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Osteoclast-activating factor (OAF) is a soluble mediator found in supernates of human peripheral leukocytes which have been cultured with antigens or phytomitogens. OAF is a potent stimulator of osteoclastic resorption of fetal bone in organ culture. The present studies were designed to characterize OAF chemically. Bone resorbing activity from supernates of leukocytes cultured without added plasma was not lost on dialysis using a membrane with a molecular weight cutoff of 3,500, but was lost when heated to 60 degrees C for 30 min. The activity was lost after treatment with trypsin or pronase but not after treatment with ribonuclease or neuraminidase. Papain, which inactivated parathyroid hormone at a concentration of 25 mug/ml, did not inactivate OAF at 250 mug/ml. OAF did not react with an antibody to bovine parathyroid hormone which cross-reacts with human parathyroid hormone. OAF was also distinguished from active metabolites of vitamin D and from prostaglandin by extraction procedures and immunoassay for prostaglandin E(2). When the medium from activated leukocytes cultured with autologous plasma was fractionated by gel filtration on Sephadex, bone resorbing activity eluated both with plasma proteins and in lower molecular weight fractions. However, when medium from leukocytes cultured without added plasma was chromatographed, all the OAF activity was eluted in a sharp low molecular weight peak located between chymotrypsinogen (25,000 molecular weight) and ribonuclease A (13,700 molecular weight). This peak contained about 4% of the total protein originally present in the supernate. Its activity was destroyed by overnight incubation at 37 degrees C at pH 6 or 8, but not at pH 7.2. After incubation at 4 degrees C, the activity was lost at pH 3 or 10, but not at pH 4-9. The active fraction from Sephadex G-100 was therefore chromatographed at pH 7.2 on DEAE cellulose and carboxymethyl cellulose. The active material was not adsorbed; however, about sevenfold further purification was achieved by removal of contaminants. The material obtained after sequential Sephadex, DEAE and, carboxymethyl cellulose chromatography stimulated resorption of fetal rat bone in culture at concentrations of 0.75-3 mug protein/ml, indicating that this preparation of OAF was nearly as potent as bovine parathyroid hormone in this system.

The central 95 percentile estimates of the normal white cell types (as determined by a standard differential count) were calculated from 777 normal individuals. The results were divided into groups by age and sex and expressed both as percentages and as absolute numbers of cells.A similar survey was made using the prototype of a new automated differential counting method (Hemalog-D(TM)). The two sets of laboratory values showed no statistically significant differences regarding age or sex and were strikingly similar in most cell types with the exception of band and juvenile forms of granulocytes. As expected, the normal range using the manual technique was somewhat broader than that found with the automated method. In addition to being the first comparison of machine counts with visual differentials this study provides additional insight into the age distributions of normal cells.

Heterozygotes for Tay-Sachs disease can be distinguished by measuring the serum hexosaminidase activity and calculating the percentage of the heat-labile (A) form. We tested 7565 Ashkenazi Jews in Metropolitan Toronto and found a carrier frequency of 0.071. This figure was similar to the predicted frequency on the basis of caseload over a five-year period. We also found that 15% of women taking oral contraceptives were false-positive carriers. As with pregnant women, these false-positive carriers could be distinguished from true carriers by assaying leukocyte hexosaminidases.

Dialyzable transfer factor from human leukocytes was found to be strongly chemotactic for granulocytes and weakly chemotactic for monocytes in vitro. Chemotactic properties were also demonstrated in vivo in rhesus monkey skin. Initial purification of the dialyzable transfer factor by Sephadex G-25 chromatography revealed multiple fractions containing material with 255-nm absorbance. The fractions containing chemotactic activity were also capable of transferring delayed hypersensitivity to rhesus monkeys. This chemotactic material has an apparent molecular weight of 5000 daltons or less and was not inactivated by goat antibody to components C3 or C5 of human complement; chemotactic activity was lost after storage for two weeks at 4 degrees and the activity of two of three preparations was decreased by heating for 30 min at 56 degrees . This previously undescribed chemotactic activity of dialyzable transfer factor may have significance in relation to cell-mediated immune responses.

(3)H-labelled Pseudomonas endotoxin was incubated in vitro with blood from nontolerant and endotoxin tolerant calves. Formed elements were separated from serial samples of the incubated mixtures. The labelled endotoxin became associated with neutrophils, monocytes, lymphocytes, platelets and erythrocytes. Association of (3)H-endotoxin with formed elements of the blood occurred during the first five minutes of incubation and did not significantly change over the course of a three hour incubation period. Tolerance did not result in increased uptake of (3)H-endotoxin by formed elements of the blood. Tolerance of calves to endotoxin is apparently not due to increased uptake of endotoxin by formed elements of the blood. The Limulus amebocyte lysate assay was unreliable for the detection of endotoxin which was present in calf blood in vitro and requires further modification.