To evaluate the effects of activated protein C therapy in a rabbit model of meningococcal endotoxin-induced shock, we performed a prospective, blinded, placebo-controlled animal trial. Forty New Zealand White rabbits were challenged with intravenous meningococcal endotoxin (lipooligosaccharide) 100 microg/kg. Ten minutes before endotoxin challenge, animals were administered either activated protein C 1600 microg/mL (n = 20) or an equal volume of saline (n = 20) as an initial bolus. After endotoxin challenge, activated protein C treated animals were administered a continuous infusion of activated protein C 160 microg/kg/h and saline-treated animals were administered an equal volume infusion of saline. Both activated protein C treated and saline control animals demonstrated evidence of shock after endotoxin challenge; mean arterial pressure and serum bicarbonate significantly (p < .01) declined, and heart rate significantly (p < .01) increased from baseline. In activated protein C treated animals, mean plasma activated protein C activity was 5.69 microg/mL (+/- 3.2) 1 h after challenge, whereas plasma protein C activity was not detected in controls. Mean prothrombin and activated partial thromboplastin times were significantly (p < or = .01) prolonged compared with saline-treated controls. Other hematologic and chemical measurements did not differ between groups. Fifteen of 20 (75%) animals treated with activated protein C concentrate survived to 24 h, while 9 of 20 (45%) control animals survived to 24 h (p = .05). Those animals treated with activated protein C had improved survival, which corroborates the findings of early clinical studies in which replacement of protein C improved outcome.

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 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 evaluate maternal responses to Haemophilus influenzae type b (Hib) polysaccharide-tetanus protein conjugate (polyribosylribitol phosphate-tetanus or PRP-T) given to pregnant Gambian women, the transplacental transfer of antibody, and the effect of maternal immunization on infant responses to the vaccine.DESIGN:An open, randomized immunogenicity study.SETTING:A busy urban health center in The Gambia.STUDY PARTICIPANTS:A total of 451 pregnant women enrolled during the third trimester of pregnancy.INTERVENTION:Study participants were randomized to three groups. In one group, mothers were given PRP-T during the third trimester and their infants were given PRP-T at 2, 3, and 4 months of age. In the second group, mothers received PRP-T and infants were given inactivated poliovirus vaccine. In the third group, mothers received meningococcal A and C vaccine, and their infants received PRP-T.MAIN OUTCOME MEASURES:Anti-PRP antibody measurements of maternal cord, and infant blood.RESULTS:Vaccinated women had a marked increase in total anti-PRP antibody (geometric mean titer 9.0 micrograms/mL), which was greatest in women in their first or second pregnancy. Previous tetanus vaccination during the same pregnancy and high concentrations of antitetanus antibody were associated with lower anti-PRP responses. In infants of PRP-T recipients, cord blood anti-PRP IgG concentrations were 61% of simultaneous maternal concentrations. In vaccinated infants of vaccinated mothers, geometric mean anti-PRP antibody concentrations at birth, 2 months of age, and 5 months of age were 1.92, 0.35 and 2.84 micrograms/mL, respectively, while in vaccinated infants of unvaccinated mothers, the corresponding concentrations were 0.29, 0.12, and 3.91 micrograms/mL. At 2 months of age, 60% of infants of vaccinated mothers and 26% of infants of unvaccinated mothers had anti-PRP antibody concentrations considered to be protective (>0.15 micrograms/mL).CONCLUSIONS:In areas where much invasive Hib disease occurs in infants younger than 6 months, maternal immunization may help to reduce the risk of Hib disease in infants too young for immunization.

ENDOTOXIN NEUTRALIZING PROTEIN INHIBITS THE RESPONSE OF HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS TO LPS AND NON-LPS STIMULI. • 1045

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.

Gram-negative bacterial sepsis is associated with endotoxemia and a high mortality rate. In previous studies, we demonstrated the therapeutic benefit of an anti-lipopolysaccharide factor isolated from amebocytes of Limulus polyphemus, and of a recombinant version of this protein, termed endotoxin neutralizing protein (ENP), in rabbits challenged with purified lipopolysaccharides. To assess the benefit of ENP in treating a live bacterial infection, we established a rabbit model of Escherichia coli (E. coli) peritonitis and bacteremia with high mortality despite gentamicin treatment. Twenty-four pairs of New Zealand white rabbits were challenged intraperitoneally (IP) with E. coli O18ac K1 in 5% porcine mucin (mean bacteria per dose = 2.5 x 10(8)). The animals were treated with intravenous (i.v.) gentamicin (2.5 mg/kg), and with either ENP (5 mg/kg) or saline i.v. at 1 hr after E. coli challenge. All rabbits were bacteremic 1 hr after challenge (geometric mean 4.1 +/- 1.2 x 10(4) cfu/mL). Peak geometric mean serum endotoxin (2.62 v 10.54 EU/mL, P = .013) and tumor necrosis factor (TNF) (2540 v 6438 TNF units/mL, P = .046) concentrations were lower in ENP-treated animals as compared to control animals. Seven of 24 animals treated with ENP survived 24 hr compared with 4 of 24 controls (Kaplan-Meier analysis, P = .19). However, in the subgroup of 13 paired animals in whom bacteremia was eliminated by gentamicin treatment, 5 of 13 ENP-treated animals survived 24 hr, compared with 1 of 13 controls (Kaplan-Meier analysis, P = .032).(ABSTRACT TRUNCATED AT 250 WORDS)

