Sepsis and Pathophysiology of Anthrax in a Nonhuman Primate Model
2006; Elsevier BV; Volume: 169; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2006.051330
ISSN1525-2191
AutoresDeborah J. Stearns-Kurosawa, Florea Lupu, Fletcher B. Taylor, Gary T. Kinasewitz, Shinichiro Kurosawa,
Tópico(s)Zoonotic diseases and public health
ResumoStudies that define natural responses to bacterial sepsis assumed new relevance after the lethal bioterrorist attacks with Bacillus anthracis (anthrax), a spore-forming, toxigenic gram-positive bacillus. Considerable effort has focused on identifying adjunctive therapeutics and vaccines to prevent future deaths, but translation of promising compounds into the clinical setting necessitates an animal model that recapitulates responses observed in humans. Here we describe a nonhuman primate (Papio c. cynocephalus) model of B. anthracis infection using infusion of toxigenic B. anthracis Sterne 34F2 bacteria (5 × 105 to 6.5 × 109 CFU/kg). Similar to that seen in human patients, we observed changes in vascular permeability, disseminated intravascular coagulation, and systemic inflammation. The lung was a primary target organ with serosanguinous pleural effusions, intra-alveolar edema, and hemorrhagic lesions. This animal model reveals that a fatal outcome is dominated by the host septic response, thereby providing important insights into approaches for treatment and prevention of anthrax in humans. Studies that define natural responses to bacterial sepsis assumed new relevance after the lethal bioterrorist attacks with Bacillus anthracis (anthrax), a spore-forming, toxigenic gram-positive bacillus. Considerable effort has focused on identifying adjunctive therapeutics and vaccines to prevent future deaths, but translation of promising compounds into the clinical setting necessitates an animal model that recapitulates responses observed in humans. Here we describe a nonhuman primate (Papio c. cynocephalus) model of B. anthracis infection using infusion of toxigenic B. anthracis Sterne 34F2 bacteria (5 × 105 to 6.5 × 109 CFU/kg). Similar to that seen in human patients, we observed changes in vascular permeability, disseminated intravascular coagulation, and systemic inflammation. The lung was a primary target organ with serosanguinous pleural effusions, intra-alveolar edema, and hemorrhagic lesions. This animal model reveals that a fatal outcome is dominated by the host septic response, thereby providing important insights into approaches for treatment and prevention of anthrax in humans. 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.1Jernigan JA Stephens DS Ashford DA Omenaca C Topiel MS Galbraith M Tapper M Fisk TL Zaki S Popovic T Meyer RF Quinn CP Harper SA Fridkin SK Sejvar JJ Shepard CW McConnell M Guarner J Shieh WJ Malecki JM Gerberding JL Hughes JM Perkins BA Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States.Emerg Infect Dis. 2001; 7: 933-944Crossref PubMed Scopus (834) Google Scholar, 2Freedman A Afonja O Chang MW Mostashari F Blaser M Perez-Perez G Lazarus H Schacht R Guttenberg J Traister M Borkowsky W Cutaneous anthrax associated with microangiopathic hemolytic anemia and coagulopathy in a 7-month-old infant.JAMA. 2002; 287: 869-874Crossref PubMed Scopus (72) Google Scholar, 3Borio L Frank D Mani V Chiriboga C Pollanen M Ripple M Ali S DiAngelo C Lee J Arden J Titus J Fowler D O'Toole T Masur H Bartlett J Inglesby T Death due to bioterrorism-related inhalational anthrax: report of 2 patients.JAMA. 2001; 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19: 45-70Crossref PubMed Scopus (476) Google Scholar 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.17Levy MM Fink MP Marshall JC Abraham E Angus D Cook D Cohen J Opal SM Vincent JL Ramsay G 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference.Crit Care Med. 2003; 31: 1250-1256Crossref PubMed Scopus (4721) Google Scholar Clinical and pathology data from the victims of anthrax bioterrorism,1Jernigan JA Stephens DS Ashford DA Omenaca C Topiel MS Galbraith M Tapper M Fisk TL Zaki S Popovic T Meyer RF Quinn CP Harper SA Fridkin SK Sejvar JJ Shepard CW McConnell M Guarner J Shieh WJ Malecki JM Gerberding JL Hughes JM Perkins BA Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States.Emerg Infect Dis. 2001; 7: 933-944Crossref PubMed Scopus (834) Google Scholar, 18Guarner J Jernigan JA Shieh WJ Tatti K Flannagan LM Stephens DS Popovic T Ashford DA Perkins BA Zaki SR Pathology and pathogenesis of bioterrorism-related inhalational anthrax.Am J Pathol. 