Thomas J. Novitsky

Limulus Amebocyte Lysate Assay for Detection of Endotoxin in Patients with Sepsis Syndrome

Clinical predictions alone are insufficiently accurate to identify patients with specific types of bloodstream infection; laboratory assays might improve such predictions. Therefore, we performed a prospective cohort study of 356 episodes of sepsis syndrome and did Limulus amebocyte lysate (LAL) assays for endotoxin. The main outcome measures were bacteremia and infection due to gram-negative organisms; other types of infection were secondary outcomes. Assays were defined as positive if the result was > or = 0.4 enzyme-linked immunosorbent assay units per milliliter. There were positive assays in 119 (33%) of 356 episodes. Assay positivity correlated with the presence of fungal bloodstream infection (P < .003) but correlated negatively with the presence of gram-negative organisms in the bloodstream (P = .04). A trend toward higher rates of mortality in the LAL assay-positive episodes was no longer present after adjusting for severity. Thus, results of LAL assay did not correlate with the presence of bacteremia due to gram-negative organisms or with mortality after adjusting for severity but did correlate with the presence of fungal bloodstream infection.

Effect of a recombinant endotoxin-neutralizing protein on endotoxin shock in rabbits.

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)

Assay of Endotoxin by Limulus Amebocyte Lysate

Horseshoe crabs fight off infectious agents with a complex array of proteins present in amebocytes, the major cell type in their hemolymph. These amebocytes contain both large and small granules (1). When exposed to bacteria or other infectious agents the amebocytes release proteins into their surroundings by exocytosis. The small granules of Limulus amebocytes contain antibacterial proteins, including polyphemusins and the big defensins (2). The large granules contain the Limulus anti-lipopolysaccharide factor (LALF) and the clot-forming group of serine protease zymogens. Exocytosis is initiated by the reaction of amebocytes with lipopolysaccharide (LPS) from Gram-negative bacteria or other microbial components. LPS is also called endotoxin because it is found in the outer membrane of the gram-negative bacterial cell wall. A solid clot forms in response to the lipid A portion of LPS, thereby walling off the infection site or preventing the loss of blood when the animal is damaged physically (3).

Recombinant endotoxin neutralizing protein improves survival from Escherichia coli sepsis in rats.

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.

Production of recombinant endotoxin neutralizing protein in Pichia pastoris and methods for its purification

Production of recombinant Limulus endotoxin neutralizing protein (rENP) was attained with the GS115 methylotrophic strain of Pichia pastoris transformed with a plasmid, bearing multiple ENP gene copies. The synthetic gene for Limulus ENP was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter. Clones containing a single enp insert were used to construct cassettes bearing 2 and 3 tandem copies of enp. These were then integrated at the HIS locus of P. pastoris GS115 (his4). Clones were chosen for their ability to produce rENP upon methanol induction in shaker flasks, and then the 1x, 2x, and 3x-enp strains were analyzed by Southern blot for the presence of the ENP gene(s). Isolate 3 x 5q, containing a 3x-enp cassette, was the best producer of rENP. Under optimal conditions this strain grown in a fed-batch mode produced yields of >500 mg rENP/L with an average of 5.46 mg rENP/g DCW. Purification of rENP from the clarified broth resulted in a yield of 35% and a purity of >86%. Glycosylated rENP, the main contaminant, was removed with a concanavalin-A column and characterized. The pure rENP neutralized lipopolysaccharide and had the mass, amino-acid composition and N-terminal sequence expected from the cloned gene.

A comparison of bactericidal/permeability-versus increasing protein variant 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.

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.

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.

Reversible binding of heparin to the loop peptide of endotoxin neutralizing protein

Endotoxin neutralizing protein (ENP) from Limulus polyphemus is an amphipathic, 11.8 kDa protein with an isoelectric point of 10.2. ENP neutralizes lipopolysaccharide (LPS) and possesses antibacterial activity against Gram-negative bacteria. Heparin binds to ENP and blocks its LPSneutralizing activity. The relative blocking activity of heparin is equal to low molecular weight heparin and polyanetholsulfonic acid > heparan sulfate > chondroitin sulfate A > chondroitin sulfate C. Endoproteinase Glu-C hydrolysis of recombinant ENP results in four major peptides, three of which are seen following separation on reversed phase HPLC. Heparin binds to the loop peptide (31—72), which includes the heparin binding consensus sequence XBBXBX between the two cysteine residues of ENP. When heparin is added to the digest and then applied to a C18 column, the loop peptide is bound; however, it dissociates and elutes with either 5 M NaCl or 0.1 M sodium phosphate, demonstrating reversible binding to heparin. LPS and lipid A both bind to the loop peptide and remove it from digests of ENP; however, neither complex could be dissociated by salt or sodium phosphate. Heparin, LPS, and lipid A individually bind to the same site on ENP.

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

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