The binding and processing of monoclonal human IgG1 by cells of a human macrophage-like cell line (U937)

The goal of these experiments was to assess the relationship between the binding and processing of IgG by Fc-receptor-bearing cells. Cells of the U937 human macrophage-like cell line were incubated with 125I- labeled monomers, dimers, oligomers (composed of 2-4 IgG1 subunits), and HP (heavy polymers composed of 5 or more subunits per polymer) of monoclonal human IgG1 in vitro. Binding was assessed by spinning cells through a layer of phthalate oils. Internalization of IgG1 was assessed by quantitating residual binding to cells after surface-bound IgG was removed by a brief treatment with a solution containing 0.25 M acetic acid and 0.5 M sodium chloride. Catabolism was assessed by measuring the release of radioactive fragments of IgG1, which were not precipitated by 10% trichloroacetic acid. Unstimulated U937 bound about 10,000 molecules per cell of IgG1 monomer, with an equilibrium binding constant (Ka) of 5 X 10(8) M-1. After stimulation with a conditioned medium in vitro, binding per cell was increased 3-7--fold, and the Ka was decreased 2-4--fold. Both unstimulated and stimulated cells internalized and catabolized labeled IgG1 HP, but stimulated cells internalized and digested much more IgG1 HP per cell than unstimulated cells. Both monomers and dimers of IgG1 were internalized and degraded very slowly by stimulated cells, even though both preparations readily bound to cells. In contrast, oligomers and (to an even greater extent) IgG1 HP were internalized and degraded much more rapidly. Internalization of IgG1 HP was markedly inhibited by incubation at 4 degrees C, but not by incubation with a variety of metabolic inhibitors. Catabolism was inhibited by chloroquine and monensin (inhibitors of lysosomal acidification) and by cytochalasin (an inhibitor of microfilament polymerization). Binding to the surface of cells was not markedly inhibited by any agent tested. The capacity of cells to bind labeled IgG1 was markedly reduced by prior incubation in the presence of unlabeled IgG1. This reduction was in part due to the steric blockade of receptors caused by the avid, but reversible, binding of IgG1. In addition, IgG1 oligomers or HP (but not IgG1 monomers or dimers) also caused an irreversible reduction in the number of Fc receptors by a process analogous to receptor down-regulation, as observed in other receptor--ligand systems.     

Possibility of selection against mtDNA mutations in tumors

Several studies of tumors have revealed substantial numbers of clonally expanded somatic mutations in mitochondrial DNA (mtDNA), not observed in adjacent intact tissues. These findings were interpreted as indicating the involvement of mtDNA mutations in tumorigenesis. Such comparisons, however, ignore an important confounding factor: the monoclonal origin of tumors as opposed to the highly polyclonal nature of normal tissues. Analysis of recently published data on the incidence of somatic mutations in nontumor monoclonal cells suggests that, contrary to the prevailing view, the process of tumorigenesis may be accompanied by active selection against detrimental mtDNA mutations.     

Biochemical characterization and cellular localization of a formalin-resistant melanoma-associated antigen reacting with monoclonal antibody NKI/C-3

A monoclonal antibody (MAb NKI/C-3) produced against a purified membrane preparation of human melanoma cells reacts preferentially with sections of formaldehyde-fixed and paraffin-embedded tissues of melanoma, nevocellular nevi, carcinoids and medullary carcinomas of the thyroid. NKI/C-3 did not react with basal-cell carcinoma, brain tissue or brain tumors, and in only 14/196 other tumors was a clear cross-reactivity observed, e.g. with prostate carcinomas and a minority of primary breast, ovarian, lung and clear-cell carcinomas. This antibody was used in an immuno-electron microscopic study for the cellular localization of the antigen. The antigen was dispersed in the cytoplasm of melanoma cells, and more concentrated inside vacuoles and sometimes also on the melanosomes. Occasionally, the antigen was seen on the cell surface. The nature of the antigen was determined in an enzyme immunoassay (EIA). It was found that the antigen is a glycoprotein with a disulfide-dependent configuration that is essential for recognition by the MAb. The antigen was distributed heterogeneously during gel filtration as well as during SDS-polyacrylamide gel electrophoresis in the region of 25-110 kd proteins. A purified antigen preparation that was obtained after affinity chromatography on a column of MAb NKI/C-3 linked to Sepharose 4B contained a carbohydrate:protein ratio of 1:3.5.     

Comparison of color to fluorescein angiographic images from patients with early-adult onset grouped drusen suggests drusen substructure

PURPOSE: Recent structural and histochemical data from our laboratory indicate that human drusen often possess distinct, structural core domains. A similar, corelike structure is frequently noted clinically in some drusen-related conditions. To assess the nature of the corelike structures observed clinically, we evaluated color photographs and fluorescein angiograms from patients with early-adult onset grouped drusen (EAOGD). DESIGN: Retrospective case series. METHODS: The areas of the six largest drusen and hyperfluorescent lesions for eight patients confirmed to have EAOGD were estimated by two masked examiners (RRG, JCF). Data were normalized using a logarithmic transformation and evaluated using a mixed model analysis to account for intragrader, interobserver and intrasubject effects. RESULTS: The average color to hyperfluorescence drusen area ratio was 2.29 (SE, 0.06). The area of drusen viewed in color photographs significantly exceeds that of the corresponding area of hyperfluorescence (P <.0001). The average drusen area (geometric mean) was 0.014 mm(2) (SE, 0.001 mm(2)) and the average hyperfluorescent lesion area was 0.006 mm(2) (SE, 0.0005 mm(2)). The areas of the hyperfluorescent lesions do not vary among the capillary, venous, and washout angiographic phases. The lesions exceed choroidal intensity and demonstrate prominent, uniformly hyperintense fluorescence throughout all but the choroidal phases of the angiogram. CONCLUSIONS: These data show that large visible drusen in EAOGD are concentric to regions of hyperfluorescence, suggesting that drusen may possess clinically detectable, substructural domains. Ongoing studies seek to determine whether clinical evidence of drusen substructure may exist in other drusen-associated disorders, such as in age-related macular degeneration.