2003; 163: 701-709Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar as well as a 1979 inadvertent release of military-grade anthrax spores in Russia,19Abramova FA Grinberg LM Yampolskaya OV Walker DH Pathology of inhalational anthrax in 42 cases from the Sverdlovsk outbreak of 1979.Proc Natl Acad Sci USA. 1993; 90: 2291-2294Crossref PubMed Scopus (353) Google Scholar, 20Meselson M Guillemin J Hugh-Jones M Langmuir A Popova I Shelokov A Yampolskaya O The Sverdlovsk anthrax outbreak of 1979.Science. 1994; 266: 1202-1208Crossref PubMed Scopus (756) Google Scholar 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.21Gleiser CA Berdjis CC Hartman HA Gochenour WS Pathology of experimental respiratory anthrax in Macaca mulatta.Br J Exp Pathol. 1963; 44: 416-426PubMed Google Scholar, 22Albrink WS Goodlow RJ Experimental inhalation anthrax in the chimpanzee.Am J Pathol. 1959; 35: 1055-1065PubMed Google Scholar, 23Lincoln RE Hodges DR Klein F Mahlandt BG Jones Jr, WI Haines BW Rhian MA Walker JS Role of the lymphatics in the pathogenesis of anthrax.J Infect Dis. 1965; 115: 481-494Crossref PubMed Scopus (79) Google Scholar, 24Fritz DL Jaax NK Lawrence WB Davis KJ Pitt ML Ezzell JW Friedlander AM Pathology of experimental inhalation anthrax in the rhesus monkey.Lab Invest. 1995; 73: 691-702PubMed Google Scholar 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.22Albrink WS Goodlow RJ Experimental inhalation anthrax in the chimpanzee.Am J Pathol. 1959; 35: 1055-1065PubMed Google Scholar However, a consistent picture of pathophysiology progression is difficult to ascertain from these inhalational models. 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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.25Cui X Moayeri M Li Y Li X Haley M Fitz Y Correa-Araujo R Banks SM Leppla SH Eichacker PQ Lethality during continuous anthrax lethal toxin infusion is associated with circulatory shock but not inflammatory cytokine or nitric oxide release in rats.Am J Physiol. 2004; 286: R699-R709Google Scholar, 26Shoop WL Xiong Y Wiltsie J Woods A Guo J Pivnichny JV Felcetto T Michael BF Bansal A Cummings RT Cunningham BR Friedlander AM Douglas CM Patel SB Wisniewski D Scapin G Salowe SP Zaller DM Chapman KT Scolnick EM Schmatz DM Bartizal K MacCoss M Hermes JD Anthrax lethal factor inhibition.Proc Natl Acad Sci USA. 2005; 102: 7958-7963Crossref PubMed Scopus (147) Google Scholar, 27Moayeri M Haines D Young HA Leppla SH Bacillus anthracis lethal toxin induces TNF-alpha-independent hypoxia-mediated toxicity in mice.J Clin Invest. 2003; 112: 670-682Crossref PubMed Scopus (259) Google Scholar 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 characterized28Taylor Jr, FB Staging of the pathophysiologic responses of the primate microvasculature to Escherichia coli and endotoxin: examination of the elements of the compensated response and their links to the corresponding uncompensated lethal variants.Crit Care Med. 2001; 29: S78-S89Crossref PubMed Scopus (64) Google Scholar, 29Taylor Jr, FB Wada H Kinasewitz G Description of compensated and uncompensated disseminated intravascular coagulation (DIC) responses (non-overt and overt DIC) in baboon models of intravenous and intraperitoneal Escherichia coli sepsis and in the human model of endotoxemia: toward a better definition of DIC.Crit Care Med. 2000; 28: S12-S19Crossref PubMed Scopus (78) Google Scholar and has served as the basis30Taylor Jr, FB Chang A Esmon CT D'Angelo A Vigano-D'Angelo S Blick KE Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon.J Clin Invest. 