Numerous studies have been conducted on immediate and delayed hypersensitivity in mice. These studies have included skin tests, dye extravasation (1)(2)(3)(4)(5) and caliper measurements of paw swelling (6). Studies of hypersensitivity reactions in mice are particularly suitable because of the large number of inbred strains available and the economy of maintenance. Van Arman et al. (7) and Winter et al. (8) have utilized a plethysmographic measurement for the study of irritative agents on swelling of foot paws of rats. This procedure also provides a novel, quantitative, and delicate method for studying dermal allergic reactions in mice, as was shown by the present studies which were designed to answer two questions: Can plethysmographic measurements be performed with precision and reliability on volumes as small as mouse paws; will immunological swelling of mouse paws be of sufficient magnitude to enable one to quantitatively assess the degree of reactivity? In large measure these questions have been answered in our present study.The present communication deals with (i) the application of the plethysmographic method to the study of small volumes, (ii) experiments dealing with the precision and reliability of the method, and (iii) the application of the method to the study of mouse paw sweIling induced by dextran and methylated human serum albumin (MHSA) , agents which produce edema as a result of previous immunization. This technique permits one to distinguish between swelling induced by a toxic or irritative component and swelling immunologically induced by an agent. Recently, Axelrad (9) described an assay for delayed hypersensitivity by immersing rat paw in a mercury-filled beaker placed on a top-loading balance. Materials and Methods.Male LAFl (C57L X A/He) mice purchased from Jackson Memorial Laboratories, Bar Harbor, Maine, 6-16 weeks of age, were used for these experiments. Native dextran, 5-40 milion in molecular weight, and crystalline human serum albumin were purchased from Nutritional Biochemicals, Cleveland, Ohio. Methylated human serum albumin (MHSA) was prepared according to the procedure of Crowle and Hu ( 5 ) . Microliter syringes for subplantar injections were purchased from Hamilton Co., Whittier, California.Plethysmograph measurement of small volumes. The plethysmographic instrumentation was a modification of the method of Van Arman et al. (7). The apparatus consists of a plastic cup filled with mercury in which mouse paws are immersed; small pressure changes, caused by displacement of the mercury level by the immersed object, are pro-portionaIly converted by a low-pressure transducer to electrical signals which are recorded on a chart recorder. The instrument consisted of the following: (i) Gilson model M5 polygraph, Gilson Medical Electronics, Middleton, Wisconsin. (ii) Statham model P23BB transducer, 0.5 cm Hg, Statham Laboratories, Inc., Hato Rey, Puerto Rico. (iii) Plastic cup of 12 mm i.d. The size of the cup was of a convenient diameter to allow for paw immersion without touching the si...

Summary The effects of 500 r whole body γ radiation on direct and developed hemolysin plaque-forming cells in spleen tissue and on serum hemolysin levels of LAF1 mice were investigated. The major findings were: Fourteen-day assay involving a single antigen injection: When radiation was administered 2 days before or 3 days after antigen, spleen hemolysin plaque-forming cell production was markedly lowered when compared with spleen hemolysin plaque-forming cells of non-radiated animals. With this injection scheme, the principal cell type was the developed hemolysin plaque-forming cell. Spleen cellularity of radiated animals was significantly below that of non-radiated control animals.Fourteen-day assay involving two antigen injections: A second antigen injection 6 days after radiation stimulated antibody-forming cell production so that the total spleen hemolysin plaque-forming cells of the once-radiated animals were comparable to non-radiated control animals. Radiation, administered after the second antigen, markedly inhibited hemolysin plaque-forming cell production as well as spleen cellularity.Twenty-eight-day observation period involving multiple antigen injections: The effects of a single dose of 500 r administered to LAF1 mice 3 days after an initial antigen injection and given a total of two, three and four antigen injections at weekly intervals and analyzed at 14, 21 and 28 days, respectively, showed no difference from non-radiated animals in their ability to respond to antigenic stimulation. In these animals, spleen cellularity was consistently lower than in non-radiated animals.

Summary A technique is described for determining serum and tissue hemolysin titers in microliter quantities using a modification of the Jerne technique for hemolysin plaque-forming cells. The technique permits the visualization of augmented hemolysis resulting from the addition of rabbit anti-mouse sera. The enhanced antibody is a γ-globulin as characterized by several criteria. A γ-globulin or 7 S hemolysin was demonstrated in serum collected from animals injected 5 days previously with sheep red blood cells. A comparison of hemolysin plaque-forming cells and serum hemolysin titers indicated that peak levels of the developed hemolysin plaque-forming cells appeared 1 week prior to peak serum levels of developed hemolysin.