Semi-synthesis of polymyxin-B conjugated ovalbumin: Evaluation of lipopolysaccharide binding avidity and neutralization of induced tnf-α synthesis

Abstract

A method is described in these investigations for the semi-synthetic production of polymyxin-B conjugated ovalbumin in the form of polymyxin-B.Sulfo-SMCC.ovalbumin (PSO). The heterobifunctional "cross-linking" agent, Sulfo-SMCC was first reacted with polymyxin-B to produce a relatively pure reactive intermediate in the form of polymyxin-B.Sulfo-SMCC. Highly purified ovalbumin was then combined with the polymyxin-B.Sulfo-SMCC reactive intermediate and contaminants removed from the final PSO end product by exhaustive microdialysis. Purity of PSO was established with by high-performance cellulose acetate electrophoresis (HPCAE), and high-performance thin layer chromatography (HPTLC) analyses. Verification of polymyxin-B.Sulfo-SMCC.ovalbumin binding avidity for lipopolysaccharide (LPS) was determined by DotBLot analysis applying fluorescein isothiocyanate labeled E. coli (055:B5) LPS fractions (FITC-LPS). Efficacy of PSO to inhibit in vitro LPS-induced synthesis of tumor necrosis factor-alpha (TNF-alpha) was assessed with a tissue culture based biological assay system. In this context, semi-synthetic conjugates of PSO (0.349 microgram/ml) effectively inhibited Salmonella minnesota (RS) LPS (2.5 ng/ml well) induced TNF-alpha synthesis and corresponding cytoprotection (100%) to WEHI 164 clone 13 cell populations.

Endotoxin Contamination in Commercially Available Pokeweed Mitogen Contributes to the Activation of Murine Macrophages and Human Dendritic Cell Maturation

“…On the other hand, endotoxins are frequent contam- inants of various biochemical preparations because gram-negative bacteria are ubiquitous and endotoxins are very stable in nature. Commercial preparations of ovalbumin and lactoferrin were found to be highly contaminated with an endotoxin (4, 31), which may not be surprising because both substances have high affinity to endotoxin (5,22). Furthermore, commercial preparations of LTA also turned out to contain endotoxins (8).…”

Effects of pentoxifylline and polymyxin B on the acute‐phase‐response to Escherichia coli endotoxin in dwarf goats

“…However, the drug did not prevent the LPSinduced reduction in the number of circulating lymphocytes (Table 2). Higher doses were not tested, because polymyxin B is a well known neuro-and nephrotoxic agent ( van Miert & van Duin, 1977, 1978Coyne et al, 1995). Infusion of polymyxin B alone caused tachycardia, a slight inhibition of ruminal contractions (Fig.…”

“…Polymyxin B is a cationic, polypeptide antibiotic which possesses bactericidal activity against gram-negative bacteria. This drug interacts with the lipid A-keto-3-deoxyoctulosonate region of the LPS molecule and, by forming inactive polymyxin B-LPS complexes, it reduces the production of pro-inflammatory cytokines (Coyne & Fenwick, 1993;Coyne & Moritz, 1994;Durando et al, 1994;Coyne et al, 1995). Consistent with this effect on circulating TNF-a and IL-6, polymyxin B-treated rabbits ( van Miert & van Duin, 1978), goats (van Miert & van Duin, 1977) and cows had less severe clinical signs of endotoxemia than did control animals.…”