IαIp (both Inter-alpha Inhibitor and Pre-alpha Inhibitor) were isolated from human fresh frozen plasma (Rhode Island Blood Center, Providence, RI) by cryo-precipitation, solid phase extraction and ion-exchange chromatography as previously described. The PA and LF were purchased from List Biological Laboratory and their activity was confirmed in a cytotoxicity assay (8 (link)) in RAW264.7 cells (ATCC # TIB-71). All other reagents used in these experiments were purchased from Sigma (St. Louis, MO).
Male AJ mice were obtained from Jackson Laboratories (Barr Harbor, ME). Animals were housed in an IUCAC- approved facility under Biosafety Level 2 safety conditions. Animal care and protocol adherence were monitored by the Brown University Veterinary staff. Animals were housed in cages with HEPA filter lids and maintained at a constant ambient temperature and humidity with twelve hour day/night cycling.
In-vivo studies of B. anthracis Sterne 34F2 was obtained from Colorado Serum. The Sterne strain has a full complement of pXO1-encoded toxins LF, EF and PA, but lacks the pXO2-encoding anti-phagocytic poly-D-glutamic acid capsule, rendering it non-lethal to humans but still highly lethal in susceptible mouse strains (14 (link)). All work was conducted under BSL-2 conditions. B. anthracis spores (103–109/animal) were used in LD50 experiments (n=5/group). IαIp were given (30 mg/kg) ip 1 hour before the spore challenge or PBS control. This dose of IαIp was chosen based upon preliminary dose-finding experiments with recombinant anthrax lethal toxin (8 (link)). Moxifloxacin (Schering, Kenilworth, NJ) was given subcutaneously (10mg/kg q24hrX3) beginning 24 hours after spore challenge. In survival experiments, IαIp’s (30mg/kg) were administered ip 1 hour after or 24 hours after the spore challenge with moxifloxacin or PBS control.
Individual parameters between groups were compared using the Mann-Whitney U test. The non-parametric, Kruskal-Wallis one way analysis of variance was used for differences between multiple groups. The survival studies were analyzed using Kaplan-Meier survival plots and differences were assessed by the log-rank test. P values of <.05 were considered significant.
Male AJ mice were obtained from Jackson Laboratories (Barr Harbor, ME). Animals were housed in an IUCAC- approved facility under Biosafety Level 2 safety conditions. Animal care and protocol adherence were monitored by the Brown University Veterinary staff. Animals were housed in cages with HEPA filter lids and maintained at a constant ambient temperature and humidity with twelve hour day/night cycling.
In-vivo studies of B. anthracis Sterne 34F2 was obtained from Colorado Serum. The Sterne strain has a full complement of pXO1-encoded toxins LF, EF and PA, but lacks the pXO2-encoding anti-phagocytic poly-D-glutamic acid capsule, rendering it non-lethal to humans but still highly lethal in susceptible mouse strains (14 (link)). All work was conducted under BSL-2 conditions. B. anthracis spores (103–109/animal) were used in LD50 experiments (n=5/group). IαIp were given (30 mg/kg) ip 1 hour before the spore challenge or PBS control. This dose of IαIp was chosen based upon preliminary dose-finding experiments with recombinant anthrax lethal toxin (8 (link)). Moxifloxacin (Schering, Kenilworth, NJ) was given subcutaneously (10mg/kg q24hrX3) beginning 24 hours after spore challenge. In survival experiments, IαIp’s (30mg/kg) were administered ip 1 hour after or 24 hours after the spore challenge with moxifloxacin or PBS control.
Individual parameters between groups were compared using the Mann-Whitney U test. The non-parametric, Kruskal-Wallis one way analysis of variance was used for differences between multiple groups. The survival studies were analyzed using Kaplan-Meier survival plots and differences were assessed by the log-rank test. P values of <.05 were considered significant.
