R. L. Anacker

PHYSICAL ASPECTS OF REVERSIBLE INACTIVATION OF ENDOTOXIN

Most early investigations of the chemistry of endotoxins were limited to gross analyses. I t soon became apparent that subtler means of degradation, such as enzymes, were necessary to determine structural configurations and intramolecular relationships within the endotoxin complex. Just such a subtle endotoxin-degrading system was suggested by the observation of Hegemann (1954) that human plasma factors inactivated the pyrogenic properties of endotoxin. The applicability of this reaction became particularly evident when further studies showed that smaller-sized antigenic units of endotoxin appeared to be produced during the plasma-endotoxin reaction (Rudbach and Johnson, 1961, 1962; Skarnes and Chedid, 1964). The kinetics of the endotoxin-altering reaction, measured by reduction in the pyrogenicity of endotoxin (Keene, Landy and Shear, 1961) and by reduction in ability of endotoxin to precipitate quantitatively with homologous antiserum (Stauch and Johnson, 1959), lent credence to the theory that a plasma enzyme was degrading the endotoxin polymer. This endotoxin-altering system was shown to be inhibited by divalent cations (Rosen et al., 1958) and by other plasma components (Yoshioka and Johnson, 1962). These and other observations (cf. Atkins, f960; Landy, 1960) supported the contention that a plasma enzyme was degrading the endotoxin polymer. However, proof of this must be the isolation of degraded products which have come from a previously intact endotoxin molecule. This evidence has not been presented to date. Furthermore, other evidence has indicated that endotoxin may not be cleaved by plasma hydrolases. Rudbach and Johnson (1964) demonstrated that, after alteration of endotoxin by plasma, activity could be restored by procedures not considered likely to reverse an enzymatic hydrolysis.

STRUCTURE AND BIOLOGICAL FUNCTIONS OF MYCOBACTERIA

The relationship between cell structure and function is obviously too large a subject to be covered in a short presentation. However, certain fundamental concepts can be considered by limiting ourselves to only one function of mycobacterial cells, that of their ability to stimulate an immune response in a suitable animal host. To investigate the relationship between acquired immunity in the host and the part of the whole bacterial cell which incites this immunity, it is necessary to accomplish two things: first, devise a suitable test system for immunity and, second, isolate the specific component of the mycobacterial cell which incites the immunity. To begin with, we must define what we mean by an immune response and, if possible, describe i t in a quantitative fashion. In our laboratory, we place greatest reliance upon a test system which involves challenge of mice by aerosol with small numbers of virulent tubercle bacilli and modification of the effects of such challenge by prior i.v. injection of experimental vaccines.13 Assessment of the response to vaccination includes: 1. The count of grossly visible lung lesions four weeks after challenge. 2. Enumeration of the bacilli in the target organ, the lung, at prescribed inter-

Preparation and properties of an aluminum citrate-endotoxin complex from Salmonella enteritidis.

Summary Treatment of conventional aqueous ether extracted endotoxins from Salmonella enteritidis with aqueous phenol, followed by high speed centrifugation, yielded material having high biological potency with a nitrogen content of 0.4–0.5% and a fatty acid content of 3–4%. Upon reacting this purified endotoxin with lithium aluminum hydride and citric acid solution, an aluminum citrate-endotoxin complex was recovered which had the desirable features of high biological potency and a rapid rate of solution in water. Non-phenolized endotoxins extracted from S. enteritidis with aqueous ether or trichloroacetic acid also formed aluminum citrate complexes under the same conditions. These were biologically potent but somewhat more difficult to dissolve. After prolonged treatment with lithium aluminum hydride, the aluminum citrate complexes still contained 1–2% esterified fatty acids. Lipid fractions isolated from acid hydrolysates of the complexes were biologically inactive.

Effects of pretreatment of mice with BCG cell walls in saline on subsequent vaccination with BCG oil droplet vaccine

Abstract Multiple intravenous injections of bacillus Calmette-Guerin cell walls (BCG CW) suspended in Tween-saline into mice produced an unresponsive state to a subsequent vaccination with BCG-CW-oil droplet mixture. This was manifest by loss of delayed hypersensitivity to PPD as measured by footpad swelling, a diminished granulomatous response in pulmonary tissue, and almost complete abrogation of protection to an aerosol challenge with virulent Mycobacterium tuberculosis , strain H37Rv. The unresponsive state disappeared 20 days after stopping pretreatment. There was evidence of immunity early after H37Rv challenge, but by 4 wk it had markedly diminished and by 6 wk there was no difference between nonvaccinated animals and the pretreated vaccinated animals. Unresponsiveness was specific since pretreated mice were capable of developing delayed type reaction to immunization with modified sheep red blood cells in the usual manner. Relationships among delayed hypersensitivity, granulomatous inflammation, and protection against aerosol infection with virulent M. tuberculosis are discussed in the light of these findings.

Immunization of mice by combinations of inactive fractions of Mycobacterium bovis strain BCG.

Summary In an attempt to identify the mycobacterial cell wall components which enhance resistance in mice to airborne infection with Mycobacterium tuberculosis H37Rv, fractions of BCG were prepared by organic solvent extraction and by alkali or lipase treatment. Although certain of these fractions, when tested individually, were impotent in our protection test, vaccines containing combinations of solvent-extracted, alkali-treated, or lipase-treated cell walls and of an inactive chloroform or methyl alcohol extract were significantly effective. The wax D fraction, but not the wax B or C fractions, of the chloroform extract in combination with “inactive” cell walls was highly protective. Since combinations of either the “inactive” BCG cell wall residue or the BCG chloroform extract with heterologous substances failed to enhance resistance of mice, two or more specific and distinct mycobacterial components may be required in an effective vaccine.

Reaction of Escherichia coli Endotoxin with Lithium Aluminum Hydride

Summary Treatment of Boivin-type endotoxin from E. coli with LiAlH4 according to the method of Noll and Braude yielded a small amount (about 10%) of an essentially pure nonphosphorylated polysaccharide which was nontoxic and nonpyrogenic. In contrast to their RE fraction this product did not stimulate the production of antibodies in rabbits, precipitate antibody nor produce tolerance to endotoxin. The bulk of the product resulting from treatment with LiAlH4 consisted of biologically unaltered endotoxin. It was isolated as a rapidly soluble, lipid-containing aluminum citrate-endotoxin complex, the infrared spectrum of which was nearly identical to that of an RE fraction with 100-fold reduced pyrogenicity. No correlation was found between fatty acid ester content, as estimated from infrared spectra, and toxic and pyrogenic properties of the endotoxin.