A peroxidase inhibitor in leukemic AKR mouse spleen cells.

enzyme is more potent on a per guaiacol unit basis (21 , 22). The data to be presented indicate no demonstrable SPO or bactericidal activity in spleen cells obtained from leukemic AKR mice. The purpose of this report is to show that the apparent defect in both bactericidal and peroxidase activities is due to the presence of a peroxidase inhibitor in the particulate fraction of spleen cells from leukemic mice. The possible relationship between those observations and the increased susceptibility to infections experienced by patients with lymphocytic leukemia will be discussed. MATERIALS AND METHODS AKR mice were purchased from The Jackson Laboratory, Bar Harbor, Maine, and maintained in the animal facility at St. Margaret's Hospital. Mice were sacrificed by cervical disloca tion and spleens were removed aseptically. Spleen cell suspensions were prepared relatively free of red blood cells by previously described procedures (22). Where homogenates were desired homogenation in 0.25 M sucrose was carried out for 2 min at 3800 rpm in a Potter Elvehjem homogenizer with a Teflon-tipped, motor-driven pestle. Centrifugal fractionation of homogenates was carried out in a refrigerated (4°)Servall RC-2 centrifuge. Bactericidal activity was determined with E. coli from the St. Margaret's Hospital stock culture collection. The orga nisms were quantitated from a turbidimetric standard curve prepared from a turbidity versus colony count relationship. The bactericidal activity of intact spleen cells was ascertained by incubating equal numbers of bacteria and spleen cells for 60 min at 37°in Krebs-Ringer phosphate buffer, pH 7.4. At the end of the incubation period aliquots were removed from both the bacterial control and the tube containing both cells and bacteria. These aliquots were diluted 1:S in 0.5% saponin in order to lyse the spleen cells. Subsequent serial logarithmic dilutions were made in 0.9% NaCl solution. Duplicate 0.1-mi portions were taken from appropriate dilutions for plate counts in trypticase soy agar using a semimicro pour plate procedure. The experiments involving the bactericidal activity of the 20,000 X g pellet fraction of spleen cell homogenates utilized the same strain of E. coli in the logarithmic growth phase. Each tube contained 1 X l0@ bacteria per ml. A typical experiment contained 4 tubes: a bacterial control; S X l0@ M H@ 02 ‘+ bacteria; 20,000 X g pellet fraction containing 0.03 guaiacol unit of peroxidase activity + bacteria; and the complete system consisting of 11202 @ peroxidase containing pellet, and bacteria. The reactions were conducted …