The serum thymic factor (FTS) utilized in its synthetic or natural form loses its biological activity in a rosette assay after treatment with a metal ion-chelating agent, Chelex 100. This activity is restored by the addition of Zn salts and, to a lesser extent, certain other metal salts. FTS activation is secondary to the binding of the metal to the peptide. The metal-to-peptide molar ratio of 1:1 provides the best activation. These data indicate the existence of two forms of FTS. The first one lacks Zn and is biologically inactive; the second one contains Zn and is biologically active, for which we propose the name of "thymulin" (FTS-Zn). The presence of Zn in synthetic FTS was confirmed by atomic absorption spectrometry. The interaction between Zn and FTS was fiurther suggested by microanalysis demonstrating the presence of this metal in thymic reticuloepithelial cells.

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

Immunostimulant activities of muramyl dipeptide (enhancement of specific immune responses and of nonspecific resistance to infection) were retained by its N-acetylmuramyl-L-alanyl-D-glutaminyl-n-butyl ester derivative, although very large amounts administered intravenously, or even by the very sensitive intracerebroventricular route, did not elicit fever in the rabbit. This analog also appeared to be devoid of other secondary effects which have been observed after administration of muramyl dipeptide.

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Gly°The following abbreviations are also used: N" for the -lysine derivatives on peptides; Ne for the on peptides. b Descending paper chromatography in solvent VI.e-lysine derivatives 1964). In contrast to the precursor peptide, however, the carbohydrate-free peptide isolated from S. aureus cell walls had only 1 mole of D-alanine, and in addition contained 1 mole of NHS. An Edman degradation carried out on this isolated peptide demonstrated that this ammonia was a substituent of the -carboxyl group of the glutamic acid Tipper et al., 1967a).The studies reported here provide evidence that the basal peptide subunit of the cell wall peptidoglycan of the three Gram-positive bacteria: S. aureus Copenhagen, M. roseus R 27, and Streptococcus pyogenes group A, type 14, has indeed the structure V"-(L-alanyl-D-isoglutaminyl)-L-lysyl-D-alanine.