No abstract

Antigen-antibody complexes bind to the surface of macrophages even when little or no binding of antibody alone can be demonstrated (1-5). Binding of complexes is known to be mediated by the Fc region of the antibody molecules (2), and is inhibited by -F c but not -F(ab')2 fragments (6).It has been postulated that complex formation might cause an allosteric change in the antibody molecule, analogous to that observed in electron micrographs by Feinstein and Rowe (7) and Valentine and Green (8) and inferred from physical data by others (9-12). This might result in exposure of a binding site for the macrophage surface, the implication being that combination of the -F a b portions of the antibody molecule with antigen produces a conformational change in the -F c region. An alternative hypothesis, considered here, is that "free" antibody molecules may already have a binding site for the macrophage surface, but that binding is weak. Formation of a complex containing more than one antibody molecule, and thus more than one potential site of attachment, increases the strength of binding. This paper reports our studies on this problem using monovalent, divalent, and polyvalent haptens and labeled purified antibody to the hapten. It was found that monovalent hapten and polyvalent hapten in antigen excess were unable to enhance the binding of antibody to macrophages, whereas polyvalent hapten and divalent hapten at equivalence could do so. Binding of complexes was inhibited by normal rabbit gamma globulin previously cleared of aggregated molecules. Bound complex was eluted from macrophages less easily than was bound antibody alone. These results support the hypothesis that the enhancement of antibody binding to macrophages in the presence of antigen is due to increased energy of binding resulting from summation of individual binding sites, rather than to the occurrence of allosteric change. In addition, saturation studies using ultracentrifuged normal *

A B S T R A C T The level, phenotypes, and isozyme distribution of adenosine deaminase (ADA) were determined in lymphocytes from patients with chronic lymphocytic leukemia (CLL). The ADA level in lymphocytes from patients with untreated CLL was consistently lower than in lymphocytes from normal subjects. No significant differences were found in the phenotype or isozyme distribution. In untreated patients, the ADA level was inversely correlated with the lymphocyte count and the percentage of bursa-equivalent (B) cells. After therapy, a diminution in the lymphocyte count was associated with an increase of ADA activity towards normal levels. The ADA levels were slightly higher in the thymus-derived (T) than in the B lymphocytes from normal subjects. The B cells had lower activity than T cells in patients with CLL. They also had a lower activity than the B cells from normal subjects. The ADA level was 2.3-fold higher in T cells from patients with CLL than in normal T cells. The decrease in ADA levels is an anomaly that is reversible and appears to be a reflection of the proliferation of abnormal B cells in this disorder. Erythrocytes and granulocytes were separated by centrifugation through a mixture of Ficoll-Hypaque (8), while monocytes and platelets were removed on a glass wool column (9); the final preparations usually contained over 95% lymphoid cells. The criteria for selection of patients with CLL were the same as reported previously from this laboratory (10). Unless specified otherwise, the patients had never received therapy. Lymphocytes suspended in 0.05 M sodium phosphate buffer, pH 7.5, at concentrations between 5 X 10' and 2 X 108 cells/ml were disrupted by 5 cycles of .freezing and thawing followed by a 5-s exposure to ultrasound generated by a Heat Systems-Ultrasonics, Inc. cell disruptor (Plainview, N. Y.). The sonicated suspension was centrifuged at 1,100 g for 10 min and the supernatant fluid used for enzyme assays, electrophoresis, and gel-filtration studies. INTRODUCTION7The Journal of Clinical Investigation Volume 57 March 1976 -756-761 756 En.zymie assays. ADA activity was measured by the conversion of adenosine to uric acid, determined spectrophotometrically by the change in absorbance at 293 nm (11). In this assay ADA is linked to an excess of nucleoside phosphorylase and xanthine oxidase. In general, the assay, performed at 37°C in a total volume of 1 ml, contained the following: sodium phosphate buffer, pH 7.5, 50 mM; adenosine, 1.3 mM; nucleoside phosphorylase; 5 fig; xanthine oxidase, 50 ug; and lymphocyte supernatant fluid containing between 30 and 100 ug of protein. In the reaction blanks, an equal volume of 0.05 M sodium phosphate buffer, pH 7.5, was substituted for the lymphocyte supernatant fluid. Erythrocyte ADA activity was determined in a similar fashion, except for the cell disruption which was carried out by the lysis of 0.1 ml of packed erythrocytes with 4 ml of 0.01 M sodium phosphate buffer, pH 7.5, and the substitution of endogenous nucleoside phosphorylase. Protein conc...

The vast majority of human lymphoid neoplasms examined to date have been associated with a proliferation of bone marrow-dependent (B) lymphocytes. In an effort to delineate human tumors of T-cell (thymusdependent) lineage, use was made of the peripheral blood leukocytes of sixteen subjects with various forms of mycosis fungoides. The abnormal cells in the circulation of these patients are morphologically identical to those that infiltrate their nodes and skin. On electron microscopy, such neoplastic lymphocytes (S6zary cells) had "cerebriform" nuclei and an abundance of cytoplasmic fibrils not described heretofore. S6zary cells were nonadherent and nonphagocytic and usually responded to stimulation with phytohemagglutinin, refuting earlier suggestions that the cells represent monocytes or histiocytes. In contrast to chronic lymphocytic leukemia lymphocytes, the Szary cells lacked surface immunoglobulin and receptors for complement. Ultrastructural analysis identified S6zary cells in the center of directly formed rosettes (E-rosettes) characterizing the behavior of T lymphocytes in this test.Though some S6zary cells lacked both T and B cell-surface properties, in general, these observations support the view that the S6zary cell is a neoplastic variant of a thymusderived lymphocyte.