Immunodeficiency Virus Type 1 Infection and Are Characterized by a Chronic Interferon Response as Well as Extensive Transcriptional Changes in CD8(+) T Cells

Changes in T-cell function are a hallmark of human immunodeficiency virus type 1 (HIV-1) infection, but the pathogenic mechanisms leading to these changes are unclear. We examined the gene expression profiles in ex vivo human CD4(+) and CD8(+) T cells from untreated HIV-1-infected individuals at different clinical stages and rates of disease progression. Profiles of pure CD4(+) and CD8(+) T-cell subsets from HIV-1-infected nonprogressors with controlled viremia were indistinguishable from those of individuals not infected with HIV-1. Similarly, no gene clusters could distinguish T cells from individuals with early infection from those seen in chronic progressive HIV-1 infection, whereas differences were observed between uninfected individuals or nonprogressors versus early or chronic progressors. In early and chronic HIV-1 infection, three characteristic gene expression signatures were observed. (i) CD4(+) and CD8(+) T cells showed increased expression of interferon-stimulated genes (ISGs). However, some ISGs, including CXCL9, CXCL10, and CXCL11, and the interleukin-15 alpha receptor were not upregulated. (ii) CD4(+) and CD8(+) T cells showed a cluster similar to that observed in thymocytes. (iii) More genes were differentially regulated in CD8(+) T cells than in CD4(+) T cells, including a cluster of genes downregulated exclusively in CD8(+) T cells. In conclusion, HIV-1 infection induces a persistent T-cell transcriptional profile, early in infection, characterized by a dramatic but potentially aberrant interferon response and a profile suggesting an active thymic output. These findings highlight the complexity of the host-virus relationship in HIV-1 infection