Project description:We established a new protocol for negative immunomagnetic isolation of murine primary Type II alveolar epithelial cells (AEC II) yielding untouched primary murine AEC II. AEC II were collected from mice 24h after Aspergillus fumigatus or mock infection (9 replicates per experimental group) and analyzed by label-free quantitative proteomics.
Project description:Cyclic stretch of alveoli is characteristic of mechanical ventilation, and is postulated to be partly responsible for the lung injury and inflammation in ventilator induced lung injury. We propose that miRNAs may regulate some of the stretch response and, therefore, hypothesized that miRNAs would be differentially expressed between stretched and unstretched rat alveolar epithelial cells (RAECs). RAECs were isolated and cultured to express type I epithelial characteristics, after which they were equibiaxially stretched to 25% change in surface area at 15 cycles/minute for 1 or 6 hrs, or served as unstretched controls, and miRNA were extracted (n = 4 for all groups).
Project description:A GFP-expressing recombinant A/Puerto Rico/8/1934 influenza virus was used to infect C57BL/6 wild type mice and on day 3 post infection, lung alveolar epithelial cells (AEC) were isolated and sorted based on GFP expression. GFP+ AEC represent the infected AEC and GFP- AEC represent the bystander AEC. AEC were also sorted from uninfected mice to serve as controls.
Project description:Analysis of LBNF1 rat testes from controls, containing both somatic and all germ cell types and from irradiated rats in which all cells germ cells except type A spermatgogonia are eliminated. Results provide insight into distinguishing germ and somatic cell genes and identification of somatic cell genes that are upregulated after irradiation.
Project description:The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. Spores must escape through the alveolar epithelial cell (AEC) barrier and migrate to regional lymph nodes, germinate and enter the circulatory system to cause disease. Several mechanisms to explain alveolar escape have been postulated, and all these tacitly involve the AEC barrier. In this study, we incorporate our primary human type I AEC model, microarray gene profiling and gene enrichment analysis to study the response of AEC to B. anthracis, (Sterne) spores at 4 and 24 hours post-exposure. Spore exposure altered gene expression in AEC after 4 and 24 hours and differentially expressed genes (±1.3 fold, p ≤ 0.05) included CCL4/MIP-1β (4 hours), CXCL8/IL-8 (4 and 24 hours) and CXCL5/ENA-78 (24 hours). Gene enrichment analysis revealed that pathways involving cytokine or chemokine activity, receptor binding, and innate immune responses to infection were prominent. Microarray results were confirmed by qRT-PCR and multiplex ELISA assays. Chemotaxis assays demonstrated that spores induced the release of biologically active neutrophil and monocyte chemokines, and that CXCL8/IL-8 was the major neutrophil chemokine. The small or sub-chemotactic doses of CXCL5/ENA-78, CXCL3/GROββ and CCL20/MIP-3α may contribute to chemotaxis by priming effects. These data provide the first whole transcriptomic description of the human type I AEC initial response to B. anthracis spore exposure, and contribute to an increased understanding of the role of AEC in the pathogenesis of inhalational anthrax. We used microarrays to create a whole transcriptomic description of the response of primary human type I alveolar epithelial cells to B. anthracis spore exposure and demonstrated that several of the most upregulated differentially expressed genes included those for neutrophil and monocyte chemokines.
