Isolation of a nontoxic lipid A fraction containing tumor regression activity

Galanos-type endotoxin obtained from the heptose-less mutant of Salmonella typhimurium was converted to Lipid A by two cycles of treatment with sodium acetate, pH 4.5, at 100 degrees and separated on a DEAE-cellulose column into several fractions (Fractions III to VII). Tumor regression studies with strain 2 guinea pigs and syngeneic line 10 hepatocellular carcinoma showed that all fractions were effective when combined with trehalose dimycolates and an additional tumor regression factor (previously designated ACP) and incorporated into oil droplets (78 to 100% cures). A low polar fraction (Fraction IV) was relatively nontoxic [the medium lethal dose for 11-day-old chick embryos inoculated i.v. (CELD50) was more than 10 micrograms] and nonpyrogenic [the dose estimated to give a fever index (area under fever curve) of 40 sq cm in rabbits when 1 hr and 1 degrees are plotted as 1 (FI40) was 5 micrograms] as compared to the unfractionated Lipid A (CELD50 of 0.0546 micrograms; FI40 of 0.046 micrograms). All other fractions were toxic and pyrogenic and caused severe endotoxic shocks when combined with N-acetylmuramyl-L-seryl-D-isoglutamine and injected i.v. into guinea pigs. Fraction IV plus N-acetylmuramyl-L-seryl-D-isoglutamine did not cause endotoxic shock. The phosphate content of Fraction IV was about one-half of that detected in the toxic fractions.