1987; 79: 918-925Crossref PubMed Scopus (769) Google Scholar for clinical studies that culminated in Food and Drug Administration approval of an adjunct therapy for patients with severe sepsis.31Bernard GR Vincent JL Laterre PF LaRosa SP Dhainaut JF Lopez-Rodriguez A Steingrub JS Garber GE Helterbrand JD Ely EW Fisher Jr, CJ Efficacy and safety of recombinant human activated protein C for severe sepsis.N Engl J Med. 2001; 344: 699-709Crossref PubMed Scopus (5092) Google Scholar 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,14Drysdale M Heninger S Hutt J Chen Y Lyons CR Koehler TM Capsule synthesis by Bacillus anthracis is required for dissemination in murine inhalation anthrax.EMBO J. 2005; 24: 221-227Crossref PubMed Scopus (143) Google Scholar, 32Lyons CR Lovchik J Hutt J Lipscomb MF Wang E Heninger S Berliba L Garrison K Murine model of pulmonary anthrax: kinetics of dissemination, histopathology, and mouse strain susceptibility.Infect Immun. 2004; 72: 4801-4809Crossref PubMed Scopus (86) Google Scholar and overwhelming.23Lincoln RE Hodges DR Klein F Mahlandt BG Jones Jr, WI Haines BW Rhian MA Walker JS Role of the lymphatics in the pathogenesis of anthrax.J Infect Dis. 1965; 115: 481-494Crossref PubMed Scopus (79) Google Scholar 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.33Leppla SH Production and purification of anthrax toxin.Methods Enzymol. 1988; 165: 103-116Crossref PubMed Scopus (124) Google Scholar 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. Infusion methods were essentially identical to those used for E. coli34Kaneko T Stearns-Kurosawa DJ Taylor FBJ Twigg M Osaki K Kinasewitz G Peer G Kurosawa S Reduced neutrophil CD10 expression in non-human primates and humans after in vivo challenge with E. coli or lipopolysaccharide.Shock. 2003; 20: 130-137Crossref PubMed Scopus (23) Google Scholar and Shiga toxin 1.35Taylor Jr, FB Tesh VL DeBault L Li A Chang AC Kosanke SD Pysher TJ Siegler RL Characterization of the baboon responses to Shiga-like toxin: descriptive study of a new primate model of toxic responses to Stx-1.Am J Pathol. 1999; 154: 1285-1299Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar 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. 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. 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. Blood samples were taken at various time points for assay purposes and to confirm bacteremia. Except for samples taken for colony counts, blood samples were collected into 1/100 vol of 5000 U/ml penicillin and 500 μg/ml streptomycin to kill circulating vegetative bacteria. Bacteremia was confirmed by traditional plating methods using blood obtained at T+2 hours just after finishing the infusion and T+4 hours before antibiotics. Colony counts varied according to the loading dose. For a 108 CFU/kg challenge, colony counts were near 104 CFU/ml at T+2 hours and 200 CFU/ml at T+4 hours. Colony counts on blood sampled between days 2 to 7 were consistently negative. Blood pressure and rectal temperature were measured with a Critikon monitor (Critikon, Inc., Tampa, FL) and a YSI thermometer (Yellow Springs Instrument Co., Yellow Springs, OH), respectively. Complete blood counts and hematocrits were determined and blood smears were done for differential counts. Routine blood chemistries, fibrinogen, fibrin degradation products (FDP), and activated partial thromboplastin times (APTT) were determined.34Kaneko T Stearns-Kurosawa DJ Taylor FBJ Twigg M Osaki K Kinasewitz G Peer G Kurosawa S Reduced neutrophil CD10 expression in non-human primates and humans after in vivo challenge with E. coli or lipopolysaccharide.Shock. 2003; 20: 130-137Crossref PubMed Scopus (23) Google Scholar, 36Taylor FB Chang AC Peer G Li A Ezban M Hedner U Active site inhibited factor VIIa (DEGR VIIa) attenuates the coagulant and interleukin-6 and -8, but not tumor necrosis factor, responses of the baboon to LD100 Escherichia coli.Blood. 1998; 91: 1609-1615Crossref PubMed Google Scholar, 37Taylor Jr, FB Stearns-Kurosawa DJ Kurosawa S Ferrell G Chang AC Laszik Z Kosanke S Peer G Esmon CT The endothelial cell protein C receptor aids in host defense against Escherichia coli sepsis.Blood. 2000; 95: 1680-1686Crossref PubMed Google Scholar Fibrin degradation products and APTT assays were run on-line during the experiments. Plasma interleukin (IL)-1β levels were determined by enzyme-linked immunosorbent assay (ELISA) using the hIL-1Beta/IL-1F2 DuoSet kit (R&D Systems, Minneapolis, MN). Plasma IL-6 levels were determined by ELISA.36Taylor FB Chang AC Peer G Li A Ezban M Hedner U Active site inhibited factor VIIa (DEGR VIIa) attenuates the coagulant and interleukin-6 and -8, but not tumor necrosis factor, responses of the baboon to LD100 Escherichia coli.Blood. 