Heterotrimeric G proteins physically associated with the lipopolysaccharide receptor CD14 modulate both in vivo and in vitro responses to lipopolysaccharide.

Septic shock induced by lipopolysaccharide (LPS) triggering of cytokine production from monocytes/macrophages is a major cause of morbidity and mortality. The major monocyte/macrophage LPS receptor is the glycosylphosphatidylinositol (GPI)-anchored glycoprotein CD14. Here we demonstrate that CD14 coimmunoprecipitates with Gi/Go heterotrimeric G proteins. Furthermore, we demonstrate that heterotrimeric G proteins specifically regulate CD14-mediated, LPS-induced mitogen-activated protein kinase (MAPK) activation and cytokine production in normal human monocytes and cultured cells. We report here that a G protein binding peptide protects rats from LPS-induced mortality, suggesting a functional linkage between a GPI-anchored receptor and the intracellular signaling molecules with which it is physically associated.

Activated protein C concentrate for the treatment of meningococcal endotoxin shock in rabbits.

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.

Anticapsular polysaccharide antibodies and nasopharyngeal colonization with Streptococcus pneumoniae in infant rats.

To evaluate the effect of passive immunization with anticapsular antibodies on nasopharyngeal carriage, two models of Streptococcus pneumoniae colonization were developed in infant rats. In a direct inoculation model, 3- to 4-day-old infant rats were intranasally inoculated with 2 x 10(5) cfu of S. pneumoniae type 3 or 6 x 10(3) cfu of S. pneumoniae type 23F. In an intralitter transmission model, 2 infant rats were intranasally inoculated with 10(3) cfu of pneumococcus type 3 or type 19F and placed in a cage containing 10 infant rats. Pretreatment with bacterial polysaccharide immune globulin led to a significant reduction in colonization of contact animals with S. pneumoniae type 3 or 19F in the intralitter transmission model (P < .05). No effect of immune globulin could be demonstrated in the direct inoculation model. These results indicate that systemic anticapsular antibodies conferred significant protection against nasopharyngeal acquisition by intralitter spread of S. pneumoniae type 3 and 19F.

Minimum protective serum concentrations of pneumococcal anti-capsular antibodies in infant rats.

Infant rats were passively immunized to determine the protective capacity of pneumococcal anticapsular antibodies. Animal-passaged strains of Streptococcus pneumoniae serotypes 1, 4, 5, 6b, 7f, 9v, 14, 18c, 19f, and 23f were used as challenge inocula (1-1500 cfu) in a model of pulmonary infection that resulted in bacteremia, meningitis, and death. From untreated control animals, histologic sections of lung demonstrated infiltrative pneumonia and lung homogenate cultures grew S. pneumoniae at concentrations of 10(3)-10(8) cfu per gram of lung tissue. A type-specific anti-capsular antibody serum concentration of 0.1-1.15 microg/mL resulted in a statistically significant reduction in mortality compared with the reduction in untreated controls, except for serotype 14, which required 2.32 microg/mL for a significant reduction in mortality. The serum antibody level that provided 50% reduction in mortality ranged from 0.1-3.5 microg/mL for all serotypes.

Primary respiratory syncytial virus infection: pathology, immune response, and evaluation of vaccine challenge strains in a new mouse model.

Respiratory syncytial virus (RSV) is the primary cause of lower respiratory tract illness in young children. Vaccine development has been hampered by the experience of the formalin-inactivated vaccine tested in the 1960's. Currently, several vaccine candidates are under development and immune response to these candidate vaccines must be evaluated closely. We introduce a novel low-dose murine model of RSV infection and a new pathologic scoring system for the resultant pulmonary disease. We have also developed new sensitive methods for measuring cytokine expression. We then used this new model to test vaccine challenge strains of RSV in order to determine their pathogenicity.

Development of a model of low-inoculum Streptococcus pneumoniae intrapulmonary infection in infant rats.

We have developed a model of low-inoculum Streptococcus pneumoniae infection in infant rats. We challenged 4-day-old Sprague-Dawley pups via intraperitoneal or intrapulmonary injection of S. pneumoniae serotypes 1, 3, 4, 5, 6b, 7f, 9v, 14, 19f, and 23f. To achieve bacteremia with low inocula, it was necessary to passage the isolates in rats. Inocula of the 10 S. pneumoniae serotypes producing bacteremia in 50% or more animals ranged from 1 to 400 CFU. Virulence was similar by intraperitoneal and intrapulmonary routes. Lung specimens from animals challenged by the intrapulmonary route grew S. pneumoniae and demonstrated histologic evidence of focal infection. Meningitis was detected in 20 to 50% of bacteremic animals, and mortality invariably followed bacteremia within 24 to 48 h. This model of intrapulmonary infection uses low inocula of S. pneumoniae and results in bacteremia, meningitis, and death in infant rats.

High-dose recombinant endotoxin neutralizing protein improves survival in rabbits, with Escherichia coli 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.

A comparison of bactericidal/permeability-increasing protein variant versus recombinant endotoxin-neutralizing protein for the treatment of Escherichia coli sepsis in rats .

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.

Comparison of a recombinant endotoxin-neutralizing protein with a human monoclonal antibody to endotoxin for the treatment of Escherichia coli sepsis in rats.

A recombinant endotoxin-neutralizing protein (ENP) from Limulus polyphemus and a monoclonal IgM anti-lipid A antibody (HA-1A) were compared in a rat model of Escherichia coli sepsis. One hour after intraperitoneal challenge with 10(6) cfu of E. coli O18ac K1, animals were sensitized to endotoxin with lead acetate and treated with ENP, HA-1A, or saline, followed by ceftriaxone and gentamicin. Before treatment, 95% of rats had high-grade bacteremia and high serum endotoxin concentrations, which were similar in all treatment groups (P > .60). One hour after treatment, there was no bacterial growth in any blood sample, and endotoxin concentrations were significantly lower in the ENP group than in the HA-1A and saline groups (P < .01). At 24 h after challenge, survival in the ENP group was significantly higher than in the HA-1A saline group (P < .001). ENP improved survival in a rat model of E. coli sepsis with high mortality despite effective antibiotic therapy.

Comparison of early and late treatment with a recombinant endotoxin neutralizing protein in a rat model of Escherichia coli 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.

Failure of prophylactic and therapeutic use of a murine anti-tumor necrosis factor monoclonal antibody in Escherichia coli sepsis in the rabbit.

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.