Edgar Ribi

Preparation and antitumor activity of nontoxic lipid A

SummaryHighly refined, disaggregated endotoxic glycolipids (B5) from heptose-less (Re) mutant Salmonella typhimurium quantitatively converted to nontoxic (lethality for chick embryos) and nonpyrogenic (fever in rabbits) lipid A by treatment with boiling 0.1 N HCl (B5-HC1). Nontoxic B5-HCl, like toxic B5, caused regression of line-10 tumors and elimination of lymph node metastasis in 27 of 32 (84%) syngeneic strain 2 guinea pigs at a dosage of 150 μg. At this dosage, toxic B5 led to a cure in 54 of 67 (81%) tumor-bearing animals. All cured animals rejected a second line-10 tumor cell transplant. This activity depended on combining the toxic or nontoxic endotoxins with mycobacterial trehalose mycolate (P3) and an essentially nontoxic peptide-containing side-fraction (ACP) recovered during the isolation of B5. In contrast to toxic B5 or endotoxins in general, nontoxic B5-HCl did not cause endotoxic shock when combined with adjuvant dipeptide (MDP) and injected IV into guinea pigs. Chemical analysis showed that the phosphate content of nontoxic B5-HCl was about one-half that observed in toxic B5 or in toxic KDO-free lipid A, which was obtained by treating toxic B5 with sodium acetate at pH 4.5 at 100° C (B5-pH 4.5). The molar ratio of glucosamine: phosphorus: fatty acids was 2:1:4 for nontoxic B5-HCl and was 2:2:4 for toxic B5-pH 4.5. These results demonstrate that endotoxic extracts could be selectively detoxified while retaining antitumor properties. Thus, nontoxic B5-HCl may be a potential candidate for immunotherapy of human cancer.

Immunobiological activities of nontoxic lipid A: Enhancement of nonspecific resistance in combination with trehalose dimycolate against viral infection and adjuvant effects

The ability of nontoxic monophosphoryl lipid A (MPL) to stimulate nonspecific resistance against viral infection was investigated. Mice pretreated intravenously with squalane-in-water emulsions of MPL, alone or in combination with other immunostimulants, were given an aerosol of influenza virus three weeks after the pretreatment. Complete protection against lethal influenza virus infection was conferred when MPL was combined with trehalose dimycolate (TDM). The protective activity of MPL plus TDM combination was corroborated by a significant reduction of the lung virus titers. Combination of lower doses of MPL with TDM extracted from Mycobacterium bovis, but not with that of M. phlei, induced significant resistance to influenza virus. Preparations containing MPL alone, or combined with mycobacterial cell wall skeleton or muramyl dipeptide, were not effective. The adjuvant activity of MPL on bivalent influenza subunit vaccine was also studied. The primary antibody responses to influenza A and influenza B antigens were enhanced by the addition of MPL and were higher than the vaccine associated with aluminum hydroxide. The adjuvant activity of MPL was confirmed by the elevated secondary response. High levels of circulating antibodies were still present in the MPL group when antibody titers in the controls were waning.

Use of pressure cell to prepare cell walls from Mycobacteria.

Summary Use of a pressure cell for preparation of purified cell walls of mycobacteria is described. This technic eliminated difficulties encountered earlier with the Mickle procedure and the yields were considerably increased.

A new immunomodulator with potential clinical applications: Monophosphoryl lipid A, a detoxified endotoxin

I m m u n o s t i m u l a t o r y Ef fec t s of E n d o t o x i n Endotoxin is an extraordinary bacterial product because of its ability to stimulate a multitude of biologic responses in animals and in humans (3). It is isolated from the outer layer of the outer membrane of Gram-negative bacteria, (e.g., Escherichia coli), which are part of the natural intestinal flora in mammals. Endotoxin is a macromolecule composed of three regions differing in their chemical and biologic properties. The O-specific polysaccharide carries the main serologic specificity of bacteria; and is linked to a core polysaccharide common to groups of Gram-negative bacteria. This core is linked through tile 2-keto-3-deoxyoctonates to a lipid component termed lipid A, which is responsible for most of the biologic activities of endotoxin. Endotoxin is known to have numerous beneficial effects in experimental animals (3), including stimulation of lymphokine production, protection against X-radiation, enhancement of nonspecific resistance to infection, and induction of antitumor effects. In its native form, however, endotoxin cannot be used clinically because of extreme toxicity in humans, which is directly attributable to the lipid A portion of the molecule. Many investigators have explored the possibility of using various chemical methods to selectively reduce the toxicity of endotoxin, while retaining the beneficial biologic activities (8). In general, these efforts have been unsuccessful. Recently, however, a simple method of preparing nontoxic lipid A from endotoxin has been de= veloped, which has led to a better understanding of the structural relationship between lipid A and its biologic properties (4-7 , 10, 12). Detoxification of lipid A is achieved by acid hydrolysis, which removes one of the molecules two phosphate groups. The development and optimization of this approach was closely tied to the introduction of improved physical-chemical means of separating and purifying the individual components of toxic and nontoxic lipid A from complex mixtures of naturally: occurring structural homologs. The structures of the single components could then be determined by a variety of modem spectroscopic techniques; in turn, this allowed the various structural features of the toxic and nontoxic forms of lipid A to be correlated with the observed biologic and chemical properties. This review summarizes the results of these studies. In Thi s I ssue

