Project Number: 1265-32000-083-02
Project Type: Reimbursable

Start Date: Aug 01, 2005
End Date: Sep 30, 2008

Objective:
Our broad objectives are to elucidate the signaling mechanisms that mediate cellular responses to LPS and to determine whether agents that disrupt the signaling pathway(s) activated by LPS can potentially serve as therapeutic interventions that protect against the systemic complications associated with Gram-negative mastitis. Specifically, we propose to: 1) identify the bovine TLR-4 signaling molecules that promote LPS-induced NF-kappaB activation and NF-kappaB-dependent gene expression; 2) identify novel bovine TLR-4 signaling molecules; 3) identify the bovine TLR-4 signaling molecules involved in LPS-induced apoptosis; 4) identify the bovine TLR-4 signaling molecules involved in LPS-induced permeability changes of EC monolayers.

Approach:
Bovine homologues of human TLR-4 signaling molecules will be sequenced and the cDNA cloned into an expression vector. Dominant-negative mutants and siRNA strategies will be utilized to evaluate the role of bovine TLR-4 signaling molecules in promoting LPS-induced NF-kappaB signaling, apoptosis, and changes in vascular endothelial monolayer permeability. Co-immunoprecipitation and co-immunolocalization techniques, as well as a proteomics approach, will be used to identify bovine TLR-4 signaling molecule interactions.