Project description:Murine splenocytes were isolated from the spleens of C57BL/6J mice and were pretreated in vitro for three days in the presence of Pam3CSK4 (1 μg/ml), high pure LPS from E.coli O111:B4 (100 ng/ml) and R848 (5 μg/ml). PBS-treated splenocytes served as negative control. We used Qiagen Toll-like Receptor RT2 Profiler PCR Array kit to quantitate gene expression profiling of the TLR signaling pathway.
Project description:Murine splenocytes were isolated from the spleens of C57BL/6J mice and were pretreated in vitro for three days in the presence of PBS, Pam3CSK4 (1 μg/ml), high pure LPS from E.coli O111:B4 (100 ng/ml) and R848 (5 μg/ml). BALB/c irradiated recipients, were transplanted with T-cell depleted bone marrow alone, or with the aforementioned pretreated splenocytes, and 10 days after, PBMCs from all experimental groups were collected for TLR signaling pathway analysis.We used Qiagen Toll-like Receptor RT2 Profiler PCR Array kit to quantitate gene expression profiling of the TLR signaling pathway.
Project description:Murine CD3+ T-cells were immunomagnetically purified from the spleens of C57BL/6J mice and were pretreated in vitro for three days in the presence of R848 (5 μg/ml). Gene expression profile of wild type (WT) C57BL/6J TLR7-primed T-cells, was compared to unmanipulated B6 TLR7 null CD3+ Τ-cells. We used Qiagen Toll-like Receptor RT2 Profiler PCR Array kit to quantitate gene expression profiling of the TLR signaling pathway.
Project description:Infectious pneumonias exact an unacceptable mortality burden worldwide. Efforts to protect populations from pneumonia have historically focused on antibiotic development and vaccine-enhanced adaptive immunity. However, we have recently reported that the lungs’ innate defenses can be therapeutically induced by inhalation of a combination of synthetic TLR ligands that synergize to protect mice against otherwise lethal pneumonia. Simultaneous treatment with ligands for TLR2/6 and TLR9 conferred robust, synergistic protection against virulent Gram-positive and Gram-negative pathogens, as well as viruses. Protection is associated with rapid pathogen killing in the lungs, and pathogen killing can be induced from lung epithelial cells in isolation. Here we explore the mechanisms underlying this dramatic phenomenon by performing microarray gene expression analysis of mouse lungs treated by aerosol with PBS (sham treatment), Pam2CSK4 (TLR 2/6 ligand), ODN2395 (TLR9 ligand), or both TLR ligands. C57BL/6J mice were placed, unrestrained, in a aerosolization chamber and inhalationally exposed to 20 minute treatment with an 8 ml volume of PBS (sham), Pam2CSK4 10 ug/ml, ODN 2395 20 ug/ml, or the combination. 4 h after treatment, the mice were deeply anesthetized, their lungs were harvested, homogenized, and total RNA was extracted. Amplified cRNA was hybridized to Illumina Sentrix MouseRef-8 v2 Beadhips, labeled with Cy3, and scanned on an Illumina iScan. At least 8 unique samples were obtained per condition.
Project description:Murine CD3+ T-cells were immunomagnetically purified from the spleens of C57BL/6J mice and were pretreated in vitro for three days in the presence of R848 (5 μg/ml). Unmanipulated T-cells served as negative control. We used Qiagen Toll-like Receptor RT2 Profiler PCR Array kit to quantitate gene expression profiling of the TLR signaling pathway.
Project description:Because activation of the immune response is dependent on extensive changes in gene expression, it is likely that a major component of inter-individual variation in the immune response is ultimately mediated at the level of gene regulation. Here, we examine the influence of genetic variation on inducible gene expression in the murine immune response. We extracted primary CD4 splenocytes from inbred strains A/J, C57BL/6J, BALB/c, DBA/2J, and 129x1/SvJ (3 animals/strain) and measured mRNA transcript levels using microarrays in both basal state and four hours after stimulation with PMA/Ionomycin. We report strain-specific genome-wide differences in mRNA expression between A/J, Balb/c, C57BL/6J, DBA/2J and 129X1/SvJ inbred mice in CD4 splenocytes in basal, stimulated (4hr, PMA/I) states.
Project description:TLR signaling in PBMCs acquired from BALB/c mice (10 days post-BMT) after being transplanted with T-cell depleted bone marrow alone (BMTCD), or in combination with TLR2,4,7-tolerant splenocytes, over their PBS-pretreated counterparts.
