Biophysical characterization of the interaction of Limulus polyphemus endotoxin neutralizing protein with lipopolysaccharide.

Endotoxin-neutralizing protein (ENP) of the horseshoe crab is one of the most potent neutralizers of endotoxins [bacterial lipopolysaccharide (LPS)]. Here, we report on the interaction of LPS with recombinant ENP using a variety of physical and biological techniques. In biological assays (Limulus amebocyte lysate and tumour necrosis factor-alpha induction in human mononuclear cells), ENP causes a strong reduction of the immunostimulatory ability of LPS in a dose-dependent manner.

Rapid bacterial counts in metal working fluids.

A rapid (10 s) automated fluorescent method to estimate viable bacteria in metal working fluids (MWF) was compared with dip-slide cultures. The BactiFluor method compared favorably with 107 MWF (r=0.99) and with 30 other metal processing fluids.

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.

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.

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.

Limulus amebocyte lysate assay for detection of endotoxin in patients with sepsis syndrome. AMCC Sepsis Project Working Group.

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.

Endotoxin-neutralizing protein protects against endotoxin-induced endothelial barrier dysfunction.

Bacterial lipopolysaccharide induces tyrosine phosphorylation of paxillin, actin reorganization, and opening of the transendothelial paracellular pathway through which macromoles flux. In this study, lipid A was shown to be the bioactive portion of the lipopolysaccharide molecule responsible for changes in endothelial barrier function. We then studied whether endotoxin-neutralizing protein, a recombinant peptide that is derived from Limulus antilipopolysaccharide factor and targets lipid A, could block the effects of lipopolysaccharide on protein tyrosine phosphorylation, actin organization, and movement of 14C-bovine serum albumin across bovine pulmonary artery endothelial cell monolayers.

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.

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.

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.

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.

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.

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

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.

Efficacy of a recombinant endotoxin neutralizing protein in rabbits with Escherichia coli sepsis.

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.

Binding and neutralization of endotoxin by Limulus antilipopolysaccharide factor.

In order to examine the ability of Limulus antilipopolysaccharide factor (LALF) to bind lipopolysaccharide (LPS), we purified LALF to homogeneity from Limulus amoebocyte lysate and coupled it covalently to agarose beads. LALF-coupled beads captured more tritiated LPS from rough and smooth strains of gram-negative bacteria than did control human serum albumin-coupled beads. Unlabeled homologous and heterologous LPS competed for the binding of 3H-LPS to LALF-coupled beads.

Limulus antilipopolysaccharide factor protects rabbits from meningococcal endotoxin shock.

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.

Sensitivity of Limulus amebocyte lysate (LAL) to LAL-reactive glucans.

The sensitivity of Limulus amebocyte lysate (LAL) to LAL-reactive glucans (LRGs) and lipid A was tested by using commercially available and experimentally formulated LAL reagents. The glucans included two kinds of beta-(1,3)-D-glucans, laminarin and curdlan, and cellulosic material, LAL-reactive material (LAL-RM), extracted from a hollow-fiber (Cuprophan) hemodialyzer. LAL-RM loses its LAL activity when it is digested with cellulase and thus appears to be a beta-(1,4)-D-glucan or a mixed glucan containing a substantial proportion of beta-(1,4) linkages.

Plastics, endotoxins, and the Limulus amebocyte lysate test.

A variety of polypropylene and polystyrene tubes have been tested for use with the Limulus amebocyte lysate (LAL) test. Polypropylene tubes tended to be more contaminated with endotoxin than polystyrene. One brand of polypropylene tube contained a water extractable inhibitor of the LAL test.

Single-step, chromogenic Limulus amebocyte lysate assay for endotoxin.

A new reagent for the chromogenic Limulus amebocyte lysate (LAL) assay is described. LAL was formulated for optimal performance in either an endpoint procedure or a kinetic procedure with the chromogenic substrate, buffer, and LAL components colyophilized as a single reagent. The kinetic chromogenic method required an incubating microplate reader coupled to a computer for collection and analysis of data.

Labor and infection. II. Bacterial endotoxin in amniotic fluid and its relationship to the onset of preterm labor.

We have previously reported the detection of endotoxin in the amniotic fluid of patients with gram-negative intraamniotic infection. Endotoxin or lipopolysaccharide is a potent biologic product capable of inducing prostaglandin release from several cell types and, therefore, may be involved in the onset of human parturition in the presence of intraamniotic infection. This article describes a technique for the quantification of endotoxin in amniotic fluid.

Endotoxin neutralization with rabbit antisera to Escherichia coli J5 and other gram-negative bacteria.

To study the mechanisms of protection against endotoxin challenge offered by antisera to smooth and rough gram-negative organisms, we have developed an assay to quantitate endotoxin neutralization based on inhibition of the Limulus amoebocyte lysate test. Dilutions of different bacterial lipopolysaccharides (LPSs) were incubated with hyperimmune rabbit sera against Escherichia coli O113, E. coli O18, and rough mutants E.

Role of interleukin-1 in augmenting serum neutralization of bacterial lipopolysaccharide.

We have previously described an assay to quantify the serum neutralization of bacterial lipopolysaccharide which is based on a spectrophotometric Limulus amoebocyte lysate test (T.J. Novitsky, P.

Quantitation of endotoxin in products using the LAL kinetic turbidimetric assay.

The data presented here show the kinetic turbidimetric LAL assay to be a highly quantitative and effective method for determining endotoxin concentrations in products. The assay allows for the accurate assessment of inhibiting or enhancing effects in products when related to a LRW standard curve. However, designating some products as inhibitors or enhancers can be both misleading and erroneous unless qualified as to the dilution and/or endotoxin concentration.