Abstract: DESCRIPTION (provided by applicant): 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.

Public Health Relevance:
This Public Health Relevance is not available.

Thesaurus Terms:
Bacillus anthracis, anthrax, disseminated intravascular coagulation, host organism interaction, inflammation, nonhuman therapy evaluation, pathologic process, protein C, septicemia
anthrax toxin, bacteria infection mechanism, coagulation factor VII, heparin, microorganism disease chemotherapy, spore
baboon, bioterrorism /chemical warfare, confocal scanning microscopy, electron microscopy, enzyme linked immunosorbent assay, flow cytometry, immunocytochemistry, microarray technology

Institution: OKLAHOMA MEDICAL RESEARCH FOUNDATION
825 N. E. 13TH STREET
OKLAHOMA CITY, OK 731045005
Fiscal Year: 2006
Department:
Project Start: 15-JAN-2005
Project End: 31-DEC-2009
ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
IRG: SAT

Am J Pathol. 2006 August; 169(2): 433–444.
doi: 10.2353/ajpath.2006.051330. PMCID: PMC1698797
Copyright © American Society for Investigative Pathology

Sepsis and Pathophysiology of Anthrax in a Nonhuman Primate Model

Deborah J. Stearns-Kurosawa,* Florea Lupu,† Fletcher B. Taylor, Jr.,† Gary Kinasewitz,‡ and Shinichiro Kurosawa*

From the Departments of Free Radical Biology and Aging Research* and Cardiovascular Biology,† Oklahoma Medical Research Foundation, Oklahoma City; and the Department of Medicine,‡ Pulmonary and Critical Care Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
Accepted May 4, 2006.

Bacillus anthracis, a zoonotic toxigenic gram-positive, spore-forming rod, is the cause of clinical anthrax disease. There has been a significant resurgence in biomedical anthrax-related research because of the bioterrorism attacks in the United States.1–6 As a result, the genomic sequence of B. anthracis has been completed,7 exotoxin crystal structures solved,8,9 and cellular toxin receptors identified.10,11 The virulence of B. anthracis bacilli is primarily governed by products of two large plasmids that code for secreted exotoxins and an exterior capsule.12,13 The capsule, composed of poly-γ-D-glutamic acid, has anti-phagocytic properties and contributes to bacterial dissemination.14 Under the control of the atxA gene product,15 the bacteria produce exotoxin components: protective antigen (PA) serves as a conduit for translocation of lethal factor (metalloprotease) and edema factor (adenylate cyclase) into the cell for toxicity and injury.16

However, the pathophysiology of anthrax as a septic disease is less well defined. Sepsis is defined as a host systemic inflammatory response to infection and is complicated in severe sepsis with organ dysfunction, hypoperfusion, and coagulation abnormalities.17 Clinical and pathology data from the victims of anthrax bioterrorism,1,18 as well as a 1979 inadvertent release of military-grade anthrax spores in Russia,19,20 show evidence of concomitant pulmonary edema, inflammation, and disseminated intravascular coagulation (DIC). To mimic anthrax, considerable work in animal models, including rhesus monkeys and chimpanzees, has been done using administration of spores by various routes, including aerosol.21–24 These studies investigated important spore dose-response relationships and subsequent pathology observations were consistent with a general consensus that B. anthracis introduced by the respiratory route results in a fulminating septicemia rather than a primary pulmonary infection.22 However, a consistent picture of pathophysiology progression is difficult to ascertain from these inhalational models. There is significant individual variation in gross and microscopic pathology of rhesus monkeys after challenge,24 probably attributable to dose-response issues because it is difficult to know how many of the inhaled spores actually result in infection. Although organ hemorrhage, edema, and inflammatory infiltrates were noted in some animals, a systematic analysis of inflammatory or coagulation biomarkers was not available. These observations are further compounded by the current paradigm, based on toxic murine models, which describes anthrax pathogenesis as being governed by exotoxin bioactivities and host inflammatory or coagulopathic responses as playing little role.25–27 These disparities gain importance when extrapolating experimental data to patients because vaccine development and clinical management decisions are based on an understanding of disease pathogenesis.

The current study addresses whether the pathogenesis of the bacteremic phase of anthrax is governed by predominately noninflammatory pathways as suggested by toxic murine models or is represented by uncompensated inflammation and coagulation responses to the infection. We have adapted our nonhuman primate model of E. coli sepsis that has been extensively characterized28,29 and has served as the basis30 for clinical studies that culminated in Food and Drug Administration approval of an adjunct therapy for patients with severe sepsis.31 We chose infection by infusion of bacteria for reproducible dosing, because with a high B. anthracis spore infection dose, the onset of bacteremia is rapid, with dissemination within 24 ~ 48 hours,14,32 and overwhelming.23 This approach mimics the bacteremia stage during which patients become sick and seek medical attention. Unencapsulated B. anthracis 34F2 Sterne strain was used because this strain produces toxin in quantities similar to the natural fully virulent strains.33 The results illustrate the physiological, hemostatic, cellular, and inflammatory responses to anthrax, as well as distinctive lung pathology that may be a unique feature of anthrax.

Animals
Infusion methods were essentially identical to those used for E. coli34 and Shiga toxin 1.35 Papio c. cynocephalus or Papio c. anubis baboons were purchased from the breeding colony maintained at the University of Oklahoma Health Sciences Center (Dr. Gary White, Director). Baboons were free of tuberculosis, weighed 6 to 8 kg, had leukocyte concentrations of 5000/mm3 to 14,000/mm3, and hematocrits exceeding 36%. T0-hour blood samples were drawn from the cephalic vein catheter followed by bacteria infusion for 2 hours. Levofloxacin infusion (7 mg/kg) was initiated at T4 hours and repeated daily. Infusion studies were performed at the University of Oklahoma Health Sciences Center. All experiments were approved by the Institutional Animal Care and Use Committee and the Institutional Biosafety Committee of the Oklahoma Medical Research Foundation and the University of Oklahoma Health Sciences Center.

Bacteria
Vegetative bacteria germinated from Bacillus anthracis 34F2 Sterne strain spores (Colorado Serum Co., Boulder, CO) were washed and resuspended in sterile saline for infusion. Live bacteria were quantitated using the Bac-Titer-Glo microbial cell viability assay (Promega, Madison, WI). In preliminary studies, a standard curve of viable bacteria (BacTiter-Glo) versus viable bacteria obtained by traditional plating methods (CFU/ml) was established. This relationship was very reproducible (r = 0.99; n = 3), permitting use of the luminescence assay for determining viable bacteria counts, rather than counting colonies on plates, which can be difficult with B. anthracis because of chaining. CFU/kg dosage was calculated by reference to this standard curve.

Infusion Procedures
Briefly, the baboons were fasted for 24 hours before the study, with free access to water. They were immobilized the morning of the experiment with ketamine (14 mg/kg, i.m.) and sodium pentobarbital administered through a percutaneous catheter in the cephalic vein of the forearm to maintain a light level of surgical anesthesia (2 mg/kg, approximately every 20 to 40 minutes). This catheter was also used to infuse the B. anthracis bacteria and sterile saline to replace insensible loss. An additional percutaneous catheter was inserted into the saphenous vein in one hind limb and the catheter advanced to the inferior vena cava; this catheter was used for sampling blood. Baboons were orally intubated and positioned on their left side on a heat pad. Our typical infusion protocol involved blood draw at T0, followed immediately by bacteria infusion at the appropriate concentration for 2 hours, typically at 0.2 ml/minute.