Summary: Anthrax, a disease caused by the gram positive bacterium, Bacillus anthracis, poses a significant threat as an agent of biological warfare. Anthrax toxin, consisting of three distinct proteins that help the bacterium evade the immune system and kill the host during systemic infection, is a major virulence factor. The toxin consists of three proteins that act in concert and includes two enzymes, lethal factor (LF) and edema factor (EF), and a protective antigen (PA). The primary cell type infected with B.anthracis is the macrophage and the clinical syndrome is reminiscent of septic shock, which may be mediated by cytokines produced in response to LT and ET. ET has ben reported to have an inhibitory effect on human monocytes, causing he disruption of cytokine networks. LT reportedly causes lysis of murine macrophages, release of high levels of NO and TNF-a, and defects in macrophage signal transduction pathways. However, studies regarding the impact of anthrax toxins are contradictory and have primarily involved the use of murine macrophages, which have biological properties that often differ significantly from human macrophages. The focus of our laboratory concerns the role of human monocytes/macrophages in infectious diseases and the impact of these diseases on cytokine networks. We propose to study the effects of the anthrax toxin proteins alone and in combination on human monocyte/macrophage function, including cytokine production. Studies with Dr. David Frucht in DMA/LCB will determine whether differences exist between murine and human macrophages under controlled conditions and whether the murine model is appropriate for pre-clinical studies for therapeutics proposed for use in the treatment of anthrax. We have obtained E.coli expression vectors for the three anthrax toxin components from Dr. John Collier. The expression and purification of endotoxin-free anthrax proteins is ongoing in the laboratory of Dr. Steve Kozlowski in DMA. Upon completion of this process, the above studies will be initiated. The ultimate goal is to determine the therapeutic potential of cytokines and cytokine antagonists in the treatment of B. anthracis infection.