Stephen F. Carroll

Relationship between plasma levels of lipopolysaccharide (LPS) and LPS- binding protein in patients with severe sepsis and septic shock

Plasma endotoxin and lipopolysaccharide-binding protein (LBP) levels were measured in a group of 253 patients at the onset of severe sepsis and/or septic shock. Endotoxin levels were significantly greater than control levels (n=33; mean +/- SD, 5.1+/-7.3 pg/mL) in 78.3% of patients. Median endotoxin levels in patients with sepsis were 300 pg/mL (25%-75% interquartile range, 110-726 pg/mL). LBP levels were elevated in 97% of patients compared with normal control values of 4.1+/-1.65 microgram/mL. Median LBP levels in patients with sepsis were 31.2 microgram/mL (interquartile range, 22.5-47.7 microgram/mL). Median endotoxin levels at study entry were more highly elevated (515 vs. 230 pg/mL; P<.01), and LBP levels were less highly elevated (28.0 vs. 33.2 microgram/mL; P<.05) in nonsurvivors than survivors over the 28-day study period. No correlation was found between endotoxin and LBP levels. The quantitative level of both endotoxin and LBP may have prognostic significance in patients with severe sepsis.

Preliminary evaluation of recombinant amino-terminal fragment of human bactericidal/permeability-increasing protein in children with severe meningococcal sepsis

BACKGROUND Meningococcal sepsis remains an important cause of morbidity and mortality. We hypothesised that children with severe meningococcaemia might benefit from inhibition of the inflammatory processes thought responsible for fulminant disease. rBPI21 is a recombinant, N-terminal fragment of human bactericidal/permeability-increasing protein, which kills meningococci and binds to and clears bacterial endotoxin, these being the primary inducers of the systemic inflammation. The aim of this study was to determine the safety and kinetics of rBPI21 in children with severe meningococcaemia and to make a preliminary assessment of clinical outcome. METHODS In this open-label, dose-escalation, phase I/II trial in severe meningococcaemia (Glasgow meningococcal prognostic septicaemia score [GMSPS] > or = 8), 26 patients aged 1-18 years, who had received their first dose of antibiotics no more than 8 hours earlier were given rBPI21 by infusion at total doses of 1.0, 2.0, and 4.0 mg/kg. FINDINGS The patients had significantly raised plasma concentrations of bacterial endotoxin and cytokines. Peak and steady state BPI concentrations were comparable with pharmacokinetic data in healthy adults. All complications were compatible with the expected pattern for severe meningococcal sepsis. Only one patient died. This outcome was found to compare favourably with a predicted mortality of > or = 30% by GMSPS, > or = 15% by plasma endotoxin values, > or = 28% by plasma interleukin-6 concentrations, 29-49% by severity of coagulopathy, and 20% (11/54) by comparison with recent historical patients consecutively treated in participating centres before this study. INTERPRETATION This, the first clinical trial or rBPI21, shows that rBPI21 can be safely administered to children with severe meningococcaemia and that the pharmacokinetics are consistent with patterns seen in healthy adults. Predicted mortality, on the basis of GMSPS, laboratory indices of inflammation and coagulopathy, and historical controls, was for between four and eight deaths. These findings have prompted a phase III randomised trial.

Dual Role of Lipopolysaccharide (LPS)-Binding Protein in Neutralization of LPS and Enhancement of LPS-Induced Activation of Mononuclear Cells

ABSTRACT The lipopolysaccharide (LPS)-binding protein (LBP) has a concentration-dependent dual role in the pathogenesis of gram-negative sepsis: low concentrations of LBP enhance the LPS-induced activation of mononuclear cells (MNC), whereas the acute-phase rise in LBP concentrations inhibits LPS-induced cellular stimulation. In stimulation experiments, we have found that LBP mediates the LPS-induced cytokine release from MNC even under serum-free conditions. In biophysical experiments we demonstrated that LBP binds and intercalates into lipid membranes, amplified by negative charges of the latter, and that intercalated LBP can mediate the CD14-independent intercalation of LPS into membranes in a lipid-specific and temperature-dependent manner. In contrast, prior complexation of LBP and LPS inhibited binding of these complexes to membranes due to different binding of LBP to LPS or phospholipids. This results in a neutralization of LPS and, therefore, to a reduced production of tumor necrosis factor by MNC. We propose that LBP is not only present as a soluble protein in the serum but may also be incorporated as a transmembrane protein in the cytoplasmic membrane of MNC and that the interaction of LPS with membrane-associated LBP may be an important step in LBP-mediated activation of MNC, whereas LBP-LPS complexation in the serum leads to a neutralization of LPS.

Lipopolysaccharide‐binding protein mediates CD14‐independent intercalation of lipopolysaccharide into phospholipid membranes

Lipopolysaccharides (LPS, endotoxin) stimulate mononuclear cells to release cytokines which initiate endotoxic effects. Interaction of LPS at low concentrations with target cells is CD14‐independent whereas at high LPS concentrations it is CD14‐independent. Here, we demonstrate by resonance energy transfer (RET) technique that nonspecific, CD14‐independent intercalation of LPS into membrane systems can be mediated by lipopolysaccharide‐binding protein (LBP). It is proposed that in this pathway, LBP breaks down LPS aggregates, transports the smaller units to and inserts them into the phospholipid cell matrix. We furthermore show that LBP also mediates the intercalation of other negatively charged amphiphilic molecules. We propose a model explaining CD14‐independent cell activation at high endotoxin concentrations.

Anti-neutrophil cytoplasmic antibodies (ANCA) directed against bactericidal/permeability increasing protein (BPI): a new seromarker for inflammatory bowel disease and associated disorders

It was our purpose to determine the immunodiagnostic value of ANCA directed against BPI in diseases known to be associated with ANCA, such as ANCA‐associated vasculitides, inflammatory bowel disease (IBD) and the associated condition primary sclerosing cholangitis. The immunoreactivity of recombinant BPI (rBPI) was established in order to develop an ELISA specific for rBPI. By means of this assay, BPI‐ANCA were assessed in sera of 178 patients with IBD or the associated disorder primary sclerosing cholangitis, 112 patients with ANCA‐associated vasculitides, and in sera of 182 disease and 140 healthy controls. BPI‐ANCA were found to be closely associated with IBD and primary sclerosing cholangitis (34% and 44% of ANCA‐positive sera, respectively). By contrast, BPI‐ANCA positivity was low (<10%) in the double‐negative sera of patients with ANCA‐associated vasculitides and in disease and healthy controls. BPI‐ANCA appear to constitute an important marker for IBD and primary sclerosing cholangitis, but not for the ANCA‐associated vasculitides.

The three-dimensional structure of human bactericidal/permeability-increasing protein : Implications for understanding protein-lipopolysaccharide interactions

Gram-negative bacterial infections are often complicated by the inflammatory properties of lipopolysaccharides (LPS) on or released from the bacterial outer membrane. When present in the mammalian bloodstream, LPS can trigger a series of pathological changes, sometimes resulting in septic shock. Two related mammalian proteins, bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP), are known to affect the LPS-induced inflammatory response and are, therefore, of clinical interest. The recently determined three-dimensional structure of human BPI provides information on the overall protein fold, domain organization, and conserved regions of these two proteins. In addition, the discovery of two apolar lipid binding pockets in BPI indicates a possible site of interaction with LPS. The BPI structure is a powerful tool for the design of site-directed mutants, peptide mimetics/inhibitors, and BPI/LBP chimeras. These studies should help further define the functions of BPI and LBP, and their mechanism of interaction with LPS.

Measurement of bactericidal/permeability-increasing protein in human body fluids by sandwich ELISA.

A sensitive sandwich ELISA has been developed to measure levels of native bactericidal/permeability-increasing protein (BPI) as well as two recombinant forms of BPI (rBPI and rBPI23) in human body fluids. The linear range for the rBPI and rBPI23 standard curves were 100-6000 pg/ml and 25-800 pg/ml respectively. Recovery of different concentrations of rBPI spiked into pooled human plasma samples averaged 83% and ranged from 65% at 300 ng/ml to 97% at 3 ng/ml. Recovery of rBPI23 averaged 56% and ranged from 30% at 0.5 ng/ml to 90% at 50,000 ng/ml. Because LBP is present in normal human plasma and shares sequence homology with BPI, the effects of rLBP on the BPI ELISA were also evaluated. Under standard assay conditions, rLBP caused minimal interference with BPI detection. At 100 micrograms/ml, rLBP generated a signal equivalent to 3 ng/ml of rBPI and 0.6 ng/ml of rBPI23. Matched serum and plasma samples were collected from 20 healthy adults to measure endogenous levels of BPI. The range of BPI concentrations was < 0.2-2.1 ng/ml in plasma and 4.9-72.1 ng/ml in serum. Western blot analysis indicated that the BPI ELISA immunoreactivity in plasma and serum correlated with the presence of a protein doublet (M(r) approximately 60,000), which comigrated with native BPI extracted from human neutrophils. These data demonstrate that low levels of holo-BPI are present in plasma, and suggest that additional quantities of BPI were released from neutrophils during the process of coagulation.

Staghorn calculus endotoxin expression in sepsis.

Staghorn calculi are infrequent and generally are infected stones. Struvite or apatite calculi are embedded with gram-negative bacteria, which can produce endotoxin. Sepsis syndrome may occur after surgical therapy or endoscopic manipulation of infected or staghorn calculi. Sepsis, which can occur despite perioperative antibiotic use, may be due to bacteremia or endotoxemia. We present a child with an infected staghorn calculus who developed overwhelming sepsis and died after percutaneous stone manipulation. Endotoxin assay of stone fragments demonstrated an extremely high level of endotoxin despite low colony bacterial culture growth. This is the first reported case in which endotoxin was demonstrated in stone fragments from a child who died of severe sepsis syndrome after percutaneous staghorn stone manipulation.

Interaction between lipopolysaccharide (LPS), LPS-binding protein (LBP), and planar membranes.

Abstract The mechanism of interaction of the lipopolysaccharide (LPS)binding protein, LBP, with differently composed symmetric and asymmetric planar lipid bilayers was investigated in electrical measurements (membrane current, potential, capacitance). From a change of the inner membrane potential difference, binding of LBP to membranes was deduced. After addition of LBP to one side of the membrane, binding of antiLBP antibodies and LPS to LBP on both sides of the bilayer was observed. Effects resulting from an interaction of antiLBP antiserum with membranebound LBP depend on the side of addition of the antiserum, indicating a directed intercalation of LBP into the membrane. Addition of LPS to the same side as LBP may induce a change of the conformation of LBP or its orientation in the membrane. Based on these observations, we propose that LBP intercalates in a directed orientation into negativelycharged membranes and assumes a transmembrane configuration. Moreover, preincubated complexes of LPS and LBP do not interact with membranes. These experiments show that reconstituted planar membranes are a suitable tool for investigations of the interaction of non poreforming proteins that are involved in signal transduction.

ALTERATION OF THE PHARMACOKINETICS OF SMALL PROTEINS BY IODINATION

The pharmacokinetics of several proteins were investigated using two different assays. A 23 kDa recombinant protein fragment of bactericidal/permeability-increasing protein (rBPI23) was radiolabeled with 125I using Iodo-beads and administered rats. Plasma samples were collected and assayed for 125I-rBPI23 by radioactivity. In a separate experiment, rBPI23 was administered to rats and plasma samples were assayed for rBPI23 by ELISA. The clearance determined from plasma concentrations of 125I-rBPI23 measured by radioactivity was about 2.5-fold lower than that of rBPI23 determined by ELISA. In addition, the steady state volumes of distribution and mean residence times of 125I-rBPI23 measured by radioactivity were four-fold and 10-fold greater, respectively, compared to those measured by the ELISA method. By studying several proteins with a range of molecular weights, we found that the pharmacokinetics of proteins below about 60 kDa were different when assayed by radioactivity or ELISA, but those of proteins with molecular weights of at least 80 kDA revealed only minor differences. To determine which assay method yielded the correct plasma pharmacokinetic profile, rBPI23 was metabolically labeled with 35S-methionine and administered to rats, and plasma samples were assayed by radioactivity. The concentration-time profile assessed by this method was very close to that determined by ELISA. Exposing rBPI23 to chloramine-T (the oxidant used in the iodination process) and measuring its plasma concentration by ELISA revealed pharmacokinetics similar to those of the iodinated protein measured by radioactivity. In contrast, radiolabeling rBPI23 using iodinated Bolton-Hunter reagent (which avoids exposing the protein to oxidant), and measuring 125I-rBPI23 by radioactivity, yielded pharmacokinetics that were similar, although not identical, to the pharmacokinetics of rBPI23 measured by ELISA. Thus, our data suggest that directly iodinating low-molecular-weight proteins by oxidation procedures alters their clearance from the blood, preventing reliable determination of pharmacokinetic parameters.