Project description:Host response to systemic bacterial challenge (sepsis) with E. coli strains CFT073 and F11 at 6 and 12 hours post inoculum 48 hpf embryos were manually dechorianated, briefly anesthetized with 0.77 mM ethyl 3-aminobenzoate methanesulfonate salt (tricaine) (Sigma-Aldrich), and embedded in 0.8% low melt agarose (MO BIO Laboratories) without tricaine. After the agarose solidified, embryos were immersed in E3 media lacking methylene blue. Prior to injection, 1 ml of bacterial culture was pelleted, washed with 1 ml sterile PBS, and re-suspended in PBS to obtain ~1X109 CFU/ml. PBS. One nl of this bacterial suspension containing ~1000 CFU was microinjected into the bloodstream via the circulation valley using an Olympus SZ61 or SZX10 stereomicroscope together with a YOU-1 micromanipulator (Narishige), a Narishige IM-200 microinjector, and a JUN-AIR model 3-compressor. For each experiment, the average CFU per injection was determined by adding 10 1-nl drops to 1 ml of 0.7% NaCl, which was then serial diluted and plated on Luria-Bertani (LB) agar plates. Mock-infected controls were inoculated with 1 nl sterile PBS. Following injection, embryos were removed from agar and placed individually into wells of a 48-well plate (Nunc) containing E3 medium and incubated at 28.5°C. Experiments were performed in biological quadruplicate.
Project description:Severe sepsis leads to massive activation of coagulation and complement cascades that could contribute to multiple organ failure (MOF) and death. To investigate the role of the complement and its crosstalk with the hemostatic system in the pathophysiology and therapeutics of sepsis, we have used a potent inhibitor (compstatin) administered early or late post E. coli challenge in a baboon model of sepsis-induced MOF. Microarray was used to study the affect of complement pathway on global gene expression pattern in sepsis, aims on exploring and discovering the new target genes as potential drugs for the early treatment and prevention of sepsis. Lung and liver tissues were obtained from three normal healthy animals (as Ctl), three animals challenged with sublethal dose of E. coli as SLEC, three animals treated with Compstatin at different sepsis stages after E.coli challenge as SLEC-CST0 and SLEC-CST5. All the animals challenged with E. coli were sacrified at 24 hours post challenge. Total RNAs were isolated from these tissues, hybridized with Affymetrix Human Genome GeneChip U133A 2.0.
Project description:Amputation of heart tissue followed by regeneration of the heart. Samples were taken at 0 hpa (hours post-amputation), 6 hpa, 12 hpa, 24 hpa, 3 dpa and 5 dpa.
