Recent advances in the prevention of graft-versus-host disease through postthymic T-cell depletion have allowed the use of haploidentical bone marrow cells for immunologic reconstitution of severe combined immunodeficiency disease. We report a male infant with severe combined immunodeficiency (with normal adenosine deaminase) who developed two IgG kappa and one IgA lambda paraproteins 7 weeks following the administration of 1.4 X 10(9) maternal bone marrow cells depleted of postthymic T cells by soy lectin agglutination and sheep erythrocyte rosetting. Serum IgG rose from 128 to 820 mg/dl, and IgA from 0 to 2400 mg/dl, peaking at 10 weeks postgrafting. By 14 weeks posttransplantation T-cell numbers and function had risen to normal (all dividing T cells had the donor karyotype) and paraprotein concentrations began to decline. These observations strongly suggest that the later-appearing T cells regulated the B-cell clones from which the paraproteins were derived. Failure of such function to appear could account for the increased incidence of B-cell lymphomas in severe combined immunodeficiency.
(220) with an Aminco DW2 UV-VIS spectrophotometer (full absorbance scale 0.05 or 0.1), and the activity was recorded on a Hitachi Perkin Elmer Recorder 56. Kinetic studies were performed on the native hemolysate and on hemoglobin-free enzymes that had been partially purified by ACA-34 gel chromatography (2, 5). A unit of activity is defined as the amount of enzyme required to oxidize 1 ,umol of NADH per min in the standard assay.Thermostability Tests. Thermostability tests were carried out by the method of Blume et al. (7). Crude hemolysate with a protein content of-6 mg/ml prepared in 75 mM 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanedipl buffer at pH 7.4 was incubated at 53°. At 0, 20, 60, and 120 min, aliquots (0.5 ml) were chilled for 5 min before being centrifuged for 10 min at 12,000 X g. The supernatant was then assayed for PK activity.Mechanical Shaking Test. Shaking experiments were carried out in a 10 X 40 mm vial at room temperature using a TCS shaker (8). Two milliliters of the hemolysate containing 12. mg of Hb in 75 mM 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol buffer at pH 7.4 were shaken 'at a frequency of 28 Hz for various time periods. After shaking, the precipitate was removed by centrifugation and the supernatant was used for the assay of PK activity.Molecular Weight Determination. The molecular weight of normal and abnormal PK was determined by gel filtration on ACA-34 using hemoglobin (65,000), rabbit muscle aldolase (158,000), and rabbit muscle pyruvate kinase (237,000) as molecular weight standards. The column (1.5 X 99 cm) was equilibrated with a buffer containing 0.1 M KCI, 1 mM 2-morcaptoethanol, and 50 mM Tris-HCI at pH 7.5. One milliliter of crude hemolysate (1:2 hemolysis) was placed on the column and eluted with the same buffer at 4°. The enzyme activity of the eluate was determined by using 0.2-1.0 ml of each fraction as described above.
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