In 1922 Alexander Fleming described the remarkable bacteriolytic activity of an enzyme, lysozyme, which was widely distributed in tissues and secretions (1). Lysozyme (muramidase) is a cationic enzyme, tool wt 14,307, which hydrolyses N-acetyl muramic /3-1, 4 N-acetyl glucosamine linkages in the bacterial cell wall (2). Although a great deal is known about its structure and enzymology its function other than in host defence is still poorly understood.High concentrations of lysozyme are found in leukocytes, especially the polymorphonuclear leukocyte (PMN) 1 and rabbit alveolar macrophage (3). Fractionation studies of the rabbit P M N show that 70 % of its intracellular lysozyme is sedimentable and, unlike other hydrolases, it is found in both the azurophil and specific granules of the cell (4). The BCG-induced rabbit alveolar macrophage is able to release a large fraction of its intracellular lysozyme into the medium during phagocytosis (5) and may secrete lysozyme during cultivation in vitro (6). Large amounts of lysozyme accumulate in the serum and urine of patients (7) and animals (8) bearing monocytic leukemia.In this paper we report that mouse peritoneal macrophages and human monocytes synthesize and secrete substantial amounts of lysozyme in culture. We also study factors which influence the rate of lysozyme production and examine the effect of phagocytosis on its secretion. Materials and MethodsCell C u l t u r e s . -Mouse peritoneal macrophages: Female mice of the NCS (Rockefeller) strain, weighing 25-30 g were used. Peritoneal macrophages were harvested, without anticoagulants, by standard procedures (9); the cells were obtained either 4 days after stimulation by intraperitoneai injection of 0.75 ml thioglycollate medium (I0) or from control, unstimulated mice. The cell yield from unstimulated mice was 5-8 X 106 cells, of which 30-40% were macrophages and the remainder lymphocytes; thioglycollate-stimulated mice yielded 15-20 X 106 cells, consisting of 75-90% macrophages and 10-25% lymphocytes.

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Two previously reported methods (2M NaOH, 18 hr, 100°C; 3M NaOH, 16 hr, 110°C) for alkaline hydrolysis of proteins containing methionine sulfoxide (MetSO) were compared in free amino acid and model protein systems. Recoveries of MetSO from amino acid mixtures after 2M NaOH hydrolysis and ion-exchange chromatography were higher than after 3M NaOH hydrolysis. Recoveries of methionine (Met), MetSO and methionine sulfone (MetS02) from model proteins after 2M NaOH hydrolysis suggested destruction of Met, no production of Mets02 and, in the presence of glucose, possible production of small amounts of MetSO. Except for one soy isolate, measured MetSO was < 7% of total methionine (oxidized plus unoxidized) in soy products. In milk-and soy-based infant formulas, measured MetSO ranged from 7 -32% of totalmethionine.

Clonal, noniridescent mutants ofActinobaciUlus pleuropneumoniae serotypes 1 and 5 were isolated following chemical mutagenesis with ethyl methanesulfonate. The absence of any detectable capsule was confirmed by inhibition radioimmunoassay. There were no differences between the parent and mutant strains in lipopolysaccharide or protein electrophoretic profiles or in hemolytic activity. There was no detectable reversion to the encapsulated phenotype in vitro after passage in mice or pigs or in microporous capsules that were implanted subcutaneously in pigs for 6 weeks. The mutants were able to survive for more than 1 week in pigs following subcutaneous inoculation, which resulted in a strong immune response to whole cells and Apx toxins I and II. Intratracheal challenge of pigs with the serotype 5 mutant at a dose 1 log greater than the 50% lethal dose for the parent resulted in no clinical disease or lesions except in one pig that had slight pneumonia and pleuritis.Twenty-four hours after challenge, A. pleuropneumoniae could not be recovered from the respiratory tracts of any of the challenged pigs except for the one infected pig; this isolate remained noncapsulated. Immunization of pigs with one or both serotypes of noncapsulated mutants protected all pigs against clinical disease following intratracheal challenge with the virulent homologous or heterologous serotype. Nonimmunized control pigs and pigs immunized with a commercial bacterin died or had to be euthanized within 24 h of challenge. Thus, live noncapsulated mutants of A. pleuropneumoniae may provide safe and cost-effective protection against swine pleuropneumonia. These observations support the possibility that noncapsulated mutants of other encapsulated, toxin-producing bacteria may also prove to be efficacious live-vaccine candidates.