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Lymphocyte localization to inflammatory sites is paramount for developing and maintaining an immune response. Rolling is the first step in recruitment, but our knowledge of its mechanisms in Th1 and Th2 CD4(+) lymphocytes is incomplete. Whereas initial studies suggested that Th1 but not Th2 lymphocytes used P-selectin for recruitment, more recent studies have proposed that both subtypes bind selectins. We used intravital microscopy to demonstrate in vivo that polarized Th1 and Th2 lymphocytes both use P-selectin to roll and adhere to cytokine [tumor necrosis factor (TNF)-alpha or interleukin (IL)-4]-activated intestinal microvasculature. The majority of Th1 lymphocyte flux in TNF-alpha- and IL-4-treated animals was P-selectin-dependent. Th1 lymphocytes also interacted with E-selectin to control rolling velocity after TNF-alpha stimulation. Th2 lymphocytes, which make IL-4 but not interferon-gamma, bound P-selectin ex vivo, with more than 95% rolling on P-selectin in vivo. Both Th1 and Th2 lymphocytes regulated rolling velocity by interacting with alpha(4)-integrin. Furthermore, in a model of spontaneous intestinal inflammation (ie, IL-10-deficient mice), both Th1 and Th2 lymphocytes rolled, adhered, and ultimately emigrated into the local microenvironment. These results suggest that both Th1 and Th2 lymphocytes use P-selectin in the initial rolling step in vivo in response to a global activator of the vasculature (TNF), a subtle inducer of P-selectin (IL-4), and pathological inflammation (IL-10-deficient mice).
Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely characterized. Using a modified Cre-loxP-based system, we now report the creation and characterization of a collection of ∼1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity.
Accurate quantification of gene expression by qRT-PCR relies on normalization against a consistently expressed control gene. However, control genes in common use often vary greatly between samples, especially in cancer. The advent of Next Generation Sequencing technology offers the possibility to better select control genes with the least cell to cell variability in steady state transcript levels. Here we analyze the transcriptomes of 55 leukemia samples to identify the most consistent genes. This list is enriched for components of the proteasome (ex. PSMA1) and spliceosome (ex. SF3B2), and also includes the translation initiation factor EIF4H, and many heterogeneous nuclear ribonucleoprotein genes (ex. HNRNPL). We have validated the consistency of our new control genes in 1933 cancer and normal tissues using publically available RNA-seq data, and their usefulness in qRT-PCR analysis is clearly demonstrated.
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