The specific aims of this project address the hypothesis that impaired expression and/or function of leukocyte adhesive mechanisms in the neonate leads to deficits in acute inflammation and increased susceptibility to bacterial infection. The focus of this proposed research will be on the adhesive mechanisms by which neutrophils localize in tissue during acute inflammation, and the specific deficits in the neonate. The experimental design will follow the pattern of working with isolated molecular mechanisms and functions in vitro to define the specific neonatal deficits, and then examining the possible contribution of each deficit to reduced inflammation in vivo in a neonatal animal model. The studies in vitro will model leukocyte/endothelial cell adhesion under conditions of flow, leukocyte aggregation under conditions of defined shear stress, transendothelial migration with and without defined chemotactic gradients, and adherence-dependent mechanisms of leukocyte motility; and these studies will utilize specific probes for each of the known adhesive mechanisms (e.g., MAbs, Ig-chimeras, transfected cell lines, etc). The studies in vivo will utilize neonatal rabbits as a model of human neonates since we have evidence that neonatal rabbits exhibit some of the same major deficits that have been seen in neonatal human neutrophils in vitro.
Specific Aim 1. Define the specific deficits in margination of neonatal neutrophils on endothelial cell and platelet monolayers under conditions of flow. This involves investigation of the molecular basis for the L-selectin deficit, studies of P-selectin- and E-selectin-dependent pathways as they contribute to leukocyte margination (rolling adhesion), and beta2 integrin-dependent mechanisms for stopping rolling leukocytes.
Specific Aim 2. Define the specific deficits in homotypic and heterotypic aggregation of neonatal neutrophils under defined shear conditions. This involves investigation of L- selectin-, and P-selectin-dependent mechanisms as they apply to aggregation as well as studies of the kinetics of CD11b/CD18 adhesion and de-adhesion.
Specific Aim 3. Define the specific deficits in transendothelial migration of neonatal neutrophils. This involves adherence-dependent mechanisms of motility, migration in response to cytokine-stimulated endothelial cells, and migration in response to chemotactic gradients (e.g., IL-8).
Specific Aim 4. Define the contribution of each in vitro deficit in adhesive mechanisms to reduced acute inflammation in a rabbit neonatal model. This involves assessment of acute inflammation in response to nonspecific stimuli, evaluation of the adhesion cascade using intravital microscopy, and responses to specific bacteria (e.g., group B Streptococcus).
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