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Objective: To compare a recombinant bactericidal/permeability-increasing protein variant and a recombinant endotoxin-neutralizing protein., Design: Randomized, blinded, controlled study, using a rat model of sepsis., Setting: Animal research facility., Subjects: Male Wistar rats., Interventions: An inoculum of 1.5 x 10(7) to 1.8 x 10(8) Escherichia coli O18ac K1, implanted in the peritoneum, produced bacteremia in 95% of animals after 1 hr. One hour after E. coli challenge, animals received recombinant bactericidal/permeability-increasing protein variant, recombinant endotoxin-neutralizing protein, or saline intravenously, followed by ceftriaxone and gentamicin intramuscularly., Measurements and Main Results: Twenty-four (85.7%) of 28 animals receiving recombinant endotoxin-neutralizing protein (p < .001 vs. control) survived 7 days compared with nine (33.3%) of 27 recombinant bactericidal/permeability-increasing protein variant-treated (p < .001 vs. control) and two (6.5%) of 31 control animals., Conclusions: Both recombinant endotoxin-neutralizing protein and recombinant bactericidal/permeability-increasing protein variant improved survival. Recombinant endotoxin-neutralizing protein was superior to recombinant bactericidal/permeability-increasing protein variant in its protective effect at the doses tested. Our results suggest that both proteins may be useful in the treatment of human Gram-negative sepsis.

Objective: To test the efficacy of a recombinant endotoxin neutralizing protein as compared with saline in rats with Escherichia coli sepsis., Design: Prospective, controlled animal trial., Setting: Hospital animal research laboratory., Subjects: Male Wistar rats challenged with intraperitoneal E. coli, O18ac K1, and treated 1 hr later with ceftriaxone and gentamicin., Interventions: Recombinant endotoxin neutralizing protein, 50 mg/kg, was administered to rats 1, 2, or 3 hrs after E. coli challenge; saline was administered to control animals., Measurements and Main Results: Quantitative bacteremia, 1 hr after challenge and before antibiotic administration, was not significantly different between treatment groups (range geometric mean 451 to 621 colony-forming units [cfu]/mL). The endotoxin concentration, measured immediately before recombinant endotoxin neutralizing protein administration, was significantly higher in animals sampled and treated at 2 hrs (geometric mean 260 EU/mL; 95% confidence interval 140 to 480 EU/mL), or 3 hrs (geometric mean 697 EU/mL; 95% confidence interval 307 to 1585 EU/mL) after E. coli challenge, compared with animals sampled and treated at 1 hr (geometric mean 17 EU/mL; 95% confidence interval 7 to 69 EU/ mL). Survival rate was significantly greater in rats treated with recombinant endotoxin neutralizing protein at 1 hr (23/27; p < .001) or 2 hrs (8/30; p < .01) after E. coli challenge than in controls (1/32)., Conclusion: Administration of recombinant endotoxin neutralizing protein delayed up to 2 hrs after challenge with E. coli improves survival in antibiotic-treated rats with Gram-negative sepsis.

Objective: To assess the benefit of a recombinant endotoxin neutralizing protein from Limulus polyphemus in treating Gram-negative bacterial sepsis in rabbits., Design: Prospective, blinded, controlled, laboratory trial., Setting: Animal research laboratory., Subjects: New Zealand White rabbits., Interventions: We established a rabbit model of Escherichia coli peritonitis and bacteremia, with high mortality rate, despite treatment with gentamicin and ceftriaxone. Twenty-five pairs of male New Zealand White rabbits were challenged intraperitoneally with E. coli O18ac K1 in 5% porcine mucin (mean 7 x 10(1) colony-forming units). All animals were treated with intravenous gentamicin (2.5 mg/kg) and ceftriaxone (100 mg/kg), and with either intravenous endotoxin neutralizing protein (50 mg/kg) or saline 1 hr after E. coli challenge., Measurements and Main Results: All animals were bacteremic 1 hr after challenge (mean 3.6 x 10(5) colony-forming units/mL). Animals in both groups developed tachycardia, hypotension, and acidosis (NS). Geometric mean serum endotoxin and tumor necrosis factor (TNF) concentrations were significantly ( p < .001) higher 1 hr after challenge compared with baseline prechallenge concentrations in both groups. From 1 to 2 hrs after challenge, endotoxin concentrations increased 2.5-fold in control animals (95% confidence interval = 13.1 to 32.9 endotoxin units/mL, p = .024), whereas endotoxin concentrations increased only 1.2-fold in endotoxin neutralizing protein-treated animals (95% confidence interval = 20.4 to 23.6 endotoxin units/mL, NS). TNF concentrations increased significantly (p < .001) in both groups from 1 to 2 hrs after challenge. Eighteen (72%) of 25 endotoxin neutralizing protein-treated animals vs. 11 (44%) of 25 controls survived 24 hrs (p = .032)., Conclusions: Treatment with endotoxin neutralizing protein had the following effects: a) the increase in serum endotoxin was blunted, but not TNF concentrations measured 1 hr after antibiotic treatment; and b) survival in rabbits with E. Coli sepsis was improved.

Objective: To determine the efficacy of a murine anti-tumor necrosis factor (TNF) monoclonal antibody in the treatment of Escherichia coli peritonitis and sepsis in the rabbit., Design: Prospective, paired, randomized, blinded, controlled animal trial., Setting: Animal research laboratory., Subjects: Male New Zealand white rabbits., Interventions: Anesthetized rabbits were cannulated with indwelling femoral arterial and venous catheters. Peritonitis and sepsis were induced by intraperitoneal challenge using live E. coli O18ac bacteria. All animals were treated with gentamicin and ceftriaxone 1 hr after challenge. One group (prophylaxis experiment) consisting of ten rabbit pairs (the prophylaxis group), was treated with either murine anti-TNF monoclonal antibody or an equivalent volume of 5% albumin 3 hrs before E. coli challenge. A second group (therapeutic experiment) of 17 rabbit pairs, the treatment group, was also treated with murine anti-TNF monoclonal antibody or albumin control 1 hr after E. coli challenge., Measurements and Main Results: All animals were bacteremic 1 hr after challenge. Physiologic measures of sepsis (heart rate, mean arterial pressure, serum bicarbonate, and arterial pH) did not differ between control, prophylaxis, and treatment groups. Peak serum TNF concentration was significantly (p < .01) lower in animals receiving anti-TNF monoclonal antibody, in both the prophylaxis and treatment groups, than in control animals. The survival rate was not improved significantly in either the prophylaxis or treatment group., Conclusions: Prophylactic and therapeutic use of anti-TNF monoclonal antibody in a rabbit model of E. coli peritonitis and sepsis significantly lowers TNF concentrations but does not ameliorate the physiologic effects of sepsis and does not significantly improve survival.

Objective: A recombinant endotoxin neutralizing protein was evaluated for its ability to ameliorate the effects of Escherichia coli sepsis in rats., Design: Prospective, controlled animal trial., Setting: Hospital animal research laboratory., Subjects: Wistar rats, treated with gentamicin 1 hr after challenge with intraperitoneal E. coli O18ac., Interventions: The animals received a recombinant endotoxin neutralizing protein, in doses of 5, 25, or 50 mg/kg, either 30 or 60 mins after challenge; controls received saline., Measurements and Main Results: Geometric mean serum endotoxin concentrations in endotoxin neutralizing protein-treated animals did not differ from control animals. Tumor necrosis factor concentrations in animals treated with endotoxin neutralizing protein 30 mins after challenge were significantly lower than controls. Animals treated with 25 or 50 mg/kg of endotoxin neutralizing protein 30 mins after E. coli challenge had significant improvements in survival compared with controls. Animals treated with 50 mg/kg of endotoxin neutralizing protein 60 mins after E. coli challenge had significant improvements in survival compared with controls., Conclusion: Endotoxin neutralizing protein significantly reduces mortality from Gram-negative sepsis in an antibiotic-treatment model of E. coli peritonitis and bacteremia in rats, mediated by a neutralization of the biological effects of endotoxin.

Objectives: Limulus anti-lipopolysaccharide factor, an 11.8-kilodalton peptide isolated from amebocytes of Limulus polyphemus inhibits the biologic activities of endotoxin in vitro, including gelation of Limulus amebocyte lysate. A recombinant version of Limulus anti-lipopolysaccharide factor, termed endotoxin neutralizing protein, has now been expressed in yeast. Endotoxin-neutralizing protein was evaluated for its potential prophylactic and therapeutic effects in rabbits challenged with Escherichia coli endotoxin., Design: Controlled animal trial., Setting: Animal research laboratory., Subjects: A total of 112 New Zealand white rabbits were studied., Interventions: Rabbits were challenged with an LD80 dose of E. coli endotoxin (100 micrograms/kg); control animals (n = 52) were treated with saline solution at the time of endotoxin challenge; experimental animals received endotoxin-neutralizing protein 2.5 mg/kg prechallenge (n = 20), 5.0 mg/kg prechallenge (n = 20), or 5.0 mg/kg 30 mins postchallenge (n = 20).

Background and Methods: Endotoxin shock is mediated by various cytokines, including tumor necrosis factor. Treatment of patients with i.v. immunoglobulin has been shown to reduce the concentration of circulating cytokines. The purpose of this study was to determine the protective effects of immunoglobulin for i.v. use on meningococcal endotoxin-induced shock in a rabbit model. Experimental animals were challenged with i.v. meningococcal endotoxin (lipo-oligosaccharide) 10 micrograms/kg, and treated with either a 2-hr i.v. immunoglobulin infusion (400 mg/kg) or a similar saline infusion that was initiated 30 mins before endotoxin challenge. Control animals were challenged with saline alone., Results: Compared with untreated control animals, pulse rate increased (p less than .007) and mean arterial pressure and serum bicarbonate concentrations decreased (p less than .02) in both experimental groups, but did not differ between immunoglobulin-treated and saline-treated animals (p greater than .05) at any time after the endotoxin challenge. Geometric mean serum endotoxin concentrations were significantly (p less than .03) lower in the immunoglobulin-treated animals at 60, 120, 180, 240, 300, and 360 mins after the endotoxin challenge. The geometric mean serum tumor necrosis factor level at 1 hr after the endotoxin challenge in the immunoglobulin-treated experimental animals was lower than in saline-treated animals (5.53 vs. 8.47 tumor necrosis factor enzyme-linked immunosorbent assay U/mL), but not significantly so (p greater than .05). Mortality rate was similar in both experimental groups; eight (67%) of 12 saline-treated experimental rabbits and seven (70%) of ten immunoglobulin-treated rabbits died. All untreated control animals survived 24 hrs., Conclusions: In this model of circulatory shock in rabbits, i.v. immunoglobulin: a) does not significantly alter the physiologic responses to endotoxin challenge; b) significantly reduces endotoxin concentrations; c) reduces tumor necrosis factor concentrations, but not significantly; and d) does not improve survival rate.