Gene expression measurements in the context of epidemiological studies.

BACKGROUND: Gene expression measurements became an attractive tool to assess biological responses in epidemiological studies. However, collection of blood samples poses various technical problems. We used gene expression data from two epidemiological studies to evaluate differences between sampling methods, comparability of two methods for measuring RNA levels and stability of RNA samples over time. METHODS: For the PARSIFAL study, PBLC of 1155 children were collected using EDTA tubes in two countries. In the PASTURE study, tubes containing RNA-stabilizing solutions (PAXgene) Blood RNA Tubes; PreAnalytiX) were used to collect cord blood leucocytes of 982 children in five countries. Real-time PCR (conventional single tube assay and high-throughput low density arrays) was used to quantify expression of various innate immunity genes. In 77 PARSIFAL samples, gene expression was measured repeatedly during prolonged storage. RESULTS: In PARSIFAL (EDTA tubes) the median RNA yield after extraction significantly differed between the two centres (70 and 34 ng/microl). Collecting blood into an RNA-stabilizing solution markedly reduced differences in RNA yield in PASTURE (range of medians 91-107 ng/microl). The agreement [Spearman rank correlation (r)] between repeated measurements of gene expression decreased with increasing storage time [e.g., for CD14: r (first/second measurement) = 0.35; r (first/third measurement) = 0.03]. RNA levels measured with either the conventional method or low-density arrays were comparable (r > 0.9). CONCLUSION: Collecting blood samples into tubes containing an RNA-stabilizing solution increases RNA yield and reduces its variability. Long-term storage of samples may lead to RNA degradation, requiring special attention in longitudinal studies.

Human Toll-like receptor 2 mediates induction of the antimicrobial peptide human beta-defensin 2 in response to bacterial lipoprotein.

Recognition of pathogens by Drosophila Toll or human Toll-like receptors results in translocation of Dorsal or its human homologue NF-kappaB, respectively; in Drosophila, this is followed by the production of antimicrobial peptides serving as antimicrobial effector system of the innate immune response. We investigated whether human Toll-like receptors also mediate induction of the synthesis of antimicrobial peptides. We found that HEK293 cells transfected with Toll-like receptor 2, but not wild-type cells responded to stimulation with bacterial lipoprotein by production of human beta-defensin 2. Furthermore, the human lung epithelial cell line A549 was found to constitutively express Toll-like receptor 2 and to produce beta-defensin 2 in response to bacterial lipoprotein. This response was abrogated by blocking the signaling pathway activated through Toll-like receptors by transfecting the A549 cells with a dominant-negative form of IRAK-2. Thus, exposure of human cells to bacterial lipoprotein elicits production of the antimicrobial peptide beta-defensin 2 through Toll-like receptor 2.

Expression of MHC class II molecules contributes to lipopolysaccharide responsiveness.

Activation of phagocytes by lipopolysaccharide (LPS) causes synthesis and secretion of various mediators of inflammation. CD14, a glycosylphosphatidylinositol-anchored monocytic antigen serving as receptor for LPS, and members of the family of Toll-like receptors mediate cellular activation in response to LPS. Here we investigated whether expression of MHC class II molecules modified the response to LPS. Comparing LPS responsiveness of human and murine cells differing for expression of MHC class II molecules, we found that lack or a low level of expression of MHC class II molecules resulted in diminished secretion of pro-inflammatory cytokines following stimulation with LPS. Thus, expression of MHC class II molecules modifies LPS responsiveness, a finding suggesting that these molecules contribute to the pathogenesis not only of exotoxin-triggered toxic shock but also of endotoxin-triggered septic shock. Additionally to their role in antigen-specific immunity MHC class II molecules may influence the inflammatory response triggered by microbial constituents.

Sheath lengths of large motor axons in the ventral root L5 of neonatal and adult rats.

This study examines the developmental sheath elongation in the rat ventral root L5. Electron microscopic analysis of serial transverse sections through roots from newborn rats shows an average internuclear distance (IND) of 66 microns (calculated fresh length 73 microns). Light microscopic analysis of teased adult roots shows that the largest fibers have an average internodal length of some 1250 microns at 5 months. Hence, large fibers exhibit a developmental sheath elongation of 17 times. The ventral root L5 elongates 11 times only. This mismatch necessitates a myelin sheath remodelling.