T J Novitsky’s research while affiliated with Cape Cod Hospital and other places

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.

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 LPS-neutralizing 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.

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).

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.

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 ⩾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.

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. In the presence of serum, a 6-h exposure to lipopolysaccharide (10 ng/ml) increased transendothelial 14C-albumin flux compared to the simultaneous media control. Coadministration of endotoxin-neutralizing protein (> or =10 ng/ml) with lipopolysaccharide (10 ng/ml) protected against lipopolysaccharide-induced barrier dysfunction. This protection was dose dependent, conferring total protection at endotoxin-neutralizing protein/lipopolysaccharide ratios of > or =10:1. Similarly, endotoxin-neutralizing protein was capable of blocking the lipopolysaccharide-induced endothelial cell responses that are prerequisite to barrier dysfunction, including tyrosine phosphorylation of paxillin and actin depolymerization. Finally, endotoxin-neutralizing protein cross-protected against lipopolysaccharide derived from diverse gram-negative bacteria. Thus, endotoxin-neutralizing protein offers a novel therapeutic intervention for the vascular endothelial dysfunction of gram-negative sepsis and its attendant endotoxemia.

We conducted a prospective study of a chromogenic LAL assay in 346 patients with sepsis syndrome, as defined by a modification of the Bone criteria, and 131 healthy volunteers at eight member centers of the Academic Medical College Consortium (AMCC). We identified patients with endotoxemia (> 0.40 EU/ml) by measuring LAL-reactive material in whole blood, extracted by the Tamura nitric acid method, with the chromogenic LAL (Pyrochrome®) assay. The mean result in sepsis patients with detectable endotoxemia (n = 241) was 1.07 ± 1.57 EU/ml, and the mean result in 131 volunteers was 0.151 ± 0.113 EU/ml, with 73% of the volunteers' results falling below the detectable limit. The average incidence of endotoxemia in sepsis patients was 33%, but varied 2.7-fold among the clinical centers (range 16-44%). Assay results were repeatable when samples tested frozen at the clinical sites were compared to results on frozen samples tested at Associates of Cape Cod, Inc. (ACC). Multiple samples were obtained from 40 patients at 18-24 h interval(s). Fourteen multidraw patients (35%) were endotoxemic at one or more draw(s) and eight of these patients had two or more draws with endotoxin levels > 1.0 EU/ml. The presence of sulfa drugs gave false positive results in two patient samples. A positive LAL test did not correlate with culture-proven bacterial infection and did not significantly correlate with mortality. There was a correlation (P = 0.014) between a patient having a positive LAL test and the presence of a fungal infection when mixed fungal and bacterial infections were included. There was no correlation with a positive LAL test when only a fungal infection was present (P = 0.425) or when only a fungal and a Gram-positive infection was present (P = 0.087).

To compare a recombinant bactericidal/permeability-increasing protein variant and a recombinant endotoxin-neutralizing protein. Randomized, blinded, controlled study, using a rat model of sepsis. Animal research facility. Male Wistar rats. 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. 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. 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.

To test the efficacy of a recombinant endotoxin neutralizing protein as compared with saline in rats with Escherichia coli sepsis. Prospective, controlled animal trial. Hospital animal research laboratory. Male Wistar rats challenged with intraperitoneal E. coli, O18ac K1, and treated 1 hr later with ceftriaxone and gentamicin. 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. 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). 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.