Radio-Detoxified Endotoxin as a Potent Stimulator of Nonspecific Resistance

RADIO-DETOXIFIED ENDOTOXIN AS A POTENT STIMULATOR OF NONSPECIFIC RESISTANCE LÓRÁND BERTÓK* The enhancement of natural defense or the stimulation of nonspecific resistance is one of the most important unsolved problems of medicine [I]. Many authors [2] have observed that, within 24 hours of intravenous inoculation with killed typhoid bacilli, animals developed increased resistance to the live culture. The cell walls of certain gram-negative bacteria were also found to be particularly active in stimulating this nonspecific resistance. Other workers identified this active material as a lipopolysaccharide endotoxin [3, 4]. The effects of this material upon resistance have been demonstrated in infections induced by parasites, fungi, viruses, gram-positive and gram-negative bacteria, and mycobacteria [5]. It is also well known that repeated parenteral injections ofsmall doses of endotoxin will produce endotoxin tolerance [6]. Many factors are involved in the development of this endotoxin tolerance [7]. One of these could easily be the so-called lysosomal membrane condensation [8]. In support of this view, we have shown that endotoxin tolerance can inhibit the release of ribonuclease which is induced by a toxic dose of endotoxin [9]. The most important property of bacterial endotoxins is probably their capacity to stimulate the body's natural defense. Thus, in an immunologically nonspecific manner, the endotoxin shock induced by gram-negative bacteria may be prevented. Unfortunately, this beneficial influence is associated with some noxious properties (e.g., pyrogenic, hypotensive, abortifacient, etc.). This is why such endotoxins are not suitable for the enhancing of nonspecific resistance in endotoxinsensitive mammalian species. For this reason, to decrease the lethality of endotoxin while retaining the beneficial properties has become the aim of a number of researchers. The idea that such detoxification may be possible arose from the successful detoxification of bacterial protein toxins (e.g., tetanus anatoxin, diphtheria anatoxin, so-called toxoids). *Head, Department of Applied Radiation of the "Frédéric Joliot-Curie" National Research Institute for Radiobiology and Radiohygiene, Budapest, 1775, P.O. Box 101, Hungary .© 1980 by The University of Chicago. 0031-5982/81/2401-0201$01.00 Perspectives in Biology and Medicine ¦ Autumn 1980 \ 61 The structure of bacterial lipopolysaccharide endotoxins has become the subject of intensive investigations, and many researchers have demonstrated that these molecules have consisted of three main parts— polysaccharides, lipid A, and a polypeptide—and that the lipid A portion is responsible for the toxic influence of the molecule [10-15]. To date various interventions (physical, chemical, physicochemical, immunochemical, etc.) have been attempted for detoxification, with variable results. Of the chemical methods tried by researchers [16], the most successful has been the potassium methylate treatment [17, 18]. The O-phthalic anhydride treatment is also worthy of mention [19]. Another possibility employed is the use of ionizing radiation [20, 21]. For the production of a radio-detoxified preparation, the parent endotoxin was isolated by the warm phenol-water method [11] from a fermenter-grown culture of E. coli 089 and purified by repeated ultracentrifugation . For detoxification, the material was "dissolved" in distilled water at a concentration of 10 mg/ml and irradiated with different doses (5, 10, 15, 20 Mrad) in a Co60 source (Noratom-Gamma 350 C). The LD50 of the parent and irradiated preparations was then determined in appropriate animal species. The irradiation was shown to significantly increase the LD50 value of the endotoxin in a dosedependent fashion. For instance, the LD50 value of the parent preparation was 20 mg/kg in rats, but after irradiation with 5 Mrad it was increased to 50 mg/kg, with 10 Mrad to 70 mg/kg, and with 20 Mrad it was greater than 100 mg/kg [22]. The radiation alters the chemical structure of the lipopolysaccharide. This important alteration has some effect on fatty acids in the lipid fraction. The radiation also decreases the 2-keto-3-deoxyoctonic acid and glucosamine contents. It is probable that these changes in fatty acids are the principal cause of the detoxification [23, 24]. It should also be mentioned that the amino acids of the peptide fraction are altered by irradiation; this seems to be dose dependent [25]. The data summarized in table 1 demonstrate that the noxious properties of the parent...