Endotoxin-neutralizing Protein Research Articles

Endotoxin-like properties of an extract from a symbiotic, eukaryotic Chlorella-like green alga

Hot phenol-water extraction of axenic cultures of the eukaryotic symbiotic green alga, Chlorella, yielded a substance having many of the characteristics of bacterial endotoxin or lipopolysaccharide (LPS). This material caused gelation of extracts of Limulus amoebocyte lysate at concentrations similar to those manifest by LPS from Gram-negative enteric bacteria. Activity was reduced substantially by incubation with polymyxin B sulfate and Limulus endotoxin-neutralizing protein (ENP), both of which have been shown to neutralize the biological activity of LPS from Gram-negative enteric bacteria. Partially purified biologically active material was found to contain 3-deoxy-D-manno-octulosonic acid (KDO), a sugar characteristically found in LPS. Acid hydrolysis of the Chlorella extract yielded a precipitate with characteristics of lipid A. Further hydrolysis followed by methylation yielded products with retention times on gas chromatography indistinguishable from 3-hydroxylauric and 3-hydroxymyristic acids. Results of transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining are consistent with those obtained with purified enteric bacterial LPS. Exhaustive precautions against potential experimental artifacts due to cross contamination by endotoxin from Gram-negative bacteria, either in algal cultures or associated with glassware allowed the conclusion that the green alga, Chlorella (strain NC64A) synthesizes a LPS-like molecule.

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

To assess the benefit of a recombinant endotoxin neutralizing protein from Limulus polyphemus in treating Gram-negative bacterial sepsis in rabbits. Prospective, blinded, controlled, laboratory trial. Animal research laboratory. New Zealand White rabbits. 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. 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). 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.

Inhibition of Endotoxin-Induced Activation of the Coagulation and Fibrinolytic Pathways Using a Recombinant Endotoxin-Binding Protein (rBPI23)

A recombinant endotoxin-neutralizing protein, rBPI23, was shown to partially prevent endotoxin-induced activation of the fibrinolytic and coagulation systems in experimental endotoxemia in humans. In a placebo-controlled, blinded crossover study, eight volunteers were challenged twice with an intravenous bolus injection of endotoxin (40 EU/kg of body weight) and concurrently received either rBPI23 (1 mg/kg) or placebo (human serum albumin, 0.2 mg/kg). rBPI23 treatment significantly lowered the endotoxin-induced fibrinolytic response, ie, reduced the release of tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen activator inhibitor antigen, and complex formation of plasmin alpha 2-antiplasmin (P = .0078 for each). Plasminogen activator inhibitor activity was also reduced, but not significantly according to the Hochberg method (P = .0304). The endotoxin-induced activation of the procoagulant state as reflected by increase in F1 + 2 fragments and TAT complexes was blunted by rBPI23 infusion (P = .0391 [not significant according to the Hochberg method] and .0078, respectively). These results indicate that rBPI23 is capable of reducing both the activation of the fibrinolytic and the coagulation systems after low-dose endotoxin infusion in humans.

Endotoxin-Neutralizing Protein

Endotoxin-Neutralizing Protein

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

A recombinant endotoxin neutralizing protein was evaluated for its ability to ameliorate the effects of Escherichia coli sepsis in rats. Prospective, controlled animal trial. Hospital animal research laboratory. Wistar rats, treated with gentamicin 1 hr after challenge with intraperitoneal E. coli O18ac. 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. 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. 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.

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. Controlled animal trial. Animal research laboratory. A total of 112 New Zealand white rabbits were studied. 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).

Crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, Limulus anti-LPS factor, at 1.5 A resolution.

Lipopolysaccharide (LPS), or endotoxin, is the major mediator of septic shock, a serious complication of Gram-negative bacterial infections in humans. Molecules that bind LPS and neutralize its biological effects or enhance its clearance could have important clinical applications. Limulus anti-LPS factor (LALF) binds LPS tightly, and, in animal models, reduces mortality when administered before or after LPS challenge or bacterial infection. Here we present the high resolution structure of a recombinant LALF. It has a single domain consisting of three alpha-helices packed against a four-stranded beta-sheet. The wedge-shaped molecule has a striking charge distribution and amphipathicity that suggest how it can insert into membranes. The binding site for LPS probably involves an extended amphipathic loop, and we propose that two mammalian LPS-binding proteins will have a similar loop. The amphipathic loop structure may be used in the design of molecules with therapeutic properties against septic shock.

