While polymorphonuclear leukocytes (PMNs) and mononuclear phagocytes are highly effective at clearing infections, several bacteria (including the periodontal pathogen Actinobacillus actinomycetemcomitans [A.a.]) can resist phagocytic killing. Antimicrobial therapy against intracellular pathogens is complicated by the inability of many agents to penetrate phagocytes. However, phagocytes take up ciprofloxcin and other fluoroquinolones with high affinity. When loaded with these bactericidal agents, PMNs exhibit enhanced phagocytic killing and can potentially serve as vehicles for fluoroquinolone delivery as they migrate from the bloodstream to infection sites (e.g., the periodontal pocket). Little is known of the mechanism by which phagocytes take up fluoroquinolones. Recent work from this laboratory indicates that PMN ciprofloxcin transport is a Na+-independent process that is competitively inhibited by cationic amino acids, properties known to be associated with amino acid transport system y+. Agents that activate protein kinase C (PKC) induce a dramatic increase in the Vmax of ciprofloxcin transport through a mechanism that appears to involve the mitogen-activated protein kinase (MAP kinase) cascade. This proposal will test the hypothesis that system y+ is the major mechanism for fluoroquinolone accumulation in phagocytes and is regulated by PKC and MAP kinase. The long-term objective is to enhance the effectiveness of antimicrobial therapy.
Specific Aim 1 is to characterize and identify the transport system(s) by which PMNs and monocytes take up fluoroquinolones.
Specific Aim 2 is to identify agents that stimulate fluoroquinolone transport in phagocytes and define the mechanisms by which this process is regulated, emphasizing the role of PKC and MAP kinase.
Specific Aim 3 is to determine whether fluoroquinolone uptake by phagocytes contributes to enhanced delivery of fluoroquinolones to the periodontal pocket or enhanced killing of A.a.. Enhancement of phagocytic killing would be especially useful in the anaerobic environment of the pocket, where oxidative killing mechanism are ineffective. Ultimately, this work could facilitate new approaches for treating infections by A.a. and other pathogens that resist phagocytic killing (e.g., Salmonella and Chlamydia).
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