1998; 91: 1609-1615Crossref PubMed Google Scholar D-dimer levels were determined by ELISA (Diagnostica Stago, Asnières, France). Other cytokines were quantitated by flow cytometry-based Multiplex assay (Dr. J. Connolly, Ph.D., Baylor Institute for Immunology Research, Dallas, TX). Microtiter plates were coated with 50 μl of 1 μg/ml of goat anti-human TNF-α (anti-hTNF-α/TNFSF1A, R&D Systems), washed, and blocked. Plasma samples were diluted at least 1:50 and incubated in the wells for 2 hours at room temperature. Wells were washed, and bound antigen was detected with 0.2 μg/ml of biotinylated goat anti-human TNF-α (R&D Systems) followed by streptavidin-horseradish peroxidase and TMB substrate (1 Step Ultra TMB; Pierce, Rockford, IL). The reaction was stopped with 2 mol/L H2SO4 and OD450nm was determined. Linear standard curves were prepared using recombinant human TNF-α (R&D Systems); the assay was sensitive to 15 pg/ml TNF-α. Wells of 96-well microtiter plates were coated with 50 μl of 10 μg/ml goat anti-human protein C polyclonal antibody as a capture antibody, and an anti-human protein C HPC4 murine monoclonal antibody conjugated with biotin (EX-Link Sulfo-NHS-LC-biotin, final 4 μg/ml; Pierce) was the detection antibody. Antibodies were obtained from Dr. Charles Esmon (Cardiovascular Biology Research, Oklahoma Medical Research Foundation). Wells were coated overnight, washed, and blocked, and samples (50 μl of 1:2000) were incubated at 37°C for 1 hour. Wells were washed, incubated with detection antibody (4 μg/ml, 1.5 hours, room temperature) followed by streptavidin-horseradish peroxidase (1:8000, 1 hour, AMDEX streptavidin-horseradish peroxidase; Amersham Pharmacia Biotech, Arlington Heights, IL). Color was developed with TMB substrate. The reaction was stopped with 2 mol/L H2SO4 and the OD450nm was determined. Standard curves were made from dilution of normal human plasma or baboon pooled plasma and results expressed as percentage of normal for that species. The human and baboon standard curves were parallel and linear (data not shown). Baboons have slightly lower protein C levels compared to humans; the protein C antigen in a normal baboon plasma pool (from five animals) was 66.5 ± 1.2% of the human protein C level. Anthrax PA was quantitated by standard ELISA methods using goat anti-PA as coating antibody (1 μg/ml), biotinylated-goat anti-PA as detection antibody (1 μg/ml), and purified recombinant PA as standards (0 to 25 ng/ml; List Biologicals, Campbell, CA). Bound antigen from plasma (1:50) was detected with streptavidin-horseradish peroxidase and TMB substrate (450 nm). The assay was sensitive to 3 ng/ml PA antigen. Apoptotic cells were visualized using an in situ fluorescence TUNEL assay (Roche, Indianapolis, IN), according to the manufacturer's instructions. At necropsy, the gross appearance of the major organs was examined, and specimens were collected within 1 hour of death. Tissues were fixed in 10% neutral buffered formalin for at least 24 hours, processed by standard methods, and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E) or phosphotungstic acid (PTAH) for routine histopathology. Congestion, white cell influx, hemorrhage, thrombosis, and necrosis on blinded samples were quantified by Dr. Stanley Kosanke (Department of Pathology, School of Medicine, University of Oklahoma Health Sciences Center) as described.38Randolph MM White GL Kosanke SD Bild G Carr C Galluppi G Hinshaw LB Taylor Jr, FB Attenuation of tissue thrombosis and hemorrhage by ala-TFPI does not account for its protection against E. coli—a comparative study of treated and untreated non-surviving baboons challenged with LD100 E. coli.Thromb Haemost. 1998; 79: 1048-1053PubMed Google Scholar Tissues were rated according to the severity of the histopathological lesions. The scale ranged from 0 to + 4, with 4 being the most severe. Tissues were processed as described.39Lupu F Heim D Bachmann F Kruithof EK Expression of LDL receptor-related protein/alpha 2-macroglobulin receptor in human normal and atherosclerotic arteries.Arterioscler Thromb. 