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 μg/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). Measurements and Main Results: Rabbits treated with endotoxin-neutralizing protein before endotoxin challenge maintained stable mean arterial pressures and arterial pH and bicarbonate values which were significantly higher than controls during a 6–hr observation period. Geometric mean serum endotoxin concentrations were significantly lower in animals treated before endotoxin challenge as compared with controls measured at 30 mins and hourly after endotoxin challenge. Animals treated with endotoxin-neutralizing protein 30 mins after challenge had significantly lower geometric mean serum endotoxin concentrations measured 60 mins and hourly after challenge. Geometric mean serum tumor necrosis factor concentrations were lower in all treated animals compared with controls, but significantly so only in animals treated with endotoxin-neutralizing protein 2.5 mg/kg before endotoxin challenge. Survival to 24 hrs was significantly improved compared with controls in rabbits treated with endotoxin-neutralizing protein 2.5 mg/kg prechallenge (85% vs. 29%, p < .001), and with endotoxin-neutralizing protein 5.0 mg/kg prechallenge (90% vs. 29%, p < .001); animals treated with endotoxin-neutralizing protein 5.0 mg/kg postchallenge demonstrated improved survival compared with controls (55% vs. 29%, p = .055), although not significantly so. Conclusions: Endotoxin-neutralizing protein attenuates the toxic effects ofE. coli endotoxin in rabbits and improves survival, even when administered after endotoxin challenge. Endotoxin-neutralizing protein deserves further evaluation as a potential therapy for Gram-negative sepsis. (Crit Care Med 1994; 22:1211–1218)

Saladino, R; Garcia, C T; Thompson, C; Hammer, B; Parsonnet, J; Novitsky, T; Onderdonk, A; Siber, G; Fleisher, GAuthor Information

Limulus antilipopolysaccharide factor (LALF), an 11.8-kDa peptide isolated from amebocytes of Limulus polyphemus, neutralizes meningococcal lipooligosaccharide (LOS)-induced gelation of limulus amebocyte lysate. Rabbits challenged with an LD90 of LOS (10 micrograms/kg) premixed with LALF in vitro (n = 10) had significantly higher mean arterial pressure, arterial pH, serum bicarbonate concentrations, and survival (90% vs. 8%, P = .005) than did rabbits challenged with LOS alone. Relative to untreated controls, rabbits pretreated with LALF intravenously (iv) at 1.2 mg/kg (n = 21) also had significant improvements in physiologic measurements and higher survival (52% vs. 8%, P = .003). Even when LALF (1.2 mg/kg iv) was given 1/2 h after LOS challenge, animals showed significant improvements in physiologic measurements and survival (33% vs. 8% in untreated controls P = .028). LALF-treated animals also had significantly lower circulating endotoxin activity and tumor necrosis factor concentrations. Thus, LALF attenuates the toxic effects of meningococcal LOS in rabbits even when administered after LOS challenge and deserves further evaluation as a potential therapeutic agent for treating gram-negative septic shock.

Background and Methods: Endotoxin shock is mediated by various cytokines, including tumor necrosis factor. Treatment of patients with iv 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 iv use on meningococcal endotoxin-induced shock in a rabbit model. Experimental animals were challenged with iv meningococcal endotoxin (lipooligosaccharide) 10-mu-g/kg, and treated with either a 2-hr iv 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 < .007) and mean arterial pressure and serum bicarbonate concentrations decreased (p < .02) in both experimental groups, but did not differ between immunoglobulin-treated and saline-treated animals (p > .05) at any time after the endotoxin challenge. Geometric mean serum endotoxin concentrations were significantly (p < .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 > .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, iv 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.

Meningococcemia is the most frequent cause of septic shock in healthy children. To determine whether polymyxin B (PMB) might improve mortality from meningococcal shock, its protective activity was evaluated in rabbits challenged with an LD90 of meningococcal lipooligosaccharide (LOS) and compared with an LD80 of Escherichia coli O111:B4 lipopolysaccharide (LPS). PMB (5 mg/kg) administered intravenously 30 min before meningococcal LOS challenge had no significant effect on heart rate, mean arterial pressure, serum bicarbonate, serum tumor necrosis factor (TNF) levels, or survival relative to controls. However, PMB premixed with LOS in vitro increased serum bicarbonate levels (P less than .05) and improved 24-h survival (P less than .05). In contrast, PMB given before E. coli LPS challenge increased serum bicarbonate levels, decreased TNF levels, and improved 24-h survival (all, P less than .05). In vitro studies confirmed that PMB at 10 micrograms/ml neutralized E. coli LPS but not meningococcal LOS activity. Thus, pretreatment with PMB apparently protects rabbits against shock induced by E. coli LPS but not by meningococcal LOS.