When infused into the lateral cerebral ventricles of rabbits, human endogenous pyrogen (EP) preparations induced dose-dependent increases in slow-wave sleep concomitant with increasing body temperature. Heating EP to 70 degrees C destroyed its sleep-promoting and pyrogenic activity. Anisomycin (an antipyretic) prevented EP from increasing body temperature without affecting its sleep-promoting activity. Intravenous injection of EP induced fever and transient increases in slow-wave sleep but failed to induce prolonged increases in slow-wave sleep. We conclude that the somnogenic activity of EP is not secondary to its pyrogenic activity.

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N-Acetylmuramyl-L-alanyl-D-isoglutamine increases the humoral immune response of mice when given in aqueous media instead of the usual water-in-oil emulsions. Moreover, this compound is adjuvant active even by the oral route. In view of studying the relation between chemical structure and biological activity, several synthetic analogs were tested. The immune response could be modulated according to chemical modifications, and the synthetic analog with [-in place of L-alanine was shown to inhibit the immune response. It has been previously reported that water-soluble fractions can substitute for mycobacterial cells in Freund's complete adjuvant (FCA) (1-4). Subsequently, it was shown that synthetic Nacetylmuramyl-L-alanyl-D-isoglutamine (AcMur-L-Ala-DGlu-NH2) has the minimal structure required to duplicate the activity of mycobacteria in Freund's complete adjuvant (5-7). More recently, we have reported that, in contrast to mycobacterial adjuvant preparations, the synthetic compound and a second synthetic analog, N-acetylmuramyl-L-alanyl-D-glutamic acid (AcMur-L-Ala-D-Glu) increase the humoral immune response when given in aqueous media instead of the usual water-in-oil emulsion (8). This finding led us to test several synthetic analogs administered with antigen to ascertain their activity on the immune response. They were administered with the antigen, either in saline to mice or in water-in-oil emulsion to guinea pigs.In the work reported here, we also investigated the influence of various routes of administration of AcMur-L-Ala-D-Glu-NH2 and its influence on mice that have a low response to lipopolysaccharide (LPS) from a Gram-negative bacterium (9). Moreover, the adjuvant activity of a series of synthetic compounds was evaluated after administration to guinea pigs in Freund's incomplete adjuvant (FIA) with ovalbumin. The stability of the two methyl esters was tested by dissolving 2 mg in 0.5 ml of saline. Two hours later no hydrolysis was disclosed by silica gel thin-layer chromatography.Antigens. These were purified preparations of egg albumin (crystallized five times) and bovine-serum albumin (BSA) Fraction V (Miles Laboratories).Antibody Estimation. Anti-ovalbumin and anti-BSA were determined by passive hemagglutination using formalinized antigen-coated sheep erythrocytes (12). Anti-ovalbumin was also evaluated by quantitative precipitation, using Folin's method, and anti-BSA was measured by the antigen-binding Farr technique (13).Delayed-Type Hypersensitivity. Guinea pigs were sensitized to ovalbumin according to the conditions described in Results. Eighteen days later, skin tests were performed intradermally, and 48 hr later diameters of induration were measured. RESULTS

N-Acetylmuramyl-L-alanyl-D-isoglutamine and four other synthetic adjuvants that are structural analogs of part of the mycobacterial peptidoglycan monomer are shown to enhance the nonspecific immunity of mice infected by Kiebsiella pneumoniae. These compounds are active by various routes, including oral administration; they are also effective when administered after challenge. Of the seventeen other analogs tested, none is able to increase significantly resistance to infection, although seven of these molecules are adjuvant-active in saline. Previous results have shown that in contrast to lipopolysaccharides, these synthetic adjuvants are devoid of immunogenicity, mitogenicity, and toxicity in normal or adrenalectomized mice.

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