The threat of bioterrorism has become a reality since the attacks with Bacillus anthracis (anthrax) in the fall of 2001. The virulence of B.anthracis depends on a tripartite exotoxin and an anti-phagocytic bacterial capsule. In patients, clinical anthrax is both septicemia and toxemia, with features of an uncompensated inflammatory response. The victims of bioterrorism with inhaled or cutaneous anthrax developed a severe coagulopathy and experienced organ failure and other complications from disseminated intravascular coagulation. These responses are similar to the uncompensated inflammatory responses which contribute directly to the lethality and morbidity in established baboon models of E.coli-mediated sepsis. Activated protein C (APC), a member of the protein C pathway, is an important regulator of this host coagulation and inflammatory response to sepsis. First proposed by our group for the treatment of E.coli-mediated sepsis in the baboon, APC is now an FDA-approved therapy for patients with severe sepsis. The recent phase 3 clinical trial demonstrated that APC as an adjunct to standard anti-infective therapy significantly improved patient outcome. However, the effect of APC has not been tested in sepsis involving an exotoxin. We hypothesize that the protein C pathway plays a major role in modulating responses to B.anthracis septicemia-toxemia and that APC will reduce inflammation and mortality induced by B.anthracis. In order to test this, we will develop i.v. non-human primate models of B. anthracis (toxigenic, non-encapsulated Sterne strain) sepsis and anthrax toxemia. Dose-response studies will establish the concentration ranges of susceptibility. We will use these models to evaluate the contribution of protein C pathway members and other hemostatic regulators to the pathogenesis and lethality of anthrax infection. Antibodies that block the function of protein C pathway members (e.g., protein C, thrombomodulin, endothelial protein C receptor) will be infused with sub-lethal challenge to evaluate individual contributions to inflammatory responses andmortality. Changes at the tissue level (coagulation, inflammation, apoptosis, signaling) will be assessed with immunohistochemical, confocal and electron microscopic imaging and microarrays. Changes in cellular (neutrophil activation) and soluble mediators (e.g, sEPCR, TNF-alpha, IL-6, elastase, D-dimer) will be followed with flow cytometry and ELISAs. Using the baboon models, we will determine the effect of APC administration on mortality and pathological changes induced by B.anthracis or toxin challenge. These studies will establish non-human primate models of anthrax infection, identify molecular pathways unique to toxin action, identify regulatory pathways important to host defense against B.anthracis, provide clinically-relevant animal models for future evaluation of new therapeutic approaches, and determine the role of APC and the protein C pathway in regulating anthrax-mediated pathogenesis.