1994; 14: 1438-1444Crossref PubMed Google Scholar Tissues from saline-treated control animals37Taylor Jr, FB Stearns-Kurosawa DJ Kurosawa S Ferrell G Chang AC Laszik Z Kosanke S Peer G Esmon CT The endothelial cell protein C receptor aids in host defense against Escherichia coli sepsis.Blood. 2000; 95: 1680-1686Crossref PubMed Google Scholar were treated identically to those obtained in the current anthrax study. Tissues were fixed (4% paraformaldehyde), cryoprotected (5% sucrose, mounted in Tissue-Tek OCT compound), and snap-frozen in liquid nitrogen-cooled isopentane. Tissue cryosections were treated with 0.1 mol/L glycine in phosphate-buffered saline (PBS) for 15 minutes to block free aldehyde groups and with 3% bovine serum albumin and 5% normal goat serum in PBS plus 0.1% saponin, for 30 minutes at room temperature to block nonspecific binding sites. For double-immunofluorescence labeling, specimens were incubated with mixtures of monoclonal (mAb; 10 μg/ml) and polyclonal antibodies (20 μg/ml) for 1 hour at 20°C or overnight at 4°C. The following antibodies were used: anti-tissue factor mAb, (clone TF9-10H10; gift from Dr. James H. Morrissey, Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL), anti-nitrotyrosine mAb (LabVision Corp., Fremont, CA), anti-CD68 mAb (DAKO, Carpinteria, CA), rabbit polyclonal IgGs against human tissue factor pathway inhibitor (TFPI), and human inducible nitric oxide synthetase (iNOS; NeoMarkers Inc., Fremont, CA). The sections were washed 3 × 10 minutes in PBS/saponin and incubated for 1 hour at 20°C with combinations of appropriate detection antibodies conjugated with fluorescein isothiocyanate or Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:100 in 1% bovine serum albumin in the same buffer. After washing as above, segments were mounted in Vectashield (Vector Laboratories, Burlingame, CA) containing TO-PRO-3 iodine (Molecular Probes, Eugene, OR) as a nuclear counterstain. As negative controls for polyclonal antibody staining, primary antibodies were replaced with equivalent amounts of rabbit nonimmune serum. The anti-nitrotyrosine antibody specificity was confirmed by control experiments showing loss of antibody recognition after competition with excess (10 mmol/L) 3-nitrotyrosine (not shown). Anti-digoxigenin mAb, a hapten antigen that occurs only in plants, was used as negative control for mAb staining. Specimens were examined using a Nikon C1 confocal laser-scanning unit equipped with a three-laser launcher (488, 543, and 633 nm emission lines) installed on an Eclipse TE200-U inverted microscope (Nikon, Melville, NY). Images were taken with either a ×20 plan achromat objective (NA 0.46) or a ×60 apochromat oil immersion objective (NA 1.4). Data were analyzed for differences between dosage groups using the Student's t-test, assuming equal variance. APTT, d-dimer, elastase, and cyto/chemokine data were transformed to natural log before analysis to account for unequal variances. A P value <0.05 was considered to be significantly different. In all animals, except those that received the lowest doses, we observed coagulopathy, increased vascular permeability, and inflammation, in which the severity of the response was commensurate with the extent of the bacterial challenge. Eleven animals were studied; data are grouped according to the log10 bacterial load. Survival times after challenge with B. anthracis Sterne strain were dose-dependent (Figure 1). A 7-day survivor was considered to be a permanent survivor. Infusion of 5 × 105 and 5 × 107 CFU/kg was sublethal, and the upper limit of a sublethal dose was near 6 × 107 CFU/kg. Bacterial exotoxin production was confirmed by increases in PA (Figure 2), which is required for cellular intoxication.40Nassi S Collier RJ Finkelstein A PA63 channel of anthrax toxin: an extended beta-barrel.Biochemistry. 2002; 41: 1445-1450Crossref PubMed Scopus (110) Google Scholar PA antigen decreased to baseline after antibiotic treatment began.Figure 2Toxemia. Plasma levels of anthrax toxin PA were determined by ELISA after infusion of bacteria. Challenge dose in CFU/kg: 105 to 106 (▿, n = 2); 107 (□, n = 3); 108 (•, n = 3); and 109 (▴, n = 3).
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