John L. Cantrell

Augmentation of NK activity by Corynebacterium parvum fractions in vivo and in vitro.

Biochemically modified whole cell preparations and derived fractions of Corynebacterium parvum (C. parvum) were evaluated for the ability to augment natural killer cell cytoxicity in vivo and in vitro in rats. Unfractionated C. parvum enhanced peritoneal cell (Pc) NK activity in a dose dependent fashion. This activity appeared to be enriched in insoluble light residue material obtained from hot phenol water extraction. Enhancement of Pc cytotoxicity was significantly greater at all time points tested in rats injected with light residue when compared to rats injected with comparable amounts (by dry weight) of unfractionated organisms. In addition, pyridine extractable material and HCl modified preparations were capable of boosting Pc NK activity following intraperitoneal (I.P.) injection. Periodate treatment abrogated C. parvums ability to boost Pc cytotoxicity and insoluble residue material obtained from pyridine extraction was likewise devoid of NK-enhancing properties. Culture of rat spleen cells overnight with unfractionated C. parvum, light residue and pyridine residue materials enhanced NK cytotoxicity whereas HCl and periodate modified whole cell preparations as well as phenol and pyridine extractable material were incapable of boosting cytotoxicity in vitro. In vitro augmentation by culturing with light residue was dependent on the presence of adherent cells in rat spleen cell populations. Pyridine extracts boost cytotoxicity in vivo and have no effect in vitro while the opposite is true of pyridine residue material suggesting different mechanisms of NK augmentation by C. parvum between in vitro and in vivo systems.

Activation and mechanism of action of suppressor macrophages.

Abstract Intravenous administration of Corynebacterium parvum to alloimmunized mice activates splenic suppressor macrophages that effectively curtail primary and secondary generation of cytotoxic T lymphocytes (CTLs) in vitro . CTL generation was significantly inhibited in suppressed primary cultures by Day 3, the earliest time point that activity is first detected in control cultures. Suppressor macrophages had to be present during the first 24–48 hr of culture to effectively curtail the generation of CTLs. However, if suppressor macrophages were reactivated by 48-hr in vitro culture and then added to primary sensitizations that had been initiated 48 hr previously, they were capable of significant suppression. Suppressor cells produced a soluble factor that mediated the inhibition of CTL generation. The production or action of this factor could not be counteracted by indomethacin.

Different structural requirements of endotoxic glycolipid for tumor regression and endotoxic activity

Abstract Endotoxic glycolipid extracted from the heptose-less mutant of Salmonella typhimurium was treated with alkali and acid reagents. The glycolipid freed of all O-ester linked fatty acids by hydroxylamine had lost tumor regression activity and toxicity, whereas a partial removal of O-ester linked fatty acids by mild alkali did not impair with these activities. The glycolipid retained both activities after removal of 2-keto-3-deoxyotonate by sodium acetate (pH 4.5) but was rendered nontoxic while retaining antitumor activity when hydrolyzed by 0.1N HCl whereby 2-keto-3-deoxyoctonate and glycosidic phosphate was split off the glycolipid molecule. Nontoxic and tumor regressive fractions were separated by means of preparative thin layer chromatography of glycolipid hydrolyzed by mild acid. Thus, it was concluded that glycosidic bound phosphate and at least a portion of fatty acids of the lipid A moiety were essential for toxicity, but that this phosphate is not essential for tumor regression activity.