Lipopolysaccharide Detoxification by Endotoxin Neutralizing Protein

Endotoxin neutralizing protein (ENP), a recombinant form of the anti-lipopolysaccharide factor that was isolated from amebocytes of the American horseshoe crab, Limulus polyphemus, detoxifies lipopolysaccharide (LPS) both in vitro and in vivo. Using the Limulus amebocyte lysate assay, LPS was detoxified by ENP at a 1 to 1 weight ratio (1:1). When isolated rat aortic rings were preincubated for 16 hr with either LPS or LPS/ENP (1:5), only aortas in the LPS/ENP group contracted normally under norepinephrine stimulation. To show that detoxification of a lethal amount of LPS (18 mg/kg, LD 50 at 48 hr) persists in vivo, LPS/ albumin (1:1) or LPS/ENP (1:1) mixtures were preincubated (30 min, 37°C) and then injected intravenously into rats. In the 8 hr after injection, LPS/ENP challenged rats, in contrast to their LPS/albumin injected counterparts, had significantly fewer physical signs of acute LPS toxicity ( P < 0.001). At 48 hr after challenge, all LPS/ENP treated rats survived ( P < 0.01 vs LPS/albumin), and with significantly less weight loss ( P < 0.001 vs LPS/albumin challenged survivors). At necropsy, the LPS/ENP group was free of typical LPS-induced gross organ lesions, notably in the liver, spleen, gut-associated lymphoid tissue (GALT), and small intestine. By microscopic examination, lymphocytic necrosis in the spleen and GALT of the LPS/ENP treated survivors was significantly milder than that in the LPS/albumin challenged survivors, although the degree of hepatocellular necrosis and small intestinal enteritis was similar. LPS-neutralizing proteins such as ENP may be useful in treating LPS toxicity.

Human granulocytes express a 55-kDa lipopolysaccharide-binding protein on the cell surface that is identical to the bactericidal/permeability-increasing protein.

Several LPS-binding proteins have been identified on the surface of human granulocytes (polymorphonuclear leukocyte (PMN)). We describe a plasma-membrane associated ca. 55-kDa LPS-binding protein of human PMN that is indistinguishable from the bactericidal/permeability-increasing protein (BPI). To detect LPS-binding proteins on the cell surface, PMN were biotinylated before detergent solubilization and incubation with LPS-coated beads. Several biotinylated proteins bound to LPS-coated beads but not to uncoated beads and were characterized after elution with detergent by SDS-PAGE and western blotting using streptavidin-horseradish peroxidase. The spectrum of biotinylated proteins binding to and eluting from LPS-coated beads increased as the number of beads incubated with PMN lysate increased. However, at all concentrations of beads a 55-kDa protein was a dominant component of the eluate. Binding of the 55-kDa protein to LPS-coated beads was inhibited by lipid A, and both homologous and heterologous LPS, but not by peptidoglycan. Similar amounts of biotinylated 55-kDa LPS-binding protein were detected on PMN from patients with paroxysmal nocturnal hemoglobinuria who lacked membrane bound CD14, a known ca. 55-kDa plasma membrane-associated LPS-binding protein, indicating that the recovered biotinylated protein is not CD14. Several pieces of evidence, however, do indicate that the 55-kDa surface protein is BPI: 1) flow cytometry of PMN after labeling with rabbit anti-BPI serum and FITC-labeled goat anti-rabbit IgG revealed immunoreactive surface molecules on resting PMN and, in increased amounts, on PMN stimulated with FMLP or TNF; 2) This antiserum specifically and quantitatively inhibited binding of the biotinylated 55-kDa species to LPS-coated beads; 3) both BPI and the 55-kDa protein migrated as a doublet during SDS-PAGE and were both converted to single migrated species after N-glycosidase F treatment; 4) chemical cleavage of the biotinylated protein and native BPI with N-chlorosuccinimide yielded the same fragments. Thus, we have positively identified BPI as a LPS-binding protein on the surface of PMN. The role of this potent antibacterial, endotoxin neutralizing protein on the surface of PMN remains to be established.

Recovery of vascular tissue contractile function during sustained endotoxin exposure.

Rat aortic rings incubated in vitro with endotoxin (10 ng/ml, 24 h) exhibited marked suppressions of sensitivity and maximum contraction to norepinephrine as well as a significant increase in guanosine 3',5'-cyclic monophosphate (cGMP) levels. Increasing the incubation time to 42 h did not result in greater suppression; instead, these rings showed improved contractile performance and control-level cGMP. Rings incubated with endotoxin for 18 h and then allowed to recuperate in control medium for another 24 h exhibited normal maximal contractions and cGMP levels; however, these rings remained less sensitive to norepinephrine. An endotoxin-neutralizing protein (50 ng/ml) shielded rings from endotoxin-induced contractile suppression if present during incubation but was unable to restore tissue sensitivity to normal levels if present during the 24-h recuperation. These findings show that vascular tissue exposed to endotoxin has a varied and time-dependent ability to counter the effects of endotoxin on contractile function and intracellular cGMP.