Separation of the mixture of trehalose 6,6′-dimycolates comprising the mycobacterial glycolipid fraction, “P3”

Summary Ultra-purified trehalose dimycolate, “P3”, an agent with potent host-reactive and immunological properties, including the ability to enhance tumor immune responses, was isolated from various strains of Mycobacterium tuberculosis, M. bovis, M. avium , or M. phlei and subjected to per-trimethylsilylation to permit chromatographic resolution of trehalose dimycolates containing different pairs of mycolic acids. Samples of trimethylsilylated P3 from virulent strains of human and bovine tubercle bacilli were resolved into six different components based on pairs of α, β, and γ-mycolic acids, whereas P3 from avirulent or attenuated strains contained fewer components due to the absence of detectable β-mycolic acid-containing diesters. The virulence of pathogenic mycobacteria may depend to a significant extent upon the presence or absence of a given component.

Modulation of Humoral and Cell-Mediated Immune Responses by a Structurally Established Nontoxic Lipid A

In 1954 Westphal and his associates reported on the isolation of a moiety of bacterial endotoxin which they liberated by means of hydrolysis in dilute acetic or hydrochloric acid solutions (25). The water soluble phase of the hydrolysis reaction contained a haptenic polysaccharide which no longer retained the ability to stimulate physiological responses characteristic of the starting material (3,4). On the other hand, the hydrolysis products extractable with organic solvents did retain some of the endotoxic properties of the original substance, leading Westphal and coworkers to postulate that the “endotoxic” activities of LPS were attributable to a lipidic component, which they designated lipid A (5).

Marihuana: Identification of Cannaboids by Centrifugal Chromatography

Components in extracts of marihuana and hashish have been identified by a chromatographic technique in which centrifugal force is used to accelerate the migration of samples through columns of densely packed microparticulate gel. Rapid qualitative analysis and an estimate of the amounts of cannabinoids present was achieved.

Immunologic significance of the cell wall of mycobacteria.

Summary A technic was described for separation of cell wall and internal protoplasm from cells of Mycobacteria. Data are presented which demonstrated that cell walls produced lesions when injected intradermally into rabbits, whereas protoplasm failed to produce these lesions. Cell walls were also shown to be capable of inducing hypersensitivity of the delayed type. Separation of these morphological elements resulted in definite separation of one element possessing certain biological activities from material not possessing these characteristics. Such initial fractionation of the cell should facilitate purification and characterization of substances which elicit typical tissue responses to infection with tubercle bacilli.

Enhancement of Antitumor Resistance by Mycobacterial Products and Endotoxin

It has been more than 100 years since the suggestion was made that the control of cancer was mediated by immunologic methods. This hypothesis was based on the observation that tumors either partially or totally regressed in a few patients following an acute bacterial infection. In 1911, William Coley pioneered the study of mixed bacterial vaccines or their product known as “Coley’s toxin,” for treating cancers, and there is no doubt that these vaccines had some effect in many cases (Nauts et al., 1946). The effective ingredient appeared to be endotoxin, which caused hemorrhagic necrosis of the tumors. Thus, Gratia and Linz discovered in guinea pigs (1931) and Shwartzman and Michailovsky in mice (1932) that when animals with solid tumors are given single i.v. or i.p. inoculations with small doses of endotoxin, their tumors became hemorrhagic within 24 hr. This was originally done by analogy with the local Shwartzman reaction, with the idea that some hypothetical tumor virus might have prepared the site. It appeared that this type of tumor damage was mediated indirectly because little of the injected endotoxin would be likely to make contact with tumor cells, and would not exert any direct cytotoxicity in any case (Shapiro, 1940; Brailovsky et al., 1973).

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.