Design of a potent novel endotoxin antagonist.

BACKGROUND: Bactericidal permeability increasing protein (BPI) binds to and neutralizes lipopolysaccharide (LPS, endotoxin). Small synthetic peptides based on the amino acid sequence of the LPS binding domain of BPI neutralize LPS, albeit inefficiently. Although the LPS binding domain of native BPI possesses a beta-turn secondary structure, this structure is not present in small derivative peptides. The purpose of this study was to determine whether the addition of a beta-turn to a BPI-derived peptide is associated with more potent endotoxin antagonism. METHODS: We generated a hybrid peptide (BU3) on the basis of (1) a portion of the LPS binding domain from BPI and (2) amino acids known to initiate a beta-turn. BU3 folds with a beta-turn, and we tested its effects on LPS neutralization and LPS-induced tumor necrosis factor-alpha secretion, comparing it with BPI-derived peptide BG22 that lacks a beta-turn and to an irrelevant peptide (BG16). RESULTS: Compared with BG22, BU3 demonstrated enhanced LPS neutralization and inhibition of LPS-induced tumor necrosis factor-alpha secretion in vitro and a similar diminution of endotoxemia and tumor necrosis factor-alpha secretion in a murine model of endotoxemia. CONCLUSIONS: These data demonstrate the potential for enhancing the biologic activity of a BPI-derived peptide endotoxin antagonist via manipulation of its conformational structure.

Macrophages expressing a fusion protein derived from bactericidal/permeability-increasing protein and IgG are resistant to endotoxin.

OBJECTIVES: To generate a recombinant fusion protein (FP) based on the endotoxin-binding domain of bactericidal/permeability-increasing protein (BPI) and the constant domain of IgG and to test its ability to inhibit lipopolysaccharide (LPS)-induced macrophage tumor necrosis factor alpha (TNF-alpha) secretion. DESIGN: A murine macrophage cell line, RAW 264.7, was transfected with a BPI-IgG FP before incubation with LPS. The amount of LPS-induced TNF-alpha protein secreted was measured and compared with that secreted by cells transfected with a control construct. SETTING: Basic science research laboratory. MAIN OUTCOME MEASURES: Secreted TNF-alpha protein concentration. RESULTS: After transfection, RAW 264.7-cell FP expression was detected in cell lysates and supernatants. At each LPS dose tested, cells transfected with the FP gene secreted less TNF-alpha than did cells transfected with a control construct. CONCLUSIONS: The FP possesses substantial antiendotoxin activity, as delineated by inhibition of LPS-induced TNF-alpha secretion by murine macrophages transfected with the fusion gene construct. In the future, such FP may be used as a clinical reagent to reduce the morbidity and mortality associated with serious gram-negative bacterial infections in surgical patients.

Differential sensitivity to Escherichia coli infection in mice lacking tumor necrosis factor p55 or interleukin-1 p80 receptors.

OBJECTIVE: To determine the effect of targeted disruption of the cellular receptors of either tumor necrosis factor alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta) during experimental gram-negative bacterial infection and endotoxemia. DESIGN: Transgenic (knockout [KO]) mice deficient in either the p55 TNF receptor (TNF RI) or the p80 IL-1 receptor (IL-1 RI) were challenged with intravenous lipopolysaccharide (endotoxin) or intraperitoneal live Escherichia coli 0111:B4. Mortality was assessed daily for 7 days. Serum endotoxin levels and quantitative blood cultures were monitored at multiple times during infection. SETTING: Surgical infectious disease research laboratory. MAIN OUTCOME MEASURES: Mortality, results of quantitative blood cultures, and serum endotoxin levels. RESULTS: Both TNF and IL-1 RI KO mice were resistant to endotoxin challenge (0% mortality for both groups) compared with control mice (100% mortality [P < .01]). In contrast, only the IL-1 RI KO mice were resistant to infection caused by viable gram-negative bacteria (43% mortality) compared with control mice (100% mortality [P < .01]). Infection led to 100% mortality in TNF RI KO mice. The IL-1 RI KO mice exhibited less bacteremia and diminished endotoxemia compared with control and TNF RI KO mice 18 and 24 hours after infection. CONCLUSION: The absence of either the TNF or the IL-1 RI receptor prevents cellular activation by each respective cytokine. Absence confers protection against intravenous endotoxin, which stimulates massive rapid release of cytokines into the systemic circulation. However, bacterial infection within the peritoneal cavity is known to cause more delayed cytokine release, and cytokines may act at the site of infection to enhance host defenses. We believe that IL-1 signaling may be more critical in provoking lethal systemic toxic effects than TNF signaling. However, TNF signaling may be an important component of host defense enhancement at the